Research article Received: 30 June 2014,

Revised: 23 August 2014,

Accepted: 4 September 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bio.2791

Fluorescent sensors based on quinolinecontaining styrylcyanine: determination of ferric ions, hydrogen peroxide, and glucose, pH-sensitive properties and bioimaging Xiaodong Yang,b Peiliang Zhao,a Jinqing Qub* and Ruiyuan Liua* ABSTRACT: A novel styrylcyanine-based fluorescent probe 1 was designed and synthesized via facile methods. Ferric ions quenched the fluorescence of probe 1, whereas the addition of ferrous ions led to only small changes in the fluorescence signal. When hydrogen peroxide was introduced into the solution containing probe 1 and Fe2+, Fe2+ was oxidized to Fe3+, resulting in the quenching of the fluorescence. The probe 1/Fe2+ solution fluorescence could also be quenched by H2O2 released from glucose oxidation by glucose oxidase (GOD), which means that probe 1/Fe2+ platform could be used to detect glucose. Probe 1 is fluorescent in basic and neutral media but almost non-fluorescent in strong acidic environments. Such behaviour enables it to work as a fluorescent pH sensor in both the solution and solid states and as a chemosensor for detecting volatile organic compounds with high acidity and basicity. Subsequently, the fluorescence microscopic images of probe 1 in live cells and in zebrafish were achieved successfully, suggesting that the probe has good cell membrane permeability and a potential application for imaging in living cells and living organisms. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: bioimaging; ferric ions; fluorescent sensor; hydrogen peroxide; pH sensitive; quinoline

Introduction Among all the kinds of detection methods, fluorescent sensors have attracted much attention owing to their sensitivity, selectivity, simplicity, high degree of specificity and low detection limit (1–4). A series of fluorescent molecules based on styrylcyanine for chemical and biological sensors was synthesized because of their large molar absorption coefficient, intense fluorescence, and response to analytes such a metal ions (5–9), explosives (10,11), saccharide (12,13), protein (14–17), enzyme (18–20), and DNA (21,22) etc. Over the past few years, fluorescent molecular bearing nitrogen heterocyclic groups, including pyridine, bipyridine, pyrrole, and quinoline, have attracted much attention due to their binding affinities to metal ions (23–28). The sensors containing these nitrogen heterocyclic compounds exhibited sensitive responses towards metal ions (29–31). At the same time, protonation of these heteroatom-containing groups significantly tuned their electron-withdrawing property, which influenced the emission colour or intensity of fluorophores with these nitrogen heterocyclic groups (32,33). In this paper, we report a novel fluorescent chemosensorbased quinoline-containing styrylcyanine (probe 1, Scheme 1) from 4-pyridinecarboxaldehyde with 1,1,2-trimethylbenz[e]indole. The interactions between probe 1 and metal ions were investigated. It was interesting to find that Fe2+ and Fe3+ showed different effects on the fluorescence of probe 1. Fe3+ could effectively quench probe 1 fluorescence, while Fe2+ at the same concentration caused nearly no change. This finding was definitely an attractive phenomenon that

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might be utilized as a potential signal transducer in bioand chemosensors. Here, we report a fluorescence assay based on the combination of the redox of Fe3+/Fe2+ and the subsequent fluorescence recovery of probe 1 to detect oxidant, as well as its related substrates. We also found that the probe’s emission could be reversely switched between bright green and a dark state by repeated protonation and deprotonation, thus enabling it to work as fluorescent sensor for acidic and basic organic vapours. Most importantly, the bright fluorescence in living cells and living organisms suggested that probe 1 has a potential application for bioimaging due to its fluorescence characteristics.

* Correspondence to: Jinqing Qu, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, P.R. China. E-mail: [email protected] Ruiyuan Liu, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou510515, P.R. China. E-mail: [email protected] a

School of Pharmaceutical Sciences, Southern Medical University, Guangzhou510515, P.R. China

b

School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou510640, P.R. China Abbreviations: DMSO, dimethyl sulphoxide; IR, infra-red; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; PL, photoluminescence; ROI, region of interest; ROS, reactive oxygen species; TEA, triethanolamine; TFA, trifluoroacetic acid; TLC, thin-layer chromatography; UV, ultraviolet-visible

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X. Yang et al. Absorption and fluorescence analysis

Scheme 1. Synthesis of probe 1.

Experimental Chemicals and reagents All chemicals and solvents were of analytical grade and were used without further purification. 1,1,2-Trimethyl-1H-benzo[e]indole and 4-quinolinecarboxaldehyde were purchased from Sigma-Aldrich. All other chemicals were commercially available from J&K Scientific Ltd. Buffer solutions with pH 1 to 12 were purchased from Merck or Sigma-Aldrich. Mixtures of probe 1 in water with various pH values were prepared by adding dimethyl sulphoxide (DMSO) solutions to buffer solutions with specific pH values. Buffers with a pH of 3–4 were prepared by acetic acid and sodium acetate and confirmed by pH meter readings. The stock solutions of metal ions for selective experiments were prepared respectively by dissolving KCl, NaCl, CaCl2, Cu(NO3) 2 · 2H2O, Pb(NO3)2, Zn(NO3)2, HgCl2, MgSO4, MnCl2 · 4H2O, AgNO3, CoCl2 · 6H2O, FeCl2 · 4H2O, FeCl3 · 6H2O in double-distilled water.

A typical experimental procedure is described as follows: stock solutions of 1 (DMSO, 2 mM) and FeCl3.6H2O (distilled water, 20 mM) were prepared in flasks equipped with stopcocks, respectively. The 1 solution (100 μL) and the FeCl3 solution (100 μL) were transferred to a vial, and then the resulting mixture was diluted to 10 mL with DMSO/water mixture (2:8, v/v) to give the sample solution, of which [1] and [Fe3+] were adjusted to be 20 μM and 200 μM, respectively. The concentration of 1 was 20 μM throughout the analysis experiments except that otherwise pointed out. The fluorescence intensity was measured with the excitation wavelength 369 nm except as otherwise noted, and the excitation and emission slits were set to 1 and 1 nm, respectively. Procedures for sample preparation in pH switching Experimental details are shown in Table S1. To avoid dilution effect, parallel samples were prepared. ‘Cycle no. 0’ means that the dye molecules are first put in water (containing 20% DMSO) without any acid and base. From no. 0.5 to no. 4, base and acid were added alternatively to adjust the pH. Additional water, if needed, was added to ensure that the final dye concentrations of all samples were identical. Cytotoxicity

Instrumentation 1

H (400 MHz) and 13C (100 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance/DMX 400-MHz NMR spectrometer with DMSO-d6 as solvents and tetramethylsilane as an internal reference. Infra-red (IR) spectra were measured using a Shimadzu FTIR-8100 spectrophotometer. Melting points (mp) was measured on a Yanaco micro-melting point apparatus. Elemental analysis was performed on an Eager 300 elemental microanalyzer. Ultraviolet-visible (UV–vis) spectra were recorded in a quartz cell (thickness: 1 cm) at room temperature using a JASCO J-820 spectropolarimeter. Fluorescence spectra were measured on a FLS-920 Edinburgh fluorescence spectrophotometer. Synthesis of 1 A mixture of 2.09 g (10 mmol) 1,1,2-trimethyl-1H-benzo[e]indole and 1.57 g (10 mmol) 4-quinolinecarboxaldehyde was refluxed in 50 ml anhydride ethanol with one drop piperidine for 12 h. After the solvent was removed under reduced pressure, the yellow solid was purified by column chromatography using methylene chloride as the eluent. From this reaction, 2.05 g of yellow product were obtained. Yield: 60%. 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 8.99 (d, J = 4.5 Hz, 1H), 8.61 (d, J = 15.9 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.12 (s, 1H), 8.10 (s, 1H), 8.06 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H), 7.76 (s, 1H), 7.72 (d, J = 5.9 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 1.70 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ = 184.33, 150.73, 150.29, 148.28, 140.55, 139.90, 132.26, 130.44, 129.68, 129.62, 129.49, 129.11, 128.06, 127.21, 126.80, 125.58, 125.38, 124.97, 123.51, 123.09, 120.27, 117.87, 54.28, 21.85. IR(v-1, LiBr): 3452, 2961, 2915, 2851, 2024, 1639, 1568, 1498, 1458, 1386, 1115, 961, 818, 756, 608. MS (MALDI-Cl): C25H20N2 m/z 348.1626 for [M + H+] 349.1699. Elemental analysis: calculated C, 86.17; H, 5.79; N, 8.04. Found C, 86.11; H, 5.82; N, 8.07. mp: 246.5–247.0°C.

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The prostatic carcinoma DU145 and HeLa cell line were provided by Nanfang Hospital Guangzhou (China). HeLa cells and DU145 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C in humidified environment of 5% CO2 and 95% air. Immediately before the experiment, the cells were placed in a 96-well plate, followed by addition of increasing concentrations of probe 1. The final concentrations of the probe were kept from 0 to 20 μM. The cells were then incubated at 37°C in an atmosphere of 5% CO2 and 95% air for 24 h, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (n = 5). Untreated assay with RPMI 1640 (n = 5) was also conducted under the same conditions. Cell culture and fluorescence imaging DU145 cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics at 37°C in humidified environment of 5% CO2. Cells were plated on 6-well plate at 5 × 104 cells per well and allowed to adhere for 12 h. Fluorescence imaging was performed with a inverted fluorescence microscope (IX71, Olympus). Before the experiments, cells were washed with phosphate-buffered saline (PBS) and then incubated with probe 1 (10 μM) in PBS for 30 min at 37°C. Cell imaging was then carried out after washing cells with physiological saline. Emission was collected at 510–550 nm for the green channel. Fluorescence imaging in zebrafishes Zebrafish eggs, 12 hpf, were incubated in respective 25 mL Petri dishes with probe 1 (20 mL fish water, 10 μM). These embryos were then kept under standard conditions at 28°C for 5 days in agreement with Home Office regulations on a 12 h light/12 h dark cycle. Tricaine methane sulphonate (MS222) was used to anesthetise the fish before any fluorescence microscopy.

Copyright © 2014 John Wiley & Sons, Ltd.

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Fluorescent pH sensors Fluorescence imaging experiments were performed on a FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan) with FV5-LAMAR for excitation at 559 nm through a × 100 0.4 NA objective. The fluorescence was collected in the range of 510– 550 nm (probe 1). Region of interest (ROI) was selected based on peripheries of zebrafishes. Image processing and analysis were performed on Olympus software (FV10-ASW), and the ratio of ROI was calculated pixel-by-pixel. All data were expressed as mean ± standard deviation.

Results and discussion Fluorescence response towards ferric ions Probe 1 was prepared by reaction 1,1,2-trimethylbenz[e]indole with 4-quinolinecarboxaldehyde in EtOH (Scheme 1). Probe 1 was characterized by NMR, mass spectroscopies and elemental analysis, which gave satisfactory data corresponding to its molecular structure (see Figs S1–S4 ESI† for details). We first measured the fluorescence response of probe 1 towards ferric ions. The fluorescence titration of Fe3+ to probe 1 was performed in water (containing 20% DMSO) (Fig. 1a). The free probe exhibited green emission with the maximum around 540 nm; upon addition of Fe3+, the fluorescence intensity at 540 nm in the emission spectra decreased. The addition of 200 μM Fe3+ quenched 96% of the original fluorescence of probe 1. As shown in Fig. 1b, a linear relationship was observed between the fluorescence intensity at 540 nm and the Fe3+ amount at concentrations lower than 200 μM. The Stern–Volmer quenching constant (ksv = 1.503 × 106 M 1, R = 0.9883), which was calculated via a linear analysis of the fluorescence intensity versus the Fe3+ concentration, suggested a strong binding ability of probe 1 to Fe3+. The detection limit for Fe3+ was determined as 6.78 μM based on S/N = 3, which is sufficiently low to allow the fluorogenic detection of micromolar concentration of Fe3+. We also carried out the fluorescence titration experiment of Fe2+ to probe 1 in water (containing 20% DMSO) (Fig. S5). The fluorescence spectrum changed slightly upon addition of Fe2+. This result indicated that Fe2+ and Fe3+ showed different effects on probe 1 fluorescence. To gain insight into the luminescent response of probe 1 towards Fe3+, 1H NMR titrations of probe 1 in the presence of

Fe3+ were performed (Fig. 2). The pattern of 1H NMR titration confirmed that the Fe3+ coordination triggers an upfield shift and broadening of aromatic proton Ha of the pyridine moiety. It suggested that the quinoline moieties participate in binding to Fe3+, which decreases proton exchange, reduces π-electron density and results in a considerable quenching in fluorescence. Moreover, a slight upfield shift and broadening signal of Ha protons indicated the direct involvement N in bonding to Fe3+. The selectivity of probe 1 towards Fe3+ over relevant metal ions was then evaluated by measuring the changes in the optical spectra upon addition of metal ions. The fluorescence response of probe 1 with various metal ions and its selectivity for Fe3+ are shown in Fig. 3. The fluorescence of probe 1 changed slightly upon addition of representative metal ions such as K+, Na+, Ca2+, Cu2+, Pb2+, Zn2+, Ag+, Mg2+, Hg2+, Co2+, Fe2+, Mn2+. In constrast, the fluorescence of probe 1 was quenched when Fe3+ was introduced. We further examined the fluorescence response of probe 1 towards Fe3+ in the presence of other potentially competing meal ions (Fig. S6). The experimental results indicated that most of the relevant metal ions only displayed minimum interference. These results confirmed the excellent selectivity of probe 1 towards Fe3+ over other competitive metal ions. Application to hydrogen peroxide and glucose sensing Hydrogen peroxide (H2O2), the simplest peroxide, is a precursor molecule of other reactive oxygen species (ROS), such as the hydroxyl radical (COH). On the one hand, hydrogen peroxide is a common contaminant in air and water due to its domestic application and in industry. On the other hand, hydrogen peroxide widely causes oxidative damage on proteins and nucleic acids, and is closely correlated with various human diseases such as cancer and Alzheimer’s disease (34,35). Furthermore, hydrogen peroxide can be generated as a byproduct from various cellular aerobic oxidation reactions such as glucose oxidation by glucose oxidase (GOx) (36). Therefore, the development of H2O2 sensors is crucial for environmental protection, clinical diagnostics and biological analysis. As mentioned above, Fe3+ could quench the fluorescence of probe 1 but Fe2+ could not. Hydrogen peroxide can oxidize Fe2+ into Fe3+, which suggests that the fluorescence of probe

3+

Figure 1. (a) Fluorescence spectra of a solution of probe 1 ([1] = 20 μM) in water (containing 20% DMSO) titration with Fe . (b) Fluorescence intensity at 540 nm 3+ with titration Fe .

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1

Figure 2. Partial H NMR spectra of 1 upon addition of Fe

3+

3+

3+

3+

in DMSO-d6. (a) [Fe ]/[1] = 0; (b) [Fe ]/[1] = 0.5; (c) [Fe ]/[1] = 1.

intensity. A linear relationship was observed between the fluorescence intensity and the H2O2 added at concentrations lower than 50 μM (Fig. 4b). The Ka (Ka = 3.734 × 107 M 1, R = 0.995), which was calculated via a linear analysis of the fluorescence intensity versus the H2O2 concentration. The detection limit for H2O2 was determined as 0.268 μM based on S/N = 3. In the presence of glucose oxidase (GOD), glucose can be specifically oxidized into gluconolactone and release an equivalent hydrogen peroxide. Immediately, the produced H2O2 can oxidize Fe2+ into Fe3+ and this leads to strong fluorescence quenching of probe 1, which can be directly correlated to the quantification of glucose. Using an aqueous solution containing probe 1 (20 μM), Fe2+ (200 μM) and GOD (1 U/mL), a fluorescence titration experiment with glucose was carried out at room temperature. Figure 5 shows the fluorescence response of the above-mentioned system upon titration, indicating that successive addition of glucose resulted in fluorescence quenching of probe 1. Similarly, the fluorescence quenching of probe 1 versus glucose concentration in the range of 0–50 μM exhibited good linearity as well (Fig. 5b), providing a detection limit of 0.96 μM. Figure 3. Visual fluorescent color variations and emission spectra of probe 1 in water (containing 20% DMSO) ([1] = 20 μM) upon the addition of various metal ions ([metal ion]/[1] = 10). The visual fluorescence was obtained with excitation at 365 nm using a hand-held UV lamp.

1/Fe2+ solution might be quenched upon the addition of hydrogen peroxide. We first investigated the influence of incubation time upon the addition of hydrogen peroxide into the probe 1/Fe2+ solution ([1] = 20 μM, [Fe2+]/[1] = 10, [H2O2]/[1] = 10, [H+]/[1] = 2). As Fig. S7 shows, the fluorescence of the probe 1/ Fe2+ solution was quenched in the presence of hydrogen peroxide. When H2O2 was introduced into the solution, Fe2+ was oxidized into Fe3+, leading to quenching of the fluorescence. Therefore, this result indicates that H2O2 could be detected through the fluorescence quenching of probe 1/Fe2+ solution. At same time, the fluorescence intensity at 540 nm decreased with increase in incubation time from 0 min to 10 min and then tends to be a constant value after 10 min. It demonstrated that the interaction of H2O2 with probe 1/Fe2+ reaches equilibrium within 10 min. Thus, the incubation time was controlled to be 10 min. For more detail, we measured the fluorescence spectra of probe 1/Fe2+ solution with various concentrations of H2O2. As shown in Fig. 4a, the titration of H2O2 into the probe 1/Fe2+ solution resulted in a continuous decrease in the fluorescence

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pH-sensitive properties Fluorescent sensors containing nitrogen heterocyclic derivatives such as pyridine, bipyridine, pyrrole, and quinoline exhibited pH sensing ability, due to the protonation of the nitrogen atom. To explore the pH-dependent optical properties of probe 1, the fluorescence and absorption pH titrations of probe 1 was performed in H2O (containing 20% DMSO) at probe concentration of 20 μM (Figs 6 and S8). As shown in Fig. 6, probe 1 was green emission at ~540 nm at high pH values and its photoluminescence (PL) intensity decreased slightly when the pH was > 5. At pH < 5, the green emission faded gradually and was completely lost. Probe 1 is a hemicyanine dye containing quinoline moieties, offering nitrogen atom for protonation. Addition of a drop of HCl into a DMSO solution of probe 1 protonates its quinoline unit and generates probe 1+. This is proved by the 1H NMR spectra shown in Fig. 7. The quinoline protons (Ha and Hb) shifted downfield and broadened after protonation because of the transformation of the quinoline ring in probe 1 to an electron-deficient quinolinium unit in probe 1+. The 1H NMR spectrum was fully restored when an excess of NaOD was injected into the solution, suggesting that the transformation between probe 1 and probe 1+ is completely reversible and non-destructive in nature.

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Fluorescent pH sensors

2+

2+

+

Figure 4. (a) Fluorescence spectra of probe 1/ Fe solution ([1] = 20 μM, [Fe ] =200 μM, [H ]/[1] = 2) in water (containing 20% DMSO) titration with H2O2. (b) Fluorescence intensity at 540 nm with titration of H2O2 [H2O2] = 0–20 μM.

2+

2+

+

Figure 5. (a) Fluorescence spectra of probe 1/Fe solution ([1] =20 μM, [Fe ]/[1] = 10, [H ]/[1] = 2, [GOD] = 1 U/mL) in water (containing 20% DMSO) titration with glucose. (b) Fluorescence intensity at 540 nm with titration of glucose [glucose] = 0–50 μM.

Figure 6. Visual fluorescent color variations and emission spectra of probe 1 in water (containing 20% DMSO) ([1 ] =20 μM) under different pHs. The visual fluorescence was obtained with excitation at 365 nm using a hand-held UV lamp.

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The fluorescence transitions were reversible upon pH change. Starting from neutral aqueous conditions, alternative addition of NaOH and HCl can adjust the resultant solution to basic (pH ~13) or acidic (pH ~3) environments. The fluorescence of probe 1 was switched turn on–off, and vice versa (Fig. 8). Such response was very fast and could be repeated for many cycles without any change in the spectral profiles. The fluorescence of probe 1 is pH-sensitive in solution, we wondered whether 1 could also work in the solid state. Due to its strong mechanical strength, we utilized a thin-layer chromatography (TLC) plate as a solid support. First, we prepared a TLC plate containing 1 by immersing a TLC plate into the acetone solution of 1 (1.0 mM) and then drying it in the air. As displayed in Fig. 9a, the dye-loaded plate emitted bright green light upon photoexcitation. After fuming with trifluoroacetic acid (TFA) vapour, the dye plate emitted dark yellow light. It, however, converted back to its green emissive form when treated with triethylamine (TEA) vapour. The switch between the dark yellow and bright green states can be repeated many times without fatigue by alternately fuming with TEA and TFA vapours as the process is non-destructive in nature. Instead of a fluorescent sensor, we also explored the application of probe 1 in other field. We used DU145 cells as a model cell to study the subcellular distribution of probe 1 by inverted

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1

Figure 7. Partial H NMR spectra of probe 1 upon addition of DCl in DMSO-d6 containing (a) 0; (b) 0.01; and (c) 0.05 mL of DCl. The spectrum in (d) is obtained by adding 0.1 mL NaOD into (c).

Figure 8. (a) Emission spectra of probe 1 in acidic, neutral, and basic conditions. (b) The emission intensity of probe 1 by repeated adjustment of its solution to acidic and basic environments. Experimental details see Table S1, ESI.† [1] = 20 μM.

Figure 9. (a) Fluorescent colour of probe 1 deposited on a TLC plate. (b) Fluorescent colour of probe 1 deposited on a TLC plate by repeated fuming with TFA and TEA vapours.

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Fluorescent pH sensors

Figure 10. (a) Bright-field imaging of DU145 cells incubated with probe 1 (10 μM). (b) Fluorescence imaging of DU145 cells; (c) overlap of (a) and (b).

Figure 11. (a) Fluorescence imaging of zebrafish (five days old) incubated with probe 1 (10 μM) from bright-field; (b) green channel imaging of (a); (c) overlap of (a) and (b).

fluorescence microscope. Firstly, to evaluate the cytotoxicity of probe 1, we performed standard 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assays using HeLa and DU145 cells at concentrations from 0–20 μM of probe 1 for 24 h, respectively. The result clearly showed that probe 1 is nontoxic to the cultured cells under the experimental conditions (Fig. S9). Herein cultured DU145 cells were incubated with probe 1 (10 μM) for 30 min at 37°C. As predicted, fluorescence images showed that probe 1 with green fluorescence was cell membrane permeable and localized in the cytoplasm region (Fig. 10). These results clearly showed that probe 1 has good cell membrane permeability and could image organelles in living cells. Encouraged by the living cell experiments, we evaluated the effectiveness of probe 1 for fluorescence imaging in a living vertebrate organism, zebrafish. Five-day-old zebrafish were incubated in solutions of probe 1 at 10 μM and the distribution of fluorescence within the zebrafish was monitored (Fig. 11). Probe 1 exhibited uptake within the fish with clear fluorescence in yolk sac and the alimentary canal, including intestinal tract and biliary system (gall bladder, liver, pancreas, solitary islet and bile ducts). In addition, probe 1 was found stable in the body of zebrafish, because no noticeable change in the averaged ratio value was observed at least within 1 h. These in vivo studies showed that probe 1 can enter zebrafish and image in a living organism.

Conclusion In conclusion, we have designed and synthesized a novel fluorescence sensor based on styrylcyanine containing quinoline. Probe 1 exhibited different fluorescence sensing properties towards Fe3+ and Fe2+. Fe3+ could effectively quench the fluorescence of

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probe 1, while Fe2+ caused nearly no change. In the presence of hydrogen peroxide, the fluorescence of the probe 1/Fe2+ solution could be quenched. The fluorescence of probe 1/Fe2+ solution could also be quenched by the H2O2 released from glucose oxidation by glucose oxidase (GOD), which suggested that the use of probe 1 could be extended to detect glucose. Probe 1 was fluorescent in basic and neutral media but showed almost no fluorescence in strong acidic environments. Such behaviour enabled it to work as a chemosensor for detecting volatile organic compounds with high acidity and basicity in solid state. Subsequently, the fluorescence microscopic images of probe 1 in live cells and in the whole body of zebrafish were achieved successfully, suggesting that probe 1 has good cell membrane permeability and potential application for imaging in live cells and living organism.

Acknowledgements We gratefully thank the National Natural Science Foundation of China (51173050 and 21376092) and Medical Scientific Research Foundation of Guangdong Province, China (A2010355) for financial support of this work.

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Luminescence 2014

Fluorescent sensors based on quinoline-containing styrylcyanine: determination of ferric ions, hydrogen peroxide, and glucose, pH-sensitive properties and bioimaging.

A novel styrylcyanine-based fluorescent probe 1 was designed and synthesized via facile methods. Ferric ions quenched the fluorescence of probe 1, whe...
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