A sensitive BODIPY-based fluorescent probe suitable for hypochlorite detection in living cells

Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72

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A sensitive BODIPY-based fluorescent probe suitable for hypochlorite detection in living cells Yuanyuan Lia , Yong Tangb , Mengmeng Gaob , Yun Wangb,* , Juan Hanc , Jinchen Xiab , Lei Wangb , Xu Tangb , Liang Nib a b c

Jingjiang College, Jiangsu University, Zhenjiang 212013, PR China School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China

A R T I C L E I N F O

Article history: Received 6 July 2017 Received in revised form 19 October 2017 Accepted 21 October 2017 Available online 28 October 2017 Keywords: Fluorescent probe Hypochlorite BODIPY Bioimaging

A B S T R A C T

A fluorescent probe composed of BODIPY and 2,4-dinitrophenylhydrazine was designed for the detection of ClO
. The oxidizing reaction between probe and ClO
resulted in a distinct fluorescence enhancement together with a color variation from pink to orange. This probe showed excellent selectivity to ClO
among various ions including common reactive oxygen species and high sensitivity with a detection limit of 228 nM. A fast response (7 min) was observed, which made the probe a promising method in real-time detection. Furthermore, this probe was successfully applied to monitor ClO
in real-life water and living cells. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Hypochlorite (ClO
), as a biologically significant part of the reactive oxygen species (ROS), plays a vital role in various physiological processes [1]. Endogenous hypochlorite, which is produced mainly from the reactivity of H2O2 and Cl
by myeloperoxidase(MPO) in leukocytes, has antibacterial effects on the biological immune-defense system [2]. A low concentration of hypochlorite can devastate invasive bacteria and pathogens, which is particularly crucial to physiological balances of proteins, DNA and RNA [3,4]. Unfortunately, the excessive hypochlorite may cause tissue damage and a variety of human diseases, including atherosclerosis, cardiovascular diseases, osteoarthritis, cystic fibrosis cancers and pulmonary lesions [5–9]. Hypochlorite is also widely used as disinfector in our daily life, for example, in swimming pool water and drinking water [10]. Owing to the biological importance of ClO
, the development of detection methods for ClO
in both real-life water and living cells has become an important issue. Fluorescent probe is a kind of powerful tool for ion detection, due to the advantages of nontoxicity, spatial resolution capability, sensitivity, simple operation, potential biological value [11]. Recently, some fluorescent probes containing functional groups

* Corresponding author: Tel: +86 158 9638 5156; Fax: +86-0511-88791800. E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.jphotochem.2017.10.037 1010-6030/© 2017 Elsevier B.V. All rights reserved.

like amide, thiosemicarbazone, p-methoxyphenol, oxime have been developed to detect ClO
[12–16]. Nonetheless, some limitations of these probes appeared during the detection process, such as delayed response time, low quantum yield, poor biocompatibility. In this work, a new fluorescent probe (Probe 1) based on C¼N bond transformation was designed and synthesized to detect ClO
. BODIPY was selected for the basic fluorophore in consideration of excellent light stability, low molecular weight, high fluorescence quantum yield, admirable cells’ penetration [17]. Probe 1 was nonfluorescent because C¼N isomerization [18,19] quenched the fluorescence of fluorophore, but exhibited significant fluorescence enhancement in response to ClO
. Excellent properties like high sensitivity and selectivity, rapid response, make Probe 1 a promising method for ClO
detection in neutral water and biological cells. 2. Experimental 2.1. Chemicals and instruments All chemicals used were of analytical grade, purchased form Aldrich and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Unless otherwise stated, these reagents were used without further purification and redistilled water were the main solvent. Mass spectrometry data were obtained with a Bruker ESQUIRE HPLC–MS AB 4000Q mass spectrometer. Ultraviolet-visible

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Scheme 1. Synthesis of Probe 1.

spectrum data were obtained with UV-2550 spectrophotometer and 1H NMR spectra were recorded on an AVANCE II 400 MHz spectrometer (Bruker Bio Spin). Varian Cary Eclipse Fluorescence spectrophotometer was used to measure fluorescence spectra. An Olympus Zeiss 710 laser scanning confocal microscopy was used for Fluorescence image of cells. All the samples were tested in DMF-PBS solution (1:1 v/v, 0.01 M PBS, pH 7.4) at room temperature. 2.2. Synthesize of Probe 1 The intermediate products, Compound 1 and 2 were prepared according to our previous work [17]. As illustrated in Scheme 1, 69.3 mg(0.35 mmol)of 2,4-dinitrophenylhydrazine and 105.5 mg (0.3 mmol)of Compound 2 were added in the mixed solution (ethyl alcohol-ethyl acetate, v:v, 1:1). Then 5 drips of acetic acid were added and the mixture was stirred for 2 h at room temperature. The reaction progress was monitored by TLC (petroleum ether- ethyl acetate, v:v, 1:2) until Compound 2 was consumed. The crude product was recrystallized from ethyl alcohol and further purified by column chromatography (petroleum ether-dichloromethane, v: v, 1:1) to give Probe 1 (139.6 mg, 87.3%). 1H NMR (400 MHz, CDCl3, ESI) d 11.21 (s, 1H), 9.12 (dd, J = 12.7, 2.5 Hz, 1H), 8.30 (dd, J = 9.6, 2.4 Hz, 1H), 8.09 (s, 1H), 7.80 (d, J = 9.6 Hz, 1H), 7.63-7.49 (m, 3H), 7.31 (dd, J = 6.5, 2.9 Hz, 2H), 6.11 (s, 1H), 2.84 (s, 3H), 2.62 (s, 3H), 1.60 (s, 3H), 1.41 (s, 3H). MS (ESI): 533.70 [M + H]+. 2.3. Preparation of test solutions The corresponding amount of Probe 1 solid was dissolved in DMF to prepare a stock solution (1 mM). The test solutions were diluted with the mixture of DMF and 0.01 M PBS (phosphate-

buffered saline, pH 7.4) (1:1, v/v) for the spectral analysis. The stock solutions of some ions (10 mM for F
, Cl
, NO2
, ClO4
, HCO3
, H2PO4
, SO42
, S2O32
, CO32
, Fe3+, Cu2+, H2O2, ONOO
, ROO, OH, NO and ClO
) and the blank solution were prepared in deionized water. NaOCl and H2O2 stock solutions were respectively obtained by diluting the commercialization solutions (14.5% and 30%). Peroxynitrite (ONOO
) was provided by nitrozation of NaNO2 and H2O2. Peroxyl radicals (ROO) was prepared from 30 min stirring of 2,20 -Azobis (2-amidinopropane) dihydrochloride(AAPH) solution at 25  C. Hydroxyl radical (OH) was derived from Fenton reaction with ferrous chloride and 10 equiv. H2O2 [20]. Nitric oxide (NO) was obtained from sodium nitroferricyanide (III) dehydrate (SNP). 2.4. Calculation of fluorescent quantum yield [21] Rhodamine B dissolved in EtOH (quantum yield Ks = 0.97) was used as the standard. The quantum yields of probe in the absence and presence of NaClO were estimated according to the following equations:    2 A FX nX FX ¼ Fs S AX FS nS Where K is the quantum yield; A is absorbance of excitation wavelength; F is integrated area under the corrected emission spectra; the subscript S and X stand for the standard and for the unknown, respectively. 2.5. Measurement of the detection limit [22] The detection limit (D) based on the fluorescence titrations was estimated according to the following equations: Detection limit =

Fig. 1. (a) Fluorescence response of Probe 1 (10 mM) in presence of different concentrations of ClO
; (b) Effect of pH value on the fluorescence intensity of Probe 1 (10 mM) in the absence and presence of 400 mM ClO
. lex = 470 nm.

Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72

3s/k, where s is the standard deviation of the blank measurement, and k is the slope for the fluorescence intensity versus the concentration of target ion. To gain the standard deviation of the blank measurements, the S/N ratio of Probe 1 was not obtained until the emission intensity of Probe 1 were measured in the absence of target ions by twenty times. 2.6. Practical application To explore the applicability of Probe 1 for the detection of ClO
, filter papers (1 1.5 cm2) were immersed in the solution of Probe 1 (10 mM) for 20 min and then desiccated in air to prepare test strips. Finally, various concentrations of ClO
were added dropwise to these strips and dried up for observation. 2.7. Cell incubation and fluorescence imaging HepG2 cells seeded into 24-well plates were incubated in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS (fetal bovine serum), and maintained in a humidified atmosphere containing 5% CO2 at 37  C for 24 h. Subsequently, Probe 1 (2 mM) was cultured with the well-grown cells for 30 min and the residue probe was washed by PBS buffer for 3 times. Then sodium hypochlorite (80 mM) was added into some plates for

67

another 10 min incubation. Finally, HepG2 cells were rinsed by the same way for fluorescence imaging. 3. Results and discussion 3.1. Effects of pH and time We sought optimal [H+] concentration while monitoring fluorescence intensity. Fig. 1b exhibited the maximum fluorescence intensity of Probe 1 solution in the absence and presence of ClO
. Probe 1 solution was found almost non-fluorescent at pH 4–12 while the mixed solution of Probe 1 and ClO
showed the maximum fluorescence at pH range from 6 to 8. This phenomenon revealed that the decreasing reactivity of ClO
under acidic or alkaline conditions could lead to the decline of fluorescence intensity. Hence, the pH 7.4 was chosen to be the test condition throughout the following experiment, which was appropriate for ClO
detection in bioimaging. Time dependence is another key factor and five types of solutions were tested. As shown in Fig. 1a, Probe 1 had no fluorescence even though the time was extended to 900 s. After the addition of ClO
, the intensity was dramatically increased and then held wave nearby a constant value within 7 min, which illustrated that Probe 1 could respond to ClO
in real time and certified the stability of Probe 1 under the same condition.

Fig. 2. (a) Absorbance spectra of Probe 1 (10 mM) with 400 mM of ClO
or other ions, the illustration is the photo of these solutions; (b) Fluorescence emission spectra of Probe 1 (10 mM) with 400 mM of ClO
or400 mM of other ions, the illustration is the photo of these solutions under UV light (365 nm) illumination; (c) Fluorescence spectra response of Probe 1 (10 mM) to ClO
(400 mM) in the presence of other anions (400 mM). The order of the ions is: 1 blank, 2 F
, 3 Cl
, 4 ClO4
, 5 HCO3
, 6 S2O32
, 7 Fe3+, 8 CO32
, 9H2O2, 10 ONOO
, 11 ROO, 12 OH, 13 NO, 14 ClO
. lex = 470 nm.

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Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72

Fig. 3. (a) Fluorescence emission spectra of Probe 1 (10 mM) with increasing amount of ClO
(0
380 mM). The illustration is the photo of the solutions under UV light (365 nm) illumination. (b) The fluorescence intensity of Probe 1 (10 mM) at 515 nm versus the ClO
concentration (0
380 mM). The illustration shows the linearity relative to the ClO
concentration: intensity = 2.70 [ClO
]
6.23, R2 = 0.9980. lex = 470 nm.

Table 1 A comparison of related probes (structures and statistics) reported for ClO
fluorescence detection. Probe

Naked-eye

Response time

Bioimaging

Detection limit

Reference

None

None

None

6.4 nM

Analyst, 2017 [23]

None

7 min

HepG2

356 nM

Chem.Commun., 2015 [24]

None

3 min

L929

3800 nM

Sen. Actuators B: Chem.,2016 [25]

Colourless to yellow

None

None

2000 nM

Tetrahedron, 2016 [26]

None

10 min

None

1400 nM

J. Fluoresc., 2017 [27]

None

90 min

Hela

200 nM

Talanta, 2017 [28]

None

5 min

HepG2

600 nM

Sen. Actuators B: Chem.,2017 [29]

Yellow to none

10 min

A549

2660 nM

Sen. Actuators B: Chem.,2017 [30]

Pink to orange

7 min

HepG2

228 nM

This work

Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72

69

Scheme 2. Specific reaction process of Probe 1 with ClO
.

the colorimetric and fluorescent sensing analysis of ClO
and been applied in the multitudinous actual systems successfully.

3.2. Selectivity studies The absorption and fluorescence spectra of Probe 1 were measured with addition of 12 kinds of ions (the first one is the blank) to Probe 1 solution, in order to evaluate the selectivity of Probe 1. In the UV–vis absorption spectrum (Fig. 2a), the maximum absorption of the blank and other competing ions emerged at 543 nm. Upon addition of ClO
, the absorption band at 543 nm for Probe 1 decreased and became blue-shifted (505 nm). The illustration showed that Probe 1 solutions in the presence of competing ions were pink while the solution with an addition of ClO
was orange. This phenomenon illustrated that Probe 1 could be utilized for visual detection of ClO
. Fig. 2b showed the emission of these solutions. The addition of competing ions (40 equiv.) did not cause obvious emission changes of the fluorescence. In contrast, the fluorescence intensity of the samples with 40 equiv. of ClO
dramatically increased and the solution emitted green fluorescence (illuminated by UV light at 365 nm in the illustration). Further studies was carried out by adding 40 equiv. of ClO
to other 13 kinds of solutions and measured the maximum fluorescence emission intensity. It could be found in Fig. 2c that the intensity was almost the same with the blank. That is to say, the fluorescence response to ClO
was impervious to other ions. Both the UV absorption spectrum and the fluorescence emission spectrum demonstrated that Probe 1 was able to selectively detect ClO
among many common ions and ROS. All in all, Probe 1 has realized

3.3. Sensitivity studies The titration experiments with addition of ClO
revealed the relationship between the emission intensity and the concentration of ClO
. Probe 1 solution was non-fluorescent (K1 = 0.059). With ClO
added, the emission intensity of Probe 1 solution dramatically went up (K2 = 0.56). Moreover, the fluorescence was gradually enhanced with the increasing concentration of ClO
(Fig. 3a). When the concentration of ClO
becomes 36 equiv. compared to Probe 1, the fluorescence intensity attains saturation. This fluorescent variation is visible under the UV light at 365 nm in the illustration of Fig. 3b. Besides, in the range of NaOCl 0–340 mM, the fluorescent intensity showed a pretty good linear relationship with the ClO
concentration and the linear coefficient was 0.998 (Fig. 3b). Detection limit based on this linearity was calculated to be 228 nM, which was more sensitive than most reported probes. These results demonstrated that the probe could be used for the accurate quantitative analysis. Comparing to the chemosensors listed in Table 1 and reported in some other studies [14,15], Probe 1 exhibited tremendous superiorities in those aspects of visual inspection, response time, detection limit and bioimaging. The probe also realized the recognition of hypochlorite by dual methods. Accompanied by the

Fig. 4. Mass spectrum for the product from the reaction of Probe 1 and ClO
.

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Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72

Fig. 5. Photos of test strips for detecting ClO
in aqueous solution, the samples are: 1 blank, 2 tap water + 30 mM ClO
, 3 tap water + 100 mM ClO
, 4 tap water + 300 mM ClO
, 5 tap water + 400 mM ClO
.

research of C¼N bond transformation, the choices of recognition site and fluorophore were the dominant key to synthesize the better chemosensor. Besides, the probe was designed for ClO
detection with the colorimetric and fluorescent sensing methods, rapid response, good selectivity, excellent sensitivity and great biocompatibility. 3.4. Proposed reaction mechanism The proposed reaction mechanism is illustrated in Scheme 2. The C¼N isomerization is thought to quench fluorescence of many fluorophore [13,14]. So Probe 1 exhibited no fluorescent on account of the existence of C¼N bond. Upon ClO
added, the C¼N isomerization was removed by oxidation reaction between Probe 1 and ClO
. As a result, the product recovered the strong fluorescence of BODIPY group. To confirm the mechanism, the reaction product was proved by mass spectrometry (Fig. 4). The peak at m/z

Fig. 6. Cell viability of Probe 1 by CKK-8 assay.

353 [M + H]+ was clearly observed, which was in accordance with the proposed product. 3.5. Application test in test strips For visual recognition of ClO
, test strips based on Probe 1 were applied to tap water. Distilled deionized water/tap water, 30 mM ClO
/tap water, 100 mM ClO
/tap water, 300 mM ClO
/tap water and 400 mM ClO
/tap water were added into the test paper and dried at room temperature. The color of paper in Fig. 5 changed from pink to orange and green fluorescence appeared under UV light (365 nm) illumination. After adding a higher concentration of ClO
, the paper revealed stronger fluorescence. Through visual observation, the ClO
concentration could be estimated roughly without fluorescent instruments. This obvious phenomenon

Fig. 7. Confocal fluorescence images of HepG2 cells. (a) and (b) are the bright-field images of HepG2 cells loaded with 2 mM Probe 1 for 30 min in the absence and presence of ClO
(80 mM) for another 10 min. (c) and (d) represent the fluorescence images of (a) and (b), respectively.

Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72

demonstrated that Probe 1 is applicable for visual detection of ClO
in real water samples.

[2]

3.6. Cytotoxicity assay In order to estimate the cytotoxicity of Probe 1, the HepG2 cells were treated with the probe of various concentrations (1.0–20 mM) for 24 h. CCK-8 assay was used for the assessment of cytotoxicity [31,32]. We chose HepG2 cell in bioimaging because it is one of the typical cancer cells, which are often used for bioimaging researches in reported researches. More importantly, this kind of cell is easy to cultivate and benefit to cell imaging[33–35]. In addition, our probe was mainly used for exogenous hypochlorite detection in living cells, so this kind of common cancer cell was chosen as an example to demonstrate the feasibility of bioapplication. As presented in Fig. 6, even though 20 mM of Probe 1 was cultivated, the great cell viability of Probe 1 could reach above 90%. The low toxicity indicated that bioimaging of Probe 1 may be worthy of consideration. 3.7. Cellular imaging To further investigate the practical applicability of Probe 1 in biological researches, the fluorescence imaging was carried out in HepG2 living cells. As expected, the HepG2 cells (Fig. 7c) loaded only with Probe 1 (2 mM) had almost no fluorescence signal, which kept consistent with the fluorescence selectivity studies. Then the HepG2 cells were incubated with sodium hypochlorite (80 mM) for another 10 min under the same condition. These cells exhibited obvious green fluorescence (Fig. 7d), verifying excellent cell permeability and stable fluorescence behavior of Probe 1 in biosystem. Hence, these results indicated evidently that Probe 1 was cell-permeable and capable of imaging exogenous ClO
in living HepG2 cells.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13] [14]

4. Conclusion [15]

In summary, a novel fluorescent probe based on the integration of 2,4-dinitrophenylhydrazine and BODIPY was designed and synthesized to monitor ClO
. Upon adding ClO
to Probe 1 solution, a naked-eye visible variation from pink to orange with a remarkable fluorescence increase were observed. Moreover, Probe 1 was suitable for the quantitation in complex ionic environment with high sensitivity and rapid response. With those attractive properties, this probe was further used for monitoring ClO
in real water samples and biological imaging. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 31470434, 21576124, 21507047 and 21676124), China Postdoctoral Science Foundation funded project (No. 2017M610308), Zhenjiang Social development project (No. SH2016019), and the Project supported by the Science Foundation of Jiangsu Entry-exit Inspection Quarantine Bureau(Nos. 2016KJ51 and 2017KJ47). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jphotochem.2017. 10.037.

[16]

[17] [18]

[19]

[20]

[21]

[22]

[23] [24]

[25]

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