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A Two-Distinctly-Separated-Emission Colorimetric NIR Fluorescent Probe for Fast Hydrazine Detection in Living Cells and Mice upon Independent Excitations Zhengliang Lu, Wenlong Fan, Xiaomin Shi, Yanan Lu, and Chunhua Fan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02149 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017

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Analytical Chemistry

A Two-Distinctly-Separated-Emission Colorimetric NIR Fluorescent Probe for Fast Hydrazine Detection in Living Cells and Mice upon Independent Excitations Zhengliang Lu*, Wenlong Fan, Xiaomin Shi, Yanan Lu, Chunhua Fan Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China. Corresponding author’s fax: +86-531-82765475 Corresponding email: [email protected]

ABSTRACT: Hydrazine is carcinogenic and highly toxic so that it can lead to serious environmental contamination and serious health risks although it has been extensively used as an effective propellant and an important reactive base in industry. Thus, the development of two-emission NIR fluorescent probes for rapid detection of hydrazine with high selectivity and sensitivity is of significance and of great challenge in both biological and environmental sciences. Here, we report a two-emission colorimetric fluorescent probe for the specific detection of hydrazine based on hydrazinolysis reaction under physiological conditions. In the presence of hydrazine, the probe showed an extremely remarkable fluorescence enhancement at 627 nm compared to the decrease at 814 nm excited at different wavelength in aqueous solution. This distinct difference of two emission intensities is suitable for detection of low concentration hydrazine with a detection limit of 0.38 ppb. Addition of hydrazine resulted in a remarkable color change from blue-green to red observed by the naked eye. Kinetic study indicated a fast response of the probe toward hydrazine in minutes. Furthermore, the probe can bioimage hydrazine in living HeLa cells and mice with low cytotoxicity and excellent biocompatibility.

It is well known that hydrazine acts as not only an effective propellant in missile and rocket propulsion systems due to its flammable and detonable characteristics, but also one of most important reactive bases in chemistry, pharmacy and agriculture.1-3 Its strong reducing power also can effectively inhibit metal corrosion of water boilers for feed and heating systems through scavenging oxygen. Most importantly, hydrazine is carcinogenic and toxic so that even trace leakage potentially leads to serious environmental contamination and serious health risks during its manufacture, transportation, application, and disposal.4 Even worse, hydrazine is much easily absorbed by biological systems through dermal and oral contact, and inhalation due to perfect water solubility.5-6 As a neurotoxin and mutagen, excess exposure to hydrazine may cause severe organ damage, including but not limited to the liver, kidneys, lungs, and the central nervous system and infections of the respiratory.7-8 Therefore, the U.S Environmental Protection Agency (EPA) suggested an allowable threshold limit value of 10 ppb of hydrazine as a carcinogenic substance.9 The traditional analytical methods including chromatography, spectrophotometry, electrochemistry and titrimetry, to monitor trace hydrazine are less effective due to low temporal resolution, labor-cost sample preparation, high requirement of expensive instruments and facilities and poor compatibility with biology, which extremely encumber the rapid and convenient detection in living cell and animals.10-13 Fluorescence methodologies based on small molecular fluorescent probes are extremely attractive because of their high specificity, sensitivity, and noninvasive detection in live cells or tissues. Therefore, to date, numerous fluorescent probes were devel-

oped based on the strong nucleophilicity of hydrazine removing protecting groups anchored on fluorophores such as acetyl,14-15 4-bromo butyrate,16-18 levulinate,19-20 phthalimide,21-22 aldehyde,23-24 and malononitrile25-28. Those groups acted as a recognizing moiety of fluorescent probes and significantly regulate electron processes including excited-state intramolecular proton transfer (ESIPT), photoinduced electron transfer (PET), or intramolecular charge transfer (ICT).29-30 It is to be pointed out that numerous probes showed fluorescence emission on excitation of ultraviolet light potentially harmful to cell or tissues, and the long response time further limiting their real-time application.31-34 Excitation of long wavelength light can also reduce auto-fluorescence, phototoxicity, low lightbleaching and deeper penetration. 16, 20, 35-38 Two-emission fluorescent probes would be more advantageous than intensitybased fluorescent ones due to its good self-calibration of two build-in excitation or emission wavelengths to eliminating the interference from environmental conditions and biological systems.39-44 Unfortunately, only few two-emission NIR fluorescent probes have been successfully applied for the detection of hydrazine.45-48 For example, Peng and coworkers developed a ratiometric NIR fluorescent probe for N2H4 based on the selective hydrazinolysis at pH 4.5.45 Chow group reported a pyridomethene-BF2 chemosensor for high detection of hydrazine in 90% DMF aqueous solution.46 Recently, Zhao and coworkers demonstrated a ratiometric NIR fluorescent probe for hydrazine based on a hybrid fluorophore of coumarin and benzopyrylium with high sensitivity, selectivity and low detection limit in DMSO-PBS solution (2:3, v/v, pH 7.4).47 Very recent-

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ly, Ni group designed another hydrazine-specific NIR fluorescent probe showing ratiometric responses according to the hydrazineinduced decomposition from the cyanine derivative to the coumarin core in DMSO-HEPES (1:1, v/v), which was not further applied to biological systems.48 Those ratiometric NIR fluorescent

probes could only exhibit excellent emissions with limitations such as high amount of organic solvent or acidic environment. The probes based on the ratios of two similar emission intensities probably are non-favorable for detection of low concentration analytes. Keeping those drawbacks in mind, herein we describe a simple two-emission colorimetric NIR fluorescent probe (CyBT) based on ketocyanine for specific detection of hydrazine with a 4-bromobutyryl group as an excellent recognition unit of hydrazine. As expected, the probe shows two distinctlyseparated emission peaks at 627 nm and 814 nm upon independent excitations at 580 nm and 780 nm, respectively. However, the removing of protecting group in Cy-BT induced by hydrazine leads to a strong enhancement at 627 nm and an obvious decrease at 814 nm. Cy-BT exhibits a fast ratiometric response for hydrazine in minutes under physiological conditions in aqueous solution. Furthermore, Cy-BT was successfully applied for bioimaging hydrazine in living cells and animals. Scheme 1. Design and synthesis of two-emission NIR fluorescent probe Cy-BT.

EXPERIMENTAL SECTION Materials and Instruments. All chemical reagents and solvents were obtained commercially and used as received without further purification unless otherwise stated. Ultrapure water was purified from Millipore. 1H NMR and 13C NMR spectra were recorded on a Bruker 400M spectrometer and referenced to the solvent signals. Mass spectra (ESI) were carried out on a Bruker Daltonics APEX II 47e FT-ICR spectrometer with ESI mode (America). UV-Vis and Fluorescence spectra were performed on a Varian Cary 100 spectrophotometer and a hitachi F-7000 luminescence spectrometer, respectively. The pH value was measured using a digital pH-meter (PHSJ-3F, Leici, Shanghai, China). The fluorescence images of cells and mice were taken using a confocal laser scanning microscope (TCS SP5, Leica, Germany) with an objective lens (×40). Ketocyanine (Cy-KT) was synthesized by a modified procedure according to literature.49 Synthesis of probe Cy-BT. Under N2 atmosphere, a mixture of 4-Dimethylaminopyridine (DMAP) (40 mg, 0.3 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) (101 mg, 0.53 mmol) and 4-bromobutyric acid (71 mg, 0.36 mmol) in anhydrous dichloromethane (50 mL) was stirred for 60 minutes. A solution of Cy-KT (148 mg, 0.3 mmol) in 10 mL of dichloromethane was added into the above mixture and the resulting mixture was continuously stirred at room temperature overnight. Solvent was removed under reduced pressure. The residue was purified by the silica

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gel chromatography (CH2Cl2/MeOH = 10:1, v/v) to afford pure Cy-BT as a green solid (89 mg, 40%). 1 H NMR (400

MHz, DMSO-d6) δ ppm: 7.62 – 7.55 (m, 4H), 7.45 – 7.40 (m, 4H), 7.27 (tt, J = 7.7, 4.6 Hz, 2H), 6.23 (d, J = 14.2 Hz, 2H), 4.22 (q, J = 7.1 Hz, 4H), 3.86 (t, J = 6.3 Hz, 2H), 3.07 (t, J = 7.5 Hz, 2H), 2.67 (t, J = 6.2 Hz, 4H), 2.22 – 2.16 (m, 2H), 1.85 (p, J = 6.3 Hz, 2H), 1.59 (s, 12H), 1.29 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ ppm: 171.10, 170.11, 159.59, 141.63, 140.89, 140.11, 128.92, 125.37, 122.35, 122.26, 49.16, 39.76, 32.95, 32.86, 31.93, 29.66, 28.25, 27.39, 24.56, 20.67, 12.38. MS (ESI, m/z) Calcd for [C38H46BrClN2O2-Cl‒]: 641.27372, found: 641.27357. General Procedure for Spectral Measurement. The stock 5.0 mM solution of probe Cy-BT was prepared in DMSO. A typical test solution was prepared by mixing 10.0 µL of probe Cy-BT (5.0 mM) and 990.0 µL of DMSO, appropriate aliquot of each analyte stock solution which was diluted to 5 mL with PBS buffer (20 mM, pH 7.4). The resulting solution was shaken well at 25oC for 10 min before the fluorescence and UV absorption spectra were recorded. Two fluorescence emissions at 627 and 814 nm were recorded upon excitation at 580 and 780 nm, respectively. In Vitro Cytotoxicity. HeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with heat-inactivated 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37oC in a 95% humidity and 5% CO2 environment. After washing with Dulbecco’s phosphate-buffered saline (DPBS) twice, HeLa cells (1×104 cells/ well) were seeded in a flatbottom 96-well plate in 100 µL of culture medium and incubated in 5% CO2 at 37oC for 12 h. The cells were treated with various concentration probe CyBT of 0.0, 2.0, 5.0, 10.0, and 20.0 µM, respectively, for 24 h. The cytotoxic effect of probe Cy-BT was determined by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assays. MTT solution (5.0 mg/mL, PBS) was then added into each well (10 µMK/well, 0.5 mg/mL) and the residual MTT solution was removed after 4 h, and then 100 µM of DMSO was added to each well to dissolve the formazan crystals. After shaking for 10 min, the absorbance values of the wells were recorded using a microplate reader. The cytotoxic effect (VR) of probe Cy-BT was assessed using the following equation: VR=A/A0×100%. The assays were performed eight replicates. And the statistic mean and standard derivation were utilized to estimate the cell viability. Confocal Microscopy Imaging. HeLa cells were seeded in 35 mm glass dishes at a density of 3×105 cells per dish in culture media. In a control experiment, after overnight culture, HeLa cells were incubated only with 10 µM probe Cy-BT at 37 oC for 10 min. In another group experiment, HeLa cells were firstly incubated with 10 µM probe Cy-BT for 10 min and then with 50 µM hydrazine for another 10 min. Fluorescence imaging (600-650 nm) was measured with excitation at 580 nm after the cells were washed with PBS twice. Animal Models and in Vivo Imaging. 4-week-old Balb/c mice were obtained from Laboratory Animal Center. All animal experiments were performed in accordance with the guidelines issued by The Ethical Committee. Two sets of experiments were performed. In a control experiment, B/c mice were given a skin-pop injection of Cy-BT (50 µL, 20 µM) into the peritoneal cavity. In other set of experiment, mice was given an injection of Cy-BT (50 µL, 20 µM) into the peritoneal cavity and then followed by injection of 50 µL of 100 µM


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hydrazine at the same region. Fluorescence images were taken at different time points (0 min, 3 min, 6 min, and 10 min) to observe the fluorescence intensity change process (from 600650 nm) by using IVIS spectrum imaging system with excitation at 580 nm. RESULTS AND DISCUSSION Synthesis of Probe Cy-BT. At present, development of specific fluorescent probes for hydrazine is mainly based on an intramolecular charge transfer process induced by the reaction of between the probes and analytes. However, the twoemission NIR probes reported for hydrazine more or less possess drawbacks limiting their further in biological systems. Thus, we focus on the design of novel two-emission NIR probes suitable for biological samples. Our strategy is that properties of probes, such as selectivity, emission wavelength and working pH, can be tuned by modification of functional groups. As shown in Scheme S1, Cy-BT was readily prepared through one-step reaction of Cy-KT with 4-bromo-butyric acid in the presence of DMAP and EDC·HCl in mild yield. Cy-KT was obtained following a previously reported procedure.49 All compounds were fully characterized by 1H NMR, 13 C NMR, and HRMS (ESI, Figures S1-S3).

Figure 1. Absorption spectral changes (a) and fluorescence spectral changes (b) of probe Cy-BT (10 µM) in the absence and presence of hydrazine (20 equiv.) in DMSO-H2O (1:4, v/v, PBS 20 mM, pH 7.4) (λem/ex = 627/580 nm and λem/ex = 814/780 nm). Inset showed the color change of probe with or without hydrazine in natural light. UV-vis Absorption and Fluorescence Spectra. With this probe in hand, we first evaluated absorption spectral properties of Cy-BT in the absence or presence of hydrazine in DMSOH2O (v/v, 1:4, 20 mM PBS buffer; pH 7.4). As shown in Figure 1a, the free Cy-BT in DMSO-H2O exhibited a strong absorption band at 782 nm. Once 20 equiv. of hydrazine was added, the band at 782 nm almost disappeared and consequently a new band at 547 nm was formed with a large blueshift of 235 nm. Addition of 20 equiv. of hydrazine to Cy-BT led to a fluorescent enhancement at 627 nm and an obvious decrease at 814 nm upon excitation at 580 nm and 780 nm, respectively (Figure 1b and its inset). Simultaneously, the color of Cy-BT was changed from blue-green to red under natural light (Inset of Figure 1a). This remarkable color change indicates probe Cy-BT can sensitively detect hydrazine by the naked eye without using any other instrumental technique. The fluorescence titration experiments of Cy-BT (10 µM) with hydrazine at different concentration in DMSO-H2O (v/v, 1:4, 20 mM HEPES buffer; pH 7.4) were performed to fully assess the viability of Cy-BT as a fluorescent probe for hydrazine. As shown in Figure 2a, the free Cy-BT solution showed two weak fluorescence emissions at 627 nm and 814 nm upon excitation at 580 nm and 780 nm, respectively. Its fluores-

cence quantum yield was evaluated to be 0.024 excited at 550 nm with rhodamine B as a reference.45, 50 Addition of hydrazine led to a strong fluorescent enhancement at 627 nm excited at 580 nm with an obvious fluorescence decrease at around 814 nm excited at 780 nm, implying that Cy-BT can quantitatively detect hydrazine in dual mode. Even 1 equiv. of hydrazine could induce a 100% fluorescence increase at 627 nm. A maximal fluorescence plateau (up to a 23-fold enhancement at 627 nm) was observed with addition of 20 equiv. of hydrazine. This strong fluorescence enhancement was mainly attributed to the formation of Cy-KT due to the leaving of protected 4bromobutyryl moiety of Cy-BT. The quantum yield of Cy-KT with emission at 627 nm was found to be 0.40, a ca. 17-fold enhancement compared to that of Cy-BT. The absorption changes of the probe in the presence of hydrazine are in good agreement with the fluorescence turn-on response.

Figure 2. a) Fluorescence spectral changes at 627 nm and 814 nm of Cy-BT (10 µM) upon addition of hydrazine (0-20 equiv.) excited at 580 nm and 780 nm, respectively. (λem/ex = 627/580 nm and λem/ex = 814/780 nm). Each spectrum was recorded at 17 min after addition of hydrazine. Inset show fluorescence response of Cy-BT at 814 nm excited at 780 nm upon addition of hydrazine (0-20 equiv.) b) Fluorescence ratio I627/I814 changes of Cy-BT as a function of hydrazine concentrations. c) Color change of Cy-BT (10 µM) upon addition of hydrazine at different concentration (0, 30, 60, 90, 120, 160, 200 µM). Photos were taken under natural light. Furthermore, as shown in Figure 2a, the two distinctly separated fluorescence emission intensities at 627 nm and 814 nm of Cy-BT have been found to be linearly proportional to hydrazine concentration ranging from 0-75 µM with R2 as 0.994. This indicates that Cy-BT owns high detection ability of hydrazine in a wide concentration range. Based on the linearity of the fluorescence titration experiment, the detection limit of the probe for hydrazine was determined to be 0.38 ppb (S/N = 3) at pH 7.4 in DMSO-H2O (1:4, v/v, PBS, 20 mM), which is sufficiently lower than the permissible EPA threshold of 10 ppb and demonstrates the probe has a higher sensitivity toward hydrazine than those reported in literature.51-54 As one of the most important fundamental parameters for reaction-based probes, as shown in Figure S4, response time of Cy-BT (10 µM) when reacted with hydrazine at different concentration (0, 40, 160 µM) was further examined in DMSO-H2O (1:4, v/v, PBS 20 mM). The addition of 10 equiv. of hydrazine led to a remarkable enhancement of fluorescence intensity at 627 nm and various concentration of hydrazine



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exhibited a fluorescent maximum almost after 17 min. Thus, this kinetic analysis showed that response time of Cy-BT is independent of the hydrazine concentration. According to that, a pseudo-first-order rate constant (k) for hydrazine calculated to be 0.147 M-1min-1 (Figure S5). All those data indicate that Cy-BT can be suitable for fast detection of hydrazine.

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Selectivity Studies. To estimate the selectivity, the fluorescence response of Cy-BT toward different analytes was performed in 20% DMSO aqueous buffer solution (PBS, 20.0 mM, pH 7.4). As shown in Figure 3a and 3b, 20.0 equiv. of various interfering anions (N3‒, PO43‒, HSO3‒, NO3‒, S2‒, HSO4‒, ClO‒, H2O2, t-BuOO‒, Cl‒, Br‒, I‒, CO32‒, SO42‒, SO32‒, AcO‒) and cations (Na+, K+, Ca2+, Cu2+, Zn2+, Mg2+, Mn2+, Ni2+, Pb2+, Fe3+, Fe2+, Cd2+, Al3+, Ag+) into the solution of CyBT did not induce any significant variations in the fluorescence spectra. Additionally, biothiols (Cys, Hcy, GSH) and common amines (methylamine, aniline, EDA, TEA, ammonia, hydroxylamine, ethanolamine) also brought about no changes of fluorescence intensity (Figure 3c). However, the addition of 20 equiv. of hydrazine led to a strong fluorescence enhancement at 627 nm excited at 580 nm with an obvious fluorescence decrease at around 814 nm. These competitive experiments demonstrated that the probe Cy-BT has prominent specificity of hydrazine over other interfering analytes under physiological conditions. Scheme 2. The proposed mechanism of two-emission NIR fluorescent probe Cy-BT toward hydrazine.

Figure 3. Fluorescence responses at 627 nm of Cy-BT (10 µM) to 20 equiv. of a) anions (1 Blank, 2 N3‒, 3 PO43‒, 4 HSO3‒, 5 NO3‒, 6 S2‒, 7 HSO4‒, 8 ClO‒, 9 H2O2, 10 t-BuOO‒, 11 Cl‒, 12 Br‒, 13 I‒, 14 CO32‒, 15 SO42‒, 16 SO32‒, 17 AcO‒), b) cations (1 Blank, 2 Na+, 3 K+, 4 Ca2+, 5 Cu2+, 6 Zn2+, 7 Mg2+, 8 Mn2+, 9 Ni2+, 10 Pb2+, 11 Fe3+, 12 Fe2+, 13 Cd2+, 14 Al3+, 15 Ag+) and c) amino compounds (1 Blank, 2 Cys, 3 Hcy, 4 GSH, 5 methylamine, 6 aniline, 7 EDA, 8 TEA, 9 ammonia, 10 hydroxylamine, 11 ethanolamine) upon excitation at 580 nm.

The Proposed Mechanism of Probe in Sensing Hydrazine. It is well-known that the selectivity, sensitivity, recognition mechanism, fluorescence emission intensity and wavelength, as well as working conditions, of fluorescent probes can be extremely tuned by difference or even slight modification of recognition groups or fluorescent groups. Based on this thought, we expect that grafting the 4-bromobutyrate group on Cy-KT could produce a new probe and make it a fast detection of hydrazine with two distinctly separated emission peaks under physiological conditions. A plausible response mechanism of Cy-BT to hydrazine is shown in Scheme 2. In the presence of hydrazine, bromide from the 4-bromobutyrate moiety anchored on Cy-KT was firstly substituted by hydrazine. Subsequently, nitrogen atom from hydrazine attacked carboxyl group and finally formed a cyclic compound and released the fluorescent dye Cy-KT. This hydrazine-triggered substitution-cyclisation-elimination reaction activates the intramolecular charge transfer process of Cy-KT, which emits red fluorescence at 627 nm excited at 580 nm. Mass spectra provided a direct evidence that a characteristic peak at m/z = 641.27357 corresponding to the species ([Cy-BT-Cl‒]: 641.27372) was obtained (Figure S3). In contrast, addition of hydrazine into the Cy-BT system resulted in a new peak at


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m/z = 493.5 corresponding to the species [Cy-KT+H]+ besides the probe peak at 643.4 which is assigned to the bromoisotope peak of the species ([Cy-BT-Cl‒]: 641.27357) (Figures S3, S6 and S7). In order to further confirm this proposed mechanism, time-dependent HRMS experiments were performed with an extremely diluted solution of Cy-BT in methanol once a drop of 80% hydrazine aqueous solution was added. Fortunately, as shown in Figures S8 and S9, besides the peak at 493.32089 of the species ([Cy-KT+H]+: 493.32189), we found a small peak at 593.38472 which was reasonably attributed to the hydrazine-substituted intermediate ([Cy-IM]+: 593.38500). The magnitude of this small peak also suggested the fast hydrazine-induced transformation from Cy-BT to CyKT. Another HRMS spectrum was taken after Cy-BT reacted with hydrazine for 15 minutes. As expected in Figure S10, the spectra showed a peak at 101.07104, which was definitely assigned to the cyclic byproduct ([BP+H]+: 101.07149). Additionally, we carried out 1H NMR titration experiments of CyBT in DMSO-d6 upon addition of 10 equiv. of aqueous hydrazine to further confirm the proposed mechanism. As showed in Figure S11a, the aromatic proton signal of H1 at 9.04 ppm was observed which is extremely affected by its neighboring cationic quaternary ammonium salt of the ethylindole group of Cy-BT. The leaving of the bromobutylate group induced by hydrazine led to that the two pairs of proton signals of H9 at 7.60 ppm and H8 at 6.24 ppm in Cy-BT shifted downfield to 7.91 ppm and upfield to 5.46 ppm, respectively (Figure S11b). Other remarkable changes of two pairs of proton upfield shifts of H5 from 4.22 ppm to 3.78 ppm and H10 from 2.67 ppm to 2.53 ppm are definitely consistent with the vanishing of quaternary ammonium cation of ethylindole moiety (Figure S11c). The disappearance of chemical shift of H14 at 3.86 ppm, H12 at 3.07 ppm and H13 at 2.20 ppm of the bromobutyrate group also accounted for the structural transformation from Cy-BT to Cy-KT. Thus, all those mass spectral and NMR titration analysis fully supports the proposed recognition mechanism of elimination of bromobutyrate protecting group of Cy-BT induced by hydrazine. pH Stability Studies. A suitable working pH range is crucial to investigate the utility of probe Cy-BT in biology. So the pH effect on fluorescence response of probe Cy-BT to hydrazine was examined at different pH values in 25% DMSO aqueous solution (Figure S12). It was found that probe Cy-BT did not show any obvious fluorescence emission change when pH < 8 but the solution emits strong NIR fluorescence in strong basic environment. This phenomenon indicated that the probe was probably hydrolyzed and a fluorescent dye Cy-KT showing red fluorescence was released. Furthermore, the solution of probe Cy-BT with hydrazine showed a remarkable fluorescence increase at 627 nm when pH > 5 and did not show obvious attenuation in this pH range. This fluorescence enhancement was mainly attributed to the release of Cy-KT by the hydrazine-triggered substitution-cyclisation-elimination reaction. Thus, Cy-BT and Cy-KT were not sensitive in the wide pH range of 5-8, which indicates probe Cy-BT is suitable for detection of hydrazine in biological samples under physiological conditions.

Figure 4. Confocal fluorescence images of HeLa cells treated with Cy-BT (20 µM) with or without of hydrazine. a) Image of HeLa cells. b) Bright field image of (a). c) Merging of (a) and (b). d) Image of HeLa cells with Cy-BT (20 µM). e) Bright field image of (d). f) Merging of (d) and (e). g) Image of HeLa cells with Cy-BT (20 µM) and hydrazine (100 µM). h) Bright field image of (g). i) Merging of (g) and (h). Detection of hydrazine in Living Cells. To extend further application of probe Cy-BT in biological system, the cytotoxicity experiments in living cells were carried out by MTT assay using standard cell viability protocols. HeLa cells were incubated with incubated with probe Cy-BT at different concentrations 0, 5, 10, 20 µM for 24 hours. As shown in Figure S13, HeLa cells did not show obvious influence and the cell viability was still more than 90% even under the incubation of 20 µM probe for as long as 24 h. These results suggested that probe Cy-BT has low cytotoxicity and good biocompatibility to the cultured cells and can be used to image hydrazine in living cells.

Figure 5. Representative fluorescence images (pseudocolor) of the mice. a) The mice were only given a skin-pop injection of 50 µL of 20 µM Cy-BT and incubated for 10 min as the negative control. b) The mice were injected with 50 µL of CyBT (20 µM) and incubated for 10 min, followed with an injection of 50 µL of hydrazine (100 µM) at the same region and incubation of another 10 min. According to the above good cytotoxicity and biocompatibility of Cy-BT, we next estimated the practical utility of this probe to visualize hydrazine in living cells with a confocal



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microscope Zeiss LSM710. As shown in Figure 4, no any intracellular fluorescence in the red channel was detected under excitation at 580 nm after HeLa cells were cultured only with 10 µM probe Cy-BT in DMEM for 30 min at 37oC. However, in the control test, the outstanding red fluorescence emission was observed when HeLa cells were treated with 10 µM of probe Cy-BT for 30 min and then further with 50 µM hydrazine for another 30 min. Bright-field images show that the CyBT-pretreated cells retained a good morphology, further confirming this probe is permeable to the cultured cell membrane. These excellent results verified that probe Cy-BT is able to detect intracellular hydrazine in living cells with low cytotoxicity and satisfactory biocompatibility.

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at 814 nm in aqueous solution under physiological conditions. The characteristic of un-symmetric emission intensities can significantly amplify signals of low concentration hydrazine. The probe can selectively respond to hydrazine in minutes over other interfering analytes with a low detection limit of 0.38 ppb. Compared with the few ratiometric NIR probes reported, Cy-BT can work well in aqueous solution containing small amount of organic solvent at physiological pH. Addition of hydrazine led to an obvious change from blue-green to red suitable for the naked-eye detection. Moreover, the probe has been successfully used to bioimage hydrazine in living HeLa cells and mice.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

Figure 6. Representative fluorescence images (pseudocolor) of the mice that were pretreated with 50 µL of Cy-BT (20 µM) and incubated for 10 min were given an injection of 50 µL of hydrazine (100 µM) after 10 min of incubation. Images were taken after incubation of hydrazine for 0, 3, 6, 10 min.

NMR spectra of the compounds, MS and chromatograms of the reaction systems, supplementary fluorescent spectra, cytotoxicity assay (PDF)

In Vivo Imaging in Mice. The excellent hydrazine-sensing performance in the living cells inspired us to further test whether the probe bioimage hydrazine in living animals. There were two groups of mice in this experiment. In the first group the shaved Balb/c mice was only given a skin-pop injection of 50 µL of 20 µM Cy-BT into the peritoneal cavity as the negative control. In the second group, the mice were intraperitoneally injected with 50 µL of Cy-BT (20 µM) and incubated for 10 min, followed with an injection of 50 µL of hydrazine (100 µM) at the same region. After 10 min of incubation, the mice were imaged with an IVIS spectral imaging system. As shown in Figure 5, there was an obvious fluorescence signal detected when mice was treated with probe Cy-BT in the negative control group, which indicates that the probe is stable in the biological system. However, the mouse image showed strong fluorescence enhancement when the mice were treated intraperitoneally with 50 µL of 20 µM Cy-BT followed by 50 µL of 100 µM hydrazine, which suggests that the strong fluorescence signals could be rationally attributed to hydrazinolysis induced by the added hydrazine. Subsequently, we performed time-dependent animal experiments (Figure 5). The fluorescent images were taken at different time intervals individually (0, 3, 6, 10 min) once the mice were given an intraperitoneal injection of 50 µL of hydrazine after the pretreation of 50 µL of Cy-BT (10 µM) for 10 min. As observed in Figure 6, the gradual fluorescence intensity changes were observed at different time after the hydrazine injection. The results further confirm that probe Cy-BT can be used to visualize low concentration hydrazine in the living mice.

Corresponding Author

AUTHOR INFORMATION *E-mail: [email protected]

ACKNOWLEDGMENT We thank for the supporting from Natural Science Foundation of China (grant No. 21101074), Shandong Provincial Natural Science Foundation of China (grant No. ZR2013BQ009 and ZR2016BL17), the Doctor’s Foundation of University of Jinan (Grant No. XBS1320).

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CONCLUSION In summary, we have developed a novel two-emission colorimetric NIR fluorescent probe for hydrazine based on ketocyanine anchored with 4-bromobutyrate group. The probe exhibited fast ratiometric responses toward hydrazine with a remarkable enhancement at 627 nm and an obvious decrease

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