A new water-soluble two-photon fluorescent probe for detection of trace benzoyl peroxide in wheat flour and in living cell and tissue imaging

Article abstract of DOI:10.1039/c8nj06543h

Benzoyl peroxide (BPO), a member of small-molecule reactive oxygen species (ROS), has attracted considerable attention because of its impact on human health and industrial importance. Herein, to quantitatively detect BPO in real samples and for fluorescen

Full text of DOI:10.1039/c8nj06543h

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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A new water-soluble two-photon fluorescent probe for detection
of trace benzoyl peroxide in wheat flour and living cells and tissues
imaging
Received 00th January 20xx,
Accepted 00th January 20xx
‡
‡
Haiyuan Ding , Gangqiang Yuan , Liyi Zhou*
DOI: 10.1039/x0xx00000x
Benzoyl peroxide (BPO), a member of small-molecule reactive oxygen species (ROS), it has attracted wide attention because
of its impact on human health and industrial importance. Herein, to quantitatively detect BPO in real samples and
fluorescence imaging in living cells and tissues, a new water-soluble two-photon (TP) fluorescent probe (CM-1) was
constructed. Specifically, CM-1 adopted the carboxyl (-COOH) as the water-soluble unit, cyano (-CN) forms the D-π-A-
structure as an acceptor to increase the two-photon action absorption cross-section and phenylboric acid (PBA) as the BPO
reaction moiety. After the PBA moiety reaction with BPO, CM-1 displayed a ~82-fold fluorescence intensity enhancement in
485nm and high sensitivity with a low detection limit of 33nM. Selectivity experiment demonstrated that probe can detect
BPO with high selectivity over other common substances, which makes it of great potential use in quantitative and simple
detection of BPO in wheat flour. Moreover, CM-1could be employed for imaging BPO in living HeLa cells and rat liver tissues
with large tissue-image depth (40-150μm) under two-photon excitation (800nm), thus demonstrating its practical
application in biological systems for the study of physiological and pathological functions of BPO.
www.rsc.org/
In the past few years, some methods have been developed
[7]
Introduction
for monitoring BPO, such as chemiluminescence
,
electrochemistry [8], UV-spectrophotometry [9], HPLC [10] and so
on. However, most of these methods require a time-consuming
sample pretreatment and separation, and thus are
inconvenient for the fast and in situ detection of BPO.
Compared with them, fluorescence-based methods could
maintain comparable efficiency and accuracy, offer
convenience, high sensitivity, noninvasive, as well as real-time
imaging [11a,11b]. Especially, two-photon (TP) probe-based
fluorescent imaging, which is an emerging technique and
employing near-infrared (NIR) light source excitation that can
provide improved spatial resolution and theoretically
remarkably increased imaging depth than traditional one-
photon (OP) imaging, might be the most attractive one for in
vivo detection of bio-related species [12]. in the past few years,
very few fluorescent probes for detection of BPO in real
samples and biosystems for OP excitation [13-15]. Unfortunately,
to the best of our knowledge, no water-soluble and TP
fluorescent probe have been reported for BPO detection so far.
Herein, for the first time, we present the design, synthesis, and
characterization of TP fluorescent probe CM-1 (Scheme 1) as a
new water-soluble TP fluorescent probe for detection of BPO in
real samples and fluorescence imaging in living HeLa cells and
tissues.
Benzoyl peroxide (BPO), a member of small-molecule reactive
oxygen species (ROS) has attracted wide attention due to its key
role in human health and disease. On one hand, BPO has been
widely used in our daily life for bleaching flour [1], treating acne
[2], and initiating polymerization [3]. On the other hand, BPO can
easily enter the human body by food intake or skin absorption,
resulting in potential risks. It can act as a tumor promoter and
is able to degrade into deleterious substances (e.g., biphenyl,
benzoic acid), inevitably leading to tissue damage and diseases
[4-5]. Besides, once it used as flour additives to improve the color
and lustre of wheat flour and corn starch would cause the
destruction of the flour nutrients [6]. Therefor, in order to get
deeper understanding of the chemical and biological properties
of BPO, highly sensitive and highly selective detection
techniques for tracking BPO are urgently desirable, since the
complex manifestations of BPO in both physiological and
pathological states, as well as its underlying molecular events
are still not fully understood.
a. College of Food Science and Technology, Central South University of Forestry and
Technology, Changsha, Hunan 410004, P. R. China. E-mail: zhouly0817@163.com.
b. Hunan Key Laboratory of Processed Food for Special Medical Purpose, Changsha,
Hunan410004, P. R. China.
Herein, we choose a coumarin derivative (CM-CN) as a
signaling unit for BPO. As a result, the new probe CM-1 (Scheme
1) was synthesized with a good yield (81%) via a two-step
reaction, whichupon reaction with BPO, will induce the
cleavage of PBA moiety to be left, resulting in the forming of the
† Electronic Supplementary Information (ESI) available: Details on cytotoxicity test
and compounds of 1HNMR and 13C NMR. See DOI: 10.1039/x0xx00000x.
‡Contributed equally.
This journal is © The Royal Society of Chemistry 20xx
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fluorophore and “turn-on” of the fluorescent signal. After BPO in the wheat flour and in vivo as its concentrations are usually in
reaction with BPO, the probe CM-1 exhibited a more than 82- submicromolar range. To explore the spectroscopic response
View Article Online
DOI: 10.1039/C8NJ06543H
fold fluorescence intensity enhancement at an emission mechanism, the reaction products of CM-1 with BPO were subjected
1
wavelength of 485nm. It also showed the probe displayed high to MS and H/13C NMR analyses, see the Scheme 1 for its possible
selectivity toward BPO over other analytes. Subsequently the reaction with BPO and see the Supporting Information for MS and
probe was successfully applied to detect BPO for real samples 1H/13C NMR results, the results exhibit that the reaction produced
and bioimaging.
the fluorophore CM-CN.
High selectivity is an important parameter to evaluate a newly
designed fluorescent probe performance. For this purpose, CM-1
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Results and discussion
was treated with
a
series of analytes such as tert-butyl
-
hydroperoxide (t-BuOO., TBHP), O2 , NO, OCl-, vitamins, NaClO4,
Design and synthesis of fluorescent probe CM-1
Previously, few of BPO probes have been designed [13-15]. However,
they are all water-insoluble and require a large amount of organic
solvent as a cosolvent. And they are constructed by one-photon,
could not be effectively used to analyze deep tissue imaging studies.
In order to acquire a better in vitro detection, biological image
resolution, and penetration depth, we decided to develop a new
water-soluble fluorescent probe for detection of BPO in wheat flour,
live cells, and tissues. As a proof-of-concept, a D-π-A-structured CM-
CN was chosen as the fluorophore for its outstanding two-photon
property, while a boronic acid unit was serviced as the recognition
moiety due to its reaction with BPO rapidly by some reference
NaClO3, BPO, amino acids (Cys, Hcy and GSH), NaNO2, KBrO3, H2O2,
1O2, ONOO-, KIO3, KMnO4, S2-, HSO3 , SO32-, and CuOOH to examine
-
its selectivity. The results showed in Fig. 2a, the probe showed
almost unchanged fluorescence intensity responses before and after
addition of other analytes to the probe resolution. More
interestingly, compared to BPO, the other oxidants tested produce a
much weaker fluorescence except H2O2 and ONOO-. These results
demonstrated that probe CM-1 could meet the selective
requirements for practical applications. Next, we have studied the
effect of pH on CM-1 in the absence and presence of BPO (Fig. 2b).
Without BPO, no obvious characteristic fluorescence could be
observed from pH 4.0-9.0. Upon reaction with BPO, the best
response towards BPO could be achieved with a pH range of 6.0-8.5.
Thus, the PBS solution (pH 7.4) was utilized throughout the
experiment. These results indicated that the probe was favourable
for applications in practical samples at different pH values.
1
reported [13-15]. All compounds were characterized by H, 13C NMR,
and ESI-MS (see the Supporting Information).
Acceptor
Water-soluble moiety
800nm Excitation
Acceptor
HOOC
CN
Br
O
CHO
O
CN
O
CHO
OH
CH3CN,K2CO3
65 oC,10h
PBS, pH7.4, BPO
O
EtOH, Piperidine
50 oC, 12h
N
N
N
O
a
b
N
Donor
CM-1
CM-1+BPO
B
O
B
O
B
O
e
BPO: 0-6
μ
M
f
485nm Emission
O
O
1
3
CM-CN
2
CM-1
CM-1
BPO response moiety
CM-1+BPO
OH-
CN
NC
COOH
NC
COOH
OH
NC
O
H2
O
PBS, pH7.4
O
N
O
N
O
d
c
N
O
O-
OH
N
O-
O
B
O
O
B
O
O
O
O
O
O
Scheme 1. The synthetic route of the fluorescent probe CM-1 and
its possible reaction with BPO.
Fig.1. (a) UV-vis normalized absorption, black line: 1μM CM-1; red
line: 1μM CM-1+6μM BPO; (b) The fluorescence spectra of probe
CM-1 (1μM) in the presence of various concentrations of BPO (0-
6μM); (c) Calibration curve of CM-1 to BPO. The curve was plotted
with fluorescence intensity vs BPO concentration (0-6μM); (d) Linear
relationship between F/F0 and BPO concentration (0-0.7μM); (e) and
(f) Change in the fluorescence of the probe CM-1 before and after
adding 5μM BPO in 1μM CM-1 under excitation of 365 nm UV light.
Optical property, selectivity and effect of pH value on fluorescent
probe CM-1
As expected, CM-1 is non-fluorescent (Fig. 1b) due to the strong
electron-withdrawing boronic acid unit that prevented the CM-1
changed to fluorophore CM-CN. Once upon reaction with BPO, the
PBA moiety converting into an electron-donor -O- group, which
results quickly converting into CM-CN and a significant off-on
fluorescence enhancement at 485nm (with a~82-fold enhancement),
and its intensity increased linearly with the concentration of BPO
ranging from 0μM to 0.7μM (Fig. 1d). Moreover, once under
excitation of 365nm UV light, the changes in the fluorescence of the
probe CM-1 before and after reaction with BPO had obviously
changed (Fig. 1e and Fig. 1f), which allow to distinguish it with the
naked eye. The detection limit (utilizing the 3σ/k method) for BPO
was determined to be 3.3nM, which is enough for direct detection of
90
b
a
CM-1
CM-1+BPO
90
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
pH values
1
23
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-
2-
Fig.2. (a) Selectivity experiments, fluorescence response of 1μM CM- 1O2, ONOO-, KIO3, KMnO4, S2-, HSO3 , SO3
,
and cumene
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DOI: 10.1039/C8NJ06543H
1 to 6μM BPO or 50μM other analytes. The numbers from 1 to 23 hydroperoxide; (b) Effects of pH (4-9) on CM-1 in the absence or
-
correspond to black (CM-1) to TBHP, O2 , NO, OCl-, vitamins, NaClO4, presence of BPO.
NaClO3, BPO, amino acids (Cys, Hcy and GSH), NaNO2, KBrO3, H2O2,
6
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Table 1. Determination of BPO in wheat flour samples with probe CM-1.
9
Sample
BPO spiked (M)
BPO determined (M) meana ± SDb
Recovery (%)
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Wheat flour 1
Wheat flour 2
Wheat flour 3
Wheat flour 4
Wheat flour 5
0.0
not detected
1.0 × 10-6
3.0 × 10-6
5.0 × 10-6
7.0 × 10-6
(9.58 ± 0.05) × 10-7
(3.15 ± 0.06) × 10-6
(4.65 ± 0.03) × 10-6
(7.21 ± 0.03) × 10-6
95.8
105
93.0
103
aMean of three determinations. bSD: standard deviation
BPO Detection in wheat flour
According to some literature reports, BPA moiety was responsive to
BPO has been extensively used as flour bleaching agents, while hydrogen peroxide, and hydrogen peroxide is a major endogenous
excessive BPO could induce allergic reactions and potential ROS, so, the green channel showed weak fluorescence intensity[16]
carcinogenicity and exert an effect on human peripheral In contrast, treating CM-1-incubated cultured cells with 5μM BPO for
lymphocytes. To evaluate the practical applicability of CM-1 for real 30min, the fluorescence intensity enhanced in the green channel
samples such as wheat flour is of great significance. The
wheat flour of the pH was adjusted to 7.4 prior to use. At first, wheat showed that CM-1 was cell membrane-penetrable and could be used
flour was added to CM-1 resolution and fluorescence signal intensity for TP images in live cells.
.
obviously (Fig. 3e) by TP image. Taking together, these results
at 485nm was recorded. However, wheat flour may contain little
Finally, CM-1 was further applied for TP-excited fluorescence
seven fluorescent whitening agents (FWA135, FWA140, FWA162, image of BPO in liver tissue slices from rat with images at different
FWA184, FWA185, FWA367 and FWA393)17-18, they absorb invisible tissue depths recorded by TP image in the Z-scan mode. The imaging
ultraviolet light and emit visible blue or blue-violet fluorescence. Due results showed that the probe could be successfully applied for
to the excitation light employed in the experiment was 400nm and image of BPO in liver tissues at a depth of 40-150μm in green
not their best excitation light. Moreover, background interference fluorescent channel (see Fig. 4). These results demonstrated that the
has been deducted during the measurement. Thus, the fluorescent probe possessed a good staining capability and high penetrating
whitening agents could be not excited to affect the detection results. ability in tissues for image of BPO.
The calculated BPO concentration in the wheat flour sample was
48mg/Kg. The BPO stock solution at different concentrations was
spiked in these wheat flour samples, and the probe CM-1 was then
added to detect its concentration. The recovered BPO
concentrations were shown in Table 1. The results show good
recovery values, which confirmed that the other coexisting species
hardly interfere with the BPO assay. Furthermore, the determination
does not require a time-consuming separation. This indicates that
probe CM-1 has good capacity to quantify BPO in real samples.
Detection of BPO in live cells and tissues
In order to evaluate the imaging performance of CM-1, we used this
probe to detect BPO in living cells and tissues. HeLa cells were chosen
as the model cell line. Before imaging, the cytotoxicity of the CM-1
was tested. The results showed that it was nearly nontoxic for living
cells under experimental conditions (Fig. S1). Then, HeLa cells were
incubated with CM-1 (1μM) at 37 oC for 30min, followed by
excitation at 800nm for TP image, the HeLa cells showed weak
fluorescence intensity in the green channel by TP image (Fig. 3b).
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in biological systems for the study of physiVoielowgAircticalel Oanlninde
pathological functions of BPO.
DOI: 10.1039/C8NJ06543H
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Experimental section
Reagents and apparatus
e
d
f
Unless or otherwise specified, all chemicals were obtained from
commercial suppliers and used without further purication.
Water used in all experiments was double-distilled and purified
by a Milli-Qsystem (Millipore, USA). LC-MS analyses were
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4.0
g
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1
performed using an Agilent 1100 HPLC/MSD spectrometer.
HNMR and 13 CNMR spectra were recorded on a BrukerDRX-400
spectrometer operating at 400 and 100MHz. All chemical shifts
are reported in the standard notation of parts per million. UV-
vis absorption spectra were recorded in 1.0cm path length
quartz cuvettes on a Shimadzu 2450 UV-visible Spectrometer.
All fluorescence measurements were carried out on a G-9800A
fluorescence spectrometer (Agilen) with both excitation and
emission slits set at 2.5nm, respectively. The pH was measured
with a Mettler-Toledo Delta 320 pH meter. Fluorescence
imaging of HeLa cells and Tissues was conducted on a confocal
laser scanning microscope (Olympus, Japan) with 800 nm
excitation.
CM-1
CM-1+BPO
Fig.3. TP fluorescence microscope (TPFM) images of HeLa cells: (a)
Bright field image; (b) HeLa cells incubated with 1μM CM-1 for
30min; (c) Merged image of (b) and bright field image (a); (d) Bright
field image; (e) HeLa cells pre-treated with 5μM BPO for 15min and
then incubated with 1μM CM-1 for 30min image; (f) Merged image
of (e) and bright field image (d). (g) Intracellular relative fluorescence
intensity of (b: CM-1) and (e: CM-1+BPO). TP images: λex=800nm,
λem=470-550nm. All images were acquired with a 40×oil immersion
objective, scale bar: 20μm.
Synthesis of water-soluble TP fluorescent probe CM-1
Synthesis and characterization of compound 3: 0.193g
(1.00mmol) 4-(diethylamino)-salicylaldehyd, 0.297g 2-(4-
(bromomethyl) phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborola-
ne (1.00mmol), 0.294g (3mmol) K2CO3 and 40mL CH3CN were
added into 100mL flask with a reflux condenser, and the mixture
stirred at 65oC for 10 hours under argon protection. And then,
the mixture was filtrated, the solvent was evaporated by rotary
evaporator. At last, the target compound was obtained by the
quickly column chromatography (petroleum ether/ethyl
acetate = 4:1, v/v) to yield 3 as a yellow solid (0.389g, 95.1%).
1H NMR (400 MHz, DMSO-d6) δ: 8.5 (s,1H), 8.24-8.22 (d, J=8Hz,
1H), 7.73-7.71 (d, J=8Hz, 1H), 7.49-7.47 (d, J=8Hz, 1H), 6.52-6.50
(d, J=8Hz, 1H), 6.26 (s, 1H), 5.30 (s, 1H), 3.55-3.43 (dd, J=24Hz,
4H), 1.11-1.08 (t, J=6Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ:
164.92, 161.57, 154.19, 147.16, 140.75, 135.00, 130.41, 126.71,
118.48, 115.12, 109.03, 106.24, 95.26, 90.38, 83.99, 70.23,
62.58, 61.71, 44.87, 12.78; ESI-MS: [M]+ calcd: 359.3, found:
360.1.
a
Fig.4. TP fluorescence images of a frozen liver tissue slice for the
Spec3 mode imaging. Rat liver tissue slice was stained with 1μM CM-
1 for 60min at 37 oC, followed by treatment with 5μM BPO and
incubated for another 60min. (a) The Spec3 mode imaging at a depth
of 92μm. TP images: λex=800nm, λem=470-550nm. All images were
acquired with a 40×oil immersion objective, scale bar: 10 μm.
Synthesis and Characterization of CM-1: Cyanoacetic acid
(0.085g, 1mmol) and compound 3 (0.205g, 0.5mmol) were
dissolved in EtOH (40mL) with piperidine (0.2mL) and acetic acid
(0.2mL) under argon protection at 50oC for 12hours. Followed
by the solvent was evaporated in vacuo, and the crude solid was
purified by column chromatography on silica gel eluting
(CH2Cl2/MeOH = 100:1, v/v) to afford a yellow solid in 85.3%
yield. 1 H NMR (400 MHz, DMSO-d6) δ: 9.67 (s, 1H), 8.88 (s, 1H),
8.43-8.40 (d, J=12Hz, 1H), 7.76-7.74 (d, J=8Hz, 1H), 7.15-7.12 (d,
J=12Hz, 1H), 6.80-6.77 (d, J=12Hz, 1H), 6.59 (s,1H), 3.48-3.43 (q,
J=10Hz, 4H), 1.14-1.10 (t, J=8Hz, 6H); 13C NMR (100 MHz, DMSO-
d6) δ: 185.94, 156.60, 156.25, 154.00, 141.47, 139.18, 134.13,
Conclusions
In summary, we rational designed and synthesized a novel
water-soluble TP fluorescent probe CM-1 to detect BPO in
wheat flour and living cells and tissues. The probe is based on a
TP excitation D-π-A-structure of coumarin derivatives
fluorophore (CM-CN), which is obtained upon removal of a
trigger moiety by the BPO of interest. The probe was
demonstrated to efficiently image BPO produced in live cells,
and tissues, we believe that probe CM-1 will be a useful tool for
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130.17, 122.29, 118.72, 115.31, 109.83, 103.55, 44.71, 12.71; 31571874), Natural Science Foundation of Hunan Province
View Article Online
ESI-MS: [M]- calcd: 426.4, found: 426.8.
(Grants 2017JJ3060), and Science and TecDhOnI:o1l0o.1g0y39In/Cn8oNvJa0t6io54n3H
Platformand Talent Project of Hunan Province (2017TP1021).
Spectroscopic materials and methods
The fluorescence intensity of fluorescent probe CM-1 measurement
experiments were conduct in 10mM PBS buffer (pH7.4). The Notes and references
fluorescent emission spectra was recorded at 460 to 650nm using
400nm excitation.
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Selectivity experiments
NO was generated from of 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-
1
oxo-1-triazene(NOC-5), O2 was produced by the reaction of H2O2
with NaOCl, O2 was generated from KO2[19], ·OH was formed from
−
Fenton reaction between Fe2+(EDTA) and H2O2 quantitatively,
Fe2+(EDTA) concentrations represented ·OH concentrations [20], and
tert-butylhydroperoxide (t-BuOOH), cumene hydroperoxide could
also use to induce ROS in biological systems [21]
.
7
8
BPO detection in wheat flour
Wheat flour was purchased from
a
local supermarket
9
(Changsha, P.R. China). By the following procedure to prepare
the BPO samples. Firstly, the phosphate-buffered saline (PBS)
solutions (10mM, pH 7.4, 10% ethanol (because of the BPO is
water-insoluble)) containing various concentrations of BPO (0,
0.1, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, and 6μM) were mixed with
wheat flour (1g). Secondly, samples were sonicated for 3min
and filtered with organic membrane (0.22 μ m). At Last, the
resulting samples were prepared with probe CM-1, and the
fluorescence spectrum was detected by G-9800A fluorescence
spectrophotometer.
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Preparation and staining of cell and tissue cultures
Prior to the image experiments, the HeLa cells were washed
with PBS, and incubated with 1μM probe CM-1 for 30minutes
at 37 oC, then it washed with PBS for three times and incubated
with 10μM BPO for another 30munites at 37 oC, finally, the Hela
cells were washed with PBS three times again before image.
Like the cell cultures and staining, the rat liver tissue sections
were prepared by frozen section machine. The sections were
o
incubated with 1μM probe CM-1 for 1hour at 37 C, then it
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washed with PBS for three times and incubated with 5μM BPO
for another 1hour at 37 C, finally, the sections were washed
o
with PBS three times again before image. Confocal fluorescence
image of BPO in HeLa cells and liver tissues was observed under
an Olympus FV 1000 laser confocal microscope. Two-photon
image: λex=800nm, λem=470-550nm. All images were acquired
with a 40×oil immersion objective, scale bar: 10μm or 20μm.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
This work was supported by National Natural Science
Foundation of China (Grants 21605046, 81501619 and
This journal is © The Royal Society of Chemistry 20xx
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