Development of a Terbium Complex-Based Luminescent Probe for Imaging Endogenous Hydrogen Peroxide Generation in Plant Tissues Zhiqiang Ye,* Jinxue Chen, Guilan Wang, and Jingli Yuan* State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, China
bS Supporting Information ABSTRACT:
A highly sensitive Tb3þ complex-based luminescent probe, N,N,N1,N1-[2,6-(30 - aminomethyl-10 -pyrazolyl)-4-(300 ,400 -diaminophenoxy)methylene-pyridine] tetrakis(acetate)-Tb3þ (BMTA-Tb3þ), has been designed and synthesized for the recognition and detection of hydrogen peroxide (H2O2) in aqueous solutions. This probe is almost nonluminescent because the Tb3þ luminescence is eﬀectively quenched by the electron-rich moiety, diaminophenyl, on the basis of the photoinduced electron transfer (PET) mechanism. In the presence of peroxidase, the probe can react with H2O2 to cause the cleavage of the diaminophenyl ether, which aﬀords a highly luminescent Tb3þ complex, N,N,N1,N1-[2,6-bis(30 -aminomethyl-10 -pyrazolyl)-4-hydroxymethyl-pyridine] tetrakis(acetate)-Tb3þ (BHTA-Tb3þ), accompanied by a 39-fold increase in luminescence quantum yield with the increase of luminescence lifetime from 1.95 to 2.76 ms. The dose-dependent luminescence enhancement of the probe shows a good linearity with a detection limit of 3.7 nM for H2O2, which is approximately 14-fold lower than those of the commonly used ﬂuorescent probes. The probe was used for the time-resolved luminescence imaging detection of the oligosaccharide-induced H2O2 generation in tobacco leaf epidermal tissues. On the basis of the probe, a background-free time-resolved luminescence imaging method for detecting H2O2 in complicated biological systems was successfully established.
or many years, hydrogen peroxide (H2O2) was viewed as the unwanted byproduct of an aerobic existence, and this toxic waste should be eliminated as soon as possible due to its potential damage to the biological cells. However, recent studies have revealed that mammalian cells produce H2O2 to mediate diverse physiological responses such as cell proliferation, diﬀerentiation, and migration.13 This has led to implications of cellular “redox” signaling in regulating normal processes and disease progression, including angiogenesis, oxidative stress and aging, and cancer.48 In a plant system, H2O2 can be generated during normal cellular metabolism after various environmental stresses, such as an excess of light, drought or cold, and mechanical wounding.9 The massive production of H2O2 can initiate a localized hypersensitive response, a form of programmed cell death, which appears to limit and block pathogen development.10 In addition, H2O2 is also involved in various developmental processes, such as seed germination, gravitropism, cell wall ligniﬁcation, and root development, indicating that it plays a key role in the regulation of plant responses to a range of endogenous signals and stimuli, r 2011 American Chemical Society
such as auxin and abscisic acid.11,12 Therefore, in order to enable a deeper insight on its biological functions as a beneﬁcial messenger, it is continuously requiring new sensitive methods for detecting H2O2 in biological systems, especially in living cells and tissues. A luminescent probe technique, combining the use of a microscopic imaging instrument, is an excellent tool to monitor H2O2 because of its high sensitivity, simplicity in data collection, and high spatial resolution. It has been known that several nonﬂuorescent probes, such as 2,7-dichlorodihydroﬂuorescein (DCFH),13,14 7-hydroxy-6-methoxy-coumarin (Scopoletin),15,16 N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red),17,18 4-hydroxy-3-methoxy-phenylacetic acid (HVA),19 and dihydrorhodamine 123 (DHR),20 could be oxidized by H2O2 in the presence of peroxidase, to yield highly ﬂuorescent products, and some of Received: February 19, 2011 Accepted: May 6, 2011 Published: May 06, 2011 4163
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Analytical Chemistry them have been applied to the monitoring of H2O2 in isolated mitochondria and various cells. Quite recently, several novel ﬂuorescent probes capable of detecting H2O2 in the absence of peroxidase, including an aminocoumarin derivative,21 7-hydroxy2-oxo-N-2-(diphenylphosphino)-2H-chromene-3-carboxamide (DPPEA-HC),22 and a family of boronate-based probes, peroxyﬂuor-1, peroxyresoruﬁn-1, and peroxyxanthone-1,2327 have been reported. These probes, especially the boronate-based probes with good cell membrane-permeability, have been demonstrated to be able to monitor the intracellular H2O2 concentration changes in living cells, which enables them to be the promising tools for investigating the physiological and pathological functions of cellular H2O2. However, a key problem for these organic ﬂuorophore-based probes to detect H2O2 is that the measurement would be signiﬁcantly interfered by strong autoﬂuorescence from complicated biological samples, such as plant tissues and biological slices. In recent years, time-resolved (or time-gated) luminescence imaging technique using lanthanide complexes as probes has been demonstrated to be a powerful tool to eliminate the shortlived background ﬂuorescence from complicated samples.2834 In comparison with the organic ﬂuorescence probes, lanthanide (mainly Eu3þ and Tb3þ) complex-based probes possess several very desirable properties including long luminescence lifetime, large Stokes shift, sharp emission proﬁle, and good photostability, which enable them to be favorably useful as luminescent probes for time-resolved luminescence detection to eliminate fast decaying autoﬂuorescence and scattering lights from the samples and nearby optics. Unfortunately, time-resolved luminescence is by far insensitive and inconvenient compared with conventional ﬂuorescence in imaging systems. Thus, though some lanthanide complex-based luminescent probes speciﬁc for small bioactive ions and molecules have been developed in recent several years,3544 only a few of them can be employed for intracellular use, especially for monitoring the changes of analytes at endogenous intracellular levels under various stimuli. Herein, we describe the design, synthesis, and application of a novel Tb3þ complex-based luminescent probe that can be used for time-resolved luminescence detection of H2O2 in the presence of peroxidase, N,N,N1,N1-[2,6-(30 -aminomethyl10 -pyrazolyl)-4-(300 ,400 -diaminophenoxy)methylene-pyridine] tetrakis(acetate)-Tb3þ (BMTA-Tb3þ). This probe is wellsoluble and almost nonluminescent in aqueous solution due to the strong luminescence quenching eﬀect of the diaminophenyl moiety in its ligand. However, in the presence of peroxidase, it can react with H2O2 to cause the cleavage of the diaminophenyl ether, which aﬀords a highly luminescent Tb3þ complex, N,N,N1,N1-[2,6-bis(30 -aminomethyl-10 -pyrazolyl)4-hydroxymethyl-pyridine] tetrakis(acetate)-Tb3þ (BHTATb3þ), accompanied by the signiﬁcant enhancement of luminescence intensity and luminescence lifetime. This luminescence response allows the probe to be used for the highly sensitive timeresolved luminescence detection of H2O2 in aqueous solutions. For monitoring the H2O2 generation at endogenous intracellular levels, the cell membrane permeable form of BMTA-Tb3þ, acetoxymethyl ester of BMTA-Tb3þ (AM-BMTA-Tb3þ), was also synthesized43,44 and used for the time-resolved luminescence imaging detection of the oligosaccharide-induced H2O2 generation in tobacco leaf epidermal tissues. The results demonstrated the practical utility of the new probe for background-free luminescence imaging of H2O2 in complicated biological samples.
’ EXPERIMENTAL SECTION Materials and Physical Measurements. Tetraethyl N,N,N1,
N -[2,6-bis(30 -aminomethyl-10 - pyrazolyl)-4-hydroxymethylpyridine] tetrakis(acetate) (compound 1) was synthesized according to the previous method.44 3-Nitro-4-amino-phenol was purchased from Acros Organics. Tetrahydrofuran (THF) and acetonitrile were used after appropriate distillation and purification. Horseradish peroxidase (HRP), catalase (CAT), and acetoxymethyl bromide were purchased from Sigma-Aldrich. Oligosaccharide prepared by enzymatic hydrolysis of chitosan (the degree of N-acetylation 420 nm), and a color CCD camera system (RET-2000RF-CLR-12-C, Qimaging Ltd.), was used for the steady-state luminescence imaging measurement with an exposure time of 2 s. The microscope, equipped with a 30 W xenon ﬂash-lamp (Pulse300, Photonic Research Systems Ltd.), UV-2A ﬁlters, and a time-gated digital black-and-white CCD camera system (Photonic Research Systems Ltd.), was used for the timeresolved luminescence imaging measurement with the conditions of delay time, 100 μs; gate time, 1000 μs; lamp pulse width, 6 μs; and exposure time, 500 s. Synthesis of BMTA and BHTA. The synthesis procedures of the new Tb3þ ligands BMTA and BHTA are shown in Scheme 1. The details are described as follows. Synthesis of Tetraethyl N,N,N1,N1-[2,6-Bis(30 -aminomethyl1 0 -pyrazolyl)-4-bromomethyl-pyridine] Tetrakis(acetate) (Compound 2). To a solution of compound 1 (0.69 g, 1.07 mmol) in 15 mL of dry THF was added dropwise 347 mg of PBr3 (1.28 mmol). After the solution was stirred at room temperature for 2 h, 150 mL of CHCl3 was added. The solution was washed 1
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Scheme 1. Synthesis Procedures of BMTA-Tb3þ and BHTA-Tb3þ
with 100 mL of water and dried with Na2SO4, and then, the solvent was evaporated. Purification by silica gel column chromatography with petroleum ether-ethyl acetate (2:1, v/v) as the eluent gave the compound 2 as a white oil (0.56 g, 73.7% yield). 1 H NMR (400 MHz, CDCl3): δ = 1.28 (t, 12H), 3.68 (s, 8H), 4.11 (s, 4H), 4.20(m, 8H), 4.47 (s, 2H), 6.60 (d, J = 2.4 Hz, 2H), 7.82 (s, 2H), 8.48 (d, J = 2.4 Hz, 2H). Synthesis of Tetraethyl N,N,N1,N1-[2,6-Bis(30 -aminomethyl-10 pyrazolyl)-4-(300 -nitro-400 -amino-phenoxy)methylene-pyridine] Tetrakis(acetate) (Compound 3). A mixture of 3-nitro-4-aminophenol (74 mg, 0.49 mmol) and NaH (16 mg, 0.67 mmol) in 8 mL of dry acetonitrile was stirred at room temperature for 15 min under an argon atmosphere. To the solution was added compound 2 (0.11 g, 0.16 mmol), and the solution was further stirred overnight under an argon atmosphere. After filtration, the solvent was evaporated. Purification by silica gel column chromatography with petroleum ether-ethyl acetate (1:1, v/v) as the eluent gave the compound 3 as a yellow oil (99 mg, 80% yield). 1 H NMR (400 MHz, CDCl3): δ = 1.27 (m, 12H), 3.64 (s, 8H), 4.07 (s, 4H), 4.19 (m, 8H), 5.10 (s, 2H), 6.56 (d, J = 2.4 Hz, 2H), 6.82 (d, J = 8.8 Hz, 1H), 7.20 (m, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.86 (s, 2H), 8.50 (d, J = 2.4 Hz, 2H). Synthesis of Tetraethyl N,N,N1,N1-[2,6-Bis(30 -aminomethyl0 1 -pyrazolyl)-4-(300 ,400 -diaminophenoxy) Methylene-pyridine] Tetrakis(acetate) (Compound 4). To a mixture of compound 3 (0.26 g, 0.33 mmol) and 10% Pd/C (14 mg) in ethanol (15 mL) was added dropwise a solution of NaH2PO2 (0.52 g, 4.9 mmol) in water (10 mL) at 50 °C under an argon atmosphere. After the mixture was refluxed for 6 h, the Pd/C catalyst was removed by filtration and the solvent was evaporated. The residue was dissolved in CHCl3 (20 mL), and the CHCl3 solution was washed with water (2 20 mL) and dried with Na2SO4. After evaporation, the crude product was purified by silica gel chromatography eluted with ethyl acetate to yield compound 4 as colorless oil (0.18 g, 70.3% yield). 1H NMR (400 MHz, CDCl3): δ =1.27 (m, 12H), 3.63(s, 8H), 4.06 (s, 4H), 4.17 (m, 8H), 5.05 (s, 2H), 6.33(m, 1H), 6.44(d, J = 2.0 Hz, 1H), 6.54 (d, J = 2.4 Hz, 2H), 6.63 (d, J = 8.8 Hz, 1H), 7.84 (s, 2H), 8.48 (d, J = 2.4 Hz, 2H).
Synthesis of BMTA. A mixture of compound 4 (0.18 g, 0.23 mmol), KOH (0.36 g, 5.4 mmol), 0.85 mL of H2O, and 10 mL of ethanol was stirred at room temperature for 20 h. After evaporation, the residue was dissolved in 3 mL of water, and pH of the solution was adjusted to ∼3 with HCl (3 M). The solution was stirred for 20 h at room temperature, and the precipitate was collected by filtration. The dried precipitate was added to 30 mL of dry acetonitrile, and the mixture was refluxed for 30 min. After the precipitate was filtered and dried, BMTA was obtained as a yellow solid (60 mg, 40.9% yield). 1H NMR (400 MHz, DMSOd6): δ= 3.47 (s, 8H), 3.94 (s, 4H), 5.16 (s, 2H), 6.12 (m, 1H), 6.30(m, 1H), 6.46 (d, J = 2.0 Hz, 1H), 6.54 (d, J = 2.4 Hz, 2H), 7.75 (s, 2H), 8.88 (d, J = 2.4 Hz, 2H). Elemental analysis calcd. (%) for C28H31N9O9 3 2H2O: C 49.92, H 5.24, N 18.71; found (%): C 50.19, H 5.17, N 18.82. ESI-MS (m/z): 636.6 [M H]. Synthesis of BHTA. A mixture of compound 1 (0.09 g, 0.14 mmol), KOH (0.18 g, 3.3 mmol), 0.54 mL of H2O, and 6 mL of ethanol was stirred at room temperature for 20 h. After evaporation, the residue was dissolved in 2 mL of water, and pH of the solution was adjusted to ∼3 with HCl (3 M). The solution was stirred for 20 h at room temperature, and the precipitate was collected by filtration. The dried precipitate was added to 30 mL of dry acetonitrile, and the mixture was refluxed for 30 min. After the precipitate was filtered and dried, BHTA was obtained as a white solid (30 mg, 37.7% yield). 1H NMR (400 MHz, DMSOd6): δ = 3.49 (s, 8H), 3.95 (s, 4H), 4.69 (s, 2H), 6.54 (d, J = 2.4 Hz, 2H), 7.71 (s, 2H), 8.85 (d, J = 2.4 Hz, 2H). Elemental analysis calcd. (%) for C22H25N7O9 3 2.5H2O: C 45.83, H 5.24, N 17.00; found (%): C 45.84, H 5.07, N 16.88. ESI-MS (m/z): 530.2 [M H]. Synthesis of AM-BMTA-Tb3þ. AM-BMTA-Tb3þ was synthesized according to our previous method.43,44 The details are as follows. To a solution of BMTA (10.44 mg, 15.5 μmol) and bromomethyl acetate (61 μL, 618 μmol) in dry DMSO (193 μL) was added dry triethylamine (89 μL, 617 μmol) under an argon atmosphere. After stirring at room temperature overnight, the precipitate was removed by centrifugation. The supernatant was carefully collected and characterized by ESI-MS. ESI-MS 4165
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Figure 1. Time-resolved excitation and emission spectra of BMTATb3þ (solid lines) and BHTA-Tb3þ (dash lines) in 0.1 M TrisHCl buﬀer of pH 7.4.
(m/z): 926.2 [M þ H]þ. After TbCl3 3 6H2O (5.78 mg, 15.5 μmol) was added, the solution was further stirred for another 0.5 h. The freshly prepared solution was used for the cell loading without further purification. Luminescence Imaging of the Oligosaccharide-Induced H2O2 Generation in Tobacco Leaf Epidermal Tissues. The tobacco (Nicotiana tabacum var. sam sun NN) leaf epidermal tissues were peeled carefully from leaves and cut into 5 mm length. After incubation in TrisKCl buffer (10 mM Tris and 50 mM KCl, pH 7.20) containing 1.0 mM of the AM-BMTATb3þ for 8 h in light, the epidermal strips were washed 3 times and immersed into 1.0 mL of water. Oligosaccharide (200 mg/L) was added, and the incubation was further kept in the dark for 5 h at room temperature. The epidermal strips were carefully washed 3 times with water and then subjected to the luminescence imaging detection.
’ RESULTS AND DISCUSSION Design and Characterization of the Probe. Recently, we have demonstrated that photoinduced electron transfer (PET) mechanism is also a useful tool for developing lanthanide luminescent probes.44 In this work, the probe was designed by incorporating a Tb3þ chelating moiety, a polyacid derivative of 2,6-bis(N-pyrazolyl)pyridine, into an electron-rich group, diaminophenyl, since the former has an excellent antenna capability for sensitizing the Tb3þ luminescence,4749 and the latter can effectively quench the Tb3þ luminescence via a PET process to make the Tb3þ luminescence be turned-off. Thus, the probe itself is almost nonluminescent. In the presence of peroxidase and H2O2, following the cleavage of the diaminophenyl ether in the probe’s ligand, a highly luminescent Tb3þ complex, N,N, N1,N1-[2,6-bis(30 -aminomethyl-10 -pyrazolyl)-4-hydroxymethylpyridine] tetrakis(acetate)-Tb3þ (BHTA-Tb3þ), can be generated; thus, the Tb3þ luminescence is turned-on. The synthesis procedures of the probe BMTA-Tb3þ and its H2O2/HRPcleavage product BHTA-Tb3þ are shown in Scheme 1. Both BMTA and BHTA were well characterized by the NMR, ESI-MS, and CHN elementary analyses. The luminescence properties of BMTA-Tb3þ and BHTATb3þ were measured in a 0.05 M borate buﬀer of pH 9.1. BMTATb3þ shows a maximum absorption wavelength at 315 nm (ε = 17 600 M1cm1) and very weak luminescence with an emission band centered at 542 nm (quantum yield φ = 0.20% ( 0.03%).
Figure 2. Absorption spectrum changes of HRP during the reaction in the BMTA-Tb3þ/H2O2/HRP system in 0.1 M TrisHCl buﬀer of pH 7.4. Spectrum 1: HRP (2.0 μM); spectrum 2: HRP (2.0 μM) in the presence of H2O2 (32 μM); spectrum 3 to 5: BMTA-Tb3þ (5.0 μM) was added to the HRP (2.0 μM)-H2O2 (32 μM) solution for 10, 40, and 60 min, respectively.
After reacting with H2O2/HRP, a remarkable luminescence enhancement was observed with a large increase of the luminescence quantum yield (φ = 7.8% ( 0.25%). The reverse-phase HPLC and ESI-MS analyses have revealed that the main reaction product is BHTA-Tb3þ (Figure S1 in Supporting Information). In addition, the same absorption spectrum pattern of BHTATb3þ (ε315 = 10 700 M1cm1) and BMTA-Tb3þ (Figure S2 in Supporting Information) suggests that the luminescence of BMTA-Tb3þ should be surely quenched via the PET mechanism. Figure 1 shows the time-resolved excitation and emission spectra of BMTA-Tb3þ and BHTA-Tb3þ in 0.1 M TrisHCl buﬀer of pH 7.4. Although the two complexes display the same Tb3þ emission pattern with a main emission peak at 542 nm and several side peaks centered at 488, 581, 618, and 642 nm, respectively, the luminescence of BHTA-Tb3þ is 30-fold stronger than that of BMTA-Tb3þ with the increase of luminescence lifetime from 1.95 to 2.76 ms. Time-Resolved Luminescence Detection of H2O2 Using BMTA-Tb3þ as a Probe. Before the detection, it was found that BMTA-Tb3þ could not react with H2O2 or HRP since no timeresolved luminescence response was observed upon the addition of H2O2 or HRP alone to the BMTA-Tb3þ solution. However, when HRP and H2O2 were added, the luminescence intensity of the solution was significantly increased. To reveal the reaction course of BMTA-Tb3þ and H2O2 in the BMTA-Tb3þ/H2O2/ HRP system, the absorption spectrum changes of HRP during the reaction were recorded in 0.1 M TrisHCl buffer of pH 7.4. As shown in Figure 2, the resting HRP showed an absorption maximum at 402 nm (spectrum 1). After reacting with H2O2, the oxidized HRP derivatives, a mixture of HRP-compound I and HRP-compound II, was formed,5052 and the absorption maximum was shifted to 418 nm (spectrum 2). Upon the addition of BMTA-Tb3þ, the absorption peak at 418 nm disappeared gradually (spectrum 3 and 4) due to the reaction between BMTA-Tb3þ and the oxidized HRP derivatives. After reaction for 1 h, the spectrum (spectrum 5) was fully returned to that of the resting HRP, indicating that the reaction between BMTATb3þ and the oxidized HRP derivatives was completed and native HRP was restored at this time. Furthermore, it was found that the HRP concentration had only a slight effect on the luminescence intensity of the BMTA-Tb3þ/H2O2/HRP system 4166
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Figure 3. Time-resolved emission spectra of BMTA-Tb3þ (5.0 μM) in the presence of HRP (0.1 μM) and diﬀerent concentrations of H2O2.
Figure 4. Calibration curve for time-resolved luminescence detection of H2 O2 .
Figure 5. Luminescence images of the oligosaccharide-induced H2O2 generation in tobacco leaf epidermal tissues (left: bright-ﬁeld image; right: luminescence image) using BMTA-Tb3þ as a probe. (A) Timeresolved luminescence image of the tissue incubated with the freshly prepared AM-BMTA-Tb3þ (1.0 mM) for 8 h. (B and C) Steady-state and time-resolved luminescence images of the BMTA-Tb3þ-loaded tissues treated with oligosaccharide (0.2 g/L) for 5 h. (D) Time-resolved luminescence image of the BMTA-Tb3þ-loaded tissue treated with oligosaccharide (0.2 g/L) and CAT (0.1 mg/mL, ∼200 units) for 5 h. Scale bar, 40 μm. The time-resolved luminescence image is shown in pseudocolor (wavelength of 545 nm) treated by a SimplePCI software.44
(Figure S3 in Supporting Information). The above results indicate that BMTA-Tb3þ can be used as a probe for the quantitative time-resolved luminescence detection of H2O2 in the presence of HRP. Time-resolved luminescence titration of H2O2 was conducted using 5.0 μM of BMTA-Tb3þ and 0.1 μM of HRP in 0.1 M TrisHCl buﬀer of pH 7.4. As shown in Figure 3, the luminescence response of the probe to H2O2 was highly sensitive. Moreover, the dose-dependent luminescence enhancement showed a good linearity against the H2O2 concentration in the range of 108106 M (Figure 4). The detection limit for H2O2, calculated as the concentration corresponding to three standard deviations of the background signal, is 3.7 109 M, which is 14 times lower than that of the Amplex Red for H2O2 under the same conditions,17 indicating that BMTA-Tb3þ can be used to quantitatively detect the H2O2 concentration with a higher sensitivity in the presence of HRP. Luminescence Imaging of the Oligosaccharide-Induced H2O2 Generation in Tobacco Leaf Epidermal Tissues. Oligosaccharide, a kind of polymer consisting of 210 molecules of monosaccharide linked by glucosidic bonds, has attracted much attention because it not only is water-soluble, nontoxic, and biocompatible but also possesses versatile functional properties, such as antimicrobial, anticancer, antioxidant, and immunostimulant effects.5358 Unfortunately, the disease resistance mechanisms of oligosaccharide in the plant system has remained unclear until now. Recent studies on the interaction between
oligosaccharide and plant cells by fluorescence microscopy suggested that oligosaccharide could induce the plant cells to generate NO and H2O2,45,59,60 which was involved in a diverse range of plant signal transduction processes and acted as a trigger signaling of plant responses against pathogens. Especially, H2O2 was considered to be the most important molecule for signaling because of its relatively long lifetime and high permeability across the cell membranes. However, fluorescence imaging of the oligosaccharide-induced H2O2 generation in plant tissues is still difficult due to the strong interference of autofluorescence from the tissues. To monitor the oligosaccharide-induced H2O2 generation at intracellular levels using BMTA-Tb3þ as an imaging probe, the cell membrane permeable form of BMTA-Tb3þ, AM-BMTATb3þ, was synthesized according to our previous method. Similar to the cell loading process of the Ca2þ luminescent probe Fura 2,61 AM-BMTA-Tb3þ can be easily transferred into the cultured cells with an ordinary incubation method, and in the cells, accompanied by the rapid hydrolysis of AM-BMTA (Figure S4 in Supporting Information) catalyzed by ubiquitous intracellular esterases to regenerate the original ligand BMTA, the stable BMTA-Tb3þ complex is formed in the cells, which enables the cells to be used for the luminescence imaging detection of intracellular H2O2. Furthermore, it should be mentioned that, in plant cells, because of the existence of enough endogenous peroxidases for reacting with various concentrations of H2O2 (from 1 pM to 5 mM),62 the addition of exogenous peroxidase 4167
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Analytical Chemistry for the luminescence response of BMTA-Tb3þ to H2O2 is not necessary. On the basis of the above principle, the BMTA-Tb3þ-loaded tobacco leaf epidermal tissues were prepared by coincubating the tissues with AM-BMTA-Tb3þ. After further incubation in the presence of oligosaccharide, the tissues were imaged both with steady-state and time-resolved luminescence modes, respectively. As shown in Figure 5, before the oligosaccharide treatment, no time-resolved luminescence signals could be observed from the tissue (Figure 5A). The oligosaccharide treatment resulted in strong luminescence from both epidermic cells and stomatal guard cells in the tissues (Figure 5B,C). These results demonstrate that oligosaccharide can induce the plant cells to generate H2O2. Compared to the steady-state luminescence images (Figure 5B and Figure S5 in Supporting Information), highly speciﬁc and background-free time-resolved luminescence images of the epidermic and stomatal guard cells with strong green luminescence signals (Figure 5C) were obtained since the autoﬂuorescence from the tobacco cells had been completely suppressed by the time-resolved mode. To further conﬁrm that the luminescence signals from the tissues were attributed to the H2O2 generation, a control experiment was carried out by incubating the BMTA-Tb3þ-loaded tobacco leaf epidermal tissues in the presence of oligosaccharide and a H2O2 scavenger, catalase (CAT), and the tissues were imaged with time-resolved mode. In this case, the luminescence signals from both epidermic cells and stomatal guard cells in the tissues almost disappeared (Figure 5D). This result clearly indicates that the luminescence signals from the oligosaccharide-treated tissues are mainly attributed to the intracellular H2O2 generation. It should be indicated that, although oligosaccharide can also induce the NO generation in the tissues, since the luminescence intensity of BMTA-Tb3þ can only be 3-fold increased in a saturated NO aqueous solution, the eﬀect of NO is slight. In addition, due to the low concentrations of other possible interfering species in plant tissues, such as hydroxyl radical and ClO, the eﬀects of these species are negligible.
’ CONCLUSION In this work, we have successfully developed a terbium complexbased luminescent probe, BMTA-Tb3þ, for the recognition and luminescence detection of H2O2 in the presence of peroxidase. This probe was designed and synthesized on the basis of the PET mechanism by incorporating a Tb3þ complex of 2,6-bis(N-pyrazolyl)pyridine polyacid derivative into a diaminophenyl moiety. The nonluminescent probe can react with H2O2 in the presence of peroxidase to yield a highly luminescent product with a long luminescence lifetime, which allows the probe to be favorably useful for the background-free time-resolved luminescence detection of H2O2 in complicated biological samples. The results shown here of time-resolved luminescence imaging to monitor the H2O2 generation in living plant tissues demonstrate the utility of the probe for in vivo H2O2 detection. The new technique, with ﬁne time and spatial resolution capacities, provides a new strategy for visualizing the temporal and spatial distribution of H2O2 in biological tissues, which would be a useful tool for investigating the biological functions of H2O2 in living systems. ’ ASSOCIATED CONTENT
Supporting Information. HPLC analysis result of the product of BMTA-Tb3þ reacted with H2O2 in the presence of
HRP, the absorption spectra of BHTA-Tb3þ and BMTA-Tb3þ, and the eﬀect of the HRP concentration on the luminescence intensity of the BMTA-Tb3þ/H2O2/HRP system. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail: [email protected]
(Z.Y.); [email protected]
yahoo.com.cn (J.Y.). Fax: þ86-411-84986041.
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