Fluorescence detection of Fe3+ ions in aqueous solution and living cells based on a high selectivity and sensitivity chemosensor

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 674–681

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Fluorescence detection of Fe3+ ions in aqueous solution and living cells based on a high selectivity and sensitivity chemosensor Hongmin Jia a, Xue Gao a, Yu Shi b, Nima Sayyadi b, Zhiqiang Zhang a,⇑, Qi Zhao a, Qingtao Meng a,⇑, Run Zhang b a b

School of Chemical Engineering, University of Science and Technology Liaoning, Anshan 114044, China Department of Chemistry and Biomolecular Sciences, Faculty of Science and Engineering, Macquarie University, Sydney, NSW 2109, Australia

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A highly sensitive and selective

A highly sensitive and selective fluorescence chemosensor, L, was developed for the detection of Fe3+ ions in aqueous solution and single intact cell.

fluorescence chemosensor (L) was developed.  L features high sensitive with the detection limit for Fe3+ ions was as low as 2 lM.  L could be used for the imaging and quantification of Fe3+ ions in single intact cell.

a r t i c l e

i n f o

Article history: Received 25 January 2015 Received in revised form 28 April 2015 Accepted 29 April 2015 Available online 8 May 2015 Keywords: Chemosensor Ferric ion Detection Fluorescence imaging Flow cytometry

a b s t r a c t Although ferric ion (Fe3+) performs critical roles in diverse biochemical processes in living systems, its physiological and pathophysiological functions have not been fully explored due to the lack of methods for quantification of Fe3+ ions in biological system. In this work, a highly sensitive and selective fluorescence chemosensor, L, was developed for the detection of Fe3+ ions in aqueous solution and in living cells. L was facile synthesized by one step reaction and well characterized by NMR, API-ES, FT-IR, and elementary analysis. The prepared chemosensor displayed excellent selectivity for Fe3+ ions detection over a wide range of tested metal ions. In the present of Fe3+ ions, the strong green fluorescence of L was substantially quenched. The 1:1 stoichiometry of the complexation was confirmed by a Job’s plot. The association constant (Ka) of L with Fe3+ was evaluated using the Benesi–Hildebrand method and was found to be 1.36  104 M1. The MTT assay determined that L exhibits low cytotoxicity toward living cells. Confocal imaging and flow cytometry studies showed that L is readily interiorized by MDA-MB-231 cells through an energy-dependent pathway and could be used to detect of Fe3+ ions in living cells. Ó 2015 Elsevier B.V. All rights reserved.

Introduction

⇑ Corresponding authors. E-mail addresses: [email protected] (Z. Zhang), [email protected] (Q. Meng). http://dx.doi.org/10.1016/j.saa.2015.04.111 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

Ferric ion (Fe3+), an essential trace element, plays significant roles in chemical and biological processes in living organisms [1,2]. In biological systems, Fe3+ ions mainly accumulates within

H. Jia et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 674–681

liver, spleen and bone marrow cells, bound to ferritin [3] to provide oxygen-carrying capacity of heme and acts as a cofactor in many enzymatic reactions [4]. More specifically, it participates in many biological processes ranging from oxygen metabolism to DNA and RNA synthesis [5–7]. The deficiency of Fe3+ ions causes anemia, hemochromatosis, liver damage, diabetes, Parkinson’s disease and cancer [8–11]. High levels of Fe3+ ions within the body have been associated with increasing incidence of certain cancers and dysfunction of certain organs, such as heart, pancreas and liver [12– 16]. Therefore, development of the sensitive and selective detection approaches in biological systems is of great importance for deeper investigating of physiological and pathophysiological functions of Fe3+ in living organisms. Recently, several of methods for the detection of Fe3+ ions have been reported, such as atomic absorption spectrometry, voltammetry, fluorometric and colorimetric method [13,17,18–20]. Among these methods, fluorometric assay with specific fluorescence chemosensor is currently attracting much attention as a method to reveal the molecular functions of the ions in living systems [21–23]. By combining with microscopy imaging, fluorescent chemosensor can be exploited as a powerful approach to investigate ions and biomolecules of interest with high temporal and spatial resolution in a noninvasive manner [24–31]. In addition, precise evaluation of ions in living cells could be realized with high sensitivity flow cytometry assay by utilizing designed florescence chemosensor [32,33]. Although much effort has been focused on the design of fluorescence chemosensor for the detection of Fe3+ ions in living systems, most of the prepared chemosensors exhibited poor selectivity toward other paramagnetic metal ions, such as Cu2+ and Ni2+ [34–50]. Therefore, we have investigated much time and effort to develop a new high sensitivity and selectivity fluorescent method for mapping Fe3+ ions in living cells. In this work, we described a 1,8-naphthalimide-based fluorescence chemosensor (L) by a straightforward synthetic route [51]. The synthesized L was well characterized by NMR, API-ES, FT-IR and elementary analysis. The photophysical properties and recognition behaviors of L have been investigated in detail through fluorescence spectra, UV–Vis absorption spectra in HEPES–THF (7:3, v/v, pH = 7.4) solution. The characteristic fluorescent response of L benefits the intracellular imaging and quantitatively detection of Fe3+ ions in living MDA-MB-231 cells.

Experiment Reagents and instruments All reagents and solvents were of AR grade and used without further purification unless otherwise noted. 1,8-naphthalene anhydride, Hydrazine hydrate and 4-diethylaminosalicylaldehyde were purchased from Sinopharm Chemical Reagent Co., Ltd. (China); Fresh stock solution of metal ions (nitrate salts, except for chloride of Cu+ and Mn2+, all 20 mM) in H2O were prepared for further experiments. 1H NMR and 13C NMR spectra were recorded with a Varian Inova-400 spectrometer with chemical shifts reported as ppm (in CD3Cl, TMS as internal standard). API-ES mass spectra were recorded on a HP1100LC/MSD spectrometer. Melt point was measured with a digital melting point apparatus WRS-2A (Shanghai Precision & Scientific Instrument Co., Ltd). FT-IR spectra were recorded on a Nicolet Magna-IR 750 spectrometer equipped with a Nic-Plan Microscope. Elemental analyses (C, H and N) were performed on an Elementary Vario EL analyzer. Fluorescence spectra were determined with LS 55 luminescence spectrometer (Perkin Elmer, USA). The absorption spectra were measured with a Lambda 900 UV/VIS/NIR spectrophotometer (Perkin Elmer, USA). Fluorescent live cell images were acquired on an Olympus

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Fluoview FV 1000 IX81 inverted confocal laser-scanning microscope with an objective lens (40). The excitation wavelength was 473 nm for imaging of L, and 405 nm for imaging of DAPI. The relative fluorescence intensities of images were analyzed by using an ImageJ software. Flow cytometric analysis was recorded on a BD FACSAria II flow cytometer with a laser at 488 nm. The data were analysed with Flowing software. Synthesis of compound L To a solution of 4-hydrazine-1,8-naphthalimide [52] (0.14 g, 0.5 mmol) in methanol (20 mL), 1.2 equiv. of 2,3-dihydroxybenzaldehyde (0.083 g, 0.6 mmol) in 10 mL methanol was added with continuous stirring. Then, the mixture was refluxed for 3 h to form an orange turbid solution. After cooled to R.T., the precipitate was washed with methanol and dried under vacuum to get the orange solid L with the yield at 88.4%. Mp: 200.9–201.8 °C. 1H NMR: (400 MHz, CDCl3) 11.47 (s, 1H), 9.52 (s, 1H), 9.45 (s, 1H), 8.80 (s, 1H), 8.47(d, J = 6.0 Hz, 1H), 8.38 (d, J = 6.8 Hz, 1H), 7.78 (t, J = 6.2 Hz, 1H), 7.60 (d, J = 6.8 Hz, 1H), 7.27(d, J = 4.0 Hz, 1H), 6.83 (d, J = 5.6 Hz, 1H), 6.73 (t, J = 5.8 Hz, 1H), 4.01–4.04 (t, 2H), 1.58 (m, 2H), 1.31–1.34 (m, 2H), 0.90–0.93 (t, 3H). 13C NMR: (100 MHz, CDCl3) d 164.22, 163.78, 155.42, 149.37, 137.16, 132.14, 131.31, 131.07, 129.00, 122.90, 122.73, 121.82, 118.34, 110.45, 105.70, 58.54, 49.36, 29.86, 18.45. IR (KBr, cm1): 3409, 2972, 2926, 1683, 1635, 1588, 1464, 1391, 1274, 1236, 1086, 1048. API-ES (negative mode, m/z) Calcd for C23H21N3O4: 403.15. Found: 402.20 (LH+). Anal. Calcd for: C 68.47; H, 5.25; N, 10.42; Found: C, 68.03; H, 5.29; N, 10.62. Cation recognition studies by UV–Vis and fluorescence spectroscopy Deionized water was used throughout all experiments. L (10 lM) was added with different metal ions (200 lM) in THF:HEPES (3:7, v/v, pH = 7.4) buffer solution. Excitation wavelength for L was 460 nm. The metal ions recognition behavior was evaluated from the change in spectrum of L upon addition of that metal salt. The light path length of cuvette was 1.0 cm. Association constant calculation Generally, for the formation of 1:1 complexation species formed by the receptor and the guest cation, the following Benesi– Hildebrand [53] equation was used to determining the association constants (Ka).

1 1 1 ¼ þ F 0  F K a ðF 0  F min Þ½Fe3þ  F 0  F min where F0 represent the fluorescence emission at 550 nm of L and F is the intensity in the presence of Fe3+ ions, Fmin is the saturated fluorescence intensity of L in the presence of excess amount of Fe3+ ions; [Fe3+] is the concentration of Fe3+ ions ion added, and Ka is the binding constant. Quantum yield measurement Fluorescence quantum yield was determined using optically matching solutions of fluorescein (Uf = 0.85 in 0.1 M NaOH) as standard at an excitation wavelength of 550 nm and the quantum yield is calculated using the equation [54]:

Uunk ¼ Ustd

 2 ðF unk =Aunk Þ gunk ðF std =Astd Þ gstd

where Uunk and Ustd are the radiative quantum yields of the sample and standard, Funk and Fstd are the integrated emission intensities of

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the corrected spectra for the sample and standard, Aunk and Astd are the absorbances of the sample and standard at the excitation wavelength, and gunk and gstd are the indices of refraction of the sample and standard solutions, respectively. Excitation and emission slit widths were modified to adjust the luminescent intensity in a suitable range. All the spectroscopic measurements were performed at least in triplicate and averaged.

experiments, the MDA-MB-231 cells were washed with PBS (3  2 mL/dish) to remove excess L. The L-deposited cells were further treated with 2 and 10 lM Fe3+ ions in PBS for 10 min, respectively. Then, the cells were washed with PBS for three times (2 mL/well) before confocal microscope imaging.

Stern–Volmer equation

MDA-MB-231 cells (1  105 cells/mL) were plated into a six-chamber culture well and incubated for 24 h. The cells were washed with PBS for three times, then stained with 500 nM L in serum free fresh RPMI-1640 medium at 37 °C in a 5% CO2 incubator for 30 min. After washed with PBS for three times, the cells were treated with 2 and 10 lM Fe3+ ions in PBS for 10 min, respectively. Then, the cells were detached from the well using 0.05% trypsin– EDTA solution and washed with PBS for three times before flow cytometry analysis. Cells incubated with RPMI-1640 for 24 h were used as the controls for all experiments.

F0 ¼ 1 þ K SV ½Fe3þ  F where F0 is the initial emission intensity of L prior to the addition of the quencher, F is the emission intensity at any given concentration [Fe3+] of the quencher and KSV is the Stern–Volmer constant [55].

Flow cytometry analysis

Cell culture Results and discussion The breast adenocarcinoma cell line, MDA-MB-231 was obtained from American Type Culture Collection (ATCCÒ HTB-26™). Cells were maintained in Roswell Park Memorial Institute’s Medium (RPMI-1640) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), L-glutamine (2 mM), penicillin (100 lg/mL), streptomycin sulphate (100 lg/mL) and HEPPS buffer (10 mM). The cells were grown in a humidified 37 °C, 5% CO2/95% air (v/v) incubator. The growth medium was changed every two days. The cells were routinely subcultured using 0.05% trypsin– EDTA solution and growth to 80% confluence prior to experiment. Cytotoxicity of L In vitro cytotoxicity of L was examined by MTT ([3-(4,5-dime thylthazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium) assay on the MDA-MB-231 cells. The cells were seeded at a density of 5  104 cells/mL in a 96-well microassay culture plate and growth 24 h at 37 °C in a 5% CO2 incubator. The culture medium was replaced by the fresh RPMI-1640 medium containing different concentrations of L (2, 4, 6, 10 and 20 lM). Control wells were prepared by the addition of culture medium, and wells containing culture medium without cells were used as blanks. After incubated at 37 °C in a 5% CO2 incubator for 24 h, RPMI-1640 was removed and cells were washed with PBS for three times. The cells were further incubated with 4 h with 100 lL, 0.5 mg/mL MTT solution in PBS. Then, the excess MTT solution was carefully removed from wells, and the formed formazan was dissolved in 100 lL of DMSO (dimethyl sulfoxide). The optical density of each well was then measured at a wavelength of 590 nm using a microplate reader (Bio-Rad, xMark). The following formula was used to calculate the viability of cell growth:

Viability ð100%Þ ¼

mean of absorbance value of treatment group mean of absorbance value of control  100%

Design, synthesis and characterization of the fluorescence chemosensor L We chose 1,8-naphthalimide dyes to design this fluorescent chemosensor primarily owing to their excellent photochemical and photophysical properties, such as long-wavelength absorption and emission, high fluorescence quantum yield and high stability against light [56,57]. These properties make it potentially invaluable as a fluorescent probe for cellular imaging. In addition, the high binding affinity between catechol and iron ensured the high selectivity and sensitivity of the designed L towards Fe3+ ions detection. As shown in Scheme 1, L was synthesized by a straightforward one-step reaction. Briefly, condensation of 2,3-dihydroxybenzaldehyde with 4-hydrazine-1,8-naphthalimide in methanol gave target compound L in 88.4% yield. The structures of the intermediate and L were confirmed by NMR spectroscopy, API-MS, FT-IR, and elementary analysis. Fluorescence emission spectral studies of L in the present of Fe3+ ions For the purpose of evaluating selectivity of the fluorescence chemosensor L, the fluorescence emission intensity changes upon addition of equal amounts of various cations including Ni2+, Pb2+, Zn2+, Fe2+, Fe3+, Ag+, Cd2+, Co2+, Cr3+, Cu2+, Mn2+, Cu+, Al3+, Mg2+, Ca2+, Ba2+, K+, Li+ and Na+ in HEPES buffered THF/H2O solution at pH 7.4 were studied. As shown in Fig. 1, in the presence of

O

O

N

MDA-MB-231 cells were typically seeded at a density of 5  104 cells/mL in a 22 mm coverglass bottom culture dishes (ProSciTech, AU) for the confocal microscope imaging. The culture medium was replaced with the serum free RPMI-1640 medium (2 mL/dish) containing 500 nM L after 24 h growth. Then, the cells were incubated in a humidified 37 °C, 5% CO2/95% air (v/v) incubator for another 30 min. Immediately prior the imaging

O CHO OH

Confocal fluorescent imaging

N

+ OH

CH3OH

HN N

Reflux

HN

OH NH2 OH L Scheme 1. Synthesis of L.

O

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L+X L+X+Fe3+

1.0

(F0-F)/F0

0.8 0.6 0.4 0.2 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Fig. 1. Fluorescence intensity changes of L (10 lM) upon the addition of various metal ions in HEPES aqueous buffer (THF: H2O = 3:7, 20 mM, pH = 7.4). The pink bars represent the emission changes of L in the presence of cations of interest (all are 200 lM). (1) Ag+, (2) Al3+, (3) Ba2+, (4) Ca2+, (5) Cd2+, (6) Co2+, (7) Cr3+, (8) Cu2+, (9) K+, (10) Li+, (11) Mg2+, (12) Na+, (13) Ni2+, (14) Pb2+, (15) Zn2+, (16) Mn2+, (17) Cu+, (18) Fe2+, (19) Fe3+. The sky blue bars represent the changes of the emission that occurs upon the subsequent addition of 200 lM of Fe3+ ions to the above solution. The intensities were recorded at 550 nm, excitation at 460 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

20 equiv. of Fe3+ ions, the fluorescence intensity of L (10 lM) was almost completely quenched (quenching efficiency at 550 nm, (F0  F)/F0  100% = 94%) [58], which could be ascribed to a paramagnetic quenching effect of Fe3+ ions [59]. Nevertheless, no obvious fluorescence intensities changes of L occurred in the presence of Ni2+, Pb2+, Zn2+, Fe2+, Ag+, Cd2+, Co2+, Cr3+, Cu2+, Mn2+, Cu+, Al3+, Mg2+, Ca2+, Ba2+, K+, Li+ and Na+ under the same conditions. In addition, upon addition of 200 lM Fe3+ ions to the above solution giving rise to drastic quenching in accordance with the addition of equal amount of Fe3+ions alone, indicating that Fe3+-specific response was not disturbed by the competitive cations. The fluorescence titration of L in the presence of different Fe3+ ions concentrations was performed in buffered THF/water mixture (3/7 v/v; HEPES, 20 mM; pH 7.4). As shown in Fig. 2, L displayed strong green fluorescence emission at 550 nm (U = 0.5753). Upon increasing the concentration of Fe3+ ions (0–200 lM), L exhibited significant quenching in the fluorescence intensity (U = 0.0531) with a 20 nm hypsochromic shift of the maximum emission. The

analysis of Stern–Volmer graph represents a plot of (F0/F) vs. Fe3+ ions was shown in Fig. 3. The apparent linearity of the Stern– Volmer plots at low concentration of Fe3+ ions indicated that the possible static quenching due to the formation of a non-fluorescent stable complex L–Fe3+ [60]. Job’s plot evaluated from the fluorescence emission spectra of the titration solution showed the inflection point at 0.5 (Fig. 4a)), which supported the formation of a 1:1 stoichiometry complex. Based on the 1:1 binding mode, the association constant (Ka) was evaluated using the Benesi–Hildebrand method and was found to be 1.4  104 M1 (Fig. 4b). Furthermore, the fluorescence changes of L at 550 nm exhibited a linear correlation to Fe3+ ions in the concentration range from 0 to 50 lM (R2 = 0.9948) (Fig. 5). The detection limit for Fe3+ ions was estimated to be 2 lM based on a 3r/slope [61], which is low enough for Fe3+ ions detection in aqueous medium and many biological systems [62]. UV–Vis spectroscopic studies of L in the present of Fe3+ ions The binding behavior of L toward different metallic cations was further investigated by UV–Vis absorption spectra. The absorption spectra of the L in THF/H2O (3:7, v/v, pH = 7.4) medium exhibited an absorption band with a maximum absorbance peak at about 470 nm. Upon an increase in the concentration of Fe3+ ions (0– 200 lM) resulted in a 15 nm bathochromic shift of the maximum absorbance peak and a gradual increase of the broad absorption band from 470 to 650 nm as shown in Fig. 6, indicating that the coordination of L to Fe3+ ions center. Furthermore, the Fe3+ ions titration solution of L exhibited an obvious color changed from yellow to orange (Fig. 6 insert), indicating that L can serve as a ‘‘naked-eye’’ Fe3+ ions indicator in aqueous medium. However, in the presence of other competitive metal ions such as Ag+, Co2+, Ni2+, Cd2+, Zn2+, Fe2+, Cr3+, Mn2+, Cu+, Al3+, Li+, Ca2+, Mg2+, Ba2+, K+, Na+ and Li+ did not induce any UV–Vis absorption responses and color changes except Pb2+ and Cu2+ (Fig. 7). Effect of pH In order to investigate the influence of the different acid concentration on the fluorescence spectra of L and find a suitable pH span in which L can selectively detect Fe3+ ions efficiently in

2.4 140

1.0

0 equiv F550

0.4

100 80

2

R = 0.99018

0.6

F0/F

Fluoresence Intensity

120

0.2

20 equiv.

Y = 1.09753 + 1.27961X

2.0

0.8

0

5

10 15 3+ [Fe ]/[L]

1.6

20

1.2

60 40

0.8

20 0 500

550

600

650

700

Wavelength (nm) Fig. 2. Changes in the fluorescence spectra of L (10 lM) in HEPES aqueous buffer (THF: H2O = 3:7, 20 mM, pH = 7.4) with different amounts of Fe3+ ions (0–200 lM). The inset exhibits a fluorescence titration profile at 550 nm upon the addition of Fe3+ ions. Excitation at 460 nm.

0

10

20

30

40

50

60

3+

[Fe ] (µM) Fig. 3. Stern–Volmer quenching plot of L by Fe3+ ions presented in the form of (F0/F) vs. [Fe3+]. The F0 and F values were determined from the integrated emission intensity in the absence and presence of Fe3+ ions quencher, respectively. Excitation at 460 nm.

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0.04

(b)

(a) 60

Y=0.00718+0.52762X 2

1/(F0-F)

F-F0(1-XFe)

0.03 40

R =0.98189 0.02

20 0.01 0 0.0

0.2

0.4 3+

0.6

0.8

1.0

0.00

0.02

0.04 3+

3+

0.06

6

1/[Fe ](10 )

Fe /[L + Fe ]

Fig. 4. (a) Job’s plot of L with Fe3+ ions. The total concentration of L and [Fe3+] was 10 lM. (b) Benesi–Hildebrand plot of the L–Fe3+ complex. The monitored emission wavelength was 550 nm and excitation wavelength was 460 nm.

60

1.0

Y = 5.4684 + 1.1131X 2 R = 0.9948

50

0.8

Absorbance

F0 −F

40 30 20

0.6

L+Fe 0.4

10 0

0.0 10

20

30

40

L+Pb

300

50

350

400

3+

Fe (µM)

500

550

600

Fig. 7. UV–Vis absorption spectra change of L (10 lM) upon addition of various metal ions (0–200 lM).

160

3+

+Fe

F550

Absorbance

450

2+

Wavelength (nm)

Fig. 5. The fluorescence intensity changes at 550 nm of L (3 lM) as a function of Fe3+ ions concentration (0–50 lM). Excitation was performed at 460 nm.

0.4

2+

L+Cu 0.2

0

3+

0.2

120 3+

Fe

80 0.0 400

450

500

550

600

650

Wavelength (nm) Fig. 6. Changes in the UV–Vis absorption spectra of L (10 lM) with various amounts of Fe3+ ions (0–200 lM). Inset: the color change of L upon addition of 200 lM Fe3+ ions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

environmental and biological fields. The fluorescence pH titrations of L were carried out. As depicted in Fig. 8, the fluorescence emission intensity (550 nm) of L remains essentially constant over a broad pH range 4.0–9.0. In the presence of 200 lM Fe3+ ions in aqueous solution, the fluorescence response is slightly affected by pH with gradually decreased fluorescence intensity from pH 4.0–9.0, which suggests that chemosensor L is suitable for application under physiological conditions [63].

40 4

5

6

7

8

9

pH Fig. 8. Effect of pH in different solutions on the fluorescence intensity of L (10 lM) in the absence and in the presence of Fe3+ ions (200 lM). Excitation at 460 nm.

Fluorescence bioimaging of Fe3+ ions in living cells using L Prior to the confocal microscopy imaging, the long-term cellular toxicity of L towards the MDA-MB-231 cell line was examined by means of a MTT assay. As shown in Fig. 9, after the cells were incubated with different concentrations of L (2, 4, 6 and 10 lM) for 24 h, the cell viabilities were still greater than 80%. Even in the high concentration of L (20 lM), the cellular viabilities were estimated

H. Jia et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 674–681

Cell viability (%)

100

75

50

25

0 0

2

4

6

10

20

Concentrations (µM) Fig. 9. Cell viability values (%) estimated by MTT proliferation test vs. incubation concentrations. MDA-MB-231 cells were incubated in the L containing RPMI-1640 at 37 °C in a 5% CO2 incubator for 24 h.

to be more than 70% after incubation for 24 h. These results reveal that L is low toxic to the cultured MDA-MB-231 cells. As described above, only addition of Fe3+ ions into an L solution induced a significant fluorescent quenching in the emission spectra. In addition, the properly biocompatibility of L to MDA-MB-231 cells was illustrated by the MTT assay. Therefore, L

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has the potential to serve as a fluorescent chemosensor to detect the Fe3+ ions in living cells. The applicability of L in the monitoring of intracellular Fe3+ ions was investigated by confocal luminescence microscopy. As shown in Fig. 10, intense intracellular emission was observed within the MDA-MB-231 cells incubated with 500 nM L for 30 min. Overlay of confocal images of L (Fig. 10a2) and DAPI ((Fig. 10a3) showed that the fluorescence was evident in the cytoplasm over the nucleus (Fig. 10a4). Then, L deposited MDA-MB-231 cells were incubated with Fe3+ ions (2 and 10 lM) in PBS for another 10 min at the same condition. As expected, an obviously fluorescent quenching from the intracellular area was observed after the treatment of Fe3+ ions (Fig. 10b and c). It is interesting to note that less interiorization was observed when MDA-MB-231 cells incubated with L at a temperature of 4 °C (Fig. 11b). As shown in Fig. 11c, the relative fluorescence intensity of cells incubated at 37 °C is approximately 3-fold higher than the cells incubated at 4 °C. The results imply that L enters cells and the subsequent localization is partially through an energy-dependent pathway such as endocytosis. Investigations on the detailed internalization mechanism are underway. Then, the fluorescence detection of intracellular Fe3+ ions was quantitatively determined by flow cytometry. We compared the different fluorescence intensity based on the mean fluorescence intensity (MFI) of the cell population (Fig. 12). The control group (MDA-MB-231 cells only) exhibit negligible background fluorescence (MFI: 3.09). When the cells stained with 500 nM L for 30 min, the fluorescence intensity of the cell population increases

Fig. 10. Representative confocal fluorescence images of L in MDA-MB-231 cells. The cells were stained with 500 nM L for 30 min (a), and then treated with 2 lM (b) and 10 lM Fe3+ ions (c) for another 10 min, respectively. Scale bar = 30 lm.

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Fig. 11. Confocal fluorescence images of MDA-MB-231 cells incubated with 500 nM L for 30 min at 37 °C (a), and 4 °C (b). Scale bar = 30 lm.

Acknowledgments This work is supported by the National Natural Science Foundation of China (No. 21301011) and the State Key Laboratory of Fine Chemicals (KF1305). Dr. Zhang wishes to acknowledge the financial support by Macquarie University Research Fellowship Scheme (MQRF-1487520). References

Fig. 12. Flow cytometry analysis of MDA-MB-231 cells stained with L and its fluorescent response to Fe3+ ions. (1) Control group, MDA-MB-231 cells only, (2) cells stained with L for 30 min, and then treated with 2 lM (3), 10 lM (4) Fe3+ ions for another 10 min.

dramatically (MFI: 2517.55). The fluorescence quenching was observed when the cells were further treated with 2 lM (MFI: 1218.97) and 10 lM (MFI: 2.39) Fe3+ ions.

Conclusions In summary, we reported a novel fluorescence chemosensor (L) for the detection of Fe3+ ions in aqueous solution and living MDA-MB-231 cells. The synthesized L displayed an excellent selectivity for Fe3+ ions over other environmentally and biologically relevant metal ions including other paramagnetic metal ions. With increasing concentrations of Fe3+ ions, the fluorescence intensity maximum at 550 nm was gradually quenched. The high sensitivity and selectivity, low cytotoxicity of L render the successful application of detection Fe3+ ions in living system. Confocal imaging and flow cytometry studies showed that L is readily interiorized by MDA-MB-231 cells and could be used to the detection of Fe3+ ions in cellular level. All these findings indicate that L is a very promising candidate as live-cell imaging reagent for the Fe3+ ions detection and could contribute to the understanding the roles of Fe3+ ions in living system.

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