A new resorufin-based spectroscopic probe for simple and sensitive detection of benzoyl peroxide via deboronation

Article abstract of DOI:10.1039/c2cc17768d

A new resorufin-based probe is developed, which exhibits a rapid and sensitive color and a fluorescence off-on response to benzoyl peroxide (BPO) in aqueous media containing 10% ethanol via deboronation. The probe has been applied to the simple detection of BPO in real samples such as wheat flour and antimicrobial agent. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc17768d

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 2809–2811
www.rsc.org/chemcomm
COMMUNICATION
A new resorufin-based spectroscopic probe for simple and sensitive
detection of benzoyl peroxide via deboronationw
Wei Chen, Zhao Li, Wen Shi and Huimin Ma*
Received 12th December 2011, Accepted 17th January 2012
DOI: 10.1039/c2cc17768d
A new resorufin-based probe is developed, which exhibits a rapid and
sensitive color and a fluorescence off–on response to benzoyl
peroxide (BPO) in aqueous media containing 10% ethanol via
deboronation. The probe has been applied to the simple detection
of BPO in real samples such as wheat flour and antimicrobial agent.
Reactive oxygen species (ROS) have attracted much attention
due to their key role in human health and disease.1 Benzoyl
peroxide (BPO), a strong oxidizer, is a ROS.2 Unlike most of
the other ROS, BPO has been extensively employed in our
daily life, as flour bleaching agents,3 antimicrobial agents to
Scheme 1 Synthesis of 1 and its possible reaction with BPO.
treat acne,4 and as polymerization initiators in various indus-
trial fields.5 The wide use of BPO is receiving increasing
strategy perhaps is to develop excellent spectroscopic probes and
utilize their rapid and sensitive spectroscopic responses to BPO to
concern because of its potential risks. According to the Inter-
accomplish the detection. Unfortunately, to the best of our
national Agency for Research on Cancer, BPO is not classified
knowledge, no fluorescent probes for BPO have been reported
as a carcinogen, but the existing studies have showed that it
so far, with the exception of indirect fluorescence determination.13
effects on human peripheral lymphocytes in vitro.6 Moreover,
Herein we present the design, synthesis, and characterization of 1
may be a potential tumor promoter and can exert genotoxic
(Scheme 1) as a new fluorescent probe for the BPO assay.
BPO can be decomposed into benzoic acid and other delete-
rious substances, like biphenyl and phenylbenzoate,7 which
In the construction of our probe, the key issue that we
confront is the choice of an appropriate signaling unit and
may further evoke tissue damage and diseases. Consequently,
recognition unit so that a latent fluorescent probe with low
the maximum usage level of BPO as either a flour additive or
fluorescence background could be prepared to afford high
an antimicrobial agent is strictly controlled by safety regula-
sensitivity.14 Here we choose resorufin as a signaling unit
tions of the respective country. For instance, in 2009 Codex
because of its good water-solubility and easy fluorescence
Alimentarius Commission set up a usage standard of BPO
quenching via 7-hydroxy substitution.15 On the other hand,
(o75 mg kgÀ1) in wheat flour;8 European Union and China
Chang and co-workers have reported that H2O2 can efficiently
even prohibited BPO to be used in wheat flour. Hence, the
and specifically trigger the deboronation of the probes bearing
development of simple and convenient methods for the BPO
arylboronate.16 We envision that BPO, having a similar
assay is of great significance for the safety of food and
property to H2O2, may also induce such a deboronation.
pharmaceutical products.
Therefore, arylboronate is used as the recognition unit for
Several methods have been proposed for detecting BPO,
such as electrochemistry,9 chemiluminescence,10 UV-spectro-
photometry,11 HPLC,12 and HPLC-MS.7 However, most of
BPO. As a result, the new probe 1 (Scheme 1) was synthesized
in good yield (60%) by treating resorufin sodium salt with
2-bromomethylphenylboronic acid pinacol ester.
these methods require a time-consuming sample pre-treatment
The spectroscopic response of 1 to BPO at varied concentrations
and separation, and thus are inconvenient for the fast detection
was investigated in 20 mM pH 7.4 KH2PO4–Na2HPO4 buffer
of BPO. To overcome this problem, a common but feasible
containing 10% (v/v) of ethanol (referred to the phosphate buffer).
As shown in Fig. 1A, 1 displays a moderate absorption peak at
Beijing National Laboratory for Molecular Sciences, Key Laboratory
of Analytical Chemistry for Living Biosystems, Institute of Chemistry,
484 nm with a shoulder at around 400 nm, but nearly no
absorption at 574 nm. Upon reaction with BPO, a strong absorp-
tion band centered at 574 nm is developed, and the absorbance at
574 nm is gradually increased with the increment of the BPO
concentration (Fig. S1, ESIw), concomitant with a distinct color
change from nearly colorless to pink (see the inset of Fig. 1A),
Chinese Academy of Sciences, Beijing 100190, China.
E-mail: mahm@iccas.ac.cn; Fax: +86-10-62559373;
Tel: +86-10-62554673
w Electronic supplementary information (ESI) available: Apparatus
and materials, detection of BPO, and other supporting data. See DOI:
10.1039/c2cc17768d
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 2809–2811 2809

View Article Online
Fig. 2 Fluorescence emission spectra (lex = 550 nm) of 1 (3 mM)
with varied concentrations of BPO (0, 0.5, 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32 mM BPO for curves 1–18, respectively). The
inset depicts the plot of fluorescence increase value (DF) of the reaction
system at lex/em = 550/585 nm against the corresponding reagent
blank (without BPO). The reactions were carried out for 15 min at
room temperature in the phosphate buffer.
showed that no noticeable fluorescence was produced when the
ethanol volume fraction was too low (o0.2%), and 10% (v/v) of
ethanol as a cosolvent may be used in this system (Fig. S3, ESIw).
Time course studies indicated that the reaction of 1 with BPO at
room temperature was fast, and the fluorescence increase could
reach a plateau in 10 min (Fig. S4, ESIw). For the purpose of
reproducibility, a reaction time of 15 min was employed in our
experiments. Interestingly, under the same conditions H2O2
produces a slower and weaker fluorescence response than BPO
(Fig. S4, ESIw), which may be ascribed to the presence of ethanol
promoting the solubility and thereby the reactivity of BPO.
Under the conditions optimized above, the fluorescence
increase value (DF) of 1 is directly proportional to the BPO
concentration in the range of 0.5 to 26 mM (inset of Fig. 2),
which gives a linear equation of DF = 39.1 + 56.2 Â [BPO]
(mM) (g = 0.997). The limit of detection17 is 23 nM BPO, also
showing a high sensitivity.
Fig. 1 (A) UV-vis and (B) fluorescence emission spectra (lex = 550 nm)
of 1 (3 mM) only and its reaction solutions with various species: 30 mM of
BPO, MgCl2, CaCl2, ZnSO4, CuCl2, Pb(AcO)2, FeCl2, FeCl3, CrCl3,
CoCl2, NiCl2, and CdCl2; 1.5 mM of vitamin B1, vitamin B6, vitamin C,
glucose, fructose, maltose, arginine, serine, glycine, NaF, NaBr, and NaI;
15 mM of NaCl and KNO3. The insets in (A) and (B) depict the color and
fluorescence change of the reaction systems in the absence and presence
of BPO, respectively. The reactions were carried out for 15 min at room
temperature in the phosphate buffer.
To assess the specificity of the reaction, the fluorescence
response of 1 to various possible coexisting substances was
examined in parallel under the same conditions. The tested
substances included inorganic salts, vitamins, saccharides,
amino acids and especially other important oxidants such as
H2O2, NaClO4, NaClO3, NaClO, NaNO2, KBrO3, KIO3,
KMnO4, K2Cr2O7, KO2, tert-butyl hydroperoxide, FeCl2/
H2O2, and CuSO4/H2O2/ascorbic acid. As shown in Fig. 1,
only BPO can produce a maximum absorption at 574 nm and
a large fluorescence enhancement at 585 nm, whereas the other
common non-oxidants do not show this behavior. More
interestingly, compared to BPO, the other oxidants tested
produce a much weaker fluorescence except H2O2 (Fig. S5,
ESIw). Fortunately, BPO is often employed as a single addi-
tive. This implies that 1 possesses high selectivity for BPO over
the other common substances. Besides, under the irradiation
of a UV Hg lamp (360 nm), a distinct fluorescence color
change of the reaction system in the absence and presence of
BPO can also be produced (inset of Fig. 1B), which is
convenient for the rapid detection of BPO.
which may be useful for the simple detection of BPO by the naked-
eye.
The fluorescence change of 1 upon reacting with BPO is
shown in Fig. 1B, which reveals that 1 itself has almost no
emission at 585 nm due to the alkylation of the 7-hydroxy
group of resorufin. This low background signal is extremely
desirable for sensitive detection. Upon gradual introduction of
BPO, an increase in fluorescence emission appears at 585 nm
(Fig. 2), which resembles the fluorescence feature of resorufin,
suggesting that the free fluorophore may be released. In
addition, 1 is rather stable, because no considerable change
in the fluorescence spectrum was observed after the probe’s
solution was stored at room temperature for two months.
The effect of pH of the reaction media varying from 4.8 to 8.3
was studied, revealing that pH 7.4 can be used for the present
system (Fig. S2, ESIw). Because the water-insoluble BPO in
different samples is usually extracted with an organic solvent
such as ethanol,10,11b,12c the effect of ethanol unavoidably intro-
duced into the detection system needs to be examined. The result
c
2810 Chem. Commun., 2012, 48, 2809–2811
This journal is The Royal Society of Chemistry 2012

View Article Online
To explore the spectroscopic response mechanism, the
reaction products of 1 with BPO were subjected to ESI-MS
and HPLC analyses. The ESI-MS spectrum of the reaction
solution of 1 with BPO (Fig. S6, ESIw) shows a peak at m/z =
211.9 ([M À Na]À), which is identical to that of resorufin. On
the other hand, resorufin is further verified as a major final
product by HPLC analysis. As shown in Fig. S7 (ESIw), the
chromatographic peaks of resorufin, BPO, and 1 are located at
4.20 min (peak a in curve A), 16.51 min (peak b in curve B),
and 19.60 min (peak c in curve C), respectively. After reaction
with BPO, the chromatographic peak of 1 decreases markedly,
and several new peaks appear (curve D), among which the
major one at 4.20 min clearly indicates the generation of
resorufin. Based on these data and the existing observation,16
we propose that the reaction of 1 with BPO may proceed
through the route depicted in Scheme 1: BPO oxidizes the
moiety of phenylboronic acid pinacol ester in 1, forming an
unstable intermediate, which is followed by hydrolysis and
1,4-elimination of o-quinone-methide to release the resorufin.
To evaluate the practical applicability of 1 for real samples
such as wheat flour and antimicrobial agent, the possible inter-
ferences of main coexisting species were studied on the detection
of BPO. The tolerable concentration was determined by the
criterion at which a species gave a relative error of no more than
5% in recovery of 15 mM of BPO. The results (Table S1, ESIw)
show that the coexisting species hardly interfere with the BPO
assay. Then, real wheat flour and antimicrobial agent samples
containing BPO were prepared (see ESIw), and the determination
of BPO content in these samples was demonstrated. The results
show that the determination can be completed in about 20 min
due to not requiring a time-consuming separation, and satis-
H. N. Kim, M. J. Kim, C. S. Cho, S. Y. Lee, W. J. Lee and
J. Yoon, Chem. Commun., 2011, 47, 4373–4375.
2 (a) S. H. Ibbotson, M. N. Moran, J. F. Nash and I. E. Kochevar,
J. Invest. Dermatol., 1999, 112, 933–938; (b) S. Fujisawa and
Y. Kadoma, Chemosphere, 2006, 62, 71–79.
3 B. P. Lamsal and J. M. Faubion, LWT–Food Sci. Technol., 2009,
42, 1461–1467.
4 S. R. Feldman, J. Tan, Y. Poulin, T. Dirschka, N. Kerrouche and
V. Manna, J. Am. Acad. Dermatol., 2011, 64, 1085–1091.
5 K. Matyjaszewski and J. H. Xia, Chem. Rev., 2001, 101,
2921–2990.
6 (a) G. Bhasin, H. Kauser and M. Athar, Arch. Toxicol., 2004, 78,
˙
139–146; (b) A. Yavuz, M. Topakta-s and E. S. Istifli, Turk. J. Biol.,
2010, 34, 15–24.
7 Y. Abe-Onishi, C. Yomota, N. Sugimoto, H. Kubota and
K. Tanamoto, J. Chromatogr., A, 2004, 1040, 209–214.
8 X. J. Jia, Y. Wu and P. Liu, Environ. Toxicol. Pharmacol., 2011, 31,
479–484.
9 (a) C. Y. Wang and X. Y. Hu, Anal. Lett., 2005, 38, 2175–2187;
(b) J. V. B. Kozan, R. P. Silva, S. H. P. Serrano, A. W. O. Lima
and L. Angnes, Biosens. Bioelectron., 2010, 25, 1143–1148; (c) M.
D. P. T. Sotomayor, I. L. T. Dias, G. O. Neto and L. T. Kubota,
Anal. Chim. Acta, 2003, 494, 199–205.
10 (a) W. P. Yang, Z. J. Zhang and X. Hun, Talanta, 2004, 62,
661–666; (b) W. Liu, Z. J. Zhang and L. Yang, Food Chem., 2006,
95, 693–698.
11 (a) I. Mori, H. Tominaga, Y. Fujita and T. Matsuo, Anal. Lett.,
1997, 30, 2433–2439; (b) H. Xie, H. Y. Wang, L. Y. Ma and
Y. Xiao, Spectrochim. Acta, Part A, 2005, 62, 197–202;
(c) A. Gupta, M. Gulati and N. K. Pandey, Rasayan J. Chem.,
2009, 2, 649–654.
12 (a) A. I. Saiz, G. D. Manrique and R. Fritz, J. Agric. Food Chem.,
2001, 49, 98–102; (b) P. Dehouck, E. V. Looy, E. Haghedooren,
K. Deckers, Y. V. Heyden, E. Adams, E. Roets and
J. Hoogmartens, J. Chromatogr., B: Anal. Technol. Biomed. Life
Sci., 2003, 794, 293–302; (c) M. Wada, K. Inoue, A. Ihara,
N. Kishikawa, K. Nakashima and N. Kuroda, J. Chromatogr.,
A, 2003, 987, 189–195; (d) Q. H. Wang, W. Z. Shi and C. Y. Hou,
J. Food Process. Preserv., 2010, 34, 414–424.
13 W. Chen, W. Shi, Z. Li, H. M. Ma, Y. Liu, J. H. Zhang and
Q. J. Liu, Anal. Chim. Acta, 2011, 708, 84–88.
factory recovery of BPO even with a low content (50 mg kgÀ1
)
can be obtained (Table S2, ESIw). This indicates that 1 is suitable
for the rapid and sensitive analysis of BPO.
14 (a) X. Q. Chen, M. Sun and H. M. Ma, Curr. Org. Chem., 2006, 10,
477–489; (b) J. X. Lu, C. D. Sun, W. Chen, H. M. Ma, W. Shi and
X. H. Li, Talanta, 2011, 83, 1050–1056; (c) A. Romieu,
D. Tavernier-Lohr, S. Pellet-Rostaing, M. Lemaire and
P.-Y. Renard, Tetrahedron Lett., 2010, 51, 3304–3308.
15 (a) M. Sun, D. H. Shangguan, H. M. Ma, L. H. Nie, X. H. Li,
S. X. Xiong, G. Q. Liu and W. Thiemann, Biopolymers, 2003, 72,
413–420; (b) W. Gao, B. Xing, R. Y. Tsien and J. Rao, J. Am.
Chem. Soc., 2003, 125, 11146–11147; (c) S. Y. Kim and J. I. Hong,
Org. Lett., 2007, 9, 3109–3112; (d) M. G. Choi, S. Cha, J. E. Park,
H. Lee, H. L. Jeon and S. K. Chang, Org. Lett., 2010, 12,
1468–1471; (e) M. G. Choi, J. Hwang, S. Eor and S. K. Chang,
Org. Lett., 2010, 12, 5624–5627.
16 (a) M. C. Y. Chang, A. Pralle, E. Y. Isacoff and C. J. Chang,
J. Am. Chem. Soc., 2004, 126, 15392–15393; (b) E. W. Miller,
A. E. Albers, A. Pralle, E. Y. Isacoff and C. J. Chang, J. Am.
Chem. Soc., 2005, 127, 16652–16659; (c) E. W. Miller,
O. Tulyathan, E. Y. Isacoff and C. J. Chang, Nat. Chem. Biol.,
2007, 3, 263–267; (d) G. C. van de Bittner, E. A. Dubikovskaya,
C. R. Bertozzi and C. J. Chang, Proc. Natl. Acad. Sci. U. S. A.,
2010, 107, 21316–21321; (e) C. Chung, D. Srikun, C. S. Lim,
C. J. Chang and B. R. Cho, Chem. Commun., 2011, 47, 9618–9620;
(f) A. R. Lippert, G. C. Van de Bittner and C. J. Chang, Acc.
Chem. Res., 2011, 44, 793–804.
In conclusion, we have developed a new resorufin-based probe
for BPO assay. The probe displays a rapid and sensitive color
and a fluorescence off–on response to BPO. Moreover, the
response of the probe is highly selective for BPO over other
common substances, which makes it of great potential use in
simple and quantitative detection of BPO in some real samples
such as wheat flour and antimicrobial agent.
We are grateful for the financial support from the National
Basic Research Program of China (No. 2010CB933502 and
2011CB935800), the NSF of China (No. 21105104 and 20935005),
and the Chinese Academy of Sciences (KJCX2-EW-N06-01).
Notes and references
1 (a) X. H. Li, G. X. Zhang, H. M. Ma, D. Q. Zhang, J. Li and
D. B. Zhu, J. Am. Chem. Soc., 2004, 126, 11543–11548;
(b) E. R. Stadtman, Free Radical Res., 2006, 40, 1250–1258;
(c) E. W. Miller and C. J. Chang, Curr. Opin. Chem. Biol., 2007,
11, 620–625; (d) X. Q. Chen, X. Z. Tian, I. Shin and J. Yoon,
Chem. Soc. Rev., 2011, 40, 4783–4804; (e) B. C. Dickinson and
C. J. Chang, Nat. Chem. Biol., 2011, 7, 504–511; (f) X. Q. Chen,
K. A. Lee, E. M. Ha, K. M. Lee, Y. Y. Seo, H. K. Choi,
17 (a) D. Feng, Y. Y. Zhang, T. T. Feng, W. Shi, X. H. Li and
H. M. Ma, Chem. Commun., 2011, 47, 10680–10682; (b) C. D. Sun,
W. Shi, Y. C. Song, W. Chen and H. M. Ma, Chem. Commun.,
2011, 47, 8638–8640.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 2809–2811 2811