Through Bond Energy Transfer: A Convenient and Universal Strategy

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Through Bond Energy Transfer: A Convenient and Universal Strategy toward Efficient Ratiometric Fluorescent Probe for Bioimaging Applications Yi-Jun Gong, Xiao-Bing Zhang,* Cui-Cui Zhang, Ai-Li Luo, Ting Fu, Weihong Tan, Guo-Li Shen, and Ru-Qin Yu Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 S Supporting Information *

ABSTRACT: Fluorescence resonance energy transfer (FRET) strategy has been widely applied in designing ratiometric probes for bioimaging applications. Unfortunately, for FRET systems, sufficiently large spectral overlap is necessary between the donor emission and the acceptor absorption, which would limit the resolution of double-channel images. The through-bond energy transfer (TBET) system does not need spectral overlap between donor and acceptor and could afford large wavelength difference between the two emissions with improved imaging resolution and higher energy transfer efficiency than that of the classical FRET system. It seems to be more favorable for designing ratiometric probes for bioimaging applications. In this paper, we have designed and synthesized a coumarin−rhodamine (CR) TBET system and demonstrated that TBET is a convenient strategy to design an efficient ratiometric fluorescent bioimaging probe for metal ions. Such TBET strategy is also universal, since no spectral overlap between the donor and the acceptor is necessary, and many more dye pairs than that of FRET could be chosen for probe design. As a proof-of-concept, Hg2+ was chosen as a model metal ion. By combining TBET strategy with dual-switch design, the proposed sensing platform shows two well-separated emission peaks with a wavelength difference of 110 nm, high energy transfer efficiency, and a large signal-to-background ratio, which affords a high sensitivity for the probe with a detection limit of 7 nM for Hg2+. Moreover, by employing an Hg2+-promoted desulfurization reaction as recognition unit, the probe also shows a high selectivity to Hg2+. All these unique features make it particularly favorable for ratiometric Hg2+ sensing and bioimaging applications. It has been preliminarily used for a ratiometric image of Hg2+ in living cells and practical detection of Hg2+ in river water samples with satisfying results.

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response feature,5b,c,e−j all these probes are based on single emission intensity changes, which tend to be affected by a variety of factors such as instrumental efficiency and environmental conditions, as well as the concentration of probe molecule. These interferences can be eliminated by employing ratiometric fluorescent probes,6−8 which allow the measurement of changes of the intensity ratio at two emission bands induced by metal ions and provide built-in correction for the above-mentioned environmental effects. Several strategies, including internal charge transfer (ICT),6 fluorescence resonance energy transfer (FRET),7 and two fluoroionphores designs,8 have been adopted to design ratiometric probes. Among them, FRET strategy could provide moderate resolution of the two emission bands and has been widely applied in designing ratiometric probes for bioimaging applications.7c,d,g−i Unfortunately, for FRET systems, to achieve the largest energy transfer efficiency, sufficiently large spectral

etal ions play either beneficial or deleterious roles in biology or in the environment. For example, Fe2+, Zn2+, and Mn2+ are indispensable transition metal ions for the human body and play vital roles in many biological processes.1 Cd2+, Pb2+, and Hg2+ are highly toxic for both humanity and environment, even at very low concentrations, and bioaccumulation of these metal ions in organisms can cause serious health problems.2 Therefore, the development of methods to detect metal ions in environmental samples, and image them in biological samples such as living cells, is of considerable importance and has become a current focus of chemical research. Due to its high sensitivity, fast analysis with spatial resolution for providing in situ and real-time information, and nondestructive sample preparation, the fluorescent probe method seems to be an ideal candidate for both sensing and bioimaging metal ions in various samples.3 As a consequence, in the past two decades, quite a few fluorescent probes have been developed for various metal ions based on the quenching mechanism4 or target-triggered fluorescent enhancement.5 Although the fluorescent enhancement-based probes are more favorable for bioimaging applications due to their off−on © 2012 American Chemical Society

Received: September 23, 2012 Accepted: November 20, 2012 Published: November 21, 2012 10777

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Scheme 1. (a) Structure and Synthetic Routes of CR; (b) Response Mechanism of the TBET Probe via a Hg2+-Catalyzed Rhodamine Spiro Ring-Opening Reaction

combining TBET strategy with dual-switch design, the proposed sensing platform shows two well-separated emission bands for ratiometric Hg2+ sensing and bioimaging application with a large SBR. It was applied in detection of Hg2+ in aqueous samples and ratiometric imaging of Hg2+ in living cells, both with satisfactory results.

overlap is necessary between the donor emission and the acceptor absorption, which would limit the wavelength difference between the two emission peaks (e.g., the difference for the classical fluorescein−rhodamine dye pair is fixed at ∼65 nm) and limit the resolution of double-channel fluorescence images. Further improving the imaging resolution will need the use of dye pairs with less spectral overlap, which will result in reduced energy transfer efficiency, to some extent, and a decreased signal-to-background ratio (SBR) for the probe. A new strategy for design of ratiometric probes with both high imaging resolution and large SBR is desired. The through-bond energy transfer (TBET) system,9,10a in which the donor and the acceptor moieties are linked by a conjugated bond, did not need spectral overlap between donor emission and acceptor absorption and could afford a large wavelength difference between the two emission peaks with improved imaging resolution by choosing suitable dye pairs. Moreover, the TBET system shows obviously higher energy transfer efficiency than that of the classical FRET system,10 which could provide larger target-triggered SBR for the designed ratiometric probe. TBET seems to be especially suitable for design of an efficient ratiometric probe for bioimaging metal ions. Several groups have employed TBET strategy to design fluorescent cassettes for intracellular imaging applications.10b,d−h However, all of these cassettes are based on single emission intensity changes rather than ratiometric measurement, and TBET was adopted to achieve large pseudo-Stokes shifts to avoid self-quenching of fluorophores.10 To our best knowledge, no TBET-based ratiometric fluorescent probes have been reported for metal ions. In this work, we employ TBET to develop ratiometric bioimaging probes for metal ions. As a proof-of-concept, we have designed and synthesized a coumarin−rhodamine (CR) TBET system as a ratiometric probe for Hg2+ (Scheme 1). We chose Hg2+ as a model metal ion, as it is one of the most toxic metal ions.11 By

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EXPERIMENTAL SECTION Reagents and Apparatus. All chemicals were obtained from commercial suppliers and used without further purification. Tetrahydrofuran (THF) was distilled from sodium prior to use. Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore, USA). LC-MS analyses were performed using an Agilent 1100 HPLC/MSD spectrometer. UV−vis absorption spectra were recorded with a Shimadzu UV-2450 spectrophotometer. 1H and 13C NMR spectra were recorded on a Bruker DRX-400 spectrometer operating at 400 and 100 MHz, respectively. All chemical shifts are reported in the standard d notation of parts per million. All fluorescence measurements were carried out on a HitachiF4500 fluorescence spectrometer with both excitation and emission slits set at 10.0 nm. Fluorescence images of HeLa cells were obtained using an Olympus FV1000 laser confocal microscope (Japan). Compounds CR and CR-P (the product of the reaction of CR with Hg2+) were efficiently synthesized following the synthetic methodology shown in Scheme 1. Rhodamine B thiohydrazide, 1,12 and 7-diethylamino-3-carboxylic chloridecoumarin, 2,13 were synthesized according to the literature methods. Synthesis of CR. Under nitrogen, a solution of 1 (470 mg, 1 mmol) in CH2Cl2 (10 mL) was added to Et3N (0.15 mL, 1.1 mmol) and a solution of 2 (280 mg, 1 mmol) in CH2Cl2 (30 mL) was added dropwise to the solution with stirring. The organic layer was washed with water (200 mL) after being 10778

dx.doi.org/10.1021/ac302762d | Anal. Chem. 2012, 84, 10777−10784

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Figure 1. (a) The UV−vis of CR (10 μM) in the presence of different concentrations of Hg2+ (0, 0.2, 0.3, 0.45, 0.6, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0, 15.0, 20.0, and 30.0 μM). (b) Change in color (left) and fluorescence (right) of CR (20 μM) upon addition of Hg2+ (50 μM) in buffered (pH 7.2) aqueous THF solution.

stirred for 24 h, dried over anhydrous Na2SO4, and filtered. The solvent was removed under reduced pressure. The crude product was purified by column chromatography on alumina to afford 400 mg of slight yellow solid CR in 56% yield. 1H NMR (CDCl3, 400 MHz) δ (ppm): 1.16 (t, J = 7.2 Hz, 12H), 1.22 (t, J = 7.2 Hz, 6H), 3.33 (q, J = 7.2 Hz, 8H), 3.44 (q, J = 7.2 Hz, 4H), 6.30−6.33 (m, 4H), 6.48 (s, 1H), 6.63 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.81 (s, 1H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 12.399, 29.671, 44.356, 45.081, 53.412, 76.687, 77.000, 77.320, 96.637, 97.338, 108.301, 108.515, 109.316, 109.964, 122.659, 126.633, 128.014, 128.777, 130.303, 131.218, 135.979, 148.566, 152.022, 152.693, 157.721, 162.329. ESI-MS [M+]: 716.2. Synthesis of CR-P. A solution of CR (21 mg, 0.03 mmol) in THF (30 mL) and HgCl2 (0.04 mmol) in distilled water (30 mL) was mixed in a round-bottom flask (250 mL) and kept stirring for 10 min. The mixture was separated with ethyl acetate, washed with water and brine, and dried over anhydrous Na2SO4. The solvent was then removed under reduced pressure to afford CR-P as a red solid (18 mg, 85% from CR). ESI-MS [M+]: 682.3. Spectrophotometric Experiments. Both the fluorescence and UV−vis absorption measurement experiments were

conduct in a 50% aqueous tetrahydrofuran solution (H2O/ THF = 1:1, v/v). The fluorescence emission spectra were recorded at excitation wavelength of 420 nm with emission wavelength range from 430 to 650 nm. A 2 × 10−5 M stock solution of CR was prepared by dissolving CR in THF. A stock standard solution of Hg2+ (0.01 M) was prepared by dissolving an appropriate amount of HgCl2 in water and adjusting the volume to 500 mL in a volumetric flask. This was further diluted to 6 × 10−5−4 × 10−8 M stepwise. The complex solution of Hg2+/CR was prepared by adding 5.0 mL of the stock solution of CR and 1.0 mL of the stock solution of Hg2+ in a 10 mL volumetric flask. Then, the mixture was diluted to 10 mL with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer solution. In the solution thus obtained, the concentrations were 1 × 10−5 M in CR and 3 × 10−5- 2 × 10−8 M in Hg2+. The solution was protected from light and kept at 4 °C for further use. A blank solution of CR was prepared under the same conditions without Hg2+. Cell Cultures and Imaging Experiments. The living Hela cells were obtained from the biomedical engineering center of Hunan University (Changsha, China). Immediately prior to the imaging experiments, the cells were washed with phosphatebuffered saline (PBS), incubated with 20 μM CR (in the culture medium containing 2% dimethylsulfoxide, DMSO) for 0.5 h at 10779

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37 °C, then washed with PBS for three times, and imaged. After incubating with 20 μM HgCl2 for another 0.5 h at 37 °C, the Hela cells were washed with PBS three times and imaged again. Confocal fluorescence imaging of intracellular Hg2+ in Hela cells was observed under an Olympus FV1000 laser confocal microscope. Excitation wavelength of the laser was 440 nm. Emissions were centered at 470 ± 10 and 580 ± 10 nm (double channel).

increased with the increase of the Hg2+ concentration, which corresponds to the absorption peak of the ring-opened form of the rhodamine B. This confirms that the addition of Hg2+ ions can promote the formation of the ring-opened form of CR from the spirolactam form, which possesses a high molar extinction coefficient at 567 nm. Meanwhile, a significant color change from slight yellow to red, with a fluorescence change from cyan to pink (excitation at 365 nm), could be observed easily by the naked eye (Figure 1b). Fluorescent Analytical Performance of the Ratiometric Probe. The fluorescence responses of probe CR to Hg2+ in buffered H2O/THF (1:1, v/v) solution were then recorded at an excitation wavelength of 420 nm and emission wavelength of 430−650 nm, with results given in Figure 2. The spectrum of

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RESULTS AND DISCUSSION Optimized Design and Synthesis of the TBET Probe. In our newly designed TBET system (Scheme 1), rhodamine B was chosen as an acceptor, since its fluorescent emission located at long wavelength region and its spirolactam derivatives have been reported to show metal ion-triggered “turn-on” fluorescence signal.14 Coumarin shows an emission at short wavelength area far away from that of rhodamine, which will afford a high resolution for double-channel bioimage. Moreover, it shows slight spectral overlap with the acceptor’s excitation with a negligible FRET process (Figure S1, Supporting Information), which is favorable for the investigation with the TBET. Therefore, coumarin was chosen as an energy donor for our TBET system. A thiosemicarbazide group, which exhibited irreversible Hg2+-promoted desulfurization reaction to form 1,3,4-oxadiazole derivatives, was fused in the probe molecule as the recognition unit. It is worthwhile to note that this group also exhibits a dual-switch function, one control for the close/open process of the rhodamine spirolactam to dominate the fluorescence off−on of the acceptor and the other control for the off−on TBET process through the formation of conjugate linker between the two dyes. Such a dual-switch function is favorable for a low fluorescence ratio background and, therefore, an improved sensitivity of the probe toward Hg2+. Moreover, such reaction-type probes usually show a higher selectivity to target analyte than coordination-based probes. Compound CR was efficiently synthesized following the synthetic methodology shown in Scheme 1, with previously reported rhodamine B thiohydrazide and 7-diethylamino-3carboxylic chloride-coumarin as intermediates. Compound CRP was synthesized by simply mixing CR with HgCl2 in THF/ H2O. Both of them were characterized using NMR and MS analytical spectroscopic techniques (see Supporting Information), which agreed well with the proposed structures. Spectrophotometric Response of the TBET Probe. The changes of the UV−vis spectra for CR with the gradual addition of Hg2+ in a buffered (0.01 M HEPES, pH 7.2) H2O/THF (1:1, v/v) solution are first investigated (Figure 1a). It can be seen from the curve in Figure 1 that the free CR ([Hg2+] = 0 M) shows an obvious absorption peak at 420 nm, accompanied with a slightly yellow color, which is dominated by the donor chromophore (coumarin). Moreover, the characteristic absorption peak for the acceptor (rhodamine B, near 560 nm) is not observed for free CR, demonstrating its existence in spirolactam form. However, when Hg2+ was added into the buffered solution, redshift was observed for the donor’s absorption band, and it shifts to 450 nm with the concentration of Hg2+ reaching 30 μM. This result might be ascribed to the Hg2+-triggered conversion of thiosemicarbazide to 1,3,4-oxadiazole, which shows stronger electron-withdrawing effect on coumarin and also increases its degree of conjugation. Besides the redshift of coumarin’s absorption peak, a new characteristic absorption peak at 567 nm also appeared with its absorption intensity

Figure 2. (a) The fluorescence emission spectra of CR (10 μM) in the presence of different concentrations of Hg2+ (0, 0.02, 0.04, 0.06, 0.2, 0.3, 0.45, 0.6, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0, 15.0, 20.0, and 30.0 μM) in buffered (pH 7.2) aqueous THF solution. (b) Calibration curve of the probe CR. The curve was plotted with the fluorescence intensity ratio (I580/I470) vs Hg2+ concentration. λex = 420 nm.

CR without Hg2+ exhibits only cyan emission at 470 nm of donor itself (Figure 2), and no characteristic emission of energy acceptor at 580 nm is observed, due to the rhodamine skeleton existing in spirolactam form with its conjugated system being broken. Upon addition of Hg2+, the donor emission at 470 nm sharply decreased, and a new emission band with a maximum at 580 nm (rhodamine B) appeared and gradually increased in intensity (Figure 2a), indicating that the ring-opened reaction of the rhodamine B spirolactam and the subsequent TBET 10780

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Figure 3. Fluorescence response of CR (10 μM) to 0.5 μM of Hg2+ or 5.0 μM of other metal ions (the black bar portion) and to the mixture of 5.0 μM of other metal ions with 0.5 μM of Hg2+ (the gray bar portion).

10−8 to 3.0 × 10−5 M (Figure 2b), with a detection limit of 7 nM (3σ/slope). The effects of pH on the fluorescence characteristics of the new probe were also investigated (see Figure S2 in the Supporting Information). It was obtained from the fluorescence titration curve that the pKa′ of probe CR is about 3.0, and in a range of pH from 5.0 to 8.0, acidity does not affect the fluorescent intensity of CR in a mixed THF−water solution of (1:1, v/v). Therefore, all of the detections of metal ions were operated in the mixed solution containing HEPES (0.01 M, pH = 7.2). A high selectivity to the target metal ions over other metal ions is a necessity for a probe with potential application in complex biological or envirmental systems. Our probe belongs to reaction-type probes, which should theoretically show a higher selectivity toward Hg2+ than that of binding-based probes. As shown in Figure 3, the addition of 0.5 μM of Hg2+ into the sensing system could induce a significant enhancement of fluorescence ratio at I580/I470 of the probe, while most of the other metal ions at 5 μM did not induce obvious changes of fluorescence ratio. Ag+ in a concentration larger than 5 μM showed a slight interference to Hg2+ detection, since Ag+ also shows a weak catalytic ability for the desulfurization reaction. Cu2+ existing at 5 μM also showed a moderate interference to Hg2+ detection due to its quenching effect on the coumarin moiety of CR. To test practical applicability of our fluorescent probe for Hg2+, competition experiments were also carried out. Ten times concentration of other metal ions (5 μM) is added to 0.5 μM of Hg2+ separately, and the fluorescence response of the probe is then detected. Results are also shown in Figure 3 (the gray bar portion). The addition of Ag+ to the Hg2+ solution can induce much more enhancement of the fluorescence ratio at I580/I470, indicating that Ag+ might assist the desulfurization of CR by Hg2+. The probe showed almost unchanged responses to Hg2+ before and after the addition of other interfering metal ions. All these selective results indicate that our proposed probe could meet the selective requirements for biomedical and environmental applications. Energy Transfer Mechanism. It has been well investigated that strong acid could trigger the ring-opened reaction of the rhodamine spirolactams;14 the fluorescence spectra of CR in the presence of Hg2+ (30 μM) or H+ (10 mM) were therefore recorded to verify the energy transfer mechanism, with results

process of CR are triggered by Hg2+ ions. Since the emissions of coumarin and rhodamine B were located at 470 and 580 nm, respectively (Figure 2a), a wavelength difference of 110 nm is calculated for them, which is larger than that of the classical fluorescein−rhodamine FRET dye pair (∼65 nm). This result indicates that the TBET strategy could provide higher resolution for the two emission bands than that of FRET, which is especially favorable for ratiometric imaging of intracellular metal ions, and also benefits for a large range of emission ratios. Another advantage for TBET is its high energy transfer efficiency. The efficiency of energy transfer (EET) between coumarin and rhodamine in the CR-based TBET system was then estimated with eq 1:9 η EET = 1 − ΦF‐dyad /ΦF‐donor

(1)

Here, ηEET denotes the efficiency of energy transfer, ΦF‑dyad is the fluorescence quantum yield of the dyad (the donor moiety in the TBET system), and ΦF‑donor is the fluorescence quantum yield of the donor in the absence of the acceptor. Since, in compound CR, the coumarin moiety is connected to the rhodamine with a spirolactam form, which shows neither absorption nor emission due to its conjugated system being broken, the ΦF for coumarin moiety in CR can be ΦF‑donor. It was estimated to be 0.078, whereas the ΦF of the coumarin moiety in CR-P (ΦF‑dyad) was greatly reduced to 0.002, with quinine sulfate (ΦF = 0.546 in 0.1 M H2SO4) as a standard.15 A ηEET value of 97.4% was obtained for CR-P, indicating a high efficiency of energy transfer of the CR-based TBET system triggered by Hg2+. The large wavelength difference and the dual-switch design, along with the high energy transfer efficiency of TBET, are favorable for a large SBR for the probe, thereby for a high sensitivity for Hg2+ detection. Experimental results show that the addition of Hg2+ to a solution of CR indeed induces a remarkable increase of fluorescence signal at 580 nm and the decrease of that at 470 nm. The emission intensity ratio, I580/ I470, was gradually increased with increasing Hg2+ concentration (Figure 2b) and varied from 0.0133 to 9.280 with the concentration of Hg 2+ changing from 0 to 30 μM, corresponding to a largest SBR of 697.7, which also indicated a high energy transfer efficiency of the TBET system. The linear response concentration range for Hg2+ covers from 2.0 × 10781

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Figure 4. (a) The fluorescence emission spectra of blank CR (10 μM, green dashed curve) and CR in the presence of Hg2+ (30 μM, red dotted curve) or H+ (10 mM, black solid curve) for 1 h. (b) Proposed energy transfer mechanism for CR in the presence of Hg2+ and H+.

Figure 5. Images of Hela cells treated with probe CR in the absence or presence of Hg2+. (a) Bright field image of Hela cells incubated with CR (20 μM) for 0.5 h. (b) Fluorescence image of (a) from cyan channel. (c) Fluorescence image of (a) from red channel; (d) merged image of frames (a) and (b). (e) Bright field image of Hela cells incubated with CR (20 μM) for 0.5 h and then treated with Hg2+ (50 μM) for another 0.5 h. (f) Fluorescence image of (e) from cyan channel. (g) Fluorescence image of (e) from red channel; (h) merged image of frames (e) and (g). Scale bar = 20 μm. Excitation was set at 440 nm.

observation of fluorescence signal changes at two different wavelengths with high resolution, which is especially favorable for ratiometric imaging of Hg2+ in biological samples. The ratiometric confocal fluorescence cellular imaging experiment for intracellular Hg2+ was then performed. The double-channel fluorescence images at 470 and 580 nm are shown in Figure 5. Hela cells incubated with CR (20 μM) for 30 min at room temperature showed a clear cyan intracellular fluorescence (Figure 5b), which indicated that CR was cell permeable. When cells prestained with CR were further incubated with HgCl2 (50 μM) in phosphate-buffered saline (PBS) for 30 min and washed, a nearly complete disappearance of the cyan fluorescence (Figure 5f) accompanied with strong red fluorescence (Figure 5g) was observed due to the Hg2+triggered TBET of CR. These preliminary experimental results demonstrate that CR could be used for ratiometric imaging of Hg2+ in biological samples with high resolution. Detection of Hg2+ in Real Samples. The practical application of the sensing system was then evaluated by determination of recovery of spiked Hg2+ in river water samples. The river water samples were obtained from Xiang River (Changsha, China). All the samples collected were simply filtered and showed that no Hg2+ was present. Hg2+ stock

shown in Figure 4. In the presence of Hg 2+ , the thiosemicarbazide group in CR was converted into 1,3,4oxadiazole derivatives through a desulfurization reaction, which brought on the opened-ring form of the rhodamine derivative with its fluorescence turned on. Meanwhile, the TBET process was also triggered through the formation of a conjugate linker between the two dyes, accompanied with a sharp decrease of the emission at 470 nm for the coumarin moiety and a moderate emission at 580 nm of rhodamine, which indicated high energy transfer efficiency for TBET. On the contrary, the introduction of acid induced remarkable enhancement of fluorescence intensity at 470 nm by blocking the photoninduced electron transfer (PET) from the thiosemicarbazide group to coumarin, while a weak fluorescence emission at 580 nm was observed for rhodamine, indicating a very low energy transfer efficiency for FRET due to the slight spectral overlap between the donor coumarin’s emission and the acceptor rhodamine’s absorption (see Figure S1, Supporting Information). All these experimental results indicate that the fluorescence response of the sensing system is through a TBET mechanism. Bioimaging of Hg2+ in Living Cells. The large wavelength difference of the two emissions for CR should be a benefit for 10782

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solution at different concentrations was spiked in these samples, and the CR-based ratiometric probe was then employed to detect its concentration, with analytical results shown in Table 1. It was observed that the results obtained for river water

river river river river a

water water water water

1 2 3 4

Hg2+ spiked (M)

Hg2+ recovered (M) meana ± SDb

recovery (%)

0.0 5.0 × 10−7 2.0 × 10−6 5.0 × 10−6

0.0 (4.92 ± 0.05) × 10−7 (1.94 ± 0.05) × 10−6 (5.17 ± 0.07) × 10−6

98.4 97.0 103.4

Mean of three determinations. bSD: standard deviation.

samples show good recovery values, which confirmed that the proposed probe was applicable for practical Hg2+ detection in real samples with other potentially competing species coexisting.

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CONCLUSIONS In summary, we have employed TBET strategy to design an efficient fluorescent probe CR for ratiometric bioimage of metal ions. By combining TBET strategy with dual-switch design, the proposed sensing platform shows two well-separated emission bands with high energy transfer efficiency and a large signal-tobackground ratio, which affords a high sensitivity of the probe for Hg2+. It also shows a high selectivity to Hg2+ due to the specificity of the Hg2+-promoted desulfurization reaction. The probe has also been successfully applied in the bioimage of Hg2+ in living cells and detection of Hg2+ in environmental water samples, further demonstrating its value in practical applications. Since no spectral overlap between the donor and the acceptor is necessary and many more dye pairs than that of FRET can be chosen for probe design, the TBET strategy is convenient and provides a universal platform for the design of ratiometric fluorescent probes for the double-channel fluorescence image of other target analytes with high resolution for biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary spectral data. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table 1. Recovery Study of Spiked Hg2+ in River Waters with Proposed Sensing System sample

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Scientific Program of China (2011CB911000), NSFC (Grants 20975034, 21177036, 21205068), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), the National Key Natural Science Foundation of China (21135001), National Instrumentation Program (2011YQ030124), the Ministry of Education of China (20100161110011), and Hunan Provincial Natural Science Foundation (Grant 11JJ1002). 10783

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