Article pubs.acs.org/ac
Through-Bond Energy Transfer-Based Ratiometric Two-Photon Probe for Fluorescent Imaging of Pd2+ Ions in Living Cells and Tissues Liyi Zhou, Qianqian Wang, Xiao-Bing Zhang,* and Weihong Tan Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China S Supporting Information *
ABSTRACT: Palladium can cause severe skin and eye irritation once it enters the human body. Ratiometric twophoton fluorescent probes can both eliminate interference from environmental factors and realize deep-tissue imaging with improved spatial localization. To quantitatively track Pd2+ in biosystems, we report here a colorimetric and two-photon ratiometric fluorescent probe, termed Np−Rh−Pd, which consists of a two-photon fluorophore (naphthalene derivative with a D-π-A structure) and a rhodamine B dye. The two fluorophores are directly linked to form a two-photon ratiometric fluorescent probe for Pd2+ based on a through-bond energy transfer (TBET) strategy. It exhibits highly efficient energy transfer (90%) with two well-resolved emission peaks (wavelength difference of 100 nm), which could efficiently diminish the cross talk between channels and is especially favorable for ratiometric bioimaging applications. A signal-to-background ratio of 31.2 was observed for the probe, which affords a high sensitivity for Pd2+ with a detection limit of 2.3 × 10−7 M. It was also found that acidity does not affect the fluorescent response of the probe to Pd2+, which is favorable for its applications in practical samples. The probe was further used for fluorescence imaging of Pd2+ ions in live cells and tissue slices under two-photon excitation, which showed significant tissue-imaging depths (90−270 μm) and a high resolution for ratiometric imaging.
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bleaching, and shallow tissue-penetration depth. Moreover, these probes are based on the intensity changes of a single emission peak, which tends to be affected by a variety of factors, including instrumental variations, environmental condition changes, and probe concentration fluctuations. Therefore, it is of great significance and necessity to develop a simple and reliable fluorescent method for the quantitative measurement of the Pd2+ ion concentration in live cells and tissues. To solve these problems, we used an effective two-photon fluorescence microscopy (TPFM) approach, which excites a two-photon (TP) fluorescence dye by a long-wavelength laser light and thus provides improved three-dimensional imaging resolution with increased tissue-imaging depth (>500 μm) and an extended observation time compared to traditional confocal microscopy.6 Particularly when combined with a ratiometric strategy, the built-in correction of the two emission bands could further eliminate interference caused by instrumental variations, environmental factor changes, and probe concentration fluctuations.7 Previously, we established a ratiometric twophoton fluorescence probe platform, termed Np−Rh, by directly connecting a two-photon naphthalene derivative with
alladium, one of the platinum-group elements, can cause severe skin and eye irritation1 when it is taken into human bodies via contaminated food, medicine, and water.2 DNA, proteins, and other biomacromolecules, such as vitamin B6, have been reported to be able to strongly bind palladium ions, which may lead to a variety of cellular dysfunction processes.3 Therefore, the development of an efficient method for detecting Pd2+ ions in biosystems is of great biological importance. Traditional analytical methods,4 such as inductively coupled plasma mass spectrometry (ICP-MS),4a atomic absorption spectrometry (AAS),4b inductively coupled plasma emission spectroscopy (ICP-AES),4c and X-ray fluorescence (XRF),4d have been developed for the efficient detection of palladium ions. These methods, however, usually require expensive instruments and highly skilled professionals. In contrast, fluorescence methods can avoid these shortcomings while maintaining comparable efficiency and accuracy and have therefore been widely exploited by researchers for the detection of a variety of targets. To date, only a few fluorescent probes that can quantitatively detect Pd2+ concentration have been reported.5 Unfortunately, most of these fluorescent probes are excited by one-photon excitation with a short wavelength, which results in some obvious drawbacks when used in bioimaging, such as high autofluorescence background, photo© XXXX American Chemical Society
Received: February 6, 2015 Accepted: March 26, 2015
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DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry a D-π-A structure with a rhodamine B fluorophore via the TBET strategy.8 Herein, we used this platform further to develop a new ratiometric two-photon fluorescent probe, Np− Rh−Pd, for Pd2+ ion detection and bioimaging applications. The probe molecule exhibited two well-separated fluorescence emission peaks and a high-efficiency energy transfer, which provided a large signal-to-background ratio of 31.2, and therefore high sensitivity of the probe with a detection limit of 2.3 × 10−7 M observed for Pd2+ ions. It showed a pronounced fluorescent response toward Pd2+ ions over other common metal ions. The probe was further used for fluorescence imaging of Pd2+ ions in live cells and tissues by TPFM, which exhibited large tissue-imaging depths with a high resolution for ratiometric imaging.
species were prepared from CaCl2, MgSO2, Co(CH3COO)2· 4H2O, CuCl2·H2O, NiCl2·6H2O, CrCl3·6H2O, Zn(NO3)2· 6H2O, Al(NO3)3·9H2O, FeSO2, HgCl2, Pb(NO3)2, NaCl, (GSH), Cys, and Glu using ultrapure water with final concentrations of 50 μM. Cell Cytotoxic Assays and Imaging. The cytotoxic effects of the probe were assessed using MTT assays.8 For fluorescent imaging, the cells were washed with PBS buffer followed by incubation with the probe (5 μM) for 30 min in culture medium (PBS buffer containing 0.5% EtOH) at 37 °C. Cell samples were then washed with PBS three times and imaged. Pd2+ ions (20 μM) were added to induce a ratiometric signal change during imaging. One-photon excitation wavelength was fixed at 405 nm, and the two-photon excitation wavelength of the femtosecond laser was fixed at 780 nm; the emission wavelengths were recorded at 470−530 and 550−650 nm, respectively. Preparation and Staining of Liver Tissue Slices from Nude Mice. Frozen tissue slices were prepared from the livers of nude mice. A side of the tissue was cut flat using a vibrating-blade microtome. The slices were cultured with 10 μM Np−Rh−Pd in an incubator at 37 °C for 1 h and then washed with PBS three times. Finally, the slices were incubated in culture medium with 100 μM Pd2+ ions and cultured in an incubator at 37 °C for 1 h. Before TPFM imaging, the samples were washed with PBS three more times. The TPFM images (with 10× magnification) were collected in two channels (green: 470−530 nm; red: 550−650 nm) upon excitation at 780 nm with a femtosecond pulse laser.
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EXPERIMENTAL SECTION Materials and Apparatus. All chemicals were of analytical grade, used without further purification, and provided by commercial suppliers. Water used in all experiments was purified via a Milli-Q water system from Millipore (Billerica, MA, USA). NMR spectra were carried out on a Bruker DRX400 spectrometer with TMS employed as an internal standard. All chemical shifts are given in the standard δ notation of parts per million. Mass spectra were recorded using an LCQ Advantage ion trap mass spectrometer (Thermo Finnigan). UV−vis absorption spectra were performed on a Shimadzu UV2450 spectrophotometer with a 1.0 cm path length quartz cuvette. The pH was measured with a Mettler-Toledo Delta 320 pH meter. Fluorescence measurements were carried out on a Hitachi-F4500 fluorescence spectrometer with excitation and emission slits both set at 2.5 nm. Fluorescence images of HeLa cells and tissue slices were taken using an Olympus FV1000MPE multiphoton laser scanning microscope (Japan). Synthesis of Probe Compound Np−Rh−Pd. Np−Rh−Pd was synthesized according to previously reported methods.5f,8 Characterization of compound Np−Rh−Pd is shown in the Supporting Information. A mixture of Np−Rh (0.15 g, 200 μmol), 9-anthraldehydede (0.10 g, 480 μmol), and 50 mL of ethanol was added to a round-bottom flask with a reflux condenser and heated with continuous refluxing for 4 h. The mixture solution was reduced under pressure distillation. A yellow solid Np−Rh−Pd (0.16 g, 85% yield) was obtained through column chromatography with petroleum ether/ethyl acetate (2:1, v/v) as eluent. 1H NMR (400 MHz, d-DMSO): δ 8.64 (s, 1H), 8.59−8.55 (t, J = 8 Hz, 1H), 8.04−7.65 (m, 6H), 7.52−7.40 (d, J = 24 Hz, 2H), 7.32−7.28 (m, 1H), 7.03 (s, 1H), 6.71−6.67 (d, J = 8 Hz, 1H), 6.65−6.44 (m, 5H), 3.40− 3.32 (dd, J = 16 Hz, 8H), 3.08 (s, 6H), 1.09−1.06 (t, J = 6 Hz, 12H). +c ESI, 931.4; calcd, 931.2. Spectrophotometric Measurements. Both fluorescence and absorption spectra were measured in 10 mM phosphate buffer solution containing EtOH as the cosolvent solution (H2O/ EtOH 1:1, v/v). The pH gradient of PBS buffer solution from 2.0 to 10.0 was achieved by adding different volumes of HCl or NaOH solution. The fluorescence emission spectra were recorded at an excitation wavelength of 415 nm with an emission wavelength range from 450 to 665 nm. A stock solution of probe (1 × 10−3 M) was prepared by dissolving the probe in EtOH. The procedure of calibration measurements of the probe toward different concentrations of the Pd2+ ions were shown as follows: 20 μL stock solution and 1980 μL PBS buffer solution with different Pd2+ ions mixed to reach a final probe concentration of 1 × 10−6 M. The solutions of various testing
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RESULTS AND DISCUSSION Design and Synthesis of Probe Molecule Np−Rh−Pd. We employ our previously reported D-π-A-structured naphthalene-rhodamine TBET platform to design an efficient ratiometric two-photon fluorescence probe for Pd2+. In our designed probe Np−Rh−Pd (Figure 1), the TBET mechanism is chosen to afford a ratiometric fluorescence response as it does not need spectral overlap between the donor and acceptor
Figure 1. Structures of RA, Np−Rh, and the ratiometric two-photon fluorescent probe Np−Rh−Pd. B
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 2. (A) Absorption and (B) fluorescence emission spectra of 5 μM Np−Rh−Pd in 10 mM phosphate buffer at different Pd2+ ions. (G) Calibration curve of Np−Rh−Pd to Pd2+ ions. The curve was plotted with the fluorescence intensity vs Pd2+ ion concentration (0−40 μM). (H) Linear relationship between F595/F495 and Pd2+ ion concentration in the 0−5.0 μM range. (C and D) Change in color of the probe before and after the addition of 50 μM Pd2+ ions in 10 μM of Np−Rh−Pd in 1:1 PBS/CH3CH2OH, pH 7.4). (E and F) Change in the fluorescence of the probe before and after the addition of 50 μM Pd2+ ions in 10 μM of Np−Rh−Pd under excitation by UV light.
Figure 3. (A) Fluorescence response of 1 μM Np−Rh−Pd to 1 μM of Pd2+ or 10 μM of other metal ions or neutral molecules (black bars) and to the mixture of 10 μM of other divalent metal ions or neutral molecules with 1 μM of Pd2+ (red bars). (B) Change in fluorescence of 5 μM Np−Rh− Pd in 1:1 CH3CH2OH/PBS (v/v, pH 7.4, 10 mM) with neutral molecules or metal ions: GSH, Cys, Glu, Pb2+, Hg2+, Ca2+, Mg2+, Co2+, Cu2+, Ni2+, Pd2+, Cr3+, Zn2+, Al3+, Na+, and Fe3+ (100 μM, from left to right). The numbers from 1 to 16 correspond to GSH to Fe3+, respectively.
procedure is described in Scheme S1 in the Supporting Information . Fluorescent Analytical Performance of Probe Np− Rh−Pd. The spectroscopic properties of Np−Rh−Pd (5 μM) were examined in phosphate buffer solution (10 mM, pH 7.4, 1:1 H2O/EtOH) with different Pd2+ ion concentrations. The probe displays sensitive absorption (Figure 2A) and fluorescence (Figure 2B) responses to changes in Pd2+ ion concentration. As shown in Figure 2A, in the absence of Pd2+ ions, only one absorption peak was observed at the maximum absorption wavelength (λ = 395 nm) as a result of the donor unit. After Pd2+ ions (0−40 μM) were added, a new absorption peak appeared at 550 nm, which belonged to the acceptor (rhodamine B moiety). At the same time, the solution’s color
and could afford higher energy transfer efficiency and a large wavelength difference between the two emissions with improved imaging resolution relative to the FRET system. Naphthalene derivative was chosen as the energy donor due to its outstanding two-photon properties, whereas an anthracene appended rhodamine spirolactam was selected as the energy acceptor because it shows a Pd2+-induced “turn-on” fluorescent response via a spiro ring-opening mechanism due to complexation between the probe and Pd2+ (Figure S1, Supporting Information).5a,g On the basis of our previous work, Np−Rh−Pd was prepared in 85% yield by the coupling of Np−Rh with 9anthraldehyde in a one-pot method. The detailed synthetic C
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 4. One-photon and TP-excited ratiometric fluorescence images of 5 μM Np−Rh−Pd in 10 mM PBS in HeLa cells stimulated with or without Pd2+ ions. One-photon images from the green channel (A) and the red channel (B) without Pd2+ ions. TP images from the green channel (C) and the red channel (D) without Pd2+ ions. One-photon images from the green channel (E) and the red channel (F) with Pd2+ ions. (G) Merged images of (E) and (F). (H) Pseudo-color images of (E) and (F). Two-photon images from the green channel (I) and the red channel (J) with Pd2+ ions. (K) Merged images of (I) and (J). (L) Pseudo-color images of (I) and (J). One-photon images: λex = 405 nm; two-photon images: λex = 780 nm. Green channel: λem = 470−530 nm; red channel 550−650 nm. Scale bar: 100 μm.
from pH 2.0 to 12.0. Upon addition of Pd2+, the best response toward Pd2+ could be achieved with a pH range of approximately 2.0−8.0. Thus, the PBS solution (pH 7.4) was used throughout the experiment. These results indicated that the probe was favorable for applications in practical samples at different pH values. One- and Two-Photon Fluorescence Imaging of HeLa Cells and Liver Tissue. To demonstrate the practical applicability of the probe in biological systems, we carried out fluorescence imaging experiments in both living cells (HeLa cells) and tissue slices (Nude mice). The cytotoxicity of the probe was first evaluated using MTT assays for both HeLa cells and HEK-293 cells (healthy cell line). Experimental results demonstrated that the Np−Rh−Pd probe exhibited negligible cytotoxicity to both cell lines (Figure S4 in the Supporting Information). Before one- and two-photon fluorescence imaging, cells were incubated with 5 μM Np−Rh−Pd at 37 °C for 30 min, which then exhibited strong one- or two-photon intracellular fluorescence in the green channel (Figure 4A, C) and weak fluorescence in the red channel (Figure 4B, D), corresponding to strong fluorescence of the donor and weak fluorescence of the acceptor due to the spirolactam form. These results also demonstrated that Np−Rh−Pd could penetrate the cell membrane. However, incubation of the cells with 20 μM of Pd2+ ions could significantly decrease the one- and two-photon excited fluorescence signal in the green channel (Figure 4E, I) and enhancement of the one- and two-photon excited fluorescence signal in the red channel (Figure 4F, J), corresponding to weak fluorescence from the donor and strong fluorescence from the acceptor due to the strong TBET process of the probe triggered by Pd2+ ions. These preliminary experimental results demonstrate that Np−Rh−Pd could be successfully applied to both one- and two-photon-excited ratiometric imaging of Pd2+ ions in live cells. Np−Rh−Pd was further applied for TP-excited fluorescence imaging of Pd2+ ions in liver tissue slices from nude mice with images at different tissue depths recorded by TPFM in the Zscan mode. Experimental results indicated that the probe could
changed from light-yellow to red, allowing colorimetric detection of Pd2+ ions by the naked eye. Further fluorescence experiments showed a dramatic increase in fluorescence intensity at λem = 595 nm and an obvious decrease in fluorescence intensity at λem= 495 nm (Figure 2B). F595/495 was gradually increased with increasing Pd2+ concentration (Figure 2G) and varied from 0.3 to 9.5 as the concentration of Pd2+ changed from 0 to 40 μM, respectively, corresponding to a signal-to-background ratio of 31.2, which provided the basis for high-performance ratiometric (F595/F495) detection and exhibited an excellent F595/F495 linearity toward different Pd2+ concentration (0−5 μM) (Figure 2H). In this study, the detection limit (3σ/slope)9 was estimated to be as low as 2.3 × 10−7 M, and the energy transfer efficiency was calculated to be 90% (see Figure S2 in the Supporting Information). A previous report demonstrated that a TBET system was sometimes accompanied by FRET, which restricted the energy transfer efficiency, causing it to be less than 100%.8,10 Selectivity and Effect of pH. High selectivity and a suitable pH working range are very important characteristics of a newly designed fluorescent probe. Selectivity experiments showed that only Pd2+ ions could induce the receptor’s openring reaction, whereas other ions in environmental and biological systems showed no perceptible effect, demonstrating high selectivity of the probe toward Pd2+ ions (Figure 3, black bars). Competition experiments were also carried out to assess the practical applicability of the probe. Ten micromolar of other metal ions or neutral molecules is added to 1 μM of Pd2+ separately, and the fluorescence response of the probe is then recorded with the results shown in Figure 3 (red bars). The probe showed almost unchanged fluorescent responses to Pd2+ before and after the addition of other interfering metal ions or neutral molecules. These results demonstrated that our Np− Rh−Pd probe could meet the selective requirements for practical applications. We also studied the effect of pH on Np− Rh−Pd in the absence and presence of Pd2+ ions (Figure S3, Supporting Information). Without Pd2+ ions, no obvious characteristic fluorescence of the acceptor could be observed D
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
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be successfully applied for ratiometric imaging of Pd2+ ions in tissue at a depth of 90−270 μm in two fluorescence channels at two well-separated wavelengths (see Figure 5 and Figure S5 in
ACKNOWLEDGMENTS This work was supported by the National Key Scientific Program of China (2011CB911000), the National Key Basic Research Program of China (2013CB932702), NSFC (Grants 21325520, 21327009, J1210040, 21177036, 21135001), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), the National Instrumentation Program (2011YQ030124), and the Hunan Provincial Natural Science Foundation (Grant 11JJ1002).
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Figure 5. TP fluorescence images of a frozen liver tissue slice from a nude mouse stained with 10 μM Np−Rh−Pd at 110 μm for 60 min followed by treatment with 100 μM Pd2+ and incubated for another 60 min (A, B). (C) Overlay image of (A) and (B). (D) Pseudo-color image of (A) and (B). The images were collected at 470−530 nm (green channel, A) and 550−650 nm (red channel, B) upon excitation at 780 nm with femtosecond pulses. Scale bar: 100 μm.
the Supporting Information). These results demonstrated that the probe possessed a good staining capability and high penetrating ability in tissue as well as high resolution for twocolor ratiometric imaging.
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CONCLUSION In summary, we have designed and synthesized a novel robust ratiometric two-photon fluorescent probe, termed Np−Rh−Pd, for detecting Pd2+ ions in living systems. Np−Rh−Pd consists of a two-photon fluorophore naphthalene derivative and rhodamine B dye, which were directly conjugated via a TBET strategy. In the presence of Pd2+ ions, the spirolactam ring of rhodamine B fluorophore will be opened and induce a highly efficient energy transfer from the naphthalene derivative to rhodamine B, causing a ratiometric fluorescence response to Pd2+ ions. The experimentsdemonstrate that Np−Rh−Pd has high selectivity and sensitivity for Pd2+ ions with a detection limit of 2.3 × 10−7 M. Moreover, the in situ experiments demonstrate that the probe possesses high ratiometric imaging resolution and large tissue-imaging depth (90−270 μm) at the cellular and tissue level. We believe that Np−Rh−Pd could find wide applications in environmental monitoring and biomedical diagnostics for Pd2+ ions.
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Article
ASSOCIATED CONTENT
* Supporting Information S
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone: +86-731-88821894. Fax: +86-731-88821894. Notes
The authors declare no competing financial interest. E
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
Through-Bond Energy Transfer-Based Ratiometric Two-Photon Probe for Fluorescent Imaging of Pd2+ Ions in Living Cells and Tissues Liyi Zhou, Qianqian Wang, Xiao-Bing Zhang,* and Weihong Tan Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China S Supporting Information *
ABSTRACT: Palladium can cause severe skin and eye irritation once it enters the human body. Ratiometric twophoton fluorescent probes can both eliminate interference from environmental factors and realize deep-tissue imaging with improved spatial localization. To quantitatively track Pd2+ in biosystems, we report here a colorimetric and two-photon ratiometric fluorescent probe, termed Np−Rh−Pd, which consists of a two-photon fluorophore (naphthalene derivative with a D-π-A structure) and a rhodamine B dye. The two fluorophores are directly linked to form a two-photon ratiometric fluorescent probe for Pd2+ based on a through-bond energy transfer (TBET) strategy. It exhibits highly efficient energy transfer (90%) with two well-resolved emission peaks (wavelength difference of 100 nm), which could efficiently diminish the cross talk between channels and is especially favorable for ratiometric bioimaging applications. A signal-to-background ratio of 31.2 was observed for the probe, which affords a high sensitivity for Pd2+ with a detection limit of 2.3 × 10−7 M. It was also found that acidity does not affect the fluorescent response of the probe to Pd2+, which is favorable for its applications in practical samples. The probe was further used for fluorescence imaging of Pd2+ ions in live cells and tissue slices under two-photon excitation, which showed significant tissue-imaging depths (90−270 μm) and a high resolution for ratiometric imaging.
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bleaching, and shallow tissue-penetration depth. Moreover, these probes are based on the intensity changes of a single emission peak, which tends to be affected by a variety of factors, including instrumental variations, environmental condition changes, and probe concentration fluctuations. Therefore, it is of great significance and necessity to develop a simple and reliable fluorescent method for the quantitative measurement of the Pd2+ ion concentration in live cells and tissues. To solve these problems, we used an effective two-photon fluorescence microscopy (TPFM) approach, which excites a two-photon (TP) fluorescence dye by a long-wavelength laser light and thus provides improved three-dimensional imaging resolution with increased tissue-imaging depth (>500 μm) and an extended observation time compared to traditional confocal microscopy.6 Particularly when combined with a ratiometric strategy, the built-in correction of the two emission bands could further eliminate interference caused by instrumental variations, environmental factor changes, and probe concentration fluctuations.7 Previously, we established a ratiometric twophoton fluorescence probe platform, termed Np−Rh, by directly connecting a two-photon naphthalene derivative with
alladium, one of the platinum-group elements, can cause severe skin and eye irritation1 when it is taken into human bodies via contaminated food, medicine, and water.2 DNA, proteins, and other biomacromolecules, such as vitamin B6, have been reported to be able to strongly bind palladium ions, which may lead to a variety of cellular dysfunction processes.3 Therefore, the development of an efficient method for detecting Pd2+ ions in biosystems is of great biological importance. Traditional analytical methods,4 such as inductively coupled plasma mass spectrometry (ICP-MS),4a atomic absorption spectrometry (AAS),4b inductively coupled plasma emission spectroscopy (ICP-AES),4c and X-ray fluorescence (XRF),4d have been developed for the efficient detection of palladium ions. These methods, however, usually require expensive instruments and highly skilled professionals. In contrast, fluorescence methods can avoid these shortcomings while maintaining comparable efficiency and accuracy and have therefore been widely exploited by researchers for the detection of a variety of targets. To date, only a few fluorescent probes that can quantitatively detect Pd2+ concentration have been reported.5 Unfortunately, most of these fluorescent probes are excited by one-photon excitation with a short wavelength, which results in some obvious drawbacks when used in bioimaging, such as high autofluorescence background, photo© XXXX American Chemical Society
Received: February 6, 2015 Accepted: March 26, 2015
A
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry a D-π-A structure with a rhodamine B fluorophore via the TBET strategy.8 Herein, we used this platform further to develop a new ratiometric two-photon fluorescent probe, Np− Rh−Pd, for Pd2+ ion detection and bioimaging applications. The probe molecule exhibited two well-separated fluorescence emission peaks and a high-efficiency energy transfer, which provided a large signal-to-background ratio of 31.2, and therefore high sensitivity of the probe with a detection limit of 2.3 × 10−7 M observed for Pd2+ ions. It showed a pronounced fluorescent response toward Pd2+ ions over other common metal ions. The probe was further used for fluorescence imaging of Pd2+ ions in live cells and tissues by TPFM, which exhibited large tissue-imaging depths with a high resolution for ratiometric imaging.
species were prepared from CaCl2, MgSO2, Co(CH3COO)2· 4H2O, CuCl2·H2O, NiCl2·6H2O, CrCl3·6H2O, Zn(NO3)2· 6H2O, Al(NO3)3·9H2O, FeSO2, HgCl2, Pb(NO3)2, NaCl, (GSH), Cys, and Glu using ultrapure water with final concentrations of 50 μM. Cell Cytotoxic Assays and Imaging. The cytotoxic effects of the probe were assessed using MTT assays.8 For fluorescent imaging, the cells were washed with PBS buffer followed by incubation with the probe (5 μM) for 30 min in culture medium (PBS buffer containing 0.5% EtOH) at 37 °C. Cell samples were then washed with PBS three times and imaged. Pd2+ ions (20 μM) were added to induce a ratiometric signal change during imaging. One-photon excitation wavelength was fixed at 405 nm, and the two-photon excitation wavelength of the femtosecond laser was fixed at 780 nm; the emission wavelengths were recorded at 470−530 and 550−650 nm, respectively. Preparation and Staining of Liver Tissue Slices from Nude Mice. Frozen tissue slices were prepared from the livers of nude mice. A side of the tissue was cut flat using a vibrating-blade microtome. The slices were cultured with 10 μM Np−Rh−Pd in an incubator at 37 °C for 1 h and then washed with PBS three times. Finally, the slices were incubated in culture medium with 100 μM Pd2+ ions and cultured in an incubator at 37 °C for 1 h. Before TPFM imaging, the samples were washed with PBS three more times. The TPFM images (with 10× magnification) were collected in two channels (green: 470−530 nm; red: 550−650 nm) upon excitation at 780 nm with a femtosecond pulse laser.
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EXPERIMENTAL SECTION Materials and Apparatus. All chemicals were of analytical grade, used without further purification, and provided by commercial suppliers. Water used in all experiments was purified via a Milli-Q water system from Millipore (Billerica, MA, USA). NMR spectra were carried out on a Bruker DRX400 spectrometer with TMS employed as an internal standard. All chemical shifts are given in the standard δ notation of parts per million. Mass spectra were recorded using an LCQ Advantage ion trap mass spectrometer (Thermo Finnigan). UV−vis absorption spectra were performed on a Shimadzu UV2450 spectrophotometer with a 1.0 cm path length quartz cuvette. The pH was measured with a Mettler-Toledo Delta 320 pH meter. Fluorescence measurements were carried out on a Hitachi-F4500 fluorescence spectrometer with excitation and emission slits both set at 2.5 nm. Fluorescence images of HeLa cells and tissue slices were taken using an Olympus FV1000MPE multiphoton laser scanning microscope (Japan). Synthesis of Probe Compound Np−Rh−Pd. Np−Rh−Pd was synthesized according to previously reported methods.5f,8 Characterization of compound Np−Rh−Pd is shown in the Supporting Information. A mixture of Np−Rh (0.15 g, 200 μmol), 9-anthraldehydede (0.10 g, 480 μmol), and 50 mL of ethanol was added to a round-bottom flask with a reflux condenser and heated with continuous refluxing for 4 h. The mixture solution was reduced under pressure distillation. A yellow solid Np−Rh−Pd (0.16 g, 85% yield) was obtained through column chromatography with petroleum ether/ethyl acetate (2:1, v/v) as eluent. 1H NMR (400 MHz, d-DMSO): δ 8.64 (s, 1H), 8.59−8.55 (t, J = 8 Hz, 1H), 8.04−7.65 (m, 6H), 7.52−7.40 (d, J = 24 Hz, 2H), 7.32−7.28 (m, 1H), 7.03 (s, 1H), 6.71−6.67 (d, J = 8 Hz, 1H), 6.65−6.44 (m, 5H), 3.40− 3.32 (dd, J = 16 Hz, 8H), 3.08 (s, 6H), 1.09−1.06 (t, J = 6 Hz, 12H). +c ESI, 931.4; calcd, 931.2. Spectrophotometric Measurements. Both fluorescence and absorption spectra were measured in 10 mM phosphate buffer solution containing EtOH as the cosolvent solution (H2O/ EtOH 1:1, v/v). The pH gradient of PBS buffer solution from 2.0 to 10.0 was achieved by adding different volumes of HCl or NaOH solution. The fluorescence emission spectra were recorded at an excitation wavelength of 415 nm with an emission wavelength range from 450 to 665 nm. A stock solution of probe (1 × 10−3 M) was prepared by dissolving the probe in EtOH. The procedure of calibration measurements of the probe toward different concentrations of the Pd2+ ions were shown as follows: 20 μL stock solution and 1980 μL PBS buffer solution with different Pd2+ ions mixed to reach a final probe concentration of 1 × 10−6 M. The solutions of various testing
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RESULTS AND DISCUSSION Design and Synthesis of Probe Molecule Np−Rh−Pd. We employ our previously reported D-π-A-structured naphthalene-rhodamine TBET platform to design an efficient ratiometric two-photon fluorescence probe for Pd2+. In our designed probe Np−Rh−Pd (Figure 1), the TBET mechanism is chosen to afford a ratiometric fluorescence response as it does not need spectral overlap between the donor and acceptor
Figure 1. Structures of RA, Np−Rh, and the ratiometric two-photon fluorescent probe Np−Rh−Pd. B
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 2. (A) Absorption and (B) fluorescence emission spectra of 5 μM Np−Rh−Pd in 10 mM phosphate buffer at different Pd2+ ions. (G) Calibration curve of Np−Rh−Pd to Pd2+ ions. The curve was plotted with the fluorescence intensity vs Pd2+ ion concentration (0−40 μM). (H) Linear relationship between F595/F495 and Pd2+ ion concentration in the 0−5.0 μM range. (C and D) Change in color of the probe before and after the addition of 50 μM Pd2+ ions in 10 μM of Np−Rh−Pd in 1:1 PBS/CH3CH2OH, pH 7.4). (E and F) Change in the fluorescence of the probe before and after the addition of 50 μM Pd2+ ions in 10 μM of Np−Rh−Pd under excitation by UV light.
Figure 3. (A) Fluorescence response of 1 μM Np−Rh−Pd to 1 μM of Pd2+ or 10 μM of other metal ions or neutral molecules (black bars) and to the mixture of 10 μM of other divalent metal ions or neutral molecules with 1 μM of Pd2+ (red bars). (B) Change in fluorescence of 5 μM Np−Rh− Pd in 1:1 CH3CH2OH/PBS (v/v, pH 7.4, 10 mM) with neutral molecules or metal ions: GSH, Cys, Glu, Pb2+, Hg2+, Ca2+, Mg2+, Co2+, Cu2+, Ni2+, Pd2+, Cr3+, Zn2+, Al3+, Na+, and Fe3+ (100 μM, from left to right). The numbers from 1 to 16 correspond to GSH to Fe3+, respectively.
procedure is described in Scheme S1 in the Supporting Information . Fluorescent Analytical Performance of Probe Np− Rh−Pd. The spectroscopic properties of Np−Rh−Pd (5 μM) were examined in phosphate buffer solution (10 mM, pH 7.4, 1:1 H2O/EtOH) with different Pd2+ ion concentrations. The probe displays sensitive absorption (Figure 2A) and fluorescence (Figure 2B) responses to changes in Pd2+ ion concentration. As shown in Figure 2A, in the absence of Pd2+ ions, only one absorption peak was observed at the maximum absorption wavelength (λ = 395 nm) as a result of the donor unit. After Pd2+ ions (0−40 μM) were added, a new absorption peak appeared at 550 nm, which belonged to the acceptor (rhodamine B moiety). At the same time, the solution’s color
and could afford higher energy transfer efficiency and a large wavelength difference between the two emissions with improved imaging resolution relative to the FRET system. Naphthalene derivative was chosen as the energy donor due to its outstanding two-photon properties, whereas an anthracene appended rhodamine spirolactam was selected as the energy acceptor because it shows a Pd2+-induced “turn-on” fluorescent response via a spiro ring-opening mechanism due to complexation between the probe and Pd2+ (Figure S1, Supporting Information).5a,g On the basis of our previous work, Np−Rh−Pd was prepared in 85% yield by the coupling of Np−Rh with 9anthraldehyde in a one-pot method. The detailed synthetic C
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 4. One-photon and TP-excited ratiometric fluorescence images of 5 μM Np−Rh−Pd in 10 mM PBS in HeLa cells stimulated with or without Pd2+ ions. One-photon images from the green channel (A) and the red channel (B) without Pd2+ ions. TP images from the green channel (C) and the red channel (D) without Pd2+ ions. One-photon images from the green channel (E) and the red channel (F) with Pd2+ ions. (G) Merged images of (E) and (F). (H) Pseudo-color images of (E) and (F). Two-photon images from the green channel (I) and the red channel (J) with Pd2+ ions. (K) Merged images of (I) and (J). (L) Pseudo-color images of (I) and (J). One-photon images: λex = 405 nm; two-photon images: λex = 780 nm. Green channel: λem = 470−530 nm; red channel 550−650 nm. Scale bar: 100 μm.
from pH 2.0 to 12.0. Upon addition of Pd2+, the best response toward Pd2+ could be achieved with a pH range of approximately 2.0−8.0. Thus, the PBS solution (pH 7.4) was used throughout the experiment. These results indicated that the probe was favorable for applications in practical samples at different pH values. One- and Two-Photon Fluorescence Imaging of HeLa Cells and Liver Tissue. To demonstrate the practical applicability of the probe in biological systems, we carried out fluorescence imaging experiments in both living cells (HeLa cells) and tissue slices (Nude mice). The cytotoxicity of the probe was first evaluated using MTT assays for both HeLa cells and HEK-293 cells (healthy cell line). Experimental results demonstrated that the Np−Rh−Pd probe exhibited negligible cytotoxicity to both cell lines (Figure S4 in the Supporting Information). Before one- and two-photon fluorescence imaging, cells were incubated with 5 μM Np−Rh−Pd at 37 °C for 30 min, which then exhibited strong one- or two-photon intracellular fluorescence in the green channel (Figure 4A, C) and weak fluorescence in the red channel (Figure 4B, D), corresponding to strong fluorescence of the donor and weak fluorescence of the acceptor due to the spirolactam form. These results also demonstrated that Np−Rh−Pd could penetrate the cell membrane. However, incubation of the cells with 20 μM of Pd2+ ions could significantly decrease the one- and two-photon excited fluorescence signal in the green channel (Figure 4E, I) and enhancement of the one- and two-photon excited fluorescence signal in the red channel (Figure 4F, J), corresponding to weak fluorescence from the donor and strong fluorescence from the acceptor due to the strong TBET process of the probe triggered by Pd2+ ions. These preliminary experimental results demonstrate that Np−Rh−Pd could be successfully applied to both one- and two-photon-excited ratiometric imaging of Pd2+ ions in live cells. Np−Rh−Pd was further applied for TP-excited fluorescence imaging of Pd2+ ions in liver tissue slices from nude mice with images at different tissue depths recorded by TPFM in the Zscan mode. Experimental results indicated that the probe could
changed from light-yellow to red, allowing colorimetric detection of Pd2+ ions by the naked eye. Further fluorescence experiments showed a dramatic increase in fluorescence intensity at λem = 595 nm and an obvious decrease in fluorescence intensity at λem= 495 nm (Figure 2B). F595/495 was gradually increased with increasing Pd2+ concentration (Figure 2G) and varied from 0.3 to 9.5 as the concentration of Pd2+ changed from 0 to 40 μM, respectively, corresponding to a signal-to-background ratio of 31.2, which provided the basis for high-performance ratiometric (F595/F495) detection and exhibited an excellent F595/F495 linearity toward different Pd2+ concentration (0−5 μM) (Figure 2H). In this study, the detection limit (3σ/slope)9 was estimated to be as low as 2.3 × 10−7 M, and the energy transfer efficiency was calculated to be 90% (see Figure S2 in the Supporting Information). A previous report demonstrated that a TBET system was sometimes accompanied by FRET, which restricted the energy transfer efficiency, causing it to be less than 100%.8,10 Selectivity and Effect of pH. High selectivity and a suitable pH working range are very important characteristics of a newly designed fluorescent probe. Selectivity experiments showed that only Pd2+ ions could induce the receptor’s openring reaction, whereas other ions in environmental and biological systems showed no perceptible effect, demonstrating high selectivity of the probe toward Pd2+ ions (Figure 3, black bars). Competition experiments were also carried out to assess the practical applicability of the probe. Ten micromolar of other metal ions or neutral molecules is added to 1 μM of Pd2+ separately, and the fluorescence response of the probe is then recorded with the results shown in Figure 3 (red bars). The probe showed almost unchanged fluorescent responses to Pd2+ before and after the addition of other interfering metal ions or neutral molecules. These results demonstrated that our Np− Rh−Pd probe could meet the selective requirements for practical applications. We also studied the effect of pH on Np− Rh−Pd in the absence and presence of Pd2+ ions (Figure S3, Supporting Information). Without Pd2+ ions, no obvious characteristic fluorescence of the acceptor could be observed D
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
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be successfully applied for ratiometric imaging of Pd2+ ions in tissue at a depth of 90−270 μm in two fluorescence channels at two well-separated wavelengths (see Figure 5 and Figure S5 in
ACKNOWLEDGMENTS This work was supported by the National Key Scientific Program of China (2011CB911000), the National Key Basic Research Program of China (2013CB932702), NSFC (Grants 21325520, 21327009, J1210040, 21177036, 21135001), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), the National Instrumentation Program (2011YQ030124), and the Hunan Provincial Natural Science Foundation (Grant 11JJ1002).
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Figure 5. TP fluorescence images of a frozen liver tissue slice from a nude mouse stained with 10 μM Np−Rh−Pd at 110 μm for 60 min followed by treatment with 100 μM Pd2+ and incubated for another 60 min (A, B). (C) Overlay image of (A) and (B). (D) Pseudo-color image of (A) and (B). The images were collected at 470−530 nm (green channel, A) and 550−650 nm (red channel, B) upon excitation at 780 nm with femtosecond pulses. Scale bar: 100 μm.
the Supporting Information). These results demonstrated that the probe possessed a good staining capability and high penetrating ability in tissue as well as high resolution for twocolor ratiometric imaging.
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CONCLUSION In summary, we have designed and synthesized a novel robust ratiometric two-photon fluorescent probe, termed Np−Rh−Pd, for detecting Pd2+ ions in living systems. Np−Rh−Pd consists of a two-photon fluorophore naphthalene derivative and rhodamine B dye, which were directly conjugated via a TBET strategy. In the presence of Pd2+ ions, the spirolactam ring of rhodamine B fluorophore will be opened and induce a highly efficient energy transfer from the naphthalene derivative to rhodamine B, causing a ratiometric fluorescence response to Pd2+ ions. The experimentsdemonstrate that Np−Rh−Pd has high selectivity and sensitivity for Pd2+ ions with a detection limit of 2.3 × 10−7 M. Moreover, the in situ experiments demonstrate that the probe possesses high ratiometric imaging resolution and large tissue-imaging depth (90−270 μm) at the cellular and tissue level. We believe that Np−Rh−Pd could find wide applications in environmental monitoring and biomedical diagnostics for Pd2+ ions.
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ASSOCIATED CONTENT
* Supporting Information S
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone: +86-731-88821894. Fax: +86-731-88821894. Notes
The authors declare no competing financial interest. E
DOI: 10.1021/acs.analchem.5b00505 Anal. Chem. XXXX, XXX, XXX−XXX
