Highly Sensitive and Selective Fluorescent Probes for the Detection of

Mar 13, 2017 - Developing highly sensitive and selective methods for HOCl/OCl– detection is of significant interest. In this work, two fluorescent p...
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Highly Sensitive and Selective Fluorescent Probes for the Detection of HOCl/OCl− Based on Fluorescein Derivatives Jing Lv, Fang Wang, Tingwen Wei, and Xiaoqiang Chen* State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Developing highly sensitive and selective methods for HOCl/OCl− detection is of significant interest. In this work, two fluorescent probes based on monoand bis-formylated fluorescein, FN-1 and FN-2, were developed. The probes exhibit rapid response and high selectivity to HOCl/OCl− over other reactive oxygen species (ROS)/ reactive nitrogen species (RNS). Furthermore, a good linearity between the fluorescent intensity at 529 nm and the concentration of HOCl/OCl− in the range 0−10 μM were presented. The probes FN-1 and FN-2 showed detection limits as low as 0.21 and 0.23 μM, respectively. The confocal laser scanning micrographs of HeLa cells confirmed cell permeability of the two probes and their abilities to detect HOCl/OCl− in living cells. Compared to compound FN-1, FN-2 has lower background fluorescence and a higher speed of the reaction with HOCl/OCl− which made it a better option for the detection of HOCl/OCl− in aqueous solution.

be introduced to create novel fluorescence sensors for various analytes.46−51 In this work, two novel probes for hypochlorite, FN-1 and FN-2, were designed and synthesized through condensation reaction between the aldehyde group and amine (Scheme 1). The resulting imine bond can be specifically cleaved by HOCl/OCl− among various ROS/RNS, producing remarkable fluorescence enhancement. The confocal laser scanning micrographs of HeLa cells demonstrated cell permeability of the

1. INTRODUCTION Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in a wide variety of biological events such as aging and immunity.1,2 Hypochlorous acid or hypochlorite (HOCl/OCl−) exists in our body as one of the most important reactive oxygen species,3−5 mainly produced by the reaction of chloride ions (Cl−) and hydrogen peroxide (H2O2) under the catalysis of myeloperoxidase (MPO).6−8 Owing to the strong oxidizing effect of HOCl/OCl−, it could defend against the invasion of bacteria and regulate the life cycle of the cell.9−12 However, excessive HOCl/OCl− can lead to tissue damage and diseases such as atherosclerosis, arthritis, cardiovascular disease, lung injury, and neuron degeneration.13−16 Therefore, highly sensitive and selective methods for HOCl/OCl− detection are urgently needed in living systems.17−20 Among the available detecting techniques, more and more attention was paid to fluorescent methods owing to their high sensitivity, simplicity, fast analysis, and nondestructive advantages.21−26 So far a number of fluorescent probes for the detection of HOCl/OCl− have been reported,27−41 but some of the probes require prolonged reaction time, a complicated synthesis procedure, or a high organic solvent ratio, so there is still an urgent need for the development of fast-responding fluorescent probes. It is known that fluorescein has unique advantages among other fluorophores owing to its excellent photophysical properties, such as visible absorption, high fluorescence quantum yield, good photostability, and water solubility.42−44 A number of fluorescein-based fluorescent probes have been developed for the detection of various analytes.45 Recently, we found formylated fluorescein possesses similar fluorescent properties. Moreover, with the existence of the aldehyde group, some sensitive sites can © XXXX American Chemical Society

Scheme 1. Syntheses of Probes FN-1 and FN-2

Received: Revised: Accepted: Published: A

January 26, 2017 March 2, 2017 March 13, 2017 March 13, 2017 DOI: 10.1021/acs.iecr.7b00381 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research two probes and their abilities to detect intracellular HOCl/OCl− in living cells.

obtained by thermolysis of AAPH (10 mM) in air-saturated aqueous solution at 300 K; 1O2 was generated by the reaction of H2O2 (10 mM) with NaOCl (5 mM); peroxynitrite (ONOO−) was provided by SIN-1 (10 mM); nitric oxide (NO) derived from the solution of S-nitroso-N-acetyl-DL-penicillamine. Test solutions were prepared by placing 30 μL of the probe stock solution into a 3.5 mL test tube, diluting the solution to 3 mL with PBS buffer (10 mM, pH 7.4). Then different analytes of corresponding concentrations were added. All UV−vis absorption and fluorescence spectra were measured at room temperature. For fluorescent study, the samples were excited at 490 nm and the fluorescence emission ranged from 495 to 650 nm, and the slit widths were 5 nm/5 nm for FN-1 and 5 nm/10 nm for FN-2, respectively. 2.4. Detection limit. The fluorescence emission spectra of FN-1 (10 μM) and FN-2 (10 μM) were measured 11 times, and the standard deviations of blank measurement were achieved. After different equivalents of HOCl/OCl− (0−1.0 equiv) were added to the probe solution, the fluorescence intensity was recorded after 5 min. Then, the fluorescence intensity at 529 nm was plotted as the concentration of HOCl/OCl−. The detection limit was calculated by using

2. EXPERIMENTAL SECTION 2.1. Materials and Instruments. Unless otherwise noted, all reagents and solvents were purchased from commercial suppliers and used without any purification. Deionized water was used throughout all experiments. Column chromatography was performed on silica gel. 1H NMR and 13C NMR spectra were collected on a Bruker 2000. Mass spectra were obtained from a Q-Tof mass spectrometer (Agilent 6530). Fluorescent spectra were measured on an RF-5301/PC spectrofluorophotometer. Absorption spectra were recorded on an α-1860A UV−vis spectrometer. MTT assays were carried out by Multiskan Go (51119200-VAN). The cell imaging experiments were carried out using a confocal scanning microscope (Leica, TCS sp5 II). 2.2. Syntheses of Probes. 2.2.1. Synthesis of Probe FN-1. The synthesis routine is shown in Scheme 1. Fluoresceinmonoaldehyde (1) was prepared according to the previous report. 51 Compound 1 (180 mg, 0.5 mmol) and 1,8diaminonaphthalene (174 mg, 1.1 mmol) were mixed in 30 mL of ethanol and refluxed for 3 h under N2 atomosphere. After the reaction, the solvent was evaporated under vacuum and the obtained precipitation was purified by silica column chromatography using CH2Cl2 to give FN-1 as a deep yellow solid (175 mg, yield 97%). 1H NMR (DMSO, 400 MHz), δ (ppm): 10.12 (s, 1H), 8.03(d, 1H, J = 7.5 Hz), 7.84 (t, 1H, J = 7.2 Hz), 7.75 (t, 1H, J = 7.4 Hz), 7.29 (d, 1H, J = 7.6 Hz), 7.20 (t, 2H, J = 7.8 Hz), 7.11 (d, 2H, J = 8.2 Hz), 6.72 (d, 2H, J = 2.0 Hz), 6.65(s, 1H), 6.57 (t, 5H, J = 8.6 Hz). 13C NMR (DMSO, 100 MHz), δ (ppm): 169.13, 159.98, 152.80, 152.14, 150.20, 143.00, 142.89, 136.18, 134.78, 130.69, 129.76, 129.35, 126.72, 125.28, 124.30, 117.50, 117.31, 113.61, 113.32, 111.83, 110.50, 109.82, 106.76, 106.66, 103.05, 83.570, 60.35. HR-MS (ESI) m/z calcd for C31H20O5N2 [M − H] + : 499.1372, found: 499.1276. (See the Supporting Information (SI), Figures S1−S3.) 2.2.2. Synthesis of Probe FN-2. The synthesis routine is shown in Scheme 1. Fluorescein-bisaldehyde (2) was prepared according to the previous report.52 Compound 2 (194 mg, 0.5 mmol) and 1,8-diaminonaphthalene (174 mg, 1.1 mmol) were mixed in 30 mL of ethanol and refluxed for 3 h under N2 atomosphere. After the reaction, the solvent was evaporated under vacuum and the obtained precipitation was purified by silica column chromatography using CH2Cl2 to give FN-2 as a faint yellow solid (167 mg, yield 86%). 1H NMR (DMSO, 400 MHz), δ (ppm): 10.14 (s, 1H), 8.07 (d, 1H, J = 7.6 Hz), 7.82 (t, 1H, J = 7.5 Hz), 7.79 (t, 1H, J = 7.5 Hz), 7.28 (t, 2H, J = 7.0 Hz), 7.23 (t, 3H, J = 8.4 Hz), 7.15 (d, 3H, J = 8.4 Hz), 6.91 (s, 1H), 6.88 (s, 1H), 6.79 (s, 1H), 6.77 (d, 3H, J = 2.8 Hz), 6.73 (s, 2H), 6.63 (t, 4H, J = 7.6 Hz), 6.24 (d, 2H, J = 7.2 Hz), 6.12 (s, 1H). 13C NMR (DMSO, 100 MHz), δ (ppm): 168.89, 167.80, 160.20, 151.54, 150.41, 142.03, 141.57, 136.25, 134.22, 133.36, 131.19, 130.98, 129.74, 129.06, 127.11, 126.90, 126.51, 125.68, 124.30, 117.27, 113.92, 113.29, 112.40, 110.93, 107.62, 107.31, 83.73, 61.14. HR-MS (ESI) m/z calcd for C42H28O5N4 [M − H]+: 667.2060, found: 667.1962. (See the SI, Figures S4−S6.) 2.3. Fluorescence and Absorbance Measurements. The probes FN-1 and FN-2 (1 mM) were dissolved in DMF and maintained at room temperature. Hypochlorite anion (HOCl/ OCl−) was provided by NaOCl (10 mM); hydroxyl radical (•OH) was generated by Fenton reaction (Fe2+/H2O2 = 10 mM/ 10 mM); superoxide anion (O2−) derived from dissolved KO2 (10 mM) in DMSO solution; alkyl peroxyl radical (ROO•) was

detection limit = 3σ /k

where σ is the standard deviation of blank sample; k is the slope of the fluorescence intensity (I529 nm) versus the concentration of HOCl/OCl−. 2.5. Detection of Fluorescence Quantum Yield. Fluorescence quantum yield of the two probes with or without HOCl/OCl− were measured by using fluorescein in 0.1 N NaOH(aq) (ϕS = 0.925) as a standard and calculated with the following expression:53 ϕf =

n f 2 ASDf ϕ nS2 A f DS S

(A ≤ 0.05)

where A represents the absorbance, n represents the refractive index of the solution, and D represents the corrected fluorescence emission spectral integral area (integrated from 450 to 650 nm). Excitation was chosen at 498 nm. 2.6. Mechanism Research. For a better understanding of the reaction mechanism, liquid chromatography−mass spectrometric (LC−MS) analyses were carried out for the solution of the probes FN-1 and FN-2 with 10 equiv of HOCl/OCl− in H2O−CH3CN solution (v/v = 99/1). Thin-layer chromatography (TLC) of mixtures and probes was checked by using CH2Cl2−CH3OH (v/v = 20/1) as eluent. 2.7. MTT Assays. The effects of probe FN-1 and FN-2 on cell viability were determined using the MTT assay. HeLa cells were seeded in 96 well culture plates at density of 2 × 104 cells per well and incubated overnight. Then the probe was added to the wells to achieve final concentrations (5, 10, 15, 20 μM). Control wells were prepared by addition of culture medium. Wells containing culture medium without cells were used as blanks. At the end of incubation, 20 μL of MTT (5.0 mg/mL) was added into each well and incubation continued for another 4 h. Then, the supernatant was removed and 150 μL of DMSO was added to each well for dissolving the MTT formazan. For each independent experiment, the assays were performed in six replicates. The optical density (OD) of formazan solutions produced was recorded on a microplate spectrophotometer at 490 nm. The cell viabilities were presented as the fold over the B

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Figure 1. Fluorescence spectral change of probes FN-1 (a) and FN-2 (b) in the presence of 10 equiv of HOCl/OCl− (excitation = 490 nm, reaction time = 5 min). Fluorescence intensity at 529 nm over time of FN-1 (c) and FN-2 (d) in the presence of 10 equiv of HOCl/OCl− in 10 mM PBS buffer. Data were collected every minute after HOCl/OCl− was added.

Figure 2. Fluorescence changes of probe FN-1 (a) and FN-2 (c) in response to various ROS/RNS (100 μM) including HOCl/OCl−, •OH, O2•−, ROO•, 1O2, ONOO−, and NO in PBS solution (pH 7.4, 10 mM, containing 1% DMF), and absorbance changes of FN-1 (b) and FN-2 (d) in response to various ROS/RNS (100 μM).

2.8. Cell Culture and Fluorescence Imaging. HeLa cells were purchased from Nanjing Cobioer Biosciences Co., Ltd., incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplement with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with

control group and were calculated according to the following formula: cell viability (%) =

ODsample − ODblank ODcontrol − ODblank

× 100 C

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Figure 3. Fluorescence and absportion spectra changes of probe FN-1 (a and b) and FN-2 (c and d) in response to different concentrations of HOCl/ OCl− (0−100 μM) in PBS solution (pH 7.4, 10 mM, containing 1% DMF). Excitation = 490 nm.

methods.51,52 The syntheses of the probes FN-1 and FN-2 were obtained through condensation reaction between mono- or bisformylated fluoroesceins and 1,8-diaminonaphthalene in ethanol (Scheme 1). Their structures were confirmed by 1H NMR, 13C NMR, and high-resolution MS (HR-MS) spectra (SI, Figures S1−S6). 3.2. Optical Responses of Probes FN-1 and FN-2 to HOCl/OCl−. We first investigated the sensing abilities of FN-1 and FN-2 toward HOCl/OCl− in PBS solution. As shown in Figure 1a,b, the initial solutions containing probes only exhibited very weak fluorescence (ϕFN‑1 = 0.008, ϕFN‑2 = 0.002). After 10 equiv of HOCl/OCl− was added to the PBS solutions containing probes (pH 7.4, 10 mM, containing 1% DMF), the fluorescent intensity increased sharply (ϕFN‑1 = 0.363, ϕFN‑2 = 0.285). The responses are so rapid in that the fluorescence of probes with HOCl/OCl− reached plateaus in 5 min (Figure 1c,d). We further studied the selectivity of the probes toward HOCl/ OCl− among various ROS/RNS; the emission spectra of the compound FN-1 (10 μM) in the presence of different ROS/RNS were recorded. As seen from Figure 2a, only the addition of HOCl/OCl− lead to the increase of fluorescent intensity, while other analytes elicited no changes in the fluorescence spectra. Besides a remarkable change of the absorbance detected after the addition of HOCl/OCl−, other analytes did not cause much change. The same phenomenon occurred with FN-2 (Figure 2b). The results indicated both FN-1 and FN-2 can be used for hypochlorite detection. Moreover, once HOCl/OCl− was added, the color of the probe solution of FN-1 turned to colorless from yellow, while FN-2 turned to light brown and strong fluorescence signals of both probe solutions were observed under a 365 nm UV lamp (SI, Figures S7 and S8). These results indicate that the probes FN-1 and FN-2 have excellent selectivity toward HOCl/ OCl− and can be used for the detection of HOCl/OCl− by the naked eye. Further, we studied the fluorescent response of probes FN-1 and FN-2 to HOCl/OCl−. As we can see from Figure 3a, the

5% CO2 in appropriate humidity. Cells were pretransferred to culture dishes and then incubated for 20 h. Three groups were studied as follows: (I) Cells were incubated with the probe (20 μM) for 10 min. (II) HeLa cells were pretreated with probe (20 μM) for 10 min and then exposed to NaOCl (150 μM) for another 10 min. (III) HeLa cells were preincubated with NaCl (250 μM) and the probe (10 μM) in MPO (1.5 U/100 mL) for 20 min and then incubated with H2O2 (10 μM) for 10 min. Cell imaging was carried out after washing the cells with PBS buffer (pH 7.4). All of the cell imagings were collected on a confocal scanning microscope.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Probes. Mono- and bis-formylated fluoroesceins 1 and 2 were synthesized according to previous

Figure 4. Optimized geometry and molecular orbitals of the two probes. D

DOI: 10.1021/acs.iecr.7b00381 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 5. (a) HPLC−MS of compound FN-1 after reaction with excessive HOCl/OCl−. Calcd for [1 − H]+: 359.0556; found 359.0248. (b) HPLC− MS of compound FN-2 after reaction with excessive HOCl/OCl−. Calcd for [2 + H+]+: 389.0617; found 389.0642.

Scheme 2. Proposed Mechanism of Probe Sensing HOCl/OCl−

fluorescent intensity of the probes FN-1 and FN-2 were enhanced along with the addition of HOCl/OCl− (0−100 μM) and were steady at about 80 μM. There were good linearity between the fluorescent intensity at 529 nm and the concentration of HOCl/OCl− in the range 0−10 μM (SI, Figure S9). Thus, the detection limits (signal-to-noise ratio S/N = 3) of probes FN-1 and FN-2 were calculated to be about 0.21 and 0.23 μM, which indicated a high sensitivity for HOCl/OCl−. The fluorescence changes of FN-1 and FN-2 for HOCl/OCl− at different pHs showed that the probe FN-2 could effectively detect HOCl/OCl− and has no significant effect on pH in a wide range from 6 to 10. Although the probe FN-1 did not show much change in acid condition, it could also effectively detect HOCl/ OCl− at neutral and weakly basic conditions (SI, Figure S10). 3.3. Proposed Optical Response Mechanism. We speculated that the weak fluorescence of the initial probes should be attributed to photoinduced electron transfer (PET) between fluorescein and 1,8-diaminonaphthalene. To verify the PET effect of the initial probes, we optimized their structures of ground state and calculated the energy of excited state by Gaussian. The calculations were conducted by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations based on the B3LYP/6-31G(d) basis set with Gaussian View5.0 software. As shown in Figure 4, the

highest occupied molecular orbital (HOMO) of the two probes localized on 1,8-diaminonaphthalene, while the lowest unoccupied molecular orbital (LUMO) delocalized on the fluorescein fluorophore which corresponded to electron cloud distribution of the PET mechanism process. In order to explore the reaction mechanism of the presented system, mass analyses were carried out for the solutions containing probes FN-1 and FN-2 with HOCl/OCl−. As shown in Figure 5a, in the mixing solution of individual probe FN-1 with excess HOCl/OCl−, there is a peak at 359.0248; it was ascribed to the product fluorescein-monoaldehyde (1) (calculated for [1 − H+]− = 359.0556), and TLC showed that a green dot appeared at the same Rf level with fluorescein-monoaldehyde (SI, Figure S11). This means HOCl/OCl− would react with FN-1 and generate fluorescein-monoaldehyde which caused a significant enhancement of the fluorescent intensity (Scheme 2). The mass and TLC results of the mixture of FN-2 and HOCl/OCl− also verified the generation of the fluorescein-dialdehyde (Figure 5 and SI, Figure S11). We proposed that HOCl/OCl− would attack the C−N bond and cause the break of the C−N bond, and the following nucleophilic attack by H2O finally interrupted the PET mechanism by breaking donor and acceptor linkage, leading to the enhancement of fluorescence (Scheme 2).50,54,55 E

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observed. In contrast, when cells were pretreated with the probe FN-1 (20 μM) for 10 min and then exposed to HOCl/OCl− (150 μM) for another 10 min, they showed strong green fluorescence. After HeLa cells were preincubated with NaCl (250 μM) and the probe (10 μM) in MPO (1.5 U/100 mL) for 20 min, and then were incubated with H2O2 (10 μM) for 10 min, a green fluorescence was also observed, which means that MPO is able to stimulate cells to generate endogenous hypochlorite, leading to fluorescence enhancement. The feasibility of the probe FN-2 for the imaging of HOCl/OCl− in living cells was also investigated, and the results were similar to those with the probe FN-1 (Figure 7). These data proved that probes FN-1 and FN-2 were suitable to display a fluorescent enhancement to HOCl/ OCl− in living cells.

4. CONCLUSIONS We have synthesized two fluorescent probes, FN-1 and FN-2, based on mono- and bis-formylated fluorescein. The probes exhibited rapid response and high selectivity to hypochlorite over other ROS/RNS. The mechanism for the detection is based on the generation of fluorescein aldehydes which blocks the effective photoinduced electron transfer (PET) process from the N atoms of 1,8-diaminonaphthalene moiety to the fluorescein moieties. Furthermore, the probes FN-1 and FN-2 can work under physiological pH conditions. Fluorescent imaging of HeLa cells also successfully demonstrated the compounds FN-1 and FN-2 to be efficient probes to detect intracellular HOCl/OCl− in living cells. Compared to FN-1, FN-2 has lower background fluorescence and a higher speed of the reaction with HOCl/ OCl−, so it would be a better option for the detection of HOCl/ OCl−.

Figure 6. Fluorescence images of HeLa cells. (a) Cells incubated with probe FN-1 (20 μM) for 10 min. (b) Subsequent treatment of the cells with NaOCl (150 μM) for 10 min. (c) Cells treated with stimulant MPO (1.5 U/100 mL) for 10 min and further incubated with probe FN-1 for 20 min.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.7b00381. 1 H NMR, 13C NMR, and MS spectra of probes, fluorescent titration assays, photographs in different ROS/RNS under white light and a 365 nm UV lamp, fluorescent responses in various pHs, MTT assays (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 025 83587856. Fax: +86 025 83587856. E-mail: [email protected].

Figure 7. Fluorescence images of HeLa cells. (a) Cells incubated with probe FN-2 (20 μM) for 10 min. (b) Subsequent treatment of the cells with NaOCl (150 μM) for 10 min. (c) Cells treated with stimulant MPO (1.5 U/100 mL) for 10 min and further incubated with probe FN-2 for 20 min.

ORCID

Xiaoqiang Chen: 0000-0003-2493-2067 Notes

The authors declare no competing financial interest.

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3.4. Fluorescence Imaging of Probes for HOCl/OCl in Living Cells. MTT assays were first performed to evaluate the cytotoxicity of FN-1 and FN-2 with their concentrations ranging from 0 to 25 μM. The MTT results indicated that the probes are of low toxicity toward HeLa cells under the experimental conditions (Figure S12). The feasibility of the probe FN-1 for fluorescent imaging of HOCl/OCl− in living cells was further investigated. As shown in Figure 6, HeLa cells were incubated with the probe FN-1 (20 μM) for 10 min; no fluorescence was

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21376117, 21406109), the Jiangsu Natural Science Funds for Distinguished Young Scholars (BK20140043), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (14KJA150005), the Qing Lan Project, and the Project of Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). F

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