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Benzoyl Peroxide Detection in Real Samples and Zebrafish Imaging by a Designed Near-Infrared Fluorescent Probe Xinwei Tian, Zhao Li, Yaxing Pang, Dongyu Li, and Xingbin Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03598 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017

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Journal of Agricultural and Food Chemistry

1

Benzoyl Peroxide Detection in Real Samples and Zebrafish Imaging by a

2

Designed Near-Infrared Fluorescent Probe

3 4

Xinwei Tian, Zhao Li*, Yaxing Pang, Dongyu Li and Xingbin Yang*

5 6

Shaanxi Engineering Laboratory for Food Green Processing and Safety Control,

7

College of Food Engineering and Nutritional Science, Shaanxi Normal University,

8

Xi'an 710062, China

9

* Corresponding author (E-mail: [email protected]; [email protected])

1

ACS Paragon Plus Environment


A

novel

near-infrared

fluorescence

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off-on

probe,

10

ABSTRACT:

11

(E)-3,3-dimethyl-1-propyl-2-(2-(6-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)be

12

nzyloxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indolium (1), is developed and

13

applied to benzoyl peroxide (BPO) detection in real samples and fluorescence

14

imaging in living cells and zebrafish. By connecting arylboronate as the recognition

15

unit to a stable hemicyanine skeleton, the probe is readily prepared, which exhibits

16

superior analytical performance such as near-infrared fluorescence emission over 700

17

nm, high sensitivity with a low detection limit of 47 nM. Upon reaction with BPO, the

18

phenylboronic acid pinacol ester is oxidized, followed by hydrolysis and

19

1,4-elimination of o-quinone-methideand to release fluorophore. In addtion, the probe

20

displays high selectivity toward BPO over other common substances, which makes it

21

of great potential use in quantitative and simple detection of BPO in wheat flour and

22

antimicrobial agent. More importantly, the probe has been successfully demonstrated

23

for monitoring BPO in living Hela cells and zebrafish. The probe with superior

24

properties could be of great potential use in other biosystems and in vivo studies.

25

KEYWORDS: Fluorescent probe, Benzoyl peroxide, Imaging analysis, Zebrafish

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INTRODUCTION

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Benzoyl peroxide (BPO) has received much attention because of its impact on human

28

health and industrial importance. It plays an important role in bleaching flour, treating

29

acne and initiating polymerization.1-4 However, the decomposition products of BPO,

30

such as benzoic acid, phenylbenzoate and biphenyl, may further evoke tissue damage

31

and diseases.5,

32

absorption, resulting in potential risks. In order to better understand the biological

33

function of BPO, sensitive and selective methods for the detection of BPO in living

34

biosystems are of great significance.

6

BPO can easily enter the human body by food intake or skin

35

In recent years, a series of analytical methods, including chemiluminescence,

36

electrochemistry, spectrophotometry, high performance liquid chromatography and

37

fluorescent probes have been developed for detection of BPO.7-15 Because of their

38

great temporal and spatial sampling capability,16-18 some excellent fluorescent probes

39

with high sensitivity have been prepared for the detection and imaging of BPO in

40

living cells.13-15 For example, Chen et al. developed a fluorescence probe based on

41

resorufin, which has been applied to simple detection of BPO in wheat flour and

42

antimicrobial agent.14 Wang et al. developed a ratiometric fluorescent probe, which

43

has been used for BPO detection in living cells.15 Hence, because they are beneficial

44

for biological imaging due to their deep tissue penetration, minimal damage and

45

interference to biological samples,19-23 near-infrared (NIR) fluorescent probes are

46

more desired for in vivo imaging studies. To the best of our knowledge, there is no

47

NIR fluorescent probe for BPO assay reported so far. Hence, NIR fluorescent probes

48

are still necessary for BPO assay.

49 50

Here we report (E)-3,3-dimethyl-1-propyl-2-(2-(6-(2-(4,4,5, 5-tetramethyl-1,3, 2-dioxaborolan-2-yl)benzyloxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indolium (1;

3



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Scheme 1) as a novel NIR fluorescence off-on probe with 706 nm emission for BPO

52

assay. We choose 2-bromomethylphenylboronic acid pinacol ester as a quenching and

53

recognizing moiety, which can distinguish BPO from other oxidation species.14,15 It is

54

known that hemicyanines (2), which can be synthesized through the decomposition of

55

IR-780 but still possesses a NIR feature.20-22 For this reason, our probe can be

56

designed by connecting 2-bromomethylphenylboronic acid pinacol ester to the

57

hemicyanine skeleton through an ether bond. Reaction of probe 1 with BPO would

58

result in the oxidation of phenylboronic acid pinacol ester, and thus the release of

59

fluorophore (2). Such a fluorescence response leads to the establishment of a highly

60

sensitive and selective method for BPO detection in real samples and imaging in

61

living cells and vertebrate animal zebrafish.

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MATERIALS AND METHODS

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Apparatus. Fluorescence spectra were obtained on a Shimadzu RF-6000

64

spectrofluorimeter in 1-cm quartz cells. UV-Vis absorption spectra were obtained

65

using a Hitachi U-3010 spectrophotometer. 1H NMR and

66

recorded on a Brucker DMX-600 spectrometer in CD3OD. Electrospray ionization

67

mass spectra (ESI-MS) was obtained using a Shimadzu LC-MS 2010A instrument

68

(Kyoto, Japan). The MTT analysis was recorded on a microplate reader (BIO-TEK

69

Synergy HT, USA). Fluorescence imaging of Hela cells and zebrafish were conducted

70

on a confocal laser scanning microscope (Leica, Germany) with 635 nm excitation.

71

Reagents.

IR-780

iodide,

Benzoyl

peroxide

13

C NMR spectra were

from

Sigma-Aldrich.

72

2-2’-Azobisisobutylonitrile, N-bromosuccinimide and Toluene-2-boronic acid were

73

purchased from J&K Scientific. The phosphate buffered saline solution and

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained

75

from Invitrogen Company. Non-additive wheat flours and antimicrobial agent were

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obtained from supermarkets. Dulbecco’s modified eagle media (DMEM), fetal bovine

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serum, penicillin and streptomycin were purchased from HyClone Company (USA).

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Synthesis

and characterization of probe

1. First, the

raw material

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2-Bromomethylphenylboronic acid pinacol ester (3, Scheme 1) was prepared using

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the previous procedure.14, 24 Second, to a stirred solution of hemicyanine 2 (40 mg, 1.0

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mmol) in CH3CN and K2CO3 (20 mg, 1.5 mmol) were mixed and the mixture was

82

stirred at 40 °C for 10 min.21 Subsequently compound 3 (20 mg, 1.0 mmol) was added

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dropwise. The reaction mixture was allowed for stirring at 45°C, then the crude

84

product was diluted with dichloromethane.The crude product was then purified using

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flash

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(E)-3,3-dimethyl-1-propyl-2-(2-(6-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)be

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nzyloxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indolium (1) as a mazarine solid

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(25 mg, 56%). The 1H NMR and 13C NMR of probe 1 are given in Figure S1 and S2,

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respectively. 1H NMR (600 MHz, 298 K, CD3OD) δ 8.72 (d, J = 14.9 Hz, 1H), 7.84

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(dd, J = 7.4, 0.9 Hz, 1H), 7.64 (d, J = 7.4 Hz, 1H), 7.58-7.43 (m, 6H), 7.39-7.34 (m,

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2H), 7.03 (d, J = 2.1 Hz, 1H), 7.00 (dd, J = 8.5, 2.3 Hz, 1H), 6.51 (d, J = 14.9 Hz, 1H),

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5.42 (s, 2H), 4.33 (t, J = 7.4 Hz, 2H), 2.81-2.75 (m, 2H), 2.72 (t, J = 6.0 Hz, 2H),

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1.98-1.91 (m, 4H), 1.81 (s, 6H), 1.28 (s, 12H), 1.08 (t, J = 7.4 Hz, 3H); 13C NMR (151

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MHz, 298 K, CD3OD) δ 179.2, 164.1, 163.1, 155.8, 146.9, 143.4, 143.3, 143.1, 137.2,

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135.2, 132.3, 130.3, 130.2, 129.6, 128.8, 128.6, 128.4, 123.8, 117.2, 115.8, 115.2,

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114.1, 104.9, 102.5, 85.2, 71.8, 52.0, 47.7, 30.1, 28.5, 25.3, 25.2, 22.3, 21.7, 11.7.

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ESI-MS, m/z calcd. for probe 1 (C41H47BNO4+, [M]+): 628.3593; found: 628.3523

98

(Figure S3).

chromatography

on

silica

gel

(CH2Cl2/MeOH

as

eluent)

to

yield

99

General procedure for BPO detection. All the fluorescence measurements were

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made in 10 mM PBS (pH 7.4) containing 10% (v/v) ethanol. In a test tube, 4 mL of

5



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PBS and 50 µL of the stock solution (1 mM) of probe 1 were mixed, followed by

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addition of ethanol and an appropriate volume of BPO sample solution. The final

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volume was adjusted to 5 mL with PBS. The reaction solution was transferred to

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measure the absorbance or fluorescence with λex/em = 670/706 nm after incubation at

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37 °C for 20 min in a shaker incubator. For comparison, the solution containing no

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BPO (control) was measured under the same conditions at the same time.

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BPO detection in wheat flour and antimicrobial agent. The BPO samples were

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prepared by following procedure. First, the PBS solutions (10 mM, pH 7.4, 10%

109

ethanol) containing various concentrations of BPO (0, 1, 2 and 4 µM) were mixed

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with wheat flour (1 g) or gel-like antimicrobial agent (1 g). The samples were

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sonicated for 2 min and filtered with organic membrane (0.22 µm). Subsequently, the

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resulting samples were prepared with probe 1 and the fluorescence spectrum was

113

recorded.

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Cytotoxicity Assay. The cytotoxicity of probe 1 or fluorophore 2 to HeLa cells was examined by standard MTT assay according to the previous report.20

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Fluorescence imaging of BPO in HeLa cells. HeLa cells were grown in DMEM

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which were supplemented with fetal bovine serum, penicillin and streptomycin at

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37 °C in a humidified 5% CO2 incubator. For fluorescence imaging, the cells were

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washed with FBS-free DMEM, and incubated with BPO (2, 4 and 6 µM) for 10 min.

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After that, the cells were treated with probe 1 (10 µM) for 20 min at 37 °C, and

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subsequently washed three times with PBS buffer. The pixel intensity at least from ten

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cells in the fluorescence image was measured by using Image J software (version

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1.37c, NIH).

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Fluorescence imaging of BPO in zebrafish. For fluorescence imaging, zebrafish

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grown in E3 embryo media for 3 days were made by pretreatment with varied

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concentration of BPO (2, 4 and 6 µM) for 10 min. After that, the zebrafish were

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incubated with 10 µM probe 1 in PBS buffer for 20 min, and then washed with PBS to

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remove the remaining probe 1.

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RESULTS AND DISCUSSION

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Spectroscopic response of probe 1 to BPO. The absorption and fluorescence

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spectra of probe 1 toward BPO in PBS containing 10% ethanol are investigated. As

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shown in Figure 1A, upon addition of BPO, the maximum absorption peak of the

133

reaction solution is red-shifted to 665 nm, and absorption band was attenuated. Most

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notably, reaction of probe 1 with BPO produces a obvious fluorescence off-on

135

response at 706nm (Figure 1B), which is the same as the characteristic absorption

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spectrum of fluorophore 2.20 The reason for the low background signal of probe 1 is

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attributed to the hydroxyl protection of fluorophore 2.20-22 These observations show

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that reaction of probe 1 with BPO would result in the oxidation of phenylboronic acid

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pinacol

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o-quinone-methideand to release fluorophore 2. In addtion, the ESI-MS analysis

141

proves the generation of fluorophore 2 (m/z 412.2 [M]+, Figure S4).

ester,

accompanied

by

hydrolysis

and

1,4-elimination

of

142

The effects of pH and the ethanol concentration on the fluorescence of probe 1

143

were examined (Figure S5). Because water-insoluble BPO in different samples is

144

extracted with the organic solvent,25,

145

experimental system. Moreover, the effect of the ethanol concentration suggests that

146

the maximum fluorescence increase is achieved in the ethanol concentration of 10%

147

(Figure S5B). Hence, in this experiment ethanol acts as cosolvent for reaction system,

148

and also promotes the reactivity of BPO through accelerating the decomposition of

149

BPO into reactive intermediates.27-29

150

26

pH 7.4 can be used for the present

Kinetic curves of probe 1 toward BPO at varied concentrations are given in Figure

7



151

S6. The fluorescence increase could reach a approximate plateau in 20 min. In

152

contrast, no significant fluorescence change was observed in the probe 1 without BPO

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during the same period of time, which indicates the probe 1 is highly stable in the

154

detection system.

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On the basis of the above observations, we choose the optimum analytical

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conditionis that reaction of probe 1 with BPO at 37 °C for 20 min in PBS solution

157

with 10% ethanol. The curve was plotted with the fluorescence intensity at 706 nm

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(Figure S7), a good linear equation of ∆F = 676.3 × [BPO] (µM) – 100.1 (R2=0.993)

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was obtained between the fluorescence increase (∆F) and the BPO concentration in

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the range of (0.5-4 µM). The detection limit is determined to be 47 nM BPO, which is

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more sensitive than that of the ratiometric fluorescent probe (80 nM).15

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Taking the complexity of the internal environment into account, the selectivity of

163

probe 1 to BPO was investigated by testing various potential interfering substances in

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three separate measurements under the same condition, such as ions, sugars, amino

165

acids, vitamins, reactive oxygen species and important oxidizing agents. As depicted

166

in Figure S8, probe 1 displays high selectivity toward BPO over the other substances

167

tested, which may attribute to the effect of solvent and structure of arylboronate

168

(recognition unit).

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BPO detection in wheat flour and antimicrobial agent. BPO has been

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extensively used as flour bleaching agents, antimicrobial agents to treat acne. While

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excessive BPO could induce allergic reactions, potential carcinogenicity, and exert

172

effect on human peripheral lymphocytes. Therefore, the real samples detection of

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BPO is of great significance. The recovered BPO concentrations were determined

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according to the low concentration plotting shown in Figure S7. The result shows

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good recovery values (average deviation=2.28 and 2.79 %, respectively; Figure 2),

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which confirmed that the other coexisting species hardly interfere the BPO assay.

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Furthermore, the determination don’t require a time-consuming separation. This

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indicates that probe 1 have a good capacity to quantify BPO in real samples.

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Toxicity of probe 1 or fluorophore 2 to HeLa cells. The cytotoxicity of probe 1 or

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fluorophore 2 to cells was tested with HeLa cells by MTT assay.20 As shown in

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Figure S9, probe 1 or fluorophore 2 exhibit good biocompatibility and low

182

cytotoxicity since the probe and fluorophore 2 in tested concentration did not produce

183

significant influence on the viability of HeLa cells after 24 h. In the following work,

184

we chose probe 1 at 10 µM to conduct the following HeLa cells and zebrafish imaging

185

study.

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Fluorescence imaging of BPO in HeLa cells. Probe 1 with excellent sensing

187

property was further utilized to image of BPO in living cells through non-invasive

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imaging. As showed in Figure 3A, HeLa cells themselves show no background

189

fluorescence, which benefits by the usage of a NIR excitation wavelength.30-31

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However, a strong fluorescence was observed in the HeLa cells treated with probe 1

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(Figure 3B), which demonstrates the good cell permeability of probe 1. Moreover,

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treatment of HeLa cells with BPO (2, 4 and 6 μM) and probe 1 caused a largely

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enhanced fluorescence (Figure 3C-3E), suggesting that BPO entered readily the cells

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and can react with probe 1 together with

195

shown in Figure S10, the fluorescence intensity from HeLa cells treated with 2, 4 and

196

6 µM of BPO inceases by ca. 0.11, 0.33 and 0.84 times, respectively, with respect to

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that without BPO (defined as 1.0). The preceding results demonstrate that probe 1 was

198

cell membrane permeable and capable of detecting BPO in living cells.

fluorescence response. Furthermore, as is

199

Fluorescence imaging of BPO in zebrafish. The prominent features of probe 1

200

encouraged us to further explore the feasibility for measuring BPO in living animals.

9



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Zebrafish is a popular vertebrate model organism due to its favorable characteristics

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such as small size, transparency of their embryos and rapid development, which

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facilitate the in vivo visualization of biologically relevant substances by NIR

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fluorescent probes.32-34 Until now no study has been reported to visualize BPO in

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zebrafish. In consideration of the excellent performance of probe 1, we chose

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zebrafish grew for 3 days in E3 embryo media subjected to fluorescence imaging. As

207

shown in Figure 4, zebrafish themselves show no background fluorescence, but the

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zebrafish treated with probe 1 give a strong fluorescence. Moreover, the zebrafish

209

treated with probe and an increasing concentration of BPO from 0 to 6 µM produced a

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gradually increase fluorescence intensity. Especially zebrafish yolk sac and tail show

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almost the same fluorescence intensity (Figure 4A, 4B). The reason for fluorescence

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enhancement is that BPO oxidizes phenylboronic acid pinacol ester, followed by

213

hydrolysis and 1,4-elimination of o-quinone-methide to release the fluorophore 2.

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Meanwhile, as show in Figure S11, the fluorescence intensity from zebrafish treated

215

with 2, 4 and 6 µM of BPO inceases by ca. 0.77, 1.11 and 1.36 times, respectively,

216

with respect to that without BPO (defined as 1.0). Also we can visually see from

217

fluorescent images, the fluorescence increase obviously while the BPO concentration

218

is 2 µM, and this is far below biological matrix that may affect the fluorescence signal,

219

which showed excellent selectivity of probe in bio-imaging. These studies indicate

220

that the probe 1 is suited for monitoring the distribution of BPO in vivo.

221

In summary, we have prepared a novel NIR fluorescent off-on probe for BPO

222

assay by connecting arylboronate to a stable hemicyanine skeleton. The probe

223

displays superior analytical performance such as 706 nm emission, high selectivity

224

with low detection limit of 47 nM, and has been successfully demonstrated in BPO

225

detection in real samples and fluorescence imaging in living Hela cells and zebrafish.

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All these features of probe 1 suggest that this NIR fluorescent probe could be applied

227

to further investigation on the biological functions and in vivo imaging studies of BPO

228

in complex systems.

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229

ASSOCIATED CONTENT

230

Supporting Information

231

The Supporting Information is available free of charge on the ACS Publications

232

website at DOI: 10.1021/acs. jafc.

233

Figure S1-S2: Synthesis of probe 1. Figure S3-S4: Electrospray ionization mass

234

spectrum. Figure S5: Effects of pH and ethanol volume fraction. Figure S6:

235

Fluorescence kinetic curves of probe 1 reacting with BPO. Figure S7: Calibration

236

curve. Figure S8: Selectivity study. Figure S9: Cytotoxicity assay. Figure S10-S11:

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Relative pixel intensity measurements obtained from the images (PDF)

238

AUTHOR INFORMATION

239

Corresponding Author

240

*(XB.Y.) Phone: +86 10-85310580. Fax: +86 10-399 85310580.

241

Email: [email protected].

242

*(Z.L.) Phone: +86 10-85310517. Fax: +86 10-85310517.

243

Email: [email protected].

244

ORCID

245

Xingbin Yang: 0000-0002-8039-0525

246

Zhao Li: 0000-0001-7702-3348

247

Funding

248

This work was financially supported by the National Natural Science Foundation of

249

China (Nos. 31671823 and 21605099), and the Fundamental Research Funds for the

250

Central Universities, China (GK201603096, 2016CSZ010).

251

Notes

252

The authors declare no competing financial interest. 12


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FIGURE CAPTIONS Scheme 1. Synthesis of probe 1 and its proposed reaction mechanism with BPO. Figure 1. (A) Absorption spectra of probe 1 (10 µM) before (a) and after (b) reaction with BPO (6 µM). (B) Fluorescence spectra (λex=670nm) of probe 1 (10 µM) reacting with BPO at different concentrations of BPO (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 and 6 µM). The reaction was performed in 10mM PBS solution (pH 7.4) with 10% ethanol at 37 °C for 20 min. λex/em = 670/706 nm. Figure 2. Spiked and measured concentrations of wheat flour (A) and antimicrobial agent (B) by probe 1 in 10 mM PBS (pH 7.4) containing 10% (v/v) ethanol. (A) Sparse bar: the spiked concentrations of BPO from left to right: 1, 2, and 4 µM; Dense bar: the measured concentrations of BPO from left to right: 1.04, 2.10, and 3.87 µM. The average deviation is 2.28%. (B) Sparse bar: the spiked concentrations of BPO from left to right: 1, 2, and 4 µM; Dense bar: the measured concentrations of BPO from left to right: 0.92, 2.13, and 4.03 µM. The average deviation is 2.79%. Figure 3. Confocal fluorescence images of Hela cells. (A) Hela cells only; (B) Hela cells were incubated with 10 µM 1 for 20 min; (C) Hela cells were pretreated with 2 µM BPO for 10 min and then incubated with 10 µM 1 for 20 min; (D) Hela cells were pretreated with 4 µM BPO for 10 min and then incubated with 10 µM 1 for 20 min. (E) Hela cells were pretreated with 6 µM BPO for 10 min and then incubated with 10 µM 1 for 20 min. The differential interference contrast (DIC) images of the corresponding samples are shown below (panels F-J). Scale bar = 50 µm. Figure 4. Fluorescence images of BPO in living 3-day-old zebrafish (A) tail and (B) yolk sac. (a) Zebrafish only (control); (b) Zebrafish were treated with probe 1 (10 µM) for 20 min; (c) Zebrafish pre-incubated with 2 µM BPO were treated with probe 1 (10 µM) for 20 min; (d) Zebrafish pre-incubated with 4 µM BPO were treated with probe

18


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1 (10 µM) for 20 min; (e) Zebrafish pre-incubated with 6 µM BPO were treated with probe 1 (10 µM) for 20 min. The DIC images of the corresponding samples are shown below (panels f-j). Scale bar = 200 µm.

19



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Scheme 1 O

HO

B

OH

O Br

Cl

N

HO

N

O

CH3CN, K2CO3

I

3 K2CO3

N

2

B O O

O

O O

B O O H2O

benzoyl peroxide O

O

N

2

N

1

Figure 1

A

B 20000

0.3

Fluorescence intensity

Absorbance

a 0.2

b 0.1

0.0 400

500

600

700

6 µM 15000

10000

0 5000

800

720

750

Figure 2

B Conc. of benzoyl peroxide(µ M)

Conc. of benzoyl peroxide(µM)

A 4

780

810

Wavelength (nm)

Wavelength (nm)

Spiked Recoverd

2

0

4

Spiked Recoverd

2

0

20


840

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Figure 3

Figure 4

21



TOC

22


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Page 23 of 25


Scheme 1

Figure 1

Figure 2

1



Figure 3

Figure 4

2


Page 24 of 25

Page 25 of 25


TOC

3


Benzoyl Peroxide Detection in Real Samples and ... - ACS Publications

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