Biosensors and Bioelectronics 61 (2014) 397–403
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A novel dual-emission ratiometric fluorescent nanoprobe for sensing and intracellular imaging of Zn2 þ Yupeng Shi a,1, Zhihua Chen a,1, Xin Cheng a, Yi Pan a, Heng Zhang a, Zhaomin Zhang a, Cheuk-Wing Li b, Changqing Yi a,n a Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong Province), School of Engineering, Sun Yat-Sen University, Guangzhou, 510006, China b Institute of Chinese Medical Sciences, University of Macau, Macau, China
art ic l e i nf o
a b s t r a c t
Article history: Received 19 February 2014 Received in revised form 2 May 2014 Accepted 21 May 2014 Available online 28 May 2014
The integration of unique characteristics of nanomaterials with highly specific recognition elements, such as biomolecules and organic molecules, are the foundation of many novel nanoprobes for bio/ chemical sensing and imaging. In the present report, branched polyethylenimine (PEI) was grafted with 8-chloroacetyl-aminoquinoline to synthesize a water-soluble and biocompatible quinoline-based Zn2 þ probe PEIQ. Then the PEIQ was covalently conjugated to [Ru(bpy)3]2 þ -encapsulated SiNPs to obtain the ratiometric fluorescence nanoprobe which exhibits a strong fluorescence emission at 600 nm and a negligible fluorescence emission at 500 nm in the absence of Zn2 þ upon a single wavelength excitation. After the addition of different amounts of Zn2 þ , the fluorescence intensity at 500 nm increased continuously while the fluorescence intensity at 600 nm remained stable, thus changing the dual emission intensity ratios and displaying continuous color changes from red to green which can be clearly observed by the naked eye. The nanoprobe exhibits good water dispersivity, biocompatibility and cell permeability, high selectivity over competing metal ions, and high sensitivity with a detection limit as low as 0.5 μM. Real-time imaging of Zn2 þ in A549 cells has also been realized using this novel nanoprobe. & 2014 Elsevier B.V. All rights reserved.
Keywords: Silica nanoparticles 8-Aminoquinoline Zn2 þ Ratiometric fluorescence Cellular imaging
1. Introduction As the second most abundant transition metal in the human body, zinc always occurs as a divalent cation [Zn (II)] and plays pivotal roles in biological systems (Falchuk, 1998; O’Halloran, 1993). For example, Zn2 þ serves as catalytic and structural cofactor when it is bound to some specific proteins, thus facilitating enzyme regulation, gene expression and neural related signal transmission (Vallee and Falchuk, 1993). Although it remains unclear about its functional role, growing evidence suggests the correlations between the disorder of Zn2 þ metabolism and many neurological diseases, including Alzheimer’s disease, infantile diarrhea, epilepsy and cerebral ischemia (Adlard and Bush, 2006; Koh et al., 1996). Because of its important biological roles, research interests to develop highly sensitive and selective detection and monitoring of Zn2 þ under physiological conditions are growing unabated (Lim et al., 2004). Fluorescence is believed to be the most effective way to determine the concentration, together with
n
Corresponding author. Tel./fax: þ 86 20 39342380. E-mail address: [email protected] (C. Yi). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.05.050 0956-5663/& 2014 Elsevier B.V. All rights reserved.
visualization of subcellular distribution of Zn2 þ in living cells (Hanaoka et al., 2004; Jiang and Guo, 2004; Walkup et al., 2000; Xie et al., 2012). Among the fluorescent probes for Zn2 þ ions detection, quinoline based molecules, especially 8-aminoquinoline and 8-hydroxy-quinoline, have been studied extensively because they are pH insensitive, ready to form strong inter-molecular hydrogen bonds with surrounding water molecules, and their metal-coordination. Moreover, quinoline based Zn2 þ sensors are suitable candidates for photofunctional molecular devices because their sensing mechanism is mainly on the basis of photo-induced electron transfer and photo-induced charge transfer processes (Du et al., 2010; Xie et al., 2011; Zhou et al., 2010). However, most of these quinoline-based probes targeting Zn2 þ ions still suffer from poor solubility, bad cell permeability, and weak sensitivity. And a few examples of ratiometric fluorescence methods for sensing and intracellular imaging of Zn2 þ have been reported (Jiang and Guo, 2004; Liu et al., 2012; Raje et al., 2013). Recently, the conjugation of fluorescent probes with nanoparticles has emerged as an attractive strategy to develop new chemosensors for intracellular monitoring of Zn2 þ , since nanoparticles can shelter the probes from interferences and can transport the probes neglecting their intrinsic solubility (Li et al., 2011; Lu et al., 2012; Rastogi et al., 2011; Ruedas-Rama and Hall,
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2008; Pal et al., 2011; Teolato et al., 2007; Xu et al., 2013). Due to their high luminescence quantum yield, good photostability and water dispersivity, biocompatibility and versatile surface modification chemistry, [Ru(bpy)3]2þ -encapsulated silica nanoparticles (SiNPs) become an excellent choice for developing bio/chemo nanosensors and diagnostic nanoprobes in bio/chemical analysis (Santra et al., 2001; Shi et al., 2013a, 2013b; Wang et al., 2013). Herein, in the present report, branched polyethylenimine (PEI) was firstly grafted with 8-chloroacetyl-aminoquinoline to synthesize a water-soluble and biocompatible quinoline-based Zn2þ probe PEIQ. Then PEIQ was covalently conjugated onto the surface of [Ru(bpy)3]2 þ -encapsulated SiNPs to develop a selective and sensitive ratiometric strategy for the sensing and monitoring of Zn2 þ both in vitro and in vivo. The nanoprobe exhibits high selectivity over competing metal ions, high sensitivity with a detection limit as low as 0.5 μM, and suitability of real-time Zn2þ imaging in A549 cells. Notably, the reported nanoprobe possesses some remarkable features: (1) the recognition moiety is synthesized by grafting branched PEI with quinoline derivative, thus reducing the possible cytotoxicity of PEI and increasing water dispersivity and cell permeability of the as-prepared Zn2 þ probe, PEIQ; (2) the internal standard is encapsulated into SiNPs and the recognition moiety is covalently conjugated onto SiNPs surface, thus providing a reliable reference signal and a stable nanoprobe; (3) the large amount of PEIQ units on the outer surface of an individual nanoparticle enable the signal amplification, hence making the immediate and highly sensitive detection of Zn2 þ possible.
vacuum. The crude product was purified by co-precipitation with ether to give yellow oil. 2.4. Synthesis and functionalization of fluorescent SiNPs
2. Materials and methods
The fluorescent [Ru(bpy)3]2 þ -encapsulated SiNPs were synthesized by a water-in-oil reverse micelle method as described in detail elsewhere. Typically, 7.5 mL of cyclohexane, 1.77 mL of Triton X-100, 1.8 mL of hexanol, and 0.34 mL of H2O were stirred for 20 mins to generate the microemulsion system, followed by the addition of 80 μL of 0.1 M [Ru(bpy)3]2 þ and 100 μL of tetraethoxy orthosilicate (TEOS). After being stirred for 30 min, silica polymerization was initiated by the addition of 60 μL aqueous ammonia and allowed to react for 24 h. Then, in order to prepare carboxylated SiNPs (SiNP-COOH), 50 μL TEOS and 50 μL carboxyethylsilanetriol sodium salt (CTES) were added to the mixture and the reaction was allowed to continue for another 24 h. Finally, acetone was added to destabilize the micro-emulsion system. The fluorescent SiNPs were isolated via centrifugation and washed in sequence with ethanol and D.I. water to remove any surfactant and unreacted reactants. Carbodiimide chemistry was employed to covalently conjugate PEIQ onto SiNPs. Briefly, 0.1 g of SiNP-COOH and 0.05 g of PEIQ were suspended in 20 mL of 0.01 M phosphate-buffered saline (PBS) buffer (pH¼7.4) containing 5 mM N-hydroxy-succinimide (NHS) and 2 mM 1-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide (EDC). Then the mixture was allowed to react at room temperature for another 16 h under gentle shaking. The final nanoprobes were obtained via centrifugation and were washed in sequence with D.I. water to remove any of the unreacted reactants.
2.1. Apparatus and reagents
2.5. Sensitivity and selectivity of the SiNPs–PEIQ nanoprobe
Morphology of the nanoparticles was examined by a transmission electron microscope (TEM, JEOL JEM-1400). DLS measurements were performed at 25 1C using a Malvern Zetasizer NanoZS90 instrument. UV–vis, FTIR, and fluorescence spectra were obtained on a Beckman DU730 UV–vis spectrometer, a VERTEX 70 spectrometer (Bruker), and a PTI Quanta-Master QM4CW spectrofluorometer, respectively. 1H NMR spectra were recorded with a Varian Mercury-400 spectrometer with Me4Si as the internal standard. The fluorescence images were acquired by an inverted fluorescence microscope (OLYMPUS IX71). The metal ions solutions were prepared from NaCl, KCl, MgCl2, CaCl2, FeCl3, Pb(NO3)2,CoCl2, NiCl2, ZnCl2, HgCl2, CrCl3 and CuSO4 in distilled water with a concentration of 0.01 M or 0.001 M.
For the detection of Zn2 þ , different concentrations of Zn2 þ (0, 1.0, 2.0, 4.0, 6.0, 10.0, 15.0, 20.0, 30.0, 50.0, and 100.0 mM) were mixed with the nanoprobe (2.0 mg mL 1) in 1.2 mL of distilled water under gentle shaking. Photographs and fluorescence spectra were taken after Zn2 þ reacted with the nanoprobe for 5 mins. To evaluate the selectivity of the nanoprobe, 20.0 μM of amino acids or 50.0 μM of competing metal ions was mixed with the SiNP–PEIQ (2.0 mg mL 1) under gentle shaking. Fluorescence spectra were taken after the metal ions or amino acids reacted with the dispersed nanoprobe for 5 mins.
2.2. Synthesis of 8-chloroacetylaminoquinoline 8-chloroacetylaminoquinoline was synthesized according to a previously reported method (Zhou et al., 2010). 288 mg of 8aminoquinoline (2 mM) and 202 mg (2.1 mM) of N(Et)3 were added into 10 mL of CH2Cl2 and mixed in a round flask for 20 min at 0 1C. Then 246 mg (2.2 mM) of chloroacetyl chloride was added dropwise. The mixture was warmed to room temperature and allowed to react overnight. The solvent was evaporated in vacuum. The crude product was further purified by column chromatography (silica gel, PE/EA at 3:1) and a pale white solid was obtained.
2.6. Intracellular imaging of Zn2 þ The A549 cells were provided by Cells Bank of the Chinese Academy of Science (Shanghai, China). Cells were grown in H-DMEM (high glucose) supplemented with 10% FBS in an atmosphere of 5% CO2 at 37 1C. Cells (105/well) were plated on 6 mm glass coverslips and allowed to adhere for 24 h. Intracellular imaging of Zn2 þ were performed in the same medium supplemented with 50 μM ZnCl2 for 0.5 h. A549 cells were washed with PBS and incubated with the nanoprobe (10.0 mg mL 1) at 37 1C for 2 h. Cell imaging was performed with an inverted fluorescence microscope after washing the cells with PBS.
3. Results and discussions
2.3. Synthesis of PEIQ
3.1. Synthetic routes for the SiNPs–PEIQ nanoprobe and its sensing mechanism
A mixture of PEI (MW ¼1800) (500 mg, 0.28 mM) and K2CO3 (38.4 mg, 0.28 mM) in 5 mL CH3CN was added to 20 mL of 8chloroacetylaminoquinoline (44 mg, 0.2 mM) in CH3CN and refluxed for 8 h under N2. Then the solvent was evaporated in
As illustrated in Scheme 1, 8-chloroacetyl-aminoquinoline firstly reacted with branched PEI to synthesize a novel receptor that selectively binds Zn2 þ , namely PEIQ. Then PEIQ was covalently conjugated to fluorescent SiNPs by the reaction between
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Scheme 1. Synthetic routes for the SiNPs–PEIQ nanoprobe and schematic principle of Zn2 þ sensing by the ratiometric fluorescent strategy using the SiNPs–PEIQ nanoprobe.
primary amine groups of PEI and carboxylic groups on the surface of SiNPs under the catalysis of EDC/NHS. The integration of SiNPs and PEIQ exhibits a strong fluorescence emission at 600 nm and a negligible fluorescence emission at 500 nm in the absence of Zn2 þ upon a single wavelength excitation. After the addition of different amounts of Zn2 þ , the fluorescence intensity at 500 nm increased continuously while the fluorescence intensity at 600 nm remained stable, thus changing the dual emission intensity ratios and displaying continuous color changes from red to green which can be clearly observed by the naked eye. Therefore, the dual emission intensity ratio and the color signature can be used in both qualitative recognition and quantitative analysis. 3.2. Synthesis and characterization of PEIQ PEI, which is a water-soluble polymer with extremely high density of ethylene diamine units, has been extensively exploited for biological purposes such as gene delivery, and for chemical purposes such as stabilizing and solubilizing nanoparticles (Neu et al., 2005; Qi et al., 2000). However, the amine groups of PEI can cause severe cytotoxicity. It has documented that the neutralization such as acetylation and carboxylation of the amine groups can significantly improve biocompatibility of PEI (Cai et al., 2013; Duan and Nie, 2007; Xia et al., 2009). In the present work, branched PEI was grafted with 8-chloroacetylaminoquinoline by the reaction between primary amine groups of PEI and chloroacetyl groups of the quinoline derivative to synthesize a water-soluble and biocompatible quinoline-based probe PEIQ for specific recognition of Zn2þ . As shown in the 1H NMR spectrum, 8-chloroacetyl-aminoquinoline displayed typical resonance peaks between 7.0 and 11.0 ppm (curve a of Fig. 1A), while the peaks of PEI appeared at 2.6 ppm which was far away from those of 8-chloroacetyl-aminoquinoline (curve b of Fig. 1A). As expected, the as-prepared PEIQ have the characteristic peaks of both PEI and 8-chloroacetylaminoquinoline (curve c of Fig. 1A), indicating the successful conjugation of PEI and the quinoline derivative. Fourier transform infrared spectroscopy (FTIR) and fluorescence spectra further confirmed the conjugation of PEI and 8-chloroacetylaminoquinoline. Compared to FTIR spectrum of pure PEI, PEIQ shows more peaks around 1500 cm 1 which can be attributed to bond stretching of CQC and/or CQN originating from quinoline (Fig. 1B). PEI has no observable fluorescence emission and no capacity for Zn2 þ response (data not shown). The as-prepared PEIQ showed a broad emission centered at 450 nm which redshifted to 500 nm and accompanied with significant increase in intensity after the addition of Zn2þ (curve a and b of Fig. 1C). Although the specific response for Zn2þ recognition is clearly observed, the sensitivity and biocompatibility of as-prepared PEIQ might not be good enough for sensing and intracellular imaging of Zn2 þ whose relevant essential concentration
ranges from 1 fM in Escherichia coli to almost 0.5 mM in some mammalian cells (Frederickson et al., 2005). 3.3. Synthesis and characterization of nanoprobe SiNP–PEIQ Owing to its advantages in terms of improved sensitivity and built-in correction for environmental effects, the development of bio/chemo sensors by ratiometric fluorescence approach has received increasing attentions in recent years (Gong et al., 2012; Haidekker et al., 2005; Yao et al., 2013; Zhu et al., 2012; Zong et al., 2011). However, the critical challenge remains in the design of new dual-emission probes which exhibits an appropriate combination of Zn2 þ selectivity, biocompatibility, and cell permeability. Due to their high luminescence quantum yield, good photostability and water dispersivity, biocompatibility and versatile surface modification chemistry, fluorescent SiNPs become an excellent choice for developing bio/chemo nanosensors and diagnostic nanoprobes in biomedical analysis (Santra et al., 2001; Shi et al., 2013a, 2013b; Wang et al., 2013). Therefore, the assembly of fluorescent SiNPs with highly specific recognition elements, such as biomolecules and organic molecules, can create novel dual-emission probes for bio/chemical sensing and imaging (Zong et al., 2011). SiNPs with surface carboxylic groups were synthesized according to the previous reports (Santra et al., 2001; Shi et al., 2013a, 2013b). TEM images (Fig. 2A) clearly showed that the spherical SiNPs with a diameter of about 70 nm were well-dispersed in aqueous solutions without aggregation. Dynamic light scattering (DLS) measurements revealed that averaged diameter of the as-prepared SiNPs was 64.46 nm, which was consistent with the TEM observations. The FTIR spectrum of SiNPs confirmed the existence of carboxylic groups on the surface of SiNPs, where the peak at 3448 cm 1 and 1655 cm 1 should be attributed to the vibration of –OH groups and –CQO groups, respectively (curve b of Fig. 2C). UV–vis absorption spectrum and fluorescence spectrum verified the successful encapsulation of [Ru(bpy)3]2 þ in the asprepared SiNPs. Two typical absorption peaks at 289 nm and 455 nm originating from [Ru(bpy)3]2 þ was displayed in the UV– vis spectrum of SiNPs (curve b of Fig. 2D). And a characteristic emission peak at 600 nm also originating from [Ru(bpy)3]2 þ in aqueous solution when excited by 360 nm wavelength was displayed in the fluorescence spectrum of SiNPs (curve c of Fig. 1C). Then, the covalent conjugation of PEIQ onto SiNPs was carried out using carbodiimide chemistry, where a solution of EDC and NHS was used to initiate cross-linking reactions between carboxylic groups on the SiNPs and free amine groups of PEIQ. The conjugation of PEIQ onto SiNPs resulted in the aggregation of SiNPs to a certain extent, as evidenced by TEM image (Fig. 2B) and increased average size of SiNPs from 64.46 to 284.82 nm based
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A
B
C
Fig. 1. (A) 1H NMR spectrum of (a) 8-chloroacetylaminoquinoline (in CDCl3), (b) PEI (in D2O), and (c) PEIQ (in D2O). (B) FTIR spectrum of (a) PEI and (b) PEIQ. (C) Fluorescence spectrum of (a) PEIQ, (b) PEIQ þ Zn2 þ , (c) SiNPs, (d) the SiNPs–PEIQ nanoprobe, and (e) the SiNPs–PEIQ nanoprobe þZn2 þ .
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B
C
D
Fig. 2. (A, B) TEM image of carboxylated SiNPs (A) and the SiNPs–PEIQ nanoprobe (B). (C) FTIR spectrum of (a) PEIQ, (b) carboxylated SiNPs, and (c) the SiNPs–PEIQ nanoprobe. (D) UV–vis spectra of (a) PEIQ, (b) SiNPs, and (c) the SiNPs–PEIQ nanoprobe.
on DLS measurements. Successful conjugation of PEIQ onto SiNPs was supported indirectly by the change in zeta potential of SiNPs from 38.4 to 18.6 mV after the surface modification. The conjugation of PEIQ onto SiNPs was also confirmed by FTIR. By comparing the spectra, the peak at 1654.9 cm 1 which is attributed to νCQO of carboxylic groups on the surface of SiNPs, was split into two peaks at 1546.9 and 1515.21 cm 1 respectively corresponding to νCQO and δN–H þ τC–N of the newly formed amide bonds between SiNPs and PEIQ (Fig. 2C). And the disappearance of the broad COOH peaks in the FTIR spectra of SiNPs–PEIQ was correlated with the cross-linking of carboxylic groups on the SiNPs and free amine groups of PEIQ (Fig. 2C). In addition, the absorption curve of SiNP–PEIQ represents a typical combination of SiNPs and PEIQ, where the peaks at 289 nm and 455 nm were originated from SiNPs and the peak at 235 nm was originated from PEIQ (Fig. 2D). From the fluorescence emission spectra, two well-resolved emission peaks centered at 500 and
600 nm under a single wavelength excitation in the presence of Zn2 þ were clearly presented, which can be ascribed to the fluorescence of PEIQ–Zn2 þ and SiNPs, respectively (curve e of Fig. 1C). Since SiNPs without the conjugation of PEIQ have no capacity for Zn2 þ response, these results further confirmed the successful preparation of the novel dual-emission ratiometric fluorescent nanoprobe, namely SiNP–PEIQ. 3.4. Sensing application of nanoprobe SiNPs–PEIQ Then the capacity of the nanoprobe SiNP–PEIQ for Zn2 þ sensing was evaluated. Upon the addition of Zn2 þ , the fluorescence intensity at 500 nm of the green is continuously increased, whereas the intensity at 600 nm of the red remains unchanged, as shown in Fig. 3A. Owing to the changes in the intensity ratio of the two emission wavelengths, the fluorescence colors of the ratiometric probe solution changed continuously from red to green as
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Fig. 3. (A) Fluorescence responses of the SiNPs–PEIQ nanoprobe (2.0 mg mL 1) in aqueous solution upon addition of 0–100 mM Zn2 þ (λex ¼365 nm). The inset image was taken under a UV lamp (excitation wavelength at 365 nm, and the concentrations of Zn2 þ are 0, 2.0, 6.0, 10.0, 20.0, and 50.0 mM, from left to right). (B) Plot of the fluorescence intensity ratio F500/F600 as a function of the Zn2 þ concentration. F500/F600 was the fluorescence intensity ratio of the SiNPs–PEIQ nanoprobe at two emission wavelengths (500 nm and 600 nm).
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Fig. 4. The selectivity of the SiNPs–PEIQ nanoprobe (2.0 mg mL 1) to various metal ions (A) and amino acids (B) in aqueous solution. The white bars represent the addition of different metal ions (50 mM) or amino acids (20 mM), and the black bar represents the addition of different metal ions (50 mM) or amino acids (20 mM) in the presence of 20 mM Zn2 þ . (C) Cell viability values (%) estimated by MTT proliferation assays versus incubation concentrations of the nanoprobe (0, 50.0, 100.0, 150.0, 200.0, and 250.0 mg mL 1).
demonstrated in the inset of Fig. 3A. Obviously, even a slightly increase in emission intensity at 500 nm could result in distinguishable color changes with respect to the original background. Therefore, it is feasible to detect Zn2 þ by the naked eye under a UV lamp. By comparing the inset picture with the fluorescence spectrum of Fig. 3A, the ratio of the fluorescence intensity is closely related to the amount of Zn2 þ , ranging from 2.0 μM to 20 μM. Therefore, the quantification of Zn2 þ can be achieved with a correlation coefficient of 0.968, and the detection limit can be as low as 0.5 μM based on the definition of three times the standard deviation of the blank signal. In comparison, the detection sensitivity of the dual-emission ratiometric fluorescent nanoprobe was found to be comparable to that of other quinoline-based molecular probes or nanosensors. More importantly, the nanoprobe shows a good applicability in the determination of Zn2 þ in aqueous
solution. As a new sensing strategy, the nanoprobe has already demonstrated its capabilities. The complicated environment of the intracellular matrix poses a great challenge to the selectivity in metal ions detection. Thus, the selectivity and competition assays were carried out for the nanoprobe. As shown in Fig. 4A, no obvious changes in the fluorescence intensity was observed for the other metal ions when compared with that obtained for Zn2 þ . Furthermore, the potential interferences show negligible effects on the signal for Zn2 þ sensing, suggesting a high selectivity of the present nanoprobe toward Zn2 þ . Taking into account that amino acids in the biological system are capable to interact with a variety of metal cations, several typical amino acids were also examined as potential interferences. As shown in Fig. 4B, no obvious change in signal intensity was observed after the addition of amino acids with the
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Fig. 5. Fluorescence images of A549 cells incubated with the 10.0 mg mL 1 SiNPs–PEIQ nanoprobe before (A, B, C, and D) and after (E, F, G, and H) the exogenous Zn source treatment (addition of 50 μM ZnCl2). (A, E) The overlay of bright-field and fluorescence images; (B, F) the fluorescence images obtained from 470 to 530 nm; (C, G) the fluorescence images obtained from 570 to 630 nm; (D, H) the overlay of the fluorescence images.
same concentration as that of Zn2 þ . On the other hand, negligible signal changes were observed when the above compounds were added into the solution in the presence of Zn2 þ . The above results indicate that the SiNPs–PEIQ nanoprobe show high selectivity toward Zn2þ in the presence of interferences such as other competing metal ions and amino acids. In addition, because of the intrinsic biocompatibility of SiNPs, outstanding biocompatibility is also demonstrated for the present nanoprobe (Fig. 4C), emphasizing its potential applications in intracellular imaging of Zn2 þ . 3.5. Intracellular imaging of Zn2 þ using nanoprobe SiNPs–PEIQ It is believed that the large number of amine groups on each PEI can arouse endosmolytic effect which results in the cell membrane permeability, intracellular organelles disruption, and thus a release of the trapped materials into the cytoplasm (Duan and Nie, 2007; Neu et al., 2005). Therefore, it is anticipated that SiNP–PEIQ is able to cross the cell membrane and thus monitoring intracellular Zn2 þ ions. As expected, a strong cellular fluorescence was observed after the SiNPs–PEIQ nanoprobe was co-incubated with A549 cells for 2 h (Fig. 5). The overlay of fluorescence and bright-field channels (Figs. 5A, E) demonstrated excellent membrane permeability of the nanoprobe as the fluorescence signal was localized in the perinuclear region of the cytosol. After the exogenous Zn treatment (50 mM ZnCl2), the fluorescence emission color of the probe turned from red to green–yellow in A549 cells (Figs. 5D, H), which agreed well with the fluorescence spectrum observed in aqueous solution (inset of Fig. 3A) and this change became more obvious by looking at separated detector channels. As depicted in Fig. 5B, C, F, and G, the green fluorescence image from the PEIQ–Zn2 þ became brighter after addition of the Zn2 þ source, whereas the red reference channel showed no obvious change. These cell based assays show the great potential of the nanoprobe system for fundamental biology research.
4. Conclusion In summary, by facilely synthesizing PEIQ as a novel Zn2 þ probe, preparing [Ru(bpy)3]2 þ -encapsulated SiNPs as an internal standard, and conjugating PEIQ onto the surface of the fluorescent SiNPs, we have developed a dual-emission ratiometric fluorescence nanoprobe for the detection of Zn2 þ with high selectivity and sensitivity. The nanoprobe displays a detection limit as low as 0.5 μM which is much lower than the allowable level of zinc ( 20 μM) in the living cells, and shows high selectivity towards Zn2 þ over competing metal ions at cellular concentrations.
The nanoprobe can be employed for quantitative monitoring of Zn2 þ in aqueous solution with a linear range from 1.0 μM to 20 μM. The fluorescent nanoprobe shows low cytotoxicity and good cell-permeability, thus it is readily applicable for sensing and intracellular imaging of Zn2 þ . This work also describes a ratiometric fluorescence strategy for sensing and in vivo imaging of other metal ions or species that are important in physiological and pathological events.
Acknowledgment The financial support from the National Scientific Foundation of China (31100723) and Guangzhou Science and Technology and Information Bureau (2013J2200053) is gratefully acknowledged.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A novel dual-emission ratiometric fluorescent nanoprobe for sensing and intracellular imaging of Zn2 þ Yupeng Shi a,1, Zhihua Chen a,1, Xin Cheng a, Yi Pan a, Heng Zhang a, Zhaomin Zhang a, Cheuk-Wing Li b, Changqing Yi a,n a Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong Province), School of Engineering, Sun Yat-Sen University, Guangzhou, 510006, China b Institute of Chinese Medical Sciences, University of Macau, Macau, China
art ic l e i nf o
a b s t r a c t
Article history: Received 19 February 2014 Received in revised form 2 May 2014 Accepted 21 May 2014 Available online 28 May 2014
The integration of unique characteristics of nanomaterials with highly specific recognition elements, such as biomolecules and organic molecules, are the foundation of many novel nanoprobes for bio/ chemical sensing and imaging. In the present report, branched polyethylenimine (PEI) was grafted with 8-chloroacetyl-aminoquinoline to synthesize a water-soluble and biocompatible quinoline-based Zn2 þ probe PEIQ. Then the PEIQ was covalently conjugated to [Ru(bpy)3]2 þ -encapsulated SiNPs to obtain the ratiometric fluorescence nanoprobe which exhibits a strong fluorescence emission at 600 nm and a negligible fluorescence emission at 500 nm in the absence of Zn2 þ upon a single wavelength excitation. After the addition of different amounts of Zn2 þ , the fluorescence intensity at 500 nm increased continuously while the fluorescence intensity at 600 nm remained stable, thus changing the dual emission intensity ratios and displaying continuous color changes from red to green which can be clearly observed by the naked eye. The nanoprobe exhibits good water dispersivity, biocompatibility and cell permeability, high selectivity over competing metal ions, and high sensitivity with a detection limit as low as 0.5 μM. Real-time imaging of Zn2 þ in A549 cells has also been realized using this novel nanoprobe. & 2014 Elsevier B.V. All rights reserved.
Keywords: Silica nanoparticles 8-Aminoquinoline Zn2 þ Ratiometric fluorescence Cellular imaging
1. Introduction As the second most abundant transition metal in the human body, zinc always occurs as a divalent cation [Zn (II)] and plays pivotal roles in biological systems (Falchuk, 1998; O’Halloran, 1993). For example, Zn2 þ serves as catalytic and structural cofactor when it is bound to some specific proteins, thus facilitating enzyme regulation, gene expression and neural related signal transmission (Vallee and Falchuk, 1993). Although it remains unclear about its functional role, growing evidence suggests the correlations between the disorder of Zn2 þ metabolism and many neurological diseases, including Alzheimer’s disease, infantile diarrhea, epilepsy and cerebral ischemia (Adlard and Bush, 2006; Koh et al., 1996). Because of its important biological roles, research interests to develop highly sensitive and selective detection and monitoring of Zn2 þ under physiological conditions are growing unabated (Lim et al., 2004). Fluorescence is believed to be the most effective way to determine the concentration, together with
n
Corresponding author. Tel./fax: þ 86 20 39342380. E-mail address: [email protected] (C. Yi). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.05.050 0956-5663/& 2014 Elsevier B.V. All rights reserved.
visualization of subcellular distribution of Zn2 þ in living cells (Hanaoka et al., 2004; Jiang and Guo, 2004; Walkup et al., 2000; Xie et al., 2012). Among the fluorescent probes for Zn2 þ ions detection, quinoline based molecules, especially 8-aminoquinoline and 8-hydroxy-quinoline, have been studied extensively because they are pH insensitive, ready to form strong inter-molecular hydrogen bonds with surrounding water molecules, and their metal-coordination. Moreover, quinoline based Zn2 þ sensors are suitable candidates for photofunctional molecular devices because their sensing mechanism is mainly on the basis of photo-induced electron transfer and photo-induced charge transfer processes (Du et al., 2010; Xie et al., 2011; Zhou et al., 2010). However, most of these quinoline-based probes targeting Zn2 þ ions still suffer from poor solubility, bad cell permeability, and weak sensitivity. And a few examples of ratiometric fluorescence methods for sensing and intracellular imaging of Zn2 þ have been reported (Jiang and Guo, 2004; Liu et al., 2012; Raje et al., 2013). Recently, the conjugation of fluorescent probes with nanoparticles has emerged as an attractive strategy to develop new chemosensors for intracellular monitoring of Zn2 þ , since nanoparticles can shelter the probes from interferences and can transport the probes neglecting their intrinsic solubility (Li et al., 2011; Lu et al., 2012; Rastogi et al., 2011; Ruedas-Rama and Hall,
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2008; Pal et al., 2011; Teolato et al., 2007; Xu et al., 2013). Due to their high luminescence quantum yield, good photostability and water dispersivity, biocompatibility and versatile surface modification chemistry, [Ru(bpy)3]2þ -encapsulated silica nanoparticles (SiNPs) become an excellent choice for developing bio/chemo nanosensors and diagnostic nanoprobes in bio/chemical analysis (Santra et al., 2001; Shi et al., 2013a, 2013b; Wang et al., 2013). Herein, in the present report, branched polyethylenimine (PEI) was firstly grafted with 8-chloroacetyl-aminoquinoline to synthesize a water-soluble and biocompatible quinoline-based Zn2þ probe PEIQ. Then PEIQ was covalently conjugated onto the surface of [Ru(bpy)3]2 þ -encapsulated SiNPs to develop a selective and sensitive ratiometric strategy for the sensing and monitoring of Zn2 þ both in vitro and in vivo. The nanoprobe exhibits high selectivity over competing metal ions, high sensitivity with a detection limit as low as 0.5 μM, and suitability of real-time Zn2þ imaging in A549 cells. Notably, the reported nanoprobe possesses some remarkable features: (1) the recognition moiety is synthesized by grafting branched PEI with quinoline derivative, thus reducing the possible cytotoxicity of PEI and increasing water dispersivity and cell permeability of the as-prepared Zn2 þ probe, PEIQ; (2) the internal standard is encapsulated into SiNPs and the recognition moiety is covalently conjugated onto SiNPs surface, thus providing a reliable reference signal and a stable nanoprobe; (3) the large amount of PEIQ units on the outer surface of an individual nanoparticle enable the signal amplification, hence making the immediate and highly sensitive detection of Zn2 þ possible.
vacuum. The crude product was purified by co-precipitation with ether to give yellow oil. 2.4. Synthesis and functionalization of fluorescent SiNPs
2. Materials and methods
The fluorescent [Ru(bpy)3]2 þ -encapsulated SiNPs were synthesized by a water-in-oil reverse micelle method as described in detail elsewhere. Typically, 7.5 mL of cyclohexane, 1.77 mL of Triton X-100, 1.8 mL of hexanol, and 0.34 mL of H2O were stirred for 20 mins to generate the microemulsion system, followed by the addition of 80 μL of 0.1 M [Ru(bpy)3]2 þ and 100 μL of tetraethoxy orthosilicate (TEOS). After being stirred for 30 min, silica polymerization was initiated by the addition of 60 μL aqueous ammonia and allowed to react for 24 h. Then, in order to prepare carboxylated SiNPs (SiNP-COOH), 50 μL TEOS and 50 μL carboxyethylsilanetriol sodium salt (CTES) were added to the mixture and the reaction was allowed to continue for another 24 h. Finally, acetone was added to destabilize the micro-emulsion system. The fluorescent SiNPs were isolated via centrifugation and washed in sequence with ethanol and D.I. water to remove any surfactant and unreacted reactants. Carbodiimide chemistry was employed to covalently conjugate PEIQ onto SiNPs. Briefly, 0.1 g of SiNP-COOH and 0.05 g of PEIQ were suspended in 20 mL of 0.01 M phosphate-buffered saline (PBS) buffer (pH¼7.4) containing 5 mM N-hydroxy-succinimide (NHS) and 2 mM 1-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide (EDC). Then the mixture was allowed to react at room temperature for another 16 h under gentle shaking. The final nanoprobes were obtained via centrifugation and were washed in sequence with D.I. water to remove any of the unreacted reactants.
2.1. Apparatus and reagents
2.5. Sensitivity and selectivity of the SiNPs–PEIQ nanoprobe
Morphology of the nanoparticles was examined by a transmission electron microscope (TEM, JEOL JEM-1400). DLS measurements were performed at 25 1C using a Malvern Zetasizer NanoZS90 instrument. UV–vis, FTIR, and fluorescence spectra were obtained on a Beckman DU730 UV–vis spectrometer, a VERTEX 70 spectrometer (Bruker), and a PTI Quanta-Master QM4CW spectrofluorometer, respectively. 1H NMR spectra were recorded with a Varian Mercury-400 spectrometer with Me4Si as the internal standard. The fluorescence images were acquired by an inverted fluorescence microscope (OLYMPUS IX71). The metal ions solutions were prepared from NaCl, KCl, MgCl2, CaCl2, FeCl3, Pb(NO3)2,CoCl2, NiCl2, ZnCl2, HgCl2, CrCl3 and CuSO4 in distilled water with a concentration of 0.01 M or 0.001 M.
For the detection of Zn2 þ , different concentrations of Zn2 þ (0, 1.0, 2.0, 4.0, 6.0, 10.0, 15.0, 20.0, 30.0, 50.0, and 100.0 mM) were mixed with the nanoprobe (2.0 mg mL 1) in 1.2 mL of distilled water under gentle shaking. Photographs and fluorescence spectra were taken after Zn2 þ reacted with the nanoprobe for 5 mins. To evaluate the selectivity of the nanoprobe, 20.0 μM of amino acids or 50.0 μM of competing metal ions was mixed with the SiNP–PEIQ (2.0 mg mL 1) under gentle shaking. Fluorescence spectra were taken after the metal ions or amino acids reacted with the dispersed nanoprobe for 5 mins.
2.2. Synthesis of 8-chloroacetylaminoquinoline 8-chloroacetylaminoquinoline was synthesized according to a previously reported method (Zhou et al., 2010). 288 mg of 8aminoquinoline (2 mM) and 202 mg (2.1 mM) of N(Et)3 were added into 10 mL of CH2Cl2 and mixed in a round flask for 20 min at 0 1C. Then 246 mg (2.2 mM) of chloroacetyl chloride was added dropwise. The mixture was warmed to room temperature and allowed to react overnight. The solvent was evaporated in vacuum. The crude product was further purified by column chromatography (silica gel, PE/EA at 3:1) and a pale white solid was obtained.
2.6. Intracellular imaging of Zn2 þ The A549 cells were provided by Cells Bank of the Chinese Academy of Science (Shanghai, China). Cells were grown in H-DMEM (high glucose) supplemented with 10% FBS in an atmosphere of 5% CO2 at 37 1C. Cells (105/well) were plated on 6 mm glass coverslips and allowed to adhere for 24 h. Intracellular imaging of Zn2 þ were performed in the same medium supplemented with 50 μM ZnCl2 for 0.5 h. A549 cells were washed with PBS and incubated with the nanoprobe (10.0 mg mL 1) at 37 1C for 2 h. Cell imaging was performed with an inverted fluorescence microscope after washing the cells with PBS.
3. Results and discussions
2.3. Synthesis of PEIQ
3.1. Synthetic routes for the SiNPs–PEIQ nanoprobe and its sensing mechanism
A mixture of PEI (MW ¼1800) (500 mg, 0.28 mM) and K2CO3 (38.4 mg, 0.28 mM) in 5 mL CH3CN was added to 20 mL of 8chloroacetylaminoquinoline (44 mg, 0.2 mM) in CH3CN and refluxed for 8 h under N2. Then the solvent was evaporated in
As illustrated in Scheme 1, 8-chloroacetyl-aminoquinoline firstly reacted with branched PEI to synthesize a novel receptor that selectively binds Zn2 þ , namely PEIQ. Then PEIQ was covalently conjugated to fluorescent SiNPs by the reaction between
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Scheme 1. Synthetic routes for the SiNPs–PEIQ nanoprobe and schematic principle of Zn2 þ sensing by the ratiometric fluorescent strategy using the SiNPs–PEIQ nanoprobe.
primary amine groups of PEI and carboxylic groups on the surface of SiNPs under the catalysis of EDC/NHS. The integration of SiNPs and PEIQ exhibits a strong fluorescence emission at 600 nm and a negligible fluorescence emission at 500 nm in the absence of Zn2 þ upon a single wavelength excitation. After the addition of different amounts of Zn2 þ , the fluorescence intensity at 500 nm increased continuously while the fluorescence intensity at 600 nm remained stable, thus changing the dual emission intensity ratios and displaying continuous color changes from red to green which can be clearly observed by the naked eye. Therefore, the dual emission intensity ratio and the color signature can be used in both qualitative recognition and quantitative analysis. 3.2. Synthesis and characterization of PEIQ PEI, which is a water-soluble polymer with extremely high density of ethylene diamine units, has been extensively exploited for biological purposes such as gene delivery, and for chemical purposes such as stabilizing and solubilizing nanoparticles (Neu et al., 2005; Qi et al., 2000). However, the amine groups of PEI can cause severe cytotoxicity. It has documented that the neutralization such as acetylation and carboxylation of the amine groups can significantly improve biocompatibility of PEI (Cai et al., 2013; Duan and Nie, 2007; Xia et al., 2009). In the present work, branched PEI was grafted with 8-chloroacetylaminoquinoline by the reaction between primary amine groups of PEI and chloroacetyl groups of the quinoline derivative to synthesize a water-soluble and biocompatible quinoline-based probe PEIQ for specific recognition of Zn2þ . As shown in the 1H NMR spectrum, 8-chloroacetyl-aminoquinoline displayed typical resonance peaks between 7.0 and 11.0 ppm (curve a of Fig. 1A), while the peaks of PEI appeared at 2.6 ppm which was far away from those of 8-chloroacetyl-aminoquinoline (curve b of Fig. 1A). As expected, the as-prepared PEIQ have the characteristic peaks of both PEI and 8-chloroacetylaminoquinoline (curve c of Fig. 1A), indicating the successful conjugation of PEI and the quinoline derivative. Fourier transform infrared spectroscopy (FTIR) and fluorescence spectra further confirmed the conjugation of PEI and 8-chloroacetylaminoquinoline. Compared to FTIR spectrum of pure PEI, PEIQ shows more peaks around 1500 cm 1 which can be attributed to bond stretching of CQC and/or CQN originating from quinoline (Fig. 1B). PEI has no observable fluorescence emission and no capacity for Zn2 þ response (data not shown). The as-prepared PEIQ showed a broad emission centered at 450 nm which redshifted to 500 nm and accompanied with significant increase in intensity after the addition of Zn2þ (curve a and b of Fig. 1C). Although the specific response for Zn2þ recognition is clearly observed, the sensitivity and biocompatibility of as-prepared PEIQ might not be good enough for sensing and intracellular imaging of Zn2 þ whose relevant essential concentration
ranges from 1 fM in Escherichia coli to almost 0.5 mM in some mammalian cells (Frederickson et al., 2005). 3.3. Synthesis and characterization of nanoprobe SiNP–PEIQ Owing to its advantages in terms of improved sensitivity and built-in correction for environmental effects, the development of bio/chemo sensors by ratiometric fluorescence approach has received increasing attentions in recent years (Gong et al., 2012; Haidekker et al., 2005; Yao et al., 2013; Zhu et al., 2012; Zong et al., 2011). However, the critical challenge remains in the design of new dual-emission probes which exhibits an appropriate combination of Zn2 þ selectivity, biocompatibility, and cell permeability. Due to their high luminescence quantum yield, good photostability and water dispersivity, biocompatibility and versatile surface modification chemistry, fluorescent SiNPs become an excellent choice for developing bio/chemo nanosensors and diagnostic nanoprobes in biomedical analysis (Santra et al., 2001; Shi et al., 2013a, 2013b; Wang et al., 2013). Therefore, the assembly of fluorescent SiNPs with highly specific recognition elements, such as biomolecules and organic molecules, can create novel dual-emission probes for bio/chemical sensing and imaging (Zong et al., 2011). SiNPs with surface carboxylic groups were synthesized according to the previous reports (Santra et al., 2001; Shi et al., 2013a, 2013b). TEM images (Fig. 2A) clearly showed that the spherical SiNPs with a diameter of about 70 nm were well-dispersed in aqueous solutions without aggregation. Dynamic light scattering (DLS) measurements revealed that averaged diameter of the as-prepared SiNPs was 64.46 nm, which was consistent with the TEM observations. The FTIR spectrum of SiNPs confirmed the existence of carboxylic groups on the surface of SiNPs, where the peak at 3448 cm 1 and 1655 cm 1 should be attributed to the vibration of –OH groups and –CQO groups, respectively (curve b of Fig. 2C). UV–vis absorption spectrum and fluorescence spectrum verified the successful encapsulation of [Ru(bpy)3]2 þ in the asprepared SiNPs. Two typical absorption peaks at 289 nm and 455 nm originating from [Ru(bpy)3]2 þ was displayed in the UV– vis spectrum of SiNPs (curve b of Fig. 2D). And a characteristic emission peak at 600 nm also originating from [Ru(bpy)3]2 þ in aqueous solution when excited by 360 nm wavelength was displayed in the fluorescence spectrum of SiNPs (curve c of Fig. 1C). Then, the covalent conjugation of PEIQ onto SiNPs was carried out using carbodiimide chemistry, where a solution of EDC and NHS was used to initiate cross-linking reactions between carboxylic groups on the SiNPs and free amine groups of PEIQ. The conjugation of PEIQ onto SiNPs resulted in the aggregation of SiNPs to a certain extent, as evidenced by TEM image (Fig. 2B) and increased average size of SiNPs from 64.46 to 284.82 nm based
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A
B
C
Fig. 1. (A) 1H NMR spectrum of (a) 8-chloroacetylaminoquinoline (in CDCl3), (b) PEI (in D2O), and (c) PEIQ (in D2O). (B) FTIR spectrum of (a) PEI and (b) PEIQ. (C) Fluorescence spectrum of (a) PEIQ, (b) PEIQ þ Zn2 þ , (c) SiNPs, (d) the SiNPs–PEIQ nanoprobe, and (e) the SiNPs–PEIQ nanoprobe þZn2 þ .
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B
C
D
Fig. 2. (A, B) TEM image of carboxylated SiNPs (A) and the SiNPs–PEIQ nanoprobe (B). (C) FTIR spectrum of (a) PEIQ, (b) carboxylated SiNPs, and (c) the SiNPs–PEIQ nanoprobe. (D) UV–vis spectra of (a) PEIQ, (b) SiNPs, and (c) the SiNPs–PEIQ nanoprobe.
on DLS measurements. Successful conjugation of PEIQ onto SiNPs was supported indirectly by the change in zeta potential of SiNPs from 38.4 to 18.6 mV after the surface modification. The conjugation of PEIQ onto SiNPs was also confirmed by FTIR. By comparing the spectra, the peak at 1654.9 cm 1 which is attributed to νCQO of carboxylic groups on the surface of SiNPs, was split into two peaks at 1546.9 and 1515.21 cm 1 respectively corresponding to νCQO and δN–H þ τC–N of the newly formed amide bonds between SiNPs and PEIQ (Fig. 2C). And the disappearance of the broad COOH peaks in the FTIR spectra of SiNPs–PEIQ was correlated with the cross-linking of carboxylic groups on the SiNPs and free amine groups of PEIQ (Fig. 2C). In addition, the absorption curve of SiNP–PEIQ represents a typical combination of SiNPs and PEIQ, where the peaks at 289 nm and 455 nm were originated from SiNPs and the peak at 235 nm was originated from PEIQ (Fig. 2D). From the fluorescence emission spectra, two well-resolved emission peaks centered at 500 and
600 nm under a single wavelength excitation in the presence of Zn2 þ were clearly presented, which can be ascribed to the fluorescence of PEIQ–Zn2 þ and SiNPs, respectively (curve e of Fig. 1C). Since SiNPs without the conjugation of PEIQ have no capacity for Zn2 þ response, these results further confirmed the successful preparation of the novel dual-emission ratiometric fluorescent nanoprobe, namely SiNP–PEIQ. 3.4. Sensing application of nanoprobe SiNPs–PEIQ Then the capacity of the nanoprobe SiNP–PEIQ for Zn2 þ sensing was evaluated. Upon the addition of Zn2 þ , the fluorescence intensity at 500 nm of the green is continuously increased, whereas the intensity at 600 nm of the red remains unchanged, as shown in Fig. 3A. Owing to the changes in the intensity ratio of the two emission wavelengths, the fluorescence colors of the ratiometric probe solution changed continuously from red to green as
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Fig. 3. (A) Fluorescence responses of the SiNPs–PEIQ nanoprobe (2.0 mg mL 1) in aqueous solution upon addition of 0–100 mM Zn2 þ (λex ¼365 nm). The inset image was taken under a UV lamp (excitation wavelength at 365 nm, and the concentrations of Zn2 þ are 0, 2.0, 6.0, 10.0, 20.0, and 50.0 mM, from left to right). (B) Plot of the fluorescence intensity ratio F500/F600 as a function of the Zn2 þ concentration. F500/F600 was the fluorescence intensity ratio of the SiNPs–PEIQ nanoprobe at two emission wavelengths (500 nm and 600 nm).
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Fig. 4. The selectivity of the SiNPs–PEIQ nanoprobe (2.0 mg mL 1) to various metal ions (A) and amino acids (B) in aqueous solution. The white bars represent the addition of different metal ions (50 mM) or amino acids (20 mM), and the black bar represents the addition of different metal ions (50 mM) or amino acids (20 mM) in the presence of 20 mM Zn2 þ . (C) Cell viability values (%) estimated by MTT proliferation assays versus incubation concentrations of the nanoprobe (0, 50.0, 100.0, 150.0, 200.0, and 250.0 mg mL 1).
demonstrated in the inset of Fig. 3A. Obviously, even a slightly increase in emission intensity at 500 nm could result in distinguishable color changes with respect to the original background. Therefore, it is feasible to detect Zn2 þ by the naked eye under a UV lamp. By comparing the inset picture with the fluorescence spectrum of Fig. 3A, the ratio of the fluorescence intensity is closely related to the amount of Zn2 þ , ranging from 2.0 μM to 20 μM. Therefore, the quantification of Zn2 þ can be achieved with a correlation coefficient of 0.968, and the detection limit can be as low as 0.5 μM based on the definition of three times the standard deviation of the blank signal. In comparison, the detection sensitivity of the dual-emission ratiometric fluorescent nanoprobe was found to be comparable to that of other quinoline-based molecular probes or nanosensors. More importantly, the nanoprobe shows a good applicability in the determination of Zn2 þ in aqueous
solution. As a new sensing strategy, the nanoprobe has already demonstrated its capabilities. The complicated environment of the intracellular matrix poses a great challenge to the selectivity in metal ions detection. Thus, the selectivity and competition assays were carried out for the nanoprobe. As shown in Fig. 4A, no obvious changes in the fluorescence intensity was observed for the other metal ions when compared with that obtained for Zn2 þ . Furthermore, the potential interferences show negligible effects on the signal for Zn2 þ sensing, suggesting a high selectivity of the present nanoprobe toward Zn2 þ . Taking into account that amino acids in the biological system are capable to interact with a variety of metal cations, several typical amino acids were also examined as potential interferences. As shown in Fig. 4B, no obvious change in signal intensity was observed after the addition of amino acids with the
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Fig. 5. Fluorescence images of A549 cells incubated with the 10.0 mg mL 1 SiNPs–PEIQ nanoprobe before (A, B, C, and D) and after (E, F, G, and H) the exogenous Zn source treatment (addition of 50 μM ZnCl2). (A, E) The overlay of bright-field and fluorescence images; (B, F) the fluorescence images obtained from 470 to 530 nm; (C, G) the fluorescence images obtained from 570 to 630 nm; (D, H) the overlay of the fluorescence images.
same concentration as that of Zn2 þ . On the other hand, negligible signal changes were observed when the above compounds were added into the solution in the presence of Zn2 þ . The above results indicate that the SiNPs–PEIQ nanoprobe show high selectivity toward Zn2þ in the presence of interferences such as other competing metal ions and amino acids. In addition, because of the intrinsic biocompatibility of SiNPs, outstanding biocompatibility is also demonstrated for the present nanoprobe (Fig. 4C), emphasizing its potential applications in intracellular imaging of Zn2 þ . 3.5. Intracellular imaging of Zn2 þ using nanoprobe SiNPs–PEIQ It is believed that the large number of amine groups on each PEI can arouse endosmolytic effect which results in the cell membrane permeability, intracellular organelles disruption, and thus a release of the trapped materials into the cytoplasm (Duan and Nie, 2007; Neu et al., 2005). Therefore, it is anticipated that SiNP–PEIQ is able to cross the cell membrane and thus monitoring intracellular Zn2 þ ions. As expected, a strong cellular fluorescence was observed after the SiNPs–PEIQ nanoprobe was co-incubated with A549 cells for 2 h (Fig. 5). The overlay of fluorescence and bright-field channels (Figs. 5A, E) demonstrated excellent membrane permeability of the nanoprobe as the fluorescence signal was localized in the perinuclear region of the cytosol. After the exogenous Zn treatment (50 mM ZnCl2), the fluorescence emission color of the probe turned from red to green–yellow in A549 cells (Figs. 5D, H), which agreed well with the fluorescence spectrum observed in aqueous solution (inset of Fig. 3A) and this change became more obvious by looking at separated detector channels. As depicted in Fig. 5B, C, F, and G, the green fluorescence image from the PEIQ–Zn2 þ became brighter after addition of the Zn2 þ source, whereas the red reference channel showed no obvious change. These cell based assays show the great potential of the nanoprobe system for fundamental biology research.
4. Conclusion In summary, by facilely synthesizing PEIQ as a novel Zn2 þ probe, preparing [Ru(bpy)3]2 þ -encapsulated SiNPs as an internal standard, and conjugating PEIQ onto the surface of the fluorescent SiNPs, we have developed a dual-emission ratiometric fluorescence nanoprobe for the detection of Zn2 þ with high selectivity and sensitivity. The nanoprobe displays a detection limit as low as 0.5 μM which is much lower than the allowable level of zinc ( 20 μM) in the living cells, and shows high selectivity towards Zn2 þ over competing metal ions at cellular concentrations.
The nanoprobe can be employed for quantitative monitoring of Zn2 þ in aqueous solution with a linear range from 1.0 μM to 20 μM. The fluorescent nanoprobe shows low cytotoxicity and good cell-permeability, thus it is readily applicable for sensing and intracellular imaging of Zn2 þ . This work also describes a ratiometric fluorescence strategy for sensing and in vivo imaging of other metal ions or species that are important in physiological and pathological events.
Acknowledgment The financial support from the National Scientific Foundation of China (31100723) and Guangzhou Science and Technology and Information Bureau (2013J2200053) is gratefully acknowledged.
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