A highly sensitive, single selective, fluorescent sensor for Al3+ detection and its application in living cell imaging

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A highly sensitive, single selective, fluorescent sensor for Al3 þ detection and its application in living cell imaging Xing-Pei Ye, Shao-bo Sun, Ying-dong Li, Li-hua Zhi, Wei-na Wu, Yuan Wang

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S0022-2313(14)00384-6 http://dx.doi.org/10.1016/j.jlumin.2014.06.050 LUMIN12777

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Journal of Luminescence

Received date: 10 February 2014 Revised date: 23 June 2014 Accepted date: 24 June 2014 Cite this article as: Xing-Pei Ye, Shao-bo Sun, Ying-dong Li, Li-hua Zhi, Wei-na Wu, Yuan Wang, A highly sensitive, single selective, fluorescent sensor for Al3 þ detection and its application in living cell imaging, Journal of Luminescence, http://dx.doi.org/10.1016/j.jlumin.2014.06.050 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 galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A highly sensitive, single selective, fluorescent sensor for Al3+ detection and its application in living cell imaging Xing-Pei Ye1 , Shao-bo Sun2, Ying-dong Li2 ,Li-hua Zhi1 , Wei-na Wu1,*, Yuan Wang1,* 1

Department of Physics and Chemistry, Henan Polytechnic University, Jiaozuo 454000, P. R. China

2

Institute of Integrated Traditional and Western Medicine, Gansu University of Traditional Chinese Medicine, Lanzhou 730000, P. R. China

ABSTRACT A

new

o-Aminophenol-based

fluorogenic

chemosensor

3,5-bis((E)-(2-hydroxyphenylimino)methyl)-4-hydroxybenzoate

1

have

Methyl been

synthesized by Schiff base condensation of Methyl 3,5-Diformyl-4-hydroxybenzoate with o-Aminophenol, which exhibits high selectivity and sensitivity toward Al3+. Fluorescence titration studies of receptors 1 with different metal cations in CH3OH medium showed highly selective and sensitive towards Al3+ ions even in the presence of other commonly coexisting metal ions. The detection limit of Al3+ ions is at the parts per billion level. Interestingly, the Al(Ⅲ) complex of 1 offered a large Stokes shift (＞120 nm), which can miximize the selfquenching effect. In addition, possible utilization of this receptor as bio-imaging fluorescent probe to detect Al3+ in human cervical HeLa cancer cell lines was also investigated by confocal fluorescence

*

Corresponding author. Tel.: +86 391 3987818; Fax: +86 391 3987811; e-mail:

[email protected] (W.N. Wu); [email protected] (Y. Wang). 1

microscopy. Keywords: Fluorescent sensor; Al3+ ion; Schiff base; cell imaging; Off-On sensor; Fluorescent probe.

1. Introduction The molecular design and construction of selective and sensitive fluorescent chemosensors for determination and bioimaging of various biologically and environmentally relevant metal ions, such as Al3+, Hg2+, Pb2+ and Cd2+, has currently attracted great attention due to the potential impact of their toxic effects [1-10]. Aluminum is the most abundant (8.3% by weight) metallic element and the third most prevalent of all elements (after oxygen and silicon) [11]. It has been widely used in many fields, including the manufacturing of cars, computers, food additives, aluminium cookware, aluminum-based pharmaceuticals and storage/cooking utensils. All of these utilization expose people to aluminum ions [12-14]. When aluminium reached a certain concentration in the human body it caused and catalysed a wide range of diseases, such as anemia, encephalopathy, dementia, gastrointestinal diseases, cardiotoxicity, Parkinson’s disease and most frequently Alzheimer’s disease [15-18]. Furthermore, it is known that 40% of the world’s acidic soils is caused by aluminum toxicity [19]. The incremental increase of Al3+ concentrations in the environment is detrimental to growing plants [20]. Therefore, the design of highly selective chemosensor for Al3+ with low detection limit, has become increasingly important in environmental and clinical chemistry. Currently, only a few fluorescent sensors have been reported for detection of Al3+

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with moderate success, due to the low coordination ability of aluminium [21-29]. In these articles, most of them have a small Stokes shift (＜60 nm), which is often detrimental to its applications [30-33]. A small Stokes shift helps to maximize the selfquenching effect, can cause crosstalk between the excitation light and the resulting fluorescence signals. a smaller separation of the excitation and emission wavelengths also reduces the signal-to-noise ratio in bioimaging applications [34-36]. Moreover, most reported Al3+ sensor often require ultraviolet excitation and laborious multistep organic synthese [37-39], which not only lead to an additional increase in autofluorescence and irreversible damages in cellular work, but also result in expensive operational cost. Therefore, it is of high interest to develop new fluorescent probes for Al3+ with large Stokes shifts, long emission wavelengths, simple and inexpensive methodology. Although many Schiff base derivatives incorporating a fluorescent moiety have been used to detect various metal ions, Schiff base-type Al3+ chemosensors that can be used for cell imaging are very rare [40]. The concentration of free aluminium varies in biological tissues. In order to unravel the physiological processes involving aluminium and the local aluminium concentration inside cellular compartments, it is necessary to design new fluorescent sensors which can monitor the concentration of aluminium at the cellular level. Herein, we design a o-Aminophenol-based Schiff base 1 named as methyl 3,5-bis((E)-(2-hydroxyphenylimino)methyl)-4- hydroxybenzoate which exhibits enhanced fluorescence upon binding to Al3+ with high selectivity. The detection limit of Al3+ ions is at the parts per billion level in CH3OH solution.

3

Surprisingly, The Al(Ⅲ) complex of 1 offered a large Stokes shift (＞120 nm) and long emission wavelengths (528 nm). Potential application of 1 in human cancer (HeLa) cells bio-imaging was also examined by confocal fluorescence microscopy. When HeLa cells were incubated with 5 μM 1 , there was a weak fluorescence from the interior of the cells. A bright fluorescence was emitted from the cells after incubating with 5μM Al(NO3)3 and 1. These results indicate that the probe 1 effectively reached into the cells and can be used to detect Al3+ in living cells.

2. Experimental 2.1. Materials and instrumentation Methyl 4-hydroxybenzoate and hexamethylenetetramine were purchased from Sigma-Aldrich.

Al(NO3)3.9H2O

and

o-Aminophenol

was

purchased

from

aladdin-reagent (China). All chemicals were used without further purification. Solutions of metal ions were prepared with nitrate salts. Methyl 3,5-Diformyl-4hydroxylbenzoate ( DFB ) was synthesized using similar method according to the literature [41]. 1

H NMR spectra were acquired with Varian 300 MHz NMR. The fluorescence

spectra were recorded on a Hitachi RF-4500 spectrofluorophotometer. Infrared spectra (4000-400 cm-1) were determined with KBr disks on a Therrno Mattson FTIR spectrometer.

Fluorescent

images

were

taken

on

Zeiss

Leica

inverted

epifluorescence/reflectance laser scanning confocal microscope.

2.2. Synthesis of o-Aminophenol Schiff-base 1 A solution of o-Aminophenol (220 mg, 2 mmol) in absolute ethanol (20 mL) was

4

added to an ethanol solution (20 mL) containing Methyl 3,5-Diformyl4-hydroxybenzoate (210 mg, 1 mmol). The mixture was refluxed for 3 h under nitrogen atmosphere. The solution was then cooled to room temperature, and the solvent was evaporated. The orange product was recrystallized from ethanol. The yield of 1 was 73%. M.p. 191-194 . Anal. Calc. for C22H19N2O5: C, 67.69; H, 4.65; N, 7.18. Found: C, 67.44; H, 4.88; N, 6.9%. ESI-MS: m/z = 391.2 for [M+H]+. 1

HNMR (300MHz, (CD3)2SO), δ(ppm): 3.32 (s, 3H), 3.88 (s, 3H), 6.88-6.93 (d, 2H, J

= 15 Hz), 6.93-6.99 (t, 2H, J = 7.5 Hz), 7.13-7.24 (t, 2H, J = 14 Hz), 7.40-7.42 (d, 2H, J = 6 Hz), 8.58 (s, 2H), 9.20 (s, 2H). IR (KBr, cm-1): υ(OH) 3246; υ(C=N) 1619. (Scheme 1)

2.3. Optical Detection of Al3+ Using 1 The receptor (10.0 μM) was mixed with different concentrations of metal ions in CH3OH in a 1 cm cell. Solutions of metal ions were prepared using nitrate salts. After equilibrium at ambient temperature for 1 min, fluorescence spectra of the mixtures were measured. Fluorescence spectra were measured at an excitation wavelength of 403 nm.

2.4. Calculation of binding constant between Al3+ and 1 In order to study the binding interaction of ligand with Al3+ in CH3OH solution, the binding constant value of the complex had been estimated from the emission intensity data following the linear Benesi–Hildebrand expression [42]: I0 I - I0

=

I0 [L]

+

I0

1

[L]Ks [M]

5

where I is the change in the fluorescence intensity at 521-526 nm, Ks is the binding constant, [L] and [M] are the concentrations of 1 and aluminium ion, respectively. I0 is the fluorescence intensity of 1 in the absence of aluminium ion. On the basis of the plot of 1/(I-I0) versus 1/[Al3+], the binding constant can be obtained.

3. Results and Discussions The synthesis of 1 is shown in Scheme 1. The reaction of Methyl 3,5-Diformyl-4hydroxylbenzoate with 2 equiv of o-Aminophenol in ethanol as solvent afforded the desired 1 in moderate yield.

3.1 Fluorescence spectra and titration A fluorescence titration of Al(NO)3 was conducted using a 10 μM solution of 1 in CH3OH. In the absence of Al3+, sensor 1 showed a weak fluorescence emission band centred at around 491 nm when excited at 403 nm , attributed to the PET process from the N-donor site of the o-Aminophenol to the DFB moiety. However, the addition of Al3+ significantly enhanced the fluorescence intensity via an intermediate CHEF process. As shown in Figure 1A, the emission peak of 1 increased with increasing Al3+ concentration. With the concentration of Al3+ up to 2 equiv of sensor 1, an 8-fold increase in fluorescence intensity was observed. The quantum yield of the complex was calculated to be 0.217. At the same time, 37 nm red-shift (491nm to 528nm) at the emission maxima was also observed. The binding constant (Ks) of 1 with Al3+ was determined to be 8.8×106 M-1 with a good linear relationship (R=0.9930, Figure 1B), as obtained by fitting the data to the Benesi-Hildebrand expression. This observation also indicates that 1c and Al3+ have a

6

1:1 binding ratio. To further elucidating the binding site, IR experiments were also carried out. As shown in Figure S1 the free ligand exhibited a broad band at 3246 cm-1, which may be assigned to the ν(OH). The band at 1619 cm-1 characteristic of ν(C=N) in the free ligand, was shifted to higher frequency region (1624 cm-1) after coordination between Schiff base ligand and metal salts. This feature indicates the involvement of the azomethine nitrogen atom in coordination and formation of metal-ligand bonds [43,44]. The band at 1217 cm-1 in the IR-spectrum of ligand is ascribed to the phenolic C-O stretching vibration in the case of salicylideneanilines . This band is found in the region 1224 cm-1 in the IR-spectra of the complexes, showing the involvement of the phenolic oxygen in coordination [45]. The non-ligand bands at 552 cm-1 were observed due to M-N, which gave conclusive evidence regarding the bonding of azomethine nitrogen of the Schiff base to the metal ion [43]. All of this reveals formation of a 1:1 stoichiometry for the 1-Al complex. The 1:1 binding mode of the sensor with Al3+ was further confirmed by the ESI-MS mass spectrum of the complex, which showed peaks at m/z = 470.3, assigned to the 1:1 complex [12-+Al3++CH3OH]++Na ( Figure S2).

(Figure 1)

3.2 Selectivity and competitive studies The fluorometric behaviour of 1 was investigated upon addition of several metal ions such as Na+, K+, Ni2+, Zn2+, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Fe3+,

7

Hg2+, Mg2+, Mn2+, Pb2+ and Al3+ in CH3OH (Figure 2 and Figure S3). The emission spectrum of a free 1 ligand showed a weak band with emission maxima positioned around 491 nm on excitation at 403 nm. The examined metal ions such as Na+, K+, Ag+, Pb2+, Mg2+, Ca2+, Ba2+, Zn2+, Cd2+ and Hg2+ hardly had any effect on the emission of 1. While Ni2+, Cu2+, Co2+, Mn2+, Cr3+, Fe2+ and Fe3+ quenched the emission intensity of the ligand to a small extent. In contrast, the addition of Al3+ resulted in a significant enhancement of the emission intensity positioned around 528 nm.

(Figure 2)

To further explore the possibility of using 1 as a practical ionselective fluorescent chemosensor for Al3+, competition experiments were carried out, in which 1 (10 μM) was frist treated with 2 equiv of Al3+, followed by adding 2 equiv of various metal ions including Na+, K+, Ni2+, Zn2+, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Fe3+, Hg2+, Mg2+, Mn2+ and Pb2+. As can be seen from Figure 3A, the enhancement in fluorescence intensity is not influenced by subsequent addition of other metal ions. The selectivity observed for Al3+ over other ions is remarkably high. The detection limit based on the formation of 1-Al was also evaluated. The concentration of 1 was fixed in 10-6 M, and then added different concentration Al3+ between 0 and 2 ppb. According to the plotting of the fluorescence intensity versus the concentration of Al3+ (Figure 3B), the detection limit of Al3+ ions is at the parts

8

per billion level. Obviously, all of these results confirmed that 1 has remarkably high selectivity and sensitivity towards Al3+ ions.

(Figure 3)

3.3. Imaging of HeLa cells incubated with Al3+ and 1 The ability of the fluorescence chemosensor 1 to detect Al3+ in Hela cells was examined. The cells were supplemented with 10 μM 1 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum for 5 h at 370C, leading to very weak intracellular fluorescence as determined by laser scanning confocal microscopy (Figure 4D), and then loaded with 20 μM Al3+ for 5 h at 370C under the same conditions, whereupon a significant increase in the fluorescence from the intracellular area was observed (Figure 4E). The fluorescence image grew brighter as the concentration of Al3+ increased (Figure 4F). The results suggest that sensor 1 can be used to image intracellular Al3+ in living cells. It should therefore be potentially useful for the study of the toxicity or bioactivity of Al3+ in living cells.

(Figure 4)

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4. Conclusion In summary, a o-Aminophenol-based chemosensor was synthesized to exhibit highly sensitive and selective binding with Al3+ over other metal ions. Obvious increases in fluorescence and large red-shift (491nm to 528nm) at the emission maxima were observed upon the addition of Al3+ into the CH3OH solution of chemosensor 1. The detection limit of Al3+ ions is at the parts per billion level in CH3OH solution. The utilization of compound 1 for the monitoring of aluminum(Ⅲ) levels in living cells was examined, and the results indicate that it has potential applications for biological toxicities. Acknowledgements This work was supported by the National Science Foundation of China (21001040).

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Figure 1. (A) Fluorescence response of 1 (10 μM) upon addition of Al3+ in CH3OH (excitation at 403 nm). Slit: excitation/emission=5.0:5.0. (B) Fitted line in the calculation of the binding constant by monitoring the fluorescence intensity changes at 491-528 nm. Figure 2. Fluorescence spectra (excitation at 403 nm) of 1 (10 μM) in CH3OH in the presence of 2 equivalent of Al3+, Fe3+, Cr3+, Zn2+, Ni2+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Pb2+, Mg2+, Mn2+, Na1+, Ag+, or K+. Slit: excitation/emission=5.0:5.0. Figure 3. (A) Fluorescence spectra of 1 (10 μM) with Al3+ (20 μM) in an methanol solution containing 20μM of various metal ions. Excitation: 403 nm. (B) Al3+ concentration (at the parts per billion level) dependent fluorescence intensity change. Figure 4. Fluorescent images of Al3+ in HeLa cells. (A and D) Fluorescence image of HeLa cells incubated with 1 (5 μM). (B and E) Fluorescence image of HeLa cells incubated with 5 μM Al(NO3)3 for 4 h and exposed with 1 (C and F) 20 μM Al(NO3)3 for 4 h and stained with PSI. (A-C) DIC images and (D-F) fluorescent images (excitation = 403 nm). Details are found in the Experimental Section. Scheme 1. Synthesis of 1.

14

Figure l

15

Figure 2

16

Figure 3

17

Figure 4

18

Scheme l

Highlights • A new Schiff base chemosensor is reported. • The sensor for Al3+ offers large Stokes shift. • The detection limit of Al3+ in CH3OH solution is at the parts per billion level. • The utilization of sensor for the monitoring of Al3+ levels in living cells was examined.

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