Turn-on Fluorescent Probe for Exogenous and Endogenous Imaging

Subscriber access provided by UNIVERSITY OF THE SUNSHINE COAST

Article

A novel turn-on fluorescent probe for exogenous and endogenous imaging of hypochlorous acid in living cells and quantitative application in flow cytometry Zixuan Zhan, Rui Liu, Li Chai, Qiuyan Li, Kexin Zhang, and Yi Lv Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02613 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A novel turn-on fluorescent probe for exogenous and endogenous imaging of hypochlorous acid in living cells and quantitative application in flow cytometry Zixuan Zhan, † Rui Liu, † Li Chai, ‡ Qiuyan Li, † Kexin Zhang,† Yi Lv† * †

Key Laboratory of Green Chemistry & Technology, Ministry of Education, College of

Chemistry, Sichuan University, Chengdu, Sichuan 610064, China ‡

Core Facility of West China Hospital, Sichuan university, Chengdu, Sichuan, 610041, China

*Corresponding Author. Email: [email protected]; Tel. & Fax +86-28-8541-2798

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Hypochlorous acid (HClO) acts as a dominant microbicidal mediator in the natural immune system and the excess production of hypochlorites is related to a series of diseases. Thus, it is vitally important and necessary to develop a highly sensitive and selective method for HClO detection in living systems and most of fluorescent probes are mainly focused on cells imaging. Besides, accurate HClO quantitative information about individual cells in a large cell population is extremely important for understanding inflammation and cellular apoptosis as well. In our work, a turn-on fluorescent probe has been synthesized, which can selectively and sensitively detect HClO with fast response time. The probe is almost nonfluorescent possibly due to both the spirolactam form of fluorescein and unbridged C=N bonds which can undergo non-radiative decay process in the excited state. Upon the addition of ClO-, the probe was oxidized to ring-opened fluorescent form and the fluorescence intensity was greatly enhanced. In live cell experiments, the probe was successfully applied to image exogenous ClO- in Hela cells and endogenous HClO in RAW 264.7 macrophage cells. In particular, the quantitative information of exogenous and endogenous HClO can also be acquired in flow cytometry. Therefore, the probe can not only image exogenous and endogenous HClO but also provide a new and promising platform to quantitative detect HClO in flow cytometry.

2


Page 2 of 25

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION SECTION It is known that reactive oxygen species (ROS) and reactive nitrogen species (RNS) cause oxidative and nitrosative stresses, respectively. These stresses play vital roles in a diversity of biological processes and are also connected to many diseases, including cancer and neurodegenerative disorders.1-4 Among various ROS, hypochlorous acid (HClO) and its conjugate base hypochlorite (ClO-) are extensively employed as bleaching agents in our daily life and also act as defense tools in the immune system.5-7 Endogenous HClO can be produced by the reaction of hydrogen peroxide (H2O2) and chloride ion (Cl-) catalyzed by the heme enzyme myeloperoxidase (MPO), which is mainly located in leukocytes including macrophages, monocytes, and neutrophils.8-11 However, the excess production of hypochlorites from phagocytes can lead to inflammation-associated tissue damage and a series of diseases, such as lung injury, hepatic ischemia-reperfusion injury, neuron degeneration, arthritis, cardiovascular disease and even cancer.12-15 Therefore, the development of a highly sensitive and selective hypochlorite detection and imaging method among H2O2 and other ROS in living systems is vitally important and necessary to unravel the fundamental processes in immune system responses. Toward this end, a number of analytical methods for the detection of ClO- have been developed, including colorimetric, electrochemical, chemiluminescence methods.16-19 In addition, compared with other detection methods, fluorescence imaging methods are generally superior in terms of sensitivity, simplicity, response time, spatial and temporal resolution. These innate advantages can offer applications for not only in vitro assays but also in vivo imaging studies. So far, a number of fluorescent probes for specific detection of ClO− have been reported

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and most of them were developed on the basis of strong oxidation properties of HClO.20-34 In particular, fluorescein and its derivatives-based fluorescent probes for ClO- detection have shown excellent photophysical properties， including good photostability， high fluorescence quantum yields and so on.35-38 Hence, the conjugation of a HClO-recognizing moiety, sensitive to hypochlorite oxidation, with an organic fluorophore, including p-methoxyphenol,39 thiol,40-45 selenide,46-48 dibenzoylhydrazine,49 hydroxamic acid50 and oxim derivatives51-53 have been widely used for probe design. The C=N isomerization as a signaling mechanism have received extensive attention since it was first designed as fluorescent chemosensor.54,55 The strong fluorescence of organic fluorophore containing unbridged C=N bonds is known to undergo non-radiative decay process in the excited state, so those compounds are often nonfluorescent.56 After the unbridged C=N was removed by specific analytes through a chemical reaction, the fluorescence intensity of compounds can be enhanced distinctly. Therefore, based on the C=N isomerization and the oxidization hydrolysis of hydrazide, a sensitive and selective turn on fluorescent probe can be established. The applications of most fluorescent probes are mainly focused on cells imaging. Besides, accurate quantitative information about individual cells in a large cell population is extremely important as well. To meet the demands, flow cytometry, as a powerful and widespread technique, can not only be utilized for the measurement of cell phenotype and function but also provide the accurate quantitative information of single-cell among several millions of cells.57 Thus, it would be pretty attractive to apply flow cytometry to various biological studies and clinical diagnoses. To date, there are a quite number of outstanding fluorescent probes for HClO detection and imaging. However, a lack of sensitive and selective fluorescent probes for

4


Page 4 of 25

Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


quantitative detection of HClO in cells has greatly hampered better understanding the role of HClO in many biological vital processes. In this work, we report a sensitive and selective turn on fluorescent probe with fast response time, for exogenous and endogenous imaging of hypochlorous acid in living cells and quantitative application in flow cytometry.

EXPERIMENTAL SECTION Materials and instrumentations. All reagents were of analytical reagent grade and used without further purification. 1H NMR,

13

C NMR spectra were measured on a Bruker AM400

NMR spectrometer. Proton Chemical shifts of NMR spectra were given in ppm relative to internals reference TMS (1H, 0.00 ppm). ESI-MS and HRMS spectral data were recorded on a Finnigan LCQDECA and a Bruke Daltonics Bio TOF mass spectrometer, respectively. Photoluminescence spectra were performed on a Hitachi F-7000 fluorescence spectrophotometer. Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. Cells imaging was performed with Zeiss inverted fluorescence microscope and flow cytometry experiment was performed in Beckman cytoflex. Fluorescence analysis. Probe was dissolved in dimethylformamide (DMF) for a stock solution (1 mM). Test solution of probe 1 (1×10-5 mol/L) was prepared in DMF-PBS solution (1: 9, v/v, 10 mM PBS, pH 7.4) and small aliquots of each testing species solution were added. The resulting solutions were shaken well at room temperature before recording spectra. Preparation of solutions of cations and anions and ROS and RNS. 1 mmol of inorganic salt (NaCl, KCl, CaCl2·2H2O, CoCl2·6H2O, NiCl2·6H2O, CdCl2,

CuCl2·2H2O, ZnCl2, PbCl2,

MgCl2·6H2O, MnCl2·4H2O, CrCl3·6H2O, FeCl3·6H2O, AlCl3·6H2O, NaF, KBr, KI, Na2S·9H2O,

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 25

Na2SO4, Na2SO3, Na2S2O4, Na2S2O8, NaHSO3, Na2CO3, Na2C2O4, NaH2PO4·2H2O, NaNO2, KSCN, NaHCO3, K2Cr2O7, NaClO3) and ascorbic acid (AA), glutathione (GSH), cysteine (Cys) were dissolved in distilled water (100 mL) to afford 1×10-2 mol/L aqueous solution. The stock solutions were diluted to desired concentrations with water when needed. Hypochlorite was prepared by dilution of commercial NaClO solution in deionized water and assayed using a spectrophotometer using ε292nm = 350 M-1cm-1. Tert-butyl hydroperoxide (TBHP) was prepared by dilution of commercial TBHP solution. Hydrogen peroxide was prepared by dilution of commercial H2O2 solution directly. Nitric oxide (NO) was prepared by treating sulfuric acid solution with sodium nitrite solution and its stock solution was prepared by bubbling NO into deoxygenated deionized water for 30 min. Peroxynitrite stock (ONOO-) was prepared by following literature procedure58 and the concentration of peroxynitrite was estimated by using a spectrophotometer ε302nm = 1670 M-1cm-1. Hydroxy radical was prepared by addition of Fe2+ solution into a solution containing excess H2O2 through Fenton chemistry. Superoxide was generated from KO2 with a saturated solution of KO2 in DMSO. Synthesis of Probe 1. The synthesis of Probe 1 is shown in Scheme S1 in the Supporting Information. The details are described as follows. Synthesis of S1. Compound S1 was synthesized using a modified literature method. Specifically,

A

solution

of

1,10-Phenanthroline-5,6-dione

(0.945

g,

4.5

mmol),

4-Hydroxybenzaldehyde (0.550 g, 4.5 mmol), aniline (0.50 mL, 5.5 mmol) and ammonium acetate (3.48 g, 45 mmol) in glacial acetic acid (20 mL) was refluxed for 24 h under an argon atmosphere, resulting in a colour change from dark-red to yellow. After being cooled to room temperature, the reaction mixture was poured into ice water (400 mL), forming a yellow

6


Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


precipitate. With the addition of ammonium hydroxide, more precipitate formed, the resulting solid was collected by filtration, washed with water and acetone, and dried in vacuum to yield compound S1. Yield: 58%. 1H-NMR (400 MHz; DMSO-d6): δ 9.90 (s, 1 Η), 9.06 (d, J = 2.40 Hz, 1 H), 8.99 (d, J = 8.00 Hz, 1 H), 8.91 (d, J = 2.40 Hz, 1 H), 7.86 (dd, J = 7.60, 4.40 Hz, 1 H), 7.77-7.67 (m, 5 H), 7.42 (dd, J = 8.40, 4.40 Hz, 1 H), 7.42-7.37 (m, 2 H), 7.31 (d, J = 8.40, 1 H), 6.75-6.70 ppm (m, 2H); 13C-NMR (100 MHz; DMSO-d6): δ 158.5, 152.3, 148.3, 147.2, 143.7, 143.5, 137.8, 135.0, 130.6, 130.5, 130.4, 129.7, 129.0, 127.1, 126.3, 123.7, 123.4, 122.4, 120.4, 119.4, 115.1 ppm; HRMS (ESI): calcd for C25H16N4O [M]- m/z =387.1246. Found: m/z = 387.1235. Synthesis of S2. Compound S2 was synthesized using a modified Duff reaction. S1 (0.390 g, 1 mmol) and hexamethylene tetraamine (0.640 g, 4 mmol) were dissolved in 6 mL trifluoroacetic acid and refluxed for three days. The mixture was allowed to cool to room temperature and 30 mL of a 4M HCl solution were added and allowed to stir for 2 hours, during which time the reaction became cloudy, the pH was adjusted to approximately three through the addition of a saturated aqueous solution of NaHCO3, forming a yellow precipitate and the resulting yellow solid was collected by filtration, washed with water, acetone, ether and dried in vacuum

to

yield

compound

S2.

Yield:

54%.

1

H-NMR

(400

MHz;

DMSO-d6):

δ 11.30 (s, 1 Η), 10.24 (s, 1 H), 9.44 (d, J = 8.40 Hz, 1 H), 9.22 (d, J = 4.40 Hz, 1 H), 9.09 (d, J = 4.40 Hz, 1 H), 8.23 (dd, J = 7.20, 4.80 Hz, 1 H), 7.87 (s, 1 H), 7.81-7.74 (m, 6 H), 7.73 (d, J = 8.40 Hz, 1 H), 7.53 (d, J = 8.40 Hz, 1 H), 7.01 (d, J = 8.80 Hz, 1 H);

13

C-NMR (100 MHz;

DMSO-d6): δ 189.6, 161.7, 157.9, 152.0, 146.7, 145.6, 138.2, 136.8, 136.2, 135.1, 134.5, 131.0,

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

130.9, 129.7, 129.2, 128.7, 127.0, 125.4, 124.7, 124.1, 122.2, 120.21, 120.17, 117.5 ppm; HRMS (ESI): calcd for C26H16N4O2 [M+H]+ m/z =417.1351. Found: m/z = 417.1358. Synthesis of S3. Compound S3 was synthesized using a literature method. To a suspension of fluorescein (6.0 g, 18.1 mmol) in ethanol (24 mL) was added excess hydrazinium hydroxide (24 mL, 80% aqueous). The reaction mixture was heated to reflux and allowed to stir for 7 h, after being cooled to room temperature, the reaction mixture was poured into ice water (400 mL) and left to sit for 2 h. The resulting yellow precipitate was filtered from the aqueous suspension and washed with water and cold ethanol. Recrystallisation of the precipitate in ethanol (250 mL) to yield the compound S3. Yield: 50%. 1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 2 H), 7.81-7.77 (m, 1 H), 7.53-7.43 (m, 2 H), 7.03-6.95 (m, 1 H), 6.59 (s, 2 H), 6.46 (d, J = 8.80, 2 H), 6.41 (d, J = 8.40 Hz, 2 H), 4.34 (s, 2 H).

13

C NMR (100 MHz, DMSO-d6) 165.5, 158.2, 152.4, 151.5,

132.6, 129.3, 128.4, 127.9, 123.4, 122.3, 112.0, 109.9, 109.6, 102.3, 64.6 ppm; HRMS (ESI): calcd for C20H14N2O4 [M]- m/z =345.0875. Found: m/z = 345.0862. Synthesis of probe 1. Probe 1 was synthesized using a literature method. A solution of S2 (0.210 g, 0.5 mmol) and S3 (0.175 g, 0.5 mmol) in absolute ethanol (15 mL) was refluxed for 6 h. The solution was cooled, concentrated to 10 mL and allowed to stand at room temperature overnight. The precipitate which appeared next day was filtered and washed 3 times with 10 mL cold ethanol, recrystallisation of the crude product from EtOH to get pure product and dry in vacuum to yield probe 1. Yield: 55%. 1H-NMR (400 MHz; DMSO-d6): δ 10.77 (s, 1 Η), 9.99 (s, 2 H), 9.08 (d, J = 2.80 Hz, 1 H), 9.04 (d, J = 8.00 Hz, 1 H), 8.94 (d, J = 2.80 Hz, 1 H), 8.80 (s, 1 H), 7.96 (d, J = 7.20 Hz, 1 H), 7.90 (dd, J = 7.60, 4.40 Hz, 1 H), 7.75-7.57 (m, 8 H), 7.49 (dd, J = 8.40 Hz, J = 4.00 Hz, 1 H), 7.34 (d, J = 8.40 Hz, 2 H), 7.13 (d, J = 7.60 Hz, 1 H), 6.76 (d, J =

8


Page 8 of 25

Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8.40 Hz, 1 H), 6.70 (s, 2 H), 6.56-6.47 (m, 4 H); 13C-NMR (100 MHz; DMSO-d6): δ 163.7.6, 158.8, 157.8, 151.9, 151.3, 151.0, 148.3, 147.1, 147.3, 147.1, 143.5, 135.0, 134.3, 132.0, 130.5, 129.2, 128.8, 128.0, 127.9, 127.3, 126.4, 123.9, 123.7, 123.4, 122.6, 121.0, 119.4, 119.1, 116.3, 112.6, 109.6, 109.2, 102.7, 64.9 ppm; HRMS (ESI): calcd for C46H28N6O5 [M]- m/z =743.2043. Found: m/z = 743.2012. Cell culture and imaging. HeLa cells and RAW264.7 cells were cultured in Dulbecco's modified

Eagle

medium

(DMEM)

containing

10%

fetal

bovine

serum

and

1%

penicillin-streptomycin at 37℃ in a 5% CO2/95% air incubator. For detection of exogenous HClO, the cultured HeLa cells in a 6-well plate were washed three times with PBS and then incubated with 20 µM of probe 1 for 1.5 h at 37 °C in a 5% CO2/95% air incubator. After washing three times with PBS, the cells were cultured for another 30 min with different concentration of ClO-. The cells were washed three times carefully with PBS and imaged. For detection of endogenous HClO, RAW264.7 cells in 6-well plate were stimulated by LPS and IFN-γfor 16 h in the culture medium and then PMA and probe 1 for another 2 h. The cells were washed three times carefully with PBS and imaged. Flow Cytometry Experiments. Cells were treated as described in Cell culture and imaging part. Before measurement by flow cytometry, cells were scraped off gently and collected into a clean 2 mL centrifuge tube. Then, cells were spun down (500 rpm, room temperature, 3 min). After discarding the supernatant, 1 mL of warm PBS was added gently to re-suspend the cell pellet. Finally, cells were analyzed on a Beckman flow cytometer equipped with 488 nm Ar laser and fluorescence was collected by PE channel.

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

RESULTS AND DISCUSSION Spectroscopic Response of Probe 1 to ClO-. Fluorescein was chosen as an ideal fluorescence reporting group due to its excellent photophysical properties and the structure of probe 1 was confirmed by 1H NMR,

13

C NMR and ESI-TOF mass spectra (Fig S1). To

investigate the response of probe 1 to ClO-, the fluorescence response behavior of probe 1 towards various concentrations ClO- was measured at room temperature. As shown in Fig 1, probe 1 exhibits very weak fluorescence due to both the spirolactam form of fluorescein and the unbridged C=N bonds which can undergo non-radiative decay process in the excited state. Upon the addition of ClO-, the fluorescence emission of probe 1 at 542 nm was greatly enhanced in the range of 0-100 µM. The reaction mechanism of probe 1 with ClO- was proposed in Scheme 1 and the result was illustrated on the ESI-MS, 1H NMR and in suit FT-IR (Fig S7- S9). To obtain efficient signaling conditions, various experimental conditions toward hypochlorite were examined, including co-solvent, buffer solution, pH and time. Firstly, we tested the signaling behavior of probe 1 toward ClO- in DMF and DMSO with different water percentages (Fig S2 and Fig S3). It was found that compared with the signal enhancement in DMF, the signal in DMSO was much lower due to the scavenging effect of DMSO for hypochlorite.59 probe 1 exhibited highest signal enhancement toward ClO- in 10% DMF-PBS buffer solution at pH 7.4. We were then make a comparison among three different buffer solutions, 10 mM PBS, 10 mM Tris-HCl and 10 mM HEPES, to see whether the enhancement of fluorescence intensity can be affected. Not surprisingly, the fluorescence responses were varied significantly quite different when treated with ClO- in Fig 2a. The fluorescence intensity was greatly enhanced in 10 mM PBS buffer solution, slightly enhanced in 10 mM HEPES and almost remain constant in 10 mM

10


Page 10 of 25

Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Tris-HCl, demonstrating that partial ClO- was consumed by Tris-HCl and HEPES. HEPES and Tris-HCl can be oxidized by ClO- and the oxidative activity of ClO- was almost lost when ClOwas incubated with HEPES or Tris-HCl prior to probe 1 addition. Thus, HEPES and Tris-HCl are inappropriate for ClO- detection and the result is in good accordance with the literature.60,61 Subsequently, the fluorescence dynamics of probe 1 were investigated in 10% DMF-PBS buffer (10 mM, pH 7.4) at room temperature. As shown in Fig. 2b, the reaction of probe 1 with ClO- was fast and the fluorescence intensity was significantly enhanced and remained stable after 1 min, indicating that probe 1 is suitable for monitoring HClO in real-time. Finally, the effect of pH on the reaction was examined carefully. As shown in Fig 3a. Probe 1 showed nearly no fluorescence at pH 5-10 and significant emission enhancement in the presence of ClOover the investigated pH range of 7.4-10, indicating that probe 1 is suitable for imaging of ClOin living cells. In order to evaluate the selectivity of probe 1 towards ClO-, the influence of anions, cations and various other biologically relevant analytes was investigated (Fig S4-S6), including ascorbic acid (AA), glutathione (GSH), cysteine (Cys), tert-butyl hydroperoxide (TBHP), hydrogen peroxide (H2O2), nitric oxide (NO), peroxynitrite anion (ONOO-), hydroxyl radical (•OH), and superoxide (•O2‑). As shown in Fig 3b, there were no observable fluorescence enhancement except with ClO-. It is worth noting that the concentrations of those analytes were much higher than ClO-. Fluorescence Imaging and flow cytometry quantitative detection of exogenous HClO in HeLa Cells. To demonstrate the potential biological applications of probe 1, we utilized it for the detection of intracellular HClO. Firstly, a MTT assay with HeLa cells and RAW264.7

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

macrophages was examined to determine the cytotoxicity of probe 1. The results in Fig 4 showed that the viability of HeLa cells and RAW264.7 macrophages remained above 80% after 4 h and 24 h incubation with different concentrations of probe 1, indicating that probe 1 has low cytotoxicity. Then HeLa cells were incubated with 20 µM probe 1 for 1.5 h, very weak fluorescence was observed (Fig S9). After the treatment of ClO- (200 µM) for 30 min, a stronger green fluorescence was detected. Meanwhile, HeLa cells were also treated with various other biologically relevant analytes. As expected in Fig S9, there were very weak fluorescence from the cells and the result of flow cytometry experiment (Fig S10) was in accordance with imaging experiment. The above result demonstrated that probe 1 has a turn-on fluorescence response and high selectivity to HClO over various other biologically relevant analytes in living cells. To evaluate the potential of visualizing HClO levels in living cells, the probe 1 loaded HeLa cells were used for the imaging of different concentrations of exogenous HClO. HeLa cells were co-incubated with 20 µM probe 1 for 1.5 h. After the cells were incubated with different concentrations of ClO- (0 µM, 50 µM, 100 µM, 200 µM, 400 µM) for another 30 min, fluorescence images were then acquired. As shown in Fig 5. In the absence of ClO-, the fluorescence of cells is weak. In contrast, HeLa cells treated with 50 µM ClO- display obvious fluorescence. Moreover, stronger fluorescence is observed when the cells were treated with a higher concentration of ClO- till 200 µM. The ClO- levels in Hela cells can also be qualitatively acquired by flow cytometry. As can be seen in Fig 6a, the geometric mean of fluorescence intensity was significantly enhanced with the increase of ClO- concentration. The above experiments proved that probe 1 has the ability to visualize and quantitatively detect ClO- in living cells.

12


Page 12 of 25

Page 13 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fluorescence Imaging and flow cytometry quantitative detection of endogenous HClO in RAW264.7 Cells. We then investigated whether the probe was capable of detecting LPS (lipopolysaccharide)-induced endogenous produced HClO in RAW264.7 cells. It is known that endogenous HClO can be produced under the stimulation of LPS, IFN-γ and phorbol myristate acetate (PMA).62 Thus, RAW264.7 macrophages were co-incubated with various concentrations of LPS (0.125 µg/ml, 0.25 µg/ml, 0.5 µg/ml) and IFN-γ (50 ng/ml) for 16 h and then PMA (1 µg/ml) and probe 1 (20 µM) for 2 h. As shown in Fig 6b, the percentage of positive cells in flow cytometry increased gradually with concentration of LPS increasing. 0.5 µg/ml LPS was used for stimulating RAW264.7 cells to produce H2O2. We further examined the applicability of probe 1 for endogenous HClO imaging. As depicted in Fig 7a and 7b, the stimulated RAW264.7 cells displayed strong fluorescence, while almost no fluorescence emission was observed in control group. Moreover, in another two groups, 4-aminobenzoic acid hydrazide (ABH) was added to decrease cellular HClO level through inhibiting the activity of MPO. The fluorescence intensity can also be reduced clearly by N-acetylcysteine (NAC), a powerful ROS scavenger. As shown in Fig 7c and 7d, the fluorescence intensity is suppressed to some extent when RAW264.7 cells were incubated with ABH and NAC, respectively. This result demonstrated that endogenous HClO can be visualized with probe 1 in RAW264.7 cells. With the above encouraging results, we decided to detect endogenous HClO in flow cytometry with probe 1. RAW264.7 macrophages were co-incubated with stimulus and probe 1 in the presence or absence of ABH or NAC. As shown in Fig 8a, only 0.3% positive cells can be acquired in unstimulated group, whereas, in the presence of the stimulus, the percentage of positive cells in flow cytometry can be significantly increased to 34.23% (Fig 8b). In addition,

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the percentage of positive cells was inhibited to 23.34% and 21.66% in the presence of ABH and NAC respectively (Fig 8c and 8d). This result indicated that probe 1 have the ability to quantitatively detect endogenous HClO in RAW264.7 cells by flow cytometry.

CONCLUSIONS In conclusion, we have successfully synthesized a turn-on fluorescent probe for highly selective and sensitive detection of hypochlorous acid with fast response time and the probe can be used for exogenous and endogenous imaging of hypochlorous acid in living cells. Furthermore, probe 1 has shown superior ability for the quantitative detection of exogenous and endogenous HClO in flow cytometry.

ACKNOELEDGEMENTS We thank Dr. Pengchi Deng and Prof. Aiming Sun in Analytical & Testing Center of Sichuan University for technical assistance. This work was supported by the National Natural Science Foundation of China (Nos. 21375089 & 21505095) and Science & Technology Department of Sichuan Province of China (2015JY0272). Supporting Information Available: The Supporting Information is available free of charge on the Internet at http://pubs.acs.org. Chemical structures of compounds, NMR and MS spectra, additional spectral data, cells imaging data and flow cytometry data.

14


Page 14 of 25

Page 15 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Lambeth, J. D. Free. Radical. Biol. Med. 2007, 43, 332-347. (2) Bove, P. F.; Vliet, A. V. D. Free. Radical. Biol. Med. 2006, 41, 515-527. (3) Chen, X.; Wang, F.; Hyun, J. Y.; Wei, T.; Qiang, J.; Ren, X.; Shin, I.; Yoon, J. Chem. Soc. Rev. 2016, 45, 2976-3016. (4) Zhang, R.; Zhao, J.; Han, G.; Liu, Z.; Liu, C.; Zhang, C.; Liu, B.; Jiang, C.; Liu, R.; Zhao, T. J. Am. Chem. Soc. 2016, 138, 3769-3778. (5) Aoki, T.; Munemori, M. Anal. Chem. 1983, 55, 209-212. (6) Hidalgo, E.; Bartolome, R.; Dominguez, C. Chem.-Biol. Interact. 2002, 139, 265-282. (7) Winterbourn, C. C.; Hampton, M. B.; Livesey, J. H.; Kettle, A. J. J. Biol. Chem. 2006, 281, 39860-39869. (8) Pattison, D. I.; Davies, M. J. Biochemistry. 2006, 45, 8152-8162. (9) Harrison, J.; Schultz, J. J. Biol. Chem. 1976, 251, 1371-1374;. (10) Yap, Y. W.; Whiteman, M.; Cheung, N. S. Cellular Signal. 2007, 19, 219-228. (11) Klebanoff, S. J. J. Leukocyte Biol. 2005, 77, 598-625. (12) Hammerschmidt, S.; Büchler, N.; Wahn, H. Chest. 2002, 121, 573-581. (13) Hasegawa, T.; Malle, E.; Farhood, A.; Jaeschke, H. Am. J. Physiol. Gastrointest. Liver Physiol. 2005, 289, 760-767. (14) Steinbeck, M. J.; Nesti, L. J.; Sharkey, P. F.; Parvizi, J. J. Orthop. Res, 2007, 25, 1128-1135. (15) Daugherty, A.; Dunn, J. L.; Rateri, D. L.; Heinecke, J. W. J. Clin. Invest. 1994, 94, 437-444.

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(16) Zhu, B.; Xu, Y.; Liu, W.; Shao, C.; Wu, H.; Jiang, H.; Du, B.; Zhang, X. Sens. Actuators. B. 2014, 191, 473-478. (17) Ordeig, O.; Mas, R.; Gonzalo, J.; Del Campo, F. J.; Muñoz, F. J.; De Haro, C. Electroanalysis. 2010, 17, 1641-1648. (18) Soto, N. O.; Horstkotte, B.; March, J. G.; Alba, P. L. L. D.; Martínez, L. L.; Martín, V. C. Anal. Chim. Acta. 2008, 611, 182-186. (19) Tang, Y.; Su, Y.; Yang, N.; Zhang, L.; Lv, Y. Anal. Chem. 2014, 86, 4528-4535. (20) Yuan, L.; Lin, W.; Song, J.; Yang, Y. Chem. Commun. 2011, 47, 12691-12693. (21) Chen, G. W., Song, F. L., Wang, J. Y., Yang, Z. G., Sun, S. G., Fan, J. L., Qiang, X. X., Wang, X., Dou, B. R., Peng, X. J. Chem. Commun, 2012, 48, 2949-2951. (22) Yuan, L.; Lin, W.; Yang, Y.; Chen, H. J. Am. Chem. Soc. 2012, 134, 1200-1211. (23) Xiao, Y. N.; Zhang. R.; Ye. Z. Q.; Dai, Z. C.; An, H. Y.; Yuan, J. L. Anal. Chem. 2012, 84, 10785−10792 (24) Zhang, Y. R.; Chen, X. P.; JingShao; Zhang, J. Y.; Yuan, Q.; Miao, J. Y.; Zhao, B. X. Chem. Commun. 2014, 50, 14241-14244. (25) Zhu, H.; Fan, J.; Wang, J.; Mu, H.; Peng, X. J. Am. Chem. Soc. 2014, 136, 12820-12823. (26) Reja, S. I.; Bhalla, V.; Sharma, A.; Kaur, G.; Kumar, M. Chem. Commun. 2014, 50, 11911-11914. (27) Hou, J. T.; Li, K.; Yang, J.; Yu, K. K.; Liao, Y. X.; Ran, Y. Z.; Liu, Y. H.; Zhou, X. D.; Yu, X. Q. Chem. Commun. 2015, 51, 6781-6784. (28) Long, L.; Wu, Y.; Wang, L.; Gong, A.; Hu, F.; Zhang, C. Chem. Commun. 2015, 51, 10435-10438.

16


Page 16 of 25

Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(29) Xu, Q.; Heo, C. H.; Kim, G.; Lee, H. W.; Kim, H. M.; Yoon, J. Angew. Chem. Int. Ed. 2015, 54, 4890-4894. (30) Yuan, L.; Wang, L.; Agrawalla, B. K.; Park, S. J.; Zhu, H.; Sivaraman, B.; Peng, J.; Xu, Q. H.; Chang, Y. T. J. Am. Chem. Soc. 2015, 137, 5930-5938. (31) Zhou, J.; Li, L.; Shi, W.; Gao, X.; Li, X.; Ma, H. Chem. Sci. 2015, 6, 4884-4888. (32) Hu, J. J.; Wong, N. K.; Lu, M. Y.; Chen, X.; Ye, S.; Zhao, A. Q.; Gao, P.; Kao, Y. T.; Shen, J.; Yang, D. Chem. Sci. 2016, 7, 2094-2099. (33) Huang, Y.; Zhang, P.; Gao, M.; Zeng, F.; Qin, A.; Wu, S.; Tang, B. Z. Chem. Commun. 2016, 52, 7288-7291. (34) Li, K.; Hou, J.-T.; Yang, J.; Yu, X.-Q. Chem. Commun.. 2017, DOI: 10.1039/c7cc01679d. (35) Huo, F. J.; Zhang, J. J.; Yang, Y. T.; Chao, J. B.; Yin, C. X.; Zhang, Y. B.; Chen, T. G. Sensors and Actuators B. 2012, 166-167, 44-49. (36) Zhang, X.; Zhang Y. X.; Zhu, Z. J. Anal. Methods. 2012, 4, 4334-4338. (37) Yin, W. Z.; Zhu, H. J.; Wang, R. Y. Dyes and Pigments. 2014, 107, 127-132. (38) Algi, M. P. Tetrahedron. 2016, 72, 1558-1565. (39) Sun, Z. N.; Liu, F. Q.; Chen, Y.; Tam, P. K.; Yang, D. Org. Lett. 2008, 10, 2171-2174. (40) Chen, X.; Lee, K. A.; Ha, E. M.; Lee, K. M.; Seo, Y. Y.; Choi, H. K.; Kim, H. N.; Kim, M. J.; Cho, C. S.; Lee, S. Y. Chem. Commun. 2011, 47, 4373-4375. (41) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 5680-5682. (42) Xu, Q.; Lee, K. A.; Lee, S.; Lee, K. M.; Lee, W. J.; Yoon, J. J. Am. Chem. Soc. 2013, 135, 9944-9949.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(43) Wu, X.; Li, Z.; Yang, L.; Han, J.; Han, S. Chem. Sci. 2013, 4, 460-467. (44) Xiao, H.; Xin, K.; Dou, H.; Yin, G.; Quan, Y.; Wang, R. Chem. Commun. 2015, 51, 1442-1445. (45) Xu, Q. L.; Heo, C. H.; Kim, J. A.; Lee, H. S.; Hu, Y.; Kim, D.; Swamy, K. M. K.; Kim, J. M.; Nam, S. J.; Kim, H. M.; Yoon, J. Anal. Chem. 2016, 88, 6615−6620. (46) Cheng, G.; Fan, J.; Sun, W.; Cao, J.; Hu, C.; Peng, X. Chem. Commun. 2014, 50, 1018-1020. (47) Liu, S. R.; Wu, S. P. Org. Lett. 2013, 15, 878-881. (48) Lou, Z.; Li, P.; Pan, Q.; Han, K. Chem. Commun. 2013, 49, 2445-2447. (49) Dr, X. C.; Wang, X.; Wang, S.; Shi, W.; Wang, K.; Ma, H. M.; Chem. Eur. J. 2008, 14, 4719–4724. (50) Yang, Y. K.; Cho, H. J.; Lee, J.; Shin, I.; Tae, J. Org. Lett. 2009, 11, 859-861. (51) Lin, W.; Long, L.; Chen, B.; Tan, W. Chem. Eur. J. 2009, 15, 2305-2309. (52) Cheng, X.; Jia, H.; Long, T.; Feng, J.; Qin, J.; Li, Z. Chem. Commun. 2011, 47, 11978-11980. (53) Emrullahoğlu, M.; M, Ü.; Karakuş, E. Chem. Commun. 2013, 49, 7836-7838. (54) Wu, J. S.; Liu, W. M.; Zhuang, X. Q.; Wang, F.; Wang, P. F.; Tao, S. L.; Zhang, X. H.; Wu, S. K.; Lee, S. T. Org. Lett. 2007, 9, 33-36. (55) Wu, J.; Liu, W.; Ge, J.; Zhang, H.; Wang, P. Chem. Soc. Rev. 2011, 40, 3483-3495. (56) Yang, G.; Morlet-Savary, F.; Peng, Z.; Wu, S.; Fouassier, J.-P. Chem. Phys. Lett. 1996, 256, 536-542. (57) Han, Y.; Yi, G.; Zhang, A. C.; Lo, Y. H. Lab Chip. 2016, 16, 4639-4647.

18


Page 18 of 25

Page 19 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(58) Rao, M. U.; Pryor, W. A. Anal. Biochem. 1996, 236, 242-249. (59) Lopez, F. C.; Shankar, A.; Thompson, M.; Shealy, B.; Locklear, D.; Rawalpally, T.; Thomas Cleary, A.; Gagliardi, C. Org. Process Res. Dev. 2005, 9, 1003-1008. (60) Prütz, W. A. Arch. Biochem. Biophys. 1996, 332, 110-120. (61) Xing, P.; Gao, K.; Wang, B.; Gao, J.; Yan, H.; Wen, J.; Li, W.; Xu, Y.; Li, H.; Chen, J. Chem. Commun. 2016, 52, 5064-5066. (62) Adachi, Y.; Kindzelskii, A. L.; Petty, A. R.; Huang, J. B.; Maeda, N.; Yotsumoto, S.; Aratani, Y.; Ohno, N.; Petty, H. R. J. Immunol. 2006, 176, 5033-5040.

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. (a) Changes in the fluorescence spectra of probe 1 (10 µM) with different concentrations of NaClO (0-100 µM). (b). Linear relationship between probe 1 and NaClO in the concentration range of 0.5−100 µM. λex/em = 480 / 542 nm

Scheme 1. The structure of probe 1, and the proposed mechanism of fluorescence response to ClO-.

20


Page 20 of 25

Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (a) Fluorescence intensity of probe 1 (10 µM) in 10 mM PBS, 10 mM Tris-HCl, 10 mM HEPES in the presence and absence of ClO- at room temperature with excitation of 480 nm. (b) Time-dependent changes in fluorescence intensity of probe 1 (10 µM) in the presence and absence of ClO- at room temperature with excitation of 480 nm

Figure 3. (a) The effects of pH values on the fluorescence intensity in the presence and absence of ClO- at room temperature with excitation of 480 nm (b) Fluorescence spectra of probe 1 before and after reaction with various biologically relevant analytes AA (1 mM), GSH (1 mM), Cys (1 mM), TBHP (200 µM), H2O2 (1 mM), NO (200 µM), ONOO- (200 µM), •OH (200 µM), •O2‑ (200 µM). and ClO- (200 µM) in PBS (pH 7.4, containg 10% DMF) at room temperature with excitation of 480 nm.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Effects of probe 1 at varied concentrations on the viability of HeLa cells and RAW264.7 cells. (a) Hela cells 4h (b) HeLa cells 24h (c) RAW264.7 cells 4h (d) RAW264.7 cells 24h

Figure 5. Fluorescence (upper) and bright field (lower) of HeLa cells treated with various concentrations of ClO-. (a) 0 µM (b) 50 µM (c) 100 µM (d) 200 µM (e) 400 µM. Scale bar: 100 µm.

22


Page 22 of 25

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. (a) Detection of exogenous ClO- in flow cytometry with probe 1. HeLa cells were co-incubated with probe 1 (20 µM) and then treated with various concentrations of ClO- for 30 min and analyzed by flow cytometry. (b) Detection of different concentrations of LPS-induced endogenous produced ClO- in RAW264.7 with probe 1 in flow cytometry. Flow cytometer equipped with 488 nm Ar laser and fluorescence was collected by PE channel.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. Fluorescence (upper) and bright field (lower) of RAW 264.7 cells treated with various stimulants (a) probe 1 (b) LPS (0.5 µg/ml), IFN-γ (50 ng/ml) for 16 h then PMA (1 µg/ml) and probe 1 (20 µM) for 2 h (c) LPS (0.5 µg/ml), IFN-γ (50 ng/ml), PMA (1 µg/ml) and ABH (200 µM) for 16 h then probe 1 (20 µM) for 2 h (d) LPS (0.5 µg/ml), IFN-γ (50 ng/ml), PMA (1 µg/ml) and NAC (1 mM) for 16 h then probe 1 (20 µM) for 2 h. Scale bar: 200 µm.

Figure 8. Detection of endogenous ClO- in flow cytometry with probe 1 (a) probe 1 (b) LPS (0.5 µg/ml), IFN-γ (50 ng/ml) for 16 h then PMA (1 µg/ml) and probe 1 (20 µM) for 2 h (c) LPS (0.5 µg/ml), IFN-γ (50 ng/ml), PMA (1 µg/ml) and ABH (200 µM) for 16 h then probe 1 (20 µM) for 2 h (d) LPS (0.5 µg/ml), IFN-γ (50 ng/ml), PMA (1 µg/ml) and NAC (1 mM) for 16 h then probe 1 (20 µM) for 2 h

24


Page 24 of 25

Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for TOC only

25


Turn-on Fluorescent Probe for Exogenous and Endogenous Imaging

Recommend Documents