Biosensors and Bioelectronics 65 (2015) 91–96
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A two-photon probe for Al3 þ in aqueous solution and its application in bioimaging Haihong Wang, Bei Wang, Zhaohua Shi, Xiaoliang Tang, Wei Dou, Qingxin Han, Yange Zhang, Weisheng Liu n Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 May 2014 Received in revised form 1 September 2014 Accepted 7 October 2014 Available online 16 October 2014
A salicylimine probe L with a simple structure has been researched more in-depth on fluorescence sensor properties based on two-photon (TP) absorption. L displays excellent selective turn-on fluorescence response for Al3 þ in hexamethylenetetramine-buffered (HMTA) aqueous solution (0.3 M, pH ¼5.8) under one-photon (OP) excitation. With the help of OP fluorescence, TP fluorescence titration, UV-spectra titration and Job's plot, the stoichiometric ratio of L with Al3 þ was determined to be 1:1. The coordination sites and the coordination mechanism of L with Al3 þ were analyzed in detail through 1H NMR data. Not only with a detection limit of 5.2 10 9 M in vitro, but also the probe has been successfully used in the live cells and tissues for the imaging of Al3 þ with TP fluorescence microscopy due to the enlarged TP cross section, providing a novel testing method for measuring Al3 þ in solution or cell tissue with low autofluorescence and cytotoxicity. & Elsevier B.V. All rights reserved.
Keywords: Two-photon fluorescence Al3 þ probe Bioimaging
1. Introduction Aluminum is the most abundant metallic element in earth's crust and its compounds are widely used in chemical industry production, from food additives, medicines, cookware, to production of light alloys, etc., which are closely associated with our daily life. It is universally known that Al is not the element people need and its accumulation can bring toxicity to the living body. When it comes to acid rain Al3 þ which comes from the soil with poor buffer ability enters into the rivers and lakes, increasing the concentration of Al3 þ in natural water and giving rise to the death of plants (Emmanuel and Ryan, 1995; Ren and Tian, 2007) and fishes (Alstad et al., 2005). In the human body Al3 þ can be a competitive inhibitor of several essential elements, such as Mg2 þ , Ca2 þ and Fe3 þ due to their similar characteristics (Das et al., 2012; Banerjee et al., 2012; Williams, 2002). An excess of Al3 þ may have a bad effect on the central nervous system, further leading to dementia, myopathy, and Alzheimer's disease (Perl et al., 1982; Perl and Brody, 1980). In order to prevent the absorbtion of Al3 þ and study in depth the key role of the metal ion in our body, it is of great importance to detect Al3 þ in vivo and in vitro with different measuring methods. n
Corresponding author. Fax: þ 86 931 8912582. E-mail address: [email protected] (W. Liu).
http://dx.doi.org/10.1016/j.bios.2014.10.018 0956-5663/& Elsevier B.V. All rights reserved.
In recent years, organic fluorescent probes were widely applied to detect biologically important ions for their generally nondestructive character, high sensitivity, instantaneous response, and the wide range of indicator dyes available (Amendola et al., 2006; Wang et al., 2010). Though many reports on the detection of Al3 þ were published (Das et al., 2013), most of them were always problematic due to the lack of spectroscopic characteristics and poor coordination ability compared to transition metals (Soroka et al., 1987). Because Al3 þ can be hydrolyzed easily to generate Al(OH)3 precipitation most of the probes cannot be applied in water let alone in buffered solution which is beneficial to practical application, such as biological imaging (Zhao et al., 2006; Li et al., 2014). So far, there have been some Al3 þ fluorescent probes being applied to cell imaging, but all of them were measured with OP microscopy (Wang et al., 2010; Kim et al., 2012; Zhi et al., 2013) which required a rather short excitation wavelength and would produced background fluorescence (Guo et al., 2013; Sun et al., 2012) as well as photo-damage. In order to overcome the above limitations, TP microscopy, as a kind of new technology, has been used in this area recently. Multiple-photon excitation technology is the progress where two or more photons emitted by a longwave light source can excite a short-wave one (Biswas et al., 2011). In contrast to OP microscopy, TP microscopy offers several distinct advantages: (1) small scattering effects can make light penetrate
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samples deeper and more easily; (2) reduced photo-damage and photobleaching can make TP microscopy more suitable for viable cell observation (Biswas et al., 2011). Previously, scientists have spared no effort in applying TP microscopy to the field of TP fluorescence imaging (Taki et al., 2004; Dong et al., 2013; Zheng et al., 2014) and developed lots of new TP fluorescence probes with large TP absorption cross sections for the use in biological imaging (Li et al., 2011; Wang et al., 2012; Stewart et al., 2008; Cao et al., 2007; Zhu et al., 2010; Yong et al., 2009; Padilha et al., 2011; Chattopadhyaya et al., 2011; Shao et al., 2011; Poronik et al., 2013; Zhang et al., 2009; Nguyen et al., 2010; Chen et al., 2009; Feng et al., 2010). Cho and Kim et al. developed a series of TP fluorescence probes for detecting Na þ (Kim et al., 2010b, 2010a), Ca2 þ (Kim et al., 2010a, 2007b), Mg2 þ (Kim et al., 2007a), Zn2 þ (Kim et al., 2008), H þ (Park et al., 2012; Lee et al., 2013), thiol (Lee et al., 2010), NO (Dong et al., 2013), etc., and applied them not only in living cells but also in tissues. In addition to this several other ions have also been studied, such as Cd2 þ (Liu et al., 2012; Li et al., 2012), Hg2 þ (Rao et al., 2012), Cu2 þ (Fu et al., 2013), F (Zhang et al., 2011), etc. Though there were several studies on the TP absorption properties of aluminum complexes, they were mainly focused on theoretical study (Liu et al., 2004a; Zhang et al., 2007; Liu et al., 2004b; Yang et al., 2011). Calixarene-based TP fluorescent sensors have been synthesized by Cho et al. to sense metal ions Pb2 þ and Al3 þ but they cannot distinguish the two ions in CH3CN (Kim et al., 2006), which is the most basic requirement probes should meet. Even so, to the best of our knowledge, there have not been any reports on TP fluorescence microscopy imaging of Al3 þ . Considering appealing applications of Schiff base derivatives in optical sensing, antitumor ability (da Silveira et al., 2008), antioxidative effects (Li and Yang, 2009), attractive electronic and photophysical property (Kasselouri et al., 1993) we researched more in-depth fluorescence sensor properties of a simple Schiff bases ligand N′-(2-hydroxybenzylidene)benzohydrazide (L), which has a large TP absorption cross section when adding Al3 þ . L displays excellent selectivity for Al3 þ in aqueous solution and it can also be used for living cell and tissue imaging.
2. Materials and methods 2.1. Materials and instrumentation All reagents and solvents employed for synthesis were commercially available and used as received. Deionized water was used as solvent. All of the solvents used were of analytical reagent grade. The solutions of metal ions were prepared from either their nitrate or their chloride salts. HMTA buffered aqueous solution (0.3 M, pH ¼5.8) was prepared in double-distilled water. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker DRX400 spectrometer in d6-DMSO and d4-CH3OH with TMS as internal standard. Mass spectra was measured on a Bruker Esquire 6000 mass spectrometer by means of the electronic spray ionization (ESI) technique. Absorption spectra were recorded using a Varian Cary 100 spectrophotometer. Fluorescence measurements were made on a Hitachi F-7000 fluorescence spectrophotometer equipped with a xenon lamp as the excitation source. The path length was 1 cm with a cell volume of 3.0 mL. Excitation and emission slits of 2.5 nm were used for the measurements of fluorescence. Elemental analyzes were conducted using an Elemental Vario (EL) instrument. Melting point was determined on an X-6 melting point apparatus without calibration (Beijing Fuka Keyi Science and Technology Co., Ltd.). Fluorescent quantum yields were determined by an absolute method using an integrating sphere on FLS920 of Edinburgh Instrument. All pH measurements
were made with a pH-10 C digital pH meter. All the measurements have been done at room temperature unless otherwise stated. 2.2. Optical detection of Al3 þ The probe L (10.0 μM) was mixed with different concentrations of metal ions, 10.0 μM Al(NO3)3 and Ga(NO3)3, 50.0 μM NaCl, KCl, AgNO3, Cr(ClO4)3, Co(ClO4)2, Ni(NO3)2, FeCl3, Cu(NO3)2, CaCl2, Mn(ClO4)2, ZnCl2, LiClO4, Cd(NO3)2, MgCl2 and Hg(ClO4)2 in HMTA buffered aqueous solution (0.3 M, pH ¼5.8). After equilibrium at ambient temperature for 25 min, absorption and fluorescence spectra of the mixtures were measured. Fluorescence spectra were measured at an excitation wavelength of 366 nm, and emission spectra were collected from 390 to 600 nm. 2.3. Measurement of TP cross section (δ) TP excitation fluorescence spectra were measured using a Steady-state and Lifetime Fluorescence Spectrometer (FLS920, Edinburgh Instruments). TP absorption cross sections (δ) at different excitation wavelengths were determined by comparing their TP excitation fluorescence with that of fluorescein (Albota et al., 1998) in aqueous solution, according to the following equation:
δ = δref Φref /Φcref /cnref /nF /Fref
(1)
In Eq. (1), the subscript ref stands for the reference molecule. δ is the TP absorption cross section value, c is the concentration of solution, n is the refractive index of the solution, F is the TP excitation fluorescence integral intensities of the solution emitted at the exciting wavelength, and Φ is the fluorescence quantum yield. The δref value of reference was taken from the literature (Li et al., 2012). 2.4. TP fluorescence imaging TP fluorescence images of L labeled cells and tissues were obtained by exciting the probes with a modelocked titanium–sapphire laser source (Mai Tai DeepSee, 80 MHz, 90 fs) set at wavelength 730 nm with AN Olympus FV1000 laser confocal microscope IΧ81 with 60 objective, numerical aperture (NA)¼0.4. The images signals in 420–470 nm range were collected by internal PMTs in 12 bit unsigned 1024 1024 pixels at 40 Hz scan speed.
3. Result and discussion 3.1. Synthesis of L L was facilely synthesized from the reaction of benzohydrazide with salicylaldehyde (Scheme 1). The molecular structure and its purity were confirmed by NMR and ESI-MS.
Scheme 1. Synthesis of the fluorescent indicator L. Reagents and conditions: (i) ethanol, reflux, 8 h, 82%.
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Fig. 1. (A) The normalized fluorescence spectra of L (10 μM) before and after addition of 50 μM Pb2 þ , Mg2 þ , Hg2 þ , Cu2 þ , Co2 þ , Ni2 þ , Li þ , Cr3 þ , Ag þ , Cd2 þ , Na þ , K þ , Fe3 þ , Ca2 þ , Zn2 þ , Mn2 þ , 10 μM Al3 þ and Ga3 þ in HMTA buffered aqueous solution (0.3 M, pH ¼ 5.8). (B) The normalized fluorescence spectra of 10 μM L after addition of increasing amounts of Al3 þ ions (0, 2, 4, 6, 8,,10, 12, 14 μM) at room temperature. Inset. The normalized fluorescence intensity at 452 nm as a function of Al3 þ concentration (λex ¼ 366 nm).
3.2. Effect of pH Fluorescence sensors based on electron donor/acceptor are usually disturbed by protons in the detection of metal ions, so it is necessary to investigate pH effect on L in the absence and presence of Al3 þ and find optimal sensing conditions. There is no change in the fluorescence spectrum of L after the addition of Al3 þ at pH below 3.0, which is because the combination of protons and N atoms in –CQN– groups inhibits the coordination of Al3 þ with L. On the other hand, the fluorescence of L has no response to Al3 þ at high pH values because the hydrolysis happens between OH and Al3 þ (Fig. S1). The best pH corresponding to the highest intensity is 5.8, which is within the biologically relevant pH range (5.5–7.5) (Shi et al., 2013), indicating that L could be applied as an Al3 þ sensor in biological organisms potentially, such as lysosomes (Dong et al., 2013). 3.3. Ion selectivity and competitiveness The selectivity of L for various common cations was investigated in HMTA buffered aqueous solution (0.3 M, pH ¼5.8) (Fig. 1A). Upon excitation at 366 nm, L (10 mM) showed weak emission intensity at ca. 450 nm with a low fluorescence quantum yield (Фf ¼ 0.05). And there was no obvious change in the spectra after adding 5 equiv other metal ions to the aqueous solution of L, such as Li þ , Na þ , Mg2 þ , K þ , Ca2 þ , Mn2 þ , Fe3 þ , Co2 þ , Ni2 þ , Cu2 þ , Zn2 þ , Ag þ , Cd2 þ , Hg2 þ and Pb2 þ . However, the addition of 1 equiv Al3 þ brought significant enhancement in the fluorescence intensity at 452 nm with a high fluorescence quantum yield (Фf ¼0.49) within 25 min (Fig. S2). This is attributed to the formation of L–Al3 þ complex and thus the increase of p–π conjunction. Ga3 þ ion, the same family as that of Al, also increased the intensity at about 465 nm, but this was considerably lower than that of Al3 þ , having little interference on the selectivity of L for Al3 þ . Moreover a little red shift, ca. 15 nm, which could be seen obviously, existed in the spectra after the treatment with 1 equiv. Ga3 þ . In order to prove that the sensor was highly selective for Al3 þ , excessive other metal ions were added in the Al3 þ determination protocol. As shown in Fig. S3, the competing ions have more or less influence on the fluorescence intensity. Among the ions, L was responsive to Al3 þ obviously, although the interference by Ga3 þ was the largest along with Cr3 þ , Cu2 þ and Ca2 þ . The interference
of the other metal ions is relatively limited, indicating that L can be applied to detect Al3 þ effectively. 3.4. Fluorescence titration Fluorescence titration was performed to study the stoichiometric ratio of the L–Al3 þ complex (Fig. 1B). With the addition of Al3 þ to L (10 mM), the fluorescence intensity increased accordingly; but when the concentration of Al3 þ was up to 10 mM, the rising tendency slowed down gradually, indicating the 1:1 binding of L to Al3 þ , which was consistent with the result from Job′s plot (Fig. S4). The association constant of L for Al3 þ was estimated to be 5.5 104 M 1 in the same media (Fig. S5). The limit of detection (LOD) was calculated to be 5.2 10 9 M (Fig. S6). According to China EPA standard the concentration of Al3 þ should be lower than 0.05 mg L 1 (1.85 10 3 M) in drinking water, which means that our proposed fluorescent method based on probe L is sensitive enough to monitor the water quality of drinking water. 3.5. Absorption spectra studies The absorption spectra of L are also studied with increasing concentration of Al3 þ to analyze the coordination mode of L–Al3 þ in solution. As shown in Fig. S7, L (10 mM) has two main absorption peaks at 288 and 325 nm; the intensity of both decreases with the addition of Al3 þ . Meanwhile, the two bands showed a red-shift accompanied by generation of a new absorption peak at 367 nm and three isosbestic points at 300, 322, 342 nm. This suggests a coordination between Al3 þ and L, which extends the conjugated system and leads to new absorption in the long wavelength region. The absorbance peak no longer changed when the concentration of Al3 þ increased from 10 μM to 14 μM, indicating 1:1 binding between L and Al3 þ . 3.6. Product analysis The determination of the coordination site of L with Al3 þ was examined by 1H NMR in d4-CH3OH. Spectra of L before and after addition of 1 equiv Al3 þ are shown in Fig. S8. The peaks of L are sharp and narrow, while the complexes are short and broad, revealing the formation of a coordination compound. From the spectrogram we can see that the imine proton (HCQN) peak (Hf)
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Scheme 2. The coordination mode and mechanism of fluorescence enhancement of L–Al3 þ .
at 8.51 ppm is shifted downfield to 8.67, attributed to the formation of L–Al3 þ complex. The participation of CQO made Hl,h, Hk,i, and Hf protons of the right benzene ring undergo overall large downfield shifts of 0.24, 0.20, and 0.15 ppm upon addition of 1.0 equiv Al(NO3)3. Similarly, the peaks corresponding to the left phenol moiety Ha, Hc, Hb, Hd were downfield shifted by 0.19, 0.16, 0.02, 0.05 ppm respectively. It was because that phenol –OH deprotonated and coordinated to Al3 þ , making the electron density of aromatic proton decrease and shifting to low magnetic field. NMR data demonstrated that it is the coordination between Al3 þ and L that resulted in the change of these chemical shifts. We made a comparison between L and L–Al3þ complex in IR (Figs. S9–10). Several main differences existed: ν(CQO) at 1672 cm 1 turned to 1626 cm 1. Large shift to low frequency suggested the coordination of CQO. In the same way, 1618 cm 1 changing to 1553 cm 1 was attributed to ν(CQN). Meanwhile, strong nitratebased vibrations at approximately 1384 cm 1 were observed in L–Al3 þ . It is well known that most Al complexes are hexacoordinated. Based on the above analysis and common sense, we come up with a proper coordination mode (Scheme 2). This complex formation was further supported by ESI/MS (Fig. S11.), peaks at m/z 283.07 [L þ Al3þ þ OH ] þ , 301.08 [L þAl3 þ þ OH þH2O] þ , 328.05 [L þ Al3þ þ NO3 ] þ , and 346.06 [L þAl3 þ þ NO3 þH2O] þ . 3.7. Mechanism of fluorescence enhancement Based on the above study, the photoinduced electron transfer (PET) mechanism (Sarkar et al., 2013; Liu et al., 2013) was
proposed to explain the Off–On properties of the probe L (Scheme 2). In the Off state the PET, which takes place from amino groups to aromatic hydrocarbons, causes fluorescence quenching of the latter. When the amino group strongly interacts with Al3 þ , PET is hindered and a very large enhancement of fluorescence is observed (On-state). Apart from PET, CQN isomerization could also be involved in the fluorescence enhancement (Li et al., 2010; Zhao et al., 2011; Jung et al., 2010; Ray and Bharadwaj, 2008; Wu et al., 2011). With the free ligands CQN isomerization is facile as a result of free rotation around this bond in the excited state. However, metal ion binding locks this bond and free rotation is prevented which is the reason why the fluorescence increases dramatically. 3.8. TP absorbtion cross section (δ) In consideration of the significant superiority of TP fluorescence sensor, we try to study the TP effects of the Al3 þ probe. The TP cross sections and the normalized TP fluorescence intensity of L and its Al3 þ complex were measured. As shown in Fig. S12, the intensity of the peak of Al3 þ complex is far greater than that of “free” L and the shape of the bands is similar to that in one-photon excited spectrum. L shows the δmax value of 21 g at 730 nm for twice one-photon excitation (Fig. 2), which increases markedly ca. 5-fold upon addition of 1.0 equiv Al3 þ . The TP property of the chemosensor can be ascribed to the increased degree of rigidity and conjunction in metal complex (Li et al., 2011). L with an enlarged TP cross section should act as a valuable tool to image Al3 þ in the living systems under TP excitation. 3.9. TP fluorescence titration Then TP fluorescence titration was conducted to research the coordinated condition under the long-wave excitation. As the concentration of Al3 þ increased, an obvious enhancement of more than 50-fold in fluorescence intensity was observed. When Al3 þ concentration reached 16 mM, the fluorescence reinforcement no longer appeared (Fig. S12). The stoichiometric ratio 1:1 of L to Al3 þ is obtained and the association constant is calculated to be 2.9 104 M 1 (Fig. S13) which is near that from OP fluorescence titration. The significant fluorescence enhancement and coordination relationship of L–Al3 þ achieved from TP fluorescence are consistent with that from OP fluorescence, illustrating that the conjecture about the coordination mode of the Al3 þ complex is reliable. 3.10. Cell cytotoxicity
Fig. 2. TP absorption cross section of L (10 mM) in the absence (black) and presence (red) of 1.0 equiv. Al3 þ (10 mM) in HMTA buffered aqueous solution (0.3 M, pH¼ 5.8).
After studying the response of Al3 þ to the probe L in vitro, we then explored its potential for live-cell imaging of Al3 þ . First, the cell viabilities of compound probe L and L Al3 þ complex on HeLa cells were evaluated using the MTT assay (Fig. S14). The HeLa cells
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displayed high viability as the concentration of L increased from 10 mM to 100 mM. On this basis equal amounts of Al3 þ were added into the cells; the content increased and the activity declined. But the viability was still over 65% when the concentrations of the probe and the Al3 þ were 50 mM, let alone the testing amount which was one fifth of it, which brought negligible toxicity to the cells. Cell cytotoxicity experiments suggest that the probe exhibits low toxicity to HeLa cells in our measure range and can be used to detect Al3 þ in vivo with little damage. 3.11. TP Bioimaging The ultimate aim of the fluorescence probe is to use it in the living system. Considering the perfect TP property the probe possessed, we conducted a TP fluorescence microscopy experiment to its higher gradation of application in complex biological systems. From Fig. 3, we can see that after incubation for 30 min, L (10 mM) emitted little fluorescence when induced by TP excitation light source. However the addition of Al3 þ (15 mM) brought strong fluorescence intensity in the living cells and the fluorescence was disappeared when EDTA, a membrane-permeable metal ion chelator that effectively removes Al3 þ , was added. The short duration of L and Al3 þ entering the cells and also the significant fluorescence emission ability of L–Al3 þ revealed that the probe has good membrane permeability and can be used to detect intramolecular Al3 þ with no autofluorescence and high resolution, which is of great value to the development of fluorescence probes. The probe L can not only detect Al3þ in the living cells qualitatively, but also estimate the Al3 þ concentration quantitatively in vivo. We obtained a series of TP fluorescence images data of HeLa cells labeled with 10 μM L and different Al3 þ concentrations (0–20 μM). As
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shown in Fig. S15A, the fluorescence intensity increased with the Al3þ concentration. When the concentration of Al3þ reached 2 μM, observable fluorescence appeared. With the addition of Al3 þ , the blue became more and more apparent. In order to analyze these results better, we use the software Imagepro-plus to calculate the average fluorescence intensity and quantify the images. The relationship between intensity and the concentration of Al3 þ was approximately linear in the range 0–10 μM (Fig. S15B). Thus, on this basis the Al3þ concentration of the cells can be estimated through calculating its average fluorescence intensity. The superiority of TP over OP is well established. In order to confirm it, we performed OP and TP fluorescence images experiments of labeled tissues (Fig. S16). When certain concentrations of L and Al3 þ were added into the tissues, very strong blue-fluorescence was presented whether it was excited by OP or TP. However, we can still observe the fluorescence from TP fluorescence microscope when the depth of penetration reaches 400 μm while it is difficult to see the signals from OP fluorescence microscope when the thickness was only up to 250 μm. The penetration depth of TP was at least 150 μm deeper than that of OP. Even so, OP penetration depth of the probe to detect the analyte was still larger than those most of the OP probes (less than 100 μm) published. By comparison, we can draw a conclusion that our probe has perfect TP fluorescence properties of sensing Al3 þ in tissues.
4. Conclusions In conclusion, we have synthesized a salicylimine-based TP probe for Al3 þ in aqueous solution and investigated its TP
Fig. 3. Bright-field images (up), TP microscope images (middle), and the overlay of fluorescence and bright-field images (down) of HeLa cells after incubation with 10 μM L for 30 min (A); 10 μM L for 30 min and then further incubated with 15 μM Al3 þ for another 25 min (B); 10 μM L for 30 min, then further incubated with 15 μM Al3 þ for another 25 min, finally added 20 μM EDTA for 1 hour at 37 °C (C).
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activities in vitro and in vivo for the first time. The response of Al3 þ to L is excellent; hundreds of times higher the fluorescence intensity than before whether it is excited by OP or TP. Moreover, the fluorescence response is free from disturbance by other ions. Ongoing efforts are underway in our group to design more novel probes to detect a variety of other substances in biological studies with less response time, better water-solubility and chelate ability.
Acknowledgments This work was supported by the NSFC (Grants no. 21431002 and 91122007), the Specialized Research Fund for the Doctoral Program of Higher Education (Grant no. 20110211130002).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.10.018.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A two-photon probe for Al3 þ in aqueous solution and its application in bioimaging Haihong Wang, Bei Wang, Zhaohua Shi, Xiaoliang Tang, Wei Dou, Qingxin Han, Yange Zhang, Weisheng Liu n Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 May 2014 Received in revised form 1 September 2014 Accepted 7 October 2014 Available online 16 October 2014
A salicylimine probe L with a simple structure has been researched more in-depth on fluorescence sensor properties based on two-photon (TP) absorption. L displays excellent selective turn-on fluorescence response for Al3 þ in hexamethylenetetramine-buffered (HMTA) aqueous solution (0.3 M, pH ¼5.8) under one-photon (OP) excitation. With the help of OP fluorescence, TP fluorescence titration, UV-spectra titration and Job's plot, the stoichiometric ratio of L with Al3 þ was determined to be 1:1. The coordination sites and the coordination mechanism of L with Al3 þ were analyzed in detail through 1H NMR data. Not only with a detection limit of 5.2 10 9 M in vitro, but also the probe has been successfully used in the live cells and tissues for the imaging of Al3 þ with TP fluorescence microscopy due to the enlarged TP cross section, providing a novel testing method for measuring Al3 þ in solution or cell tissue with low autofluorescence and cytotoxicity. & Elsevier B.V. All rights reserved.
Keywords: Two-photon fluorescence Al3 þ probe Bioimaging
1. Introduction Aluminum is the most abundant metallic element in earth's crust and its compounds are widely used in chemical industry production, from food additives, medicines, cookware, to production of light alloys, etc., which are closely associated with our daily life. It is universally known that Al is not the element people need and its accumulation can bring toxicity to the living body. When it comes to acid rain Al3 þ which comes from the soil with poor buffer ability enters into the rivers and lakes, increasing the concentration of Al3 þ in natural water and giving rise to the death of plants (Emmanuel and Ryan, 1995; Ren and Tian, 2007) and fishes (Alstad et al., 2005). In the human body Al3 þ can be a competitive inhibitor of several essential elements, such as Mg2 þ , Ca2 þ and Fe3 þ due to their similar characteristics (Das et al., 2012; Banerjee et al., 2012; Williams, 2002). An excess of Al3 þ may have a bad effect on the central nervous system, further leading to dementia, myopathy, and Alzheimer's disease (Perl et al., 1982; Perl and Brody, 1980). In order to prevent the absorbtion of Al3 þ and study in depth the key role of the metal ion in our body, it is of great importance to detect Al3 þ in vivo and in vitro with different measuring methods. n
Corresponding author. Fax: þ 86 931 8912582. E-mail address: [email protected] (W. Liu).
http://dx.doi.org/10.1016/j.bios.2014.10.018 0956-5663/& Elsevier B.V. All rights reserved.
In recent years, organic fluorescent probes were widely applied to detect biologically important ions for their generally nondestructive character, high sensitivity, instantaneous response, and the wide range of indicator dyes available (Amendola et al., 2006; Wang et al., 2010). Though many reports on the detection of Al3 þ were published (Das et al., 2013), most of them were always problematic due to the lack of spectroscopic characteristics and poor coordination ability compared to transition metals (Soroka et al., 1987). Because Al3 þ can be hydrolyzed easily to generate Al(OH)3 precipitation most of the probes cannot be applied in water let alone in buffered solution which is beneficial to practical application, such as biological imaging (Zhao et al., 2006; Li et al., 2014). So far, there have been some Al3 þ fluorescent probes being applied to cell imaging, but all of them were measured with OP microscopy (Wang et al., 2010; Kim et al., 2012; Zhi et al., 2013) which required a rather short excitation wavelength and would produced background fluorescence (Guo et al., 2013; Sun et al., 2012) as well as photo-damage. In order to overcome the above limitations, TP microscopy, as a kind of new technology, has been used in this area recently. Multiple-photon excitation technology is the progress where two or more photons emitted by a longwave light source can excite a short-wave one (Biswas et al., 2011). In contrast to OP microscopy, TP microscopy offers several distinct advantages: (1) small scattering effects can make light penetrate
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samples deeper and more easily; (2) reduced photo-damage and photobleaching can make TP microscopy more suitable for viable cell observation (Biswas et al., 2011). Previously, scientists have spared no effort in applying TP microscopy to the field of TP fluorescence imaging (Taki et al., 2004; Dong et al., 2013; Zheng et al., 2014) and developed lots of new TP fluorescence probes with large TP absorption cross sections for the use in biological imaging (Li et al., 2011; Wang et al., 2012; Stewart et al., 2008; Cao et al., 2007; Zhu et al., 2010; Yong et al., 2009; Padilha et al., 2011; Chattopadhyaya et al., 2011; Shao et al., 2011; Poronik et al., 2013; Zhang et al., 2009; Nguyen et al., 2010; Chen et al., 2009; Feng et al., 2010). Cho and Kim et al. developed a series of TP fluorescence probes for detecting Na þ (Kim et al., 2010b, 2010a), Ca2 þ (Kim et al., 2010a, 2007b), Mg2 þ (Kim et al., 2007a), Zn2 þ (Kim et al., 2008), H þ (Park et al., 2012; Lee et al., 2013), thiol (Lee et al., 2010), NO (Dong et al., 2013), etc., and applied them not only in living cells but also in tissues. In addition to this several other ions have also been studied, such as Cd2 þ (Liu et al., 2012; Li et al., 2012), Hg2 þ (Rao et al., 2012), Cu2 þ (Fu et al., 2013), F (Zhang et al., 2011), etc. Though there were several studies on the TP absorption properties of aluminum complexes, they were mainly focused on theoretical study (Liu et al., 2004a; Zhang et al., 2007; Liu et al., 2004b; Yang et al., 2011). Calixarene-based TP fluorescent sensors have been synthesized by Cho et al. to sense metal ions Pb2 þ and Al3 þ but they cannot distinguish the two ions in CH3CN (Kim et al., 2006), which is the most basic requirement probes should meet. Even so, to the best of our knowledge, there have not been any reports on TP fluorescence microscopy imaging of Al3 þ . Considering appealing applications of Schiff base derivatives in optical sensing, antitumor ability (da Silveira et al., 2008), antioxidative effects (Li and Yang, 2009), attractive electronic and photophysical property (Kasselouri et al., 1993) we researched more in-depth fluorescence sensor properties of a simple Schiff bases ligand N′-(2-hydroxybenzylidene)benzohydrazide (L), which has a large TP absorption cross section when adding Al3 þ . L displays excellent selectivity for Al3 þ in aqueous solution and it can also be used for living cell and tissue imaging.
2. Materials and methods 2.1. Materials and instrumentation All reagents and solvents employed for synthesis were commercially available and used as received. Deionized water was used as solvent. All of the solvents used were of analytical reagent grade. The solutions of metal ions were prepared from either their nitrate or their chloride salts. HMTA buffered aqueous solution (0.3 M, pH ¼5.8) was prepared in double-distilled water. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker DRX400 spectrometer in d6-DMSO and d4-CH3OH with TMS as internal standard. Mass spectra was measured on a Bruker Esquire 6000 mass spectrometer by means of the electronic spray ionization (ESI) technique. Absorption spectra were recorded using a Varian Cary 100 spectrophotometer. Fluorescence measurements were made on a Hitachi F-7000 fluorescence spectrophotometer equipped with a xenon lamp as the excitation source. The path length was 1 cm with a cell volume of 3.0 mL. Excitation and emission slits of 2.5 nm were used for the measurements of fluorescence. Elemental analyzes were conducted using an Elemental Vario (EL) instrument. Melting point was determined on an X-6 melting point apparatus without calibration (Beijing Fuka Keyi Science and Technology Co., Ltd.). Fluorescent quantum yields were determined by an absolute method using an integrating sphere on FLS920 of Edinburgh Instrument. All pH measurements
were made with a pH-10 C digital pH meter. All the measurements have been done at room temperature unless otherwise stated. 2.2. Optical detection of Al3 þ The probe L (10.0 μM) was mixed with different concentrations of metal ions, 10.0 μM Al(NO3)3 and Ga(NO3)3, 50.0 μM NaCl, KCl, AgNO3, Cr(ClO4)3, Co(ClO4)2, Ni(NO3)2, FeCl3, Cu(NO3)2, CaCl2, Mn(ClO4)2, ZnCl2, LiClO4, Cd(NO3)2, MgCl2 and Hg(ClO4)2 in HMTA buffered aqueous solution (0.3 M, pH ¼5.8). After equilibrium at ambient temperature for 25 min, absorption and fluorescence spectra of the mixtures were measured. Fluorescence spectra were measured at an excitation wavelength of 366 nm, and emission spectra were collected from 390 to 600 nm. 2.3. Measurement of TP cross section (δ) TP excitation fluorescence spectra were measured using a Steady-state and Lifetime Fluorescence Spectrometer (FLS920, Edinburgh Instruments). TP absorption cross sections (δ) at different excitation wavelengths were determined by comparing their TP excitation fluorescence with that of fluorescein (Albota et al., 1998) in aqueous solution, according to the following equation:
δ = δref Φref /Φcref /cnref /nF /Fref
(1)
In Eq. (1), the subscript ref stands for the reference molecule. δ is the TP absorption cross section value, c is the concentration of solution, n is the refractive index of the solution, F is the TP excitation fluorescence integral intensities of the solution emitted at the exciting wavelength, and Φ is the fluorescence quantum yield. The δref value of reference was taken from the literature (Li et al., 2012). 2.4. TP fluorescence imaging TP fluorescence images of L labeled cells and tissues were obtained by exciting the probes with a modelocked titanium–sapphire laser source (Mai Tai DeepSee, 80 MHz, 90 fs) set at wavelength 730 nm with AN Olympus FV1000 laser confocal microscope IΧ81 with 60 objective, numerical aperture (NA)¼0.4. The images signals in 420–470 nm range were collected by internal PMTs in 12 bit unsigned 1024 1024 pixels at 40 Hz scan speed.
3. Result and discussion 3.1. Synthesis of L L was facilely synthesized from the reaction of benzohydrazide with salicylaldehyde (Scheme 1). The molecular structure and its purity were confirmed by NMR and ESI-MS.
Scheme 1. Synthesis of the fluorescent indicator L. Reagents and conditions: (i) ethanol, reflux, 8 h, 82%.
H. Wang et al. / Biosensors and Bioelectronics 65 (2015) 91–96
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Fig. 1. (A) The normalized fluorescence spectra of L (10 μM) before and after addition of 50 μM Pb2 þ , Mg2 þ , Hg2 þ , Cu2 þ , Co2 þ , Ni2 þ , Li þ , Cr3 þ , Ag þ , Cd2 þ , Na þ , K þ , Fe3 þ , Ca2 þ , Zn2 þ , Mn2 þ , 10 μM Al3 þ and Ga3 þ in HMTA buffered aqueous solution (0.3 M, pH ¼ 5.8). (B) The normalized fluorescence spectra of 10 μM L after addition of increasing amounts of Al3 þ ions (0, 2, 4, 6, 8,,10, 12, 14 μM) at room temperature. Inset. The normalized fluorescence intensity at 452 nm as a function of Al3 þ concentration (λex ¼ 366 nm).
3.2. Effect of pH Fluorescence sensors based on electron donor/acceptor are usually disturbed by protons in the detection of metal ions, so it is necessary to investigate pH effect on L in the absence and presence of Al3 þ and find optimal sensing conditions. There is no change in the fluorescence spectrum of L after the addition of Al3 þ at pH below 3.0, which is because the combination of protons and N atoms in –CQN– groups inhibits the coordination of Al3 þ with L. On the other hand, the fluorescence of L has no response to Al3 þ at high pH values because the hydrolysis happens between OH and Al3 þ (Fig. S1). The best pH corresponding to the highest intensity is 5.8, which is within the biologically relevant pH range (5.5–7.5) (Shi et al., 2013), indicating that L could be applied as an Al3 þ sensor in biological organisms potentially, such as lysosomes (Dong et al., 2013). 3.3. Ion selectivity and competitiveness The selectivity of L for various common cations was investigated in HMTA buffered aqueous solution (0.3 M, pH ¼5.8) (Fig. 1A). Upon excitation at 366 nm, L (10 mM) showed weak emission intensity at ca. 450 nm with a low fluorescence quantum yield (Фf ¼ 0.05). And there was no obvious change in the spectra after adding 5 equiv other metal ions to the aqueous solution of L, such as Li þ , Na þ , Mg2 þ , K þ , Ca2 þ , Mn2 þ , Fe3 þ , Co2 þ , Ni2 þ , Cu2 þ , Zn2 þ , Ag þ , Cd2 þ , Hg2 þ and Pb2 þ . However, the addition of 1 equiv Al3 þ brought significant enhancement in the fluorescence intensity at 452 nm with a high fluorescence quantum yield (Фf ¼0.49) within 25 min (Fig. S2). This is attributed to the formation of L–Al3 þ complex and thus the increase of p–π conjunction. Ga3 þ ion, the same family as that of Al, also increased the intensity at about 465 nm, but this was considerably lower than that of Al3 þ , having little interference on the selectivity of L for Al3 þ . Moreover a little red shift, ca. 15 nm, which could be seen obviously, existed in the spectra after the treatment with 1 equiv. Ga3 þ . In order to prove that the sensor was highly selective for Al3 þ , excessive other metal ions were added in the Al3 þ determination protocol. As shown in Fig. S3, the competing ions have more or less influence on the fluorescence intensity. Among the ions, L was responsive to Al3 þ obviously, although the interference by Ga3 þ was the largest along with Cr3 þ , Cu2 þ and Ca2 þ . The interference
of the other metal ions is relatively limited, indicating that L can be applied to detect Al3 þ effectively. 3.4. Fluorescence titration Fluorescence titration was performed to study the stoichiometric ratio of the L–Al3 þ complex (Fig. 1B). With the addition of Al3 þ to L (10 mM), the fluorescence intensity increased accordingly; but when the concentration of Al3 þ was up to 10 mM, the rising tendency slowed down gradually, indicating the 1:1 binding of L to Al3 þ , which was consistent with the result from Job′s plot (Fig. S4). The association constant of L for Al3 þ was estimated to be 5.5 104 M 1 in the same media (Fig. S5). The limit of detection (LOD) was calculated to be 5.2 10 9 M (Fig. S6). According to China EPA standard the concentration of Al3 þ should be lower than 0.05 mg L 1 (1.85 10 3 M) in drinking water, which means that our proposed fluorescent method based on probe L is sensitive enough to monitor the water quality of drinking water. 3.5. Absorption spectra studies The absorption spectra of L are also studied with increasing concentration of Al3 þ to analyze the coordination mode of L–Al3 þ in solution. As shown in Fig. S7, L (10 mM) has two main absorption peaks at 288 and 325 nm; the intensity of both decreases with the addition of Al3 þ . Meanwhile, the two bands showed a red-shift accompanied by generation of a new absorption peak at 367 nm and three isosbestic points at 300, 322, 342 nm. This suggests a coordination between Al3 þ and L, which extends the conjugated system and leads to new absorption in the long wavelength region. The absorbance peak no longer changed when the concentration of Al3 þ increased from 10 μM to 14 μM, indicating 1:1 binding between L and Al3 þ . 3.6. Product analysis The determination of the coordination site of L with Al3 þ was examined by 1H NMR in d4-CH3OH. Spectra of L before and after addition of 1 equiv Al3 þ are shown in Fig. S8. The peaks of L are sharp and narrow, while the complexes are short and broad, revealing the formation of a coordination compound. From the spectrogram we can see that the imine proton (HCQN) peak (Hf)
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Scheme 2. The coordination mode and mechanism of fluorescence enhancement of L–Al3 þ .
at 8.51 ppm is shifted downfield to 8.67, attributed to the formation of L–Al3 þ complex. The participation of CQO made Hl,h, Hk,i, and Hf protons of the right benzene ring undergo overall large downfield shifts of 0.24, 0.20, and 0.15 ppm upon addition of 1.0 equiv Al(NO3)3. Similarly, the peaks corresponding to the left phenol moiety Ha, Hc, Hb, Hd were downfield shifted by 0.19, 0.16, 0.02, 0.05 ppm respectively. It was because that phenol –OH deprotonated and coordinated to Al3 þ , making the electron density of aromatic proton decrease and shifting to low magnetic field. NMR data demonstrated that it is the coordination between Al3 þ and L that resulted in the change of these chemical shifts. We made a comparison between L and L–Al3þ complex in IR (Figs. S9–10). Several main differences existed: ν(CQO) at 1672 cm 1 turned to 1626 cm 1. Large shift to low frequency suggested the coordination of CQO. In the same way, 1618 cm 1 changing to 1553 cm 1 was attributed to ν(CQN). Meanwhile, strong nitratebased vibrations at approximately 1384 cm 1 were observed in L–Al3 þ . It is well known that most Al complexes are hexacoordinated. Based on the above analysis and common sense, we come up with a proper coordination mode (Scheme 2). This complex formation was further supported by ESI/MS (Fig. S11.), peaks at m/z 283.07 [L þ Al3þ þ OH ] þ , 301.08 [L þAl3 þ þ OH þH2O] þ , 328.05 [L þ Al3þ þ NO3 ] þ , and 346.06 [L þAl3 þ þ NO3 þH2O] þ . 3.7. Mechanism of fluorescence enhancement Based on the above study, the photoinduced electron transfer (PET) mechanism (Sarkar et al., 2013; Liu et al., 2013) was
proposed to explain the Off–On properties of the probe L (Scheme 2). In the Off state the PET, which takes place from amino groups to aromatic hydrocarbons, causes fluorescence quenching of the latter. When the amino group strongly interacts with Al3 þ , PET is hindered and a very large enhancement of fluorescence is observed (On-state). Apart from PET, CQN isomerization could also be involved in the fluorescence enhancement (Li et al., 2010; Zhao et al., 2011; Jung et al., 2010; Ray and Bharadwaj, 2008; Wu et al., 2011). With the free ligands CQN isomerization is facile as a result of free rotation around this bond in the excited state. However, metal ion binding locks this bond and free rotation is prevented which is the reason why the fluorescence increases dramatically. 3.8. TP absorbtion cross section (δ) In consideration of the significant superiority of TP fluorescence sensor, we try to study the TP effects of the Al3 þ probe. The TP cross sections and the normalized TP fluorescence intensity of L and its Al3 þ complex were measured. As shown in Fig. S12, the intensity of the peak of Al3 þ complex is far greater than that of “free” L and the shape of the bands is similar to that in one-photon excited spectrum. L shows the δmax value of 21 g at 730 nm for twice one-photon excitation (Fig. 2), which increases markedly ca. 5-fold upon addition of 1.0 equiv Al3 þ . The TP property of the chemosensor can be ascribed to the increased degree of rigidity and conjunction in metal complex (Li et al., 2011). L with an enlarged TP cross section should act as a valuable tool to image Al3 þ in the living systems under TP excitation. 3.9. TP fluorescence titration Then TP fluorescence titration was conducted to research the coordinated condition under the long-wave excitation. As the concentration of Al3 þ increased, an obvious enhancement of more than 50-fold in fluorescence intensity was observed. When Al3 þ concentration reached 16 mM, the fluorescence reinforcement no longer appeared (Fig. S12). The stoichiometric ratio 1:1 of L to Al3 þ is obtained and the association constant is calculated to be 2.9 104 M 1 (Fig. S13) which is near that from OP fluorescence titration. The significant fluorescence enhancement and coordination relationship of L–Al3 þ achieved from TP fluorescence are consistent with that from OP fluorescence, illustrating that the conjecture about the coordination mode of the Al3 þ complex is reliable. 3.10. Cell cytotoxicity
Fig. 2. TP absorption cross section of L (10 mM) in the absence (black) and presence (red) of 1.0 equiv. Al3 þ (10 mM) in HMTA buffered aqueous solution (0.3 M, pH¼ 5.8).
After studying the response of Al3 þ to the probe L in vitro, we then explored its potential for live-cell imaging of Al3 þ . First, the cell viabilities of compound probe L and L Al3 þ complex on HeLa cells were evaluated using the MTT assay (Fig. S14). The HeLa cells
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displayed high viability as the concentration of L increased from 10 mM to 100 mM. On this basis equal amounts of Al3 þ were added into the cells; the content increased and the activity declined. But the viability was still over 65% when the concentrations of the probe and the Al3 þ were 50 mM, let alone the testing amount which was one fifth of it, which brought negligible toxicity to the cells. Cell cytotoxicity experiments suggest that the probe exhibits low toxicity to HeLa cells in our measure range and can be used to detect Al3 þ in vivo with little damage. 3.11. TP Bioimaging The ultimate aim of the fluorescence probe is to use it in the living system. Considering the perfect TP property the probe possessed, we conducted a TP fluorescence microscopy experiment to its higher gradation of application in complex biological systems. From Fig. 3, we can see that after incubation for 30 min, L (10 mM) emitted little fluorescence when induced by TP excitation light source. However the addition of Al3 þ (15 mM) brought strong fluorescence intensity in the living cells and the fluorescence was disappeared when EDTA, a membrane-permeable metal ion chelator that effectively removes Al3 þ , was added. The short duration of L and Al3 þ entering the cells and also the significant fluorescence emission ability of L–Al3 þ revealed that the probe has good membrane permeability and can be used to detect intramolecular Al3 þ with no autofluorescence and high resolution, which is of great value to the development of fluorescence probes. The probe L can not only detect Al3þ in the living cells qualitatively, but also estimate the Al3 þ concentration quantitatively in vivo. We obtained a series of TP fluorescence images data of HeLa cells labeled with 10 μM L and different Al3 þ concentrations (0–20 μM). As
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shown in Fig. S15A, the fluorescence intensity increased with the Al3þ concentration. When the concentration of Al3þ reached 2 μM, observable fluorescence appeared. With the addition of Al3 þ , the blue became more and more apparent. In order to analyze these results better, we use the software Imagepro-plus to calculate the average fluorescence intensity and quantify the images. The relationship between intensity and the concentration of Al3 þ was approximately linear in the range 0–10 μM (Fig. S15B). Thus, on this basis the Al3þ concentration of the cells can be estimated through calculating its average fluorescence intensity. The superiority of TP over OP is well established. In order to confirm it, we performed OP and TP fluorescence images experiments of labeled tissues (Fig. S16). When certain concentrations of L and Al3 þ were added into the tissues, very strong blue-fluorescence was presented whether it was excited by OP or TP. However, we can still observe the fluorescence from TP fluorescence microscope when the depth of penetration reaches 400 μm while it is difficult to see the signals from OP fluorescence microscope when the thickness was only up to 250 μm. The penetration depth of TP was at least 150 μm deeper than that of OP. Even so, OP penetration depth of the probe to detect the analyte was still larger than those most of the OP probes (less than 100 μm) published. By comparison, we can draw a conclusion that our probe has perfect TP fluorescence properties of sensing Al3 þ in tissues.
4. Conclusions In conclusion, we have synthesized a salicylimine-based TP probe for Al3 þ in aqueous solution and investigated its TP
Fig. 3. Bright-field images (up), TP microscope images (middle), and the overlay of fluorescence and bright-field images (down) of HeLa cells after incubation with 10 μM L for 30 min (A); 10 μM L for 30 min and then further incubated with 15 μM Al3 þ for another 25 min (B); 10 μM L for 30 min, then further incubated with 15 μM Al3 þ for another 25 min, finally added 20 μM EDTA for 1 hour at 37 °C (C).
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activities in vitro and in vivo for the first time. The response of Al3 þ to L is excellent; hundreds of times higher the fluorescence intensity than before whether it is excited by OP or TP. Moreover, the fluorescence response is free from disturbance by other ions. Ongoing efforts are underway in our group to design more novel probes to detect a variety of other substances in biological studies with less response time, better water-solubility and chelate ability.
Acknowledgments This work was supported by the NSFC (Grants no. 21431002 and 91122007), the Specialized Research Fund for the Doctoral Program of Higher Education (Grant no. 20110211130002).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.10.018.
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