A fluorescence turn-on H2O2 probe exhibits lysosome-localized fluorescence signals

Article abstract of DOI:10.1039/c2cc31632c

A new fluorescence turn-on probe that responds exclusively to H ₂O₂ exhibits subcellular localized fluorescence staining of lysosomes.

Full text of DOI:10.1039/c2cc31632c

View Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 5449–5451
www.rsc.org/chemcomm
COMMUNICATION
A fluorescence turn-on H2O2 probe exhibits lysosome-localized
fluorescence signalsw
Dayoung Song,a Jung Mi Lim,b Somin Cho,a Su-Jin Park,b Jaeheung Cho,a Dongmin Kang,b
Sue Goo Rhee,b Youngmin You*a and Wonwoo Nam*a
Received 5th March 2012, Accepted 3rd April 2012
DOI: 10.1039/c2cc31632c
A new fluorescence turn-on probe that responds exclusively to
H2O2 exhibits subcellular localized fluorescence staining of
lysosomes.
inspired us to develop a new H2O2 detection method using the
iron complex of a fluorophore. Paramagnetic iron in the
complex quenched the fluorescence emission of the fluoro-
phore, whereas activation by H2O2 triggered intramolecular
oxidative cleavage between the iron ionophore and the fluorophore
to produce a fluorescence turn-on signal. Oxidative interactions
between iron and H2O2 have been adopted previously for the
fluorescence detection of H2O2. In such approaches, aqueous
solutions containing a mixture of Fe ions and a fluorescent
poly(p-phenyleneethynylene) polymer displayed a fluorescence
turn-off response in the presence of enzymatically produced
H2O2.19 Another recent example is the use of an Fe3+ polypyridine
complex tethered to dihydroxyphenoxazine, the leuco form of
fluorescent resorufin.20 Although the mechanisms have not been
fully elucidated, H2O2 was proposed to oxidize the Fe center,
thereby stimulating the two-electron oxidation of dihydroxy-
phenoxazine to produce resorufin. This system, however, was
sensitive to other reactive oxygen species (ROSs), such as t-BuOOH
and O2ꢀꢁ. It should also be noted that iron-based H2O2 probes
described previously have never been applied to live specimens.
Herein, we report a novel method for the detection of fluores-
cence in response to the presence of intracellular H2O2 (Fig. 1). The
probe platform was readily constructed by complexation of iron
ions and Zinpyr-1 (ZP1), a fluorescent zinc ion probe,21 and this
idea might be extended to any ensemble of iron and fluorescent
metal ion sensors. We have proposed that the sensing mechanism
involves activation of the Fe center by H2O2, followed by oxidative
N-dealkylation of the ligand to liberate the fluorescence-quenching
Fe ionophore. The H2O2 probe exhibited exceptional selectivity for
Hydrogen peroxide (H2O2) plays a crucial role in many biological
processes in the human body, including respiration,1 intracellular
signalling,2 and immune responses.3,4 Despite the intense
investigation over the past several decades, the chemical
mechanisms of the H2O2-mediated processes have not been
fully established yet. Thus, the elucidation of these processes
would benefit from the development of sensors capable of
identifying and monitoring the trafficking and localization of
intracellular H2O2.5,6 Fluorescence probes are appropriate for
this application since fluorescence signals exhibit excellent
spatial resolution, fast response times, and high signal-to-noise
ratios. These benefits have been exploited in the development
of fluorescent H2O2 probes based on chemical reactions with
H2O2,7 including oxidation of phosphine,8 dihydrodichloro-
fluorescein,9 dihydrorhodamine,10 and dihydrophenoxazine.11
In addition, H2O2-induced cleavage of boronate,12 arylsulfonyl,13,14
benzyl,15 p-aminophenol,16 and hydroquinone16 has been
employed as a viable means for turning on fluorescence signals
by H2O2. Chelation of H2O2 by a Eu3+–tetracycline complex has
also been evaluated as a means for fluorescent detection.17
Although some of these probes have permitted the detection and
quantification of intracellular H2O2, such probe systems lack
full applicability to biological systems due to modest selectivity,
requirements for external enzymes, slow response time, or
incompatibility with biological milieus.
ꢁ
H2O2 over other ROSs, such as ꢀOH, t-BuOOH, and O2
.
ꢀ
An alternative approach to fluorescent H2O2 detection is the
mimicking of bioredox processes between H2O2 and metallo-
enzymes, such as the interaction between iron porphyrin centers of
heme enzymes and H2O2, which catalyzes the oxidation of a wide
variety of biological substrates.18 Thus, biomimetic reactions have
Interestingly, the probe localized at the lysosomes of live HeLa
cells, producing an organelle-specific response to intracellular H2O2.
a Department of Bioinspired Science, Ewha Womans University, Seoul
120-750, Korea. E-mail: odds2@ewha.ac.kr, wwnam@ewha.ac.kr;
Fax: +82-2-3277-4441; Tel: +82-2-3277-2392
b Division of Life and Pharmaceutical Sciences, Ewha Womans
University, Seoul 120-750, Korea
w Electronic supplementary information (ESI) available: Experimental
details and more spectroscopic data. CCDC 865223. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c2cc31632c
Fig. 1 Structure of the H2O2 probe, ZP1Fe2, and its fluorescence
turn-on response. Photographs showing changes in the absorption
(left panel; under room light) and fluorescence emission (right panel;
under 365 nm UV light) after the addition of H2O2.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 5449–5451 5449

View Online
The H2O2 probe ZP1Fe2 was prepared by mixing a 20 mM
aqueous FeSO4 solution with a DMSO solution containing
10 mM ZP1. The probe was fully soluble in a pH 7.0 aqueous
buffer (25 mM PIPES) and was stable under ambient conditions.
The ZP1Fe2 solution displayed an absorption peak at 507 nm
(e = 22 000 Mꢁ1 cmꢁ1) with a shoulder band around 533 nm in
the UV-vis absorption spectrum (ESIw, Fig. S1). The absorption
peak was blue-shifted relative to the absorption band of the
metal-free form (ZP1; labs = 513 nm). Similarly, the fluorescence
emission peak wavelength (lems = 528 nm) for ZP1Fe2 was blue-
shifted relative to ZP1 (lems = 536 nm). ZP1Fe2 was weakly
fluorescent (F = 0.017), and fluorescence titration isotherms and
Job’s plots unambiguously indicated a 1 : 2 binding stoichiometry
for ZP1 and Fe complexation (ESIw, Fig. S2). The fluorescence
properties were not affected by the presence of biologically
relevant metal ions, including Na, Mg, K, Ca, Mn, Ni, Zn,
Cu, Cd, and Hg (ESIw, Fig. S3). As expected based on the
Irving–Williams series, Cu and Co ions seemed to displace the
Fe ions in the ZP1Fe2 complex, turning off the fluorescence.
The fluorescence intensity of ZP1Fe2 was also resistant to pH
changes between 6.25 and 7.81 (ESIw, Fig. S4).
Fig. 3 The selective fluorescence responseꢀof ZP1Fe2 (10 mM) to H2O2
relative to other reactive oxygen species (O2 ꢁ, 1 mM KO2; OClꢁ, 1 mM
NaOCl; 1 mM t-BuOOH; t-BuOꢀ, 1 mM FeSO4 + 100 mM t-BuOOH;
ꢀOH, 1 mM FeSO4 + 100 mM H2O2; ꢀNO, excess NO gas; 1O2,
photosensitization by 1 mM methylene blue)z and chemical oxidants
(CAN, 1 mM (NH4)2Ce(NO3)6; DDQ, 1 mM 2,3-dichloro-5,6-dicyano-p-
benzoquinone). Conditions: lex = 512 nm; pH 7.0 buffer (25 mM PIPES).
did not increase in proportion to the amount of ꢀOH present
(0–1 mM Fe2EDTA + 1.5 mM H2O2, pH 7.0 PIPES buffer,
25 1C; ESIw, Fig. S6). Furthermore, the fluorescence intensity of
a solution containing 10 mM iron-free ZP1 was not affected by
the presence of ꢀOH (0–100 mM FeSO4 + 0–100 mM K2EDTA +
1.5 mM H2O2, pH 7.0 PIPES buffer, 25 1C; ESIw, Fig. S7). These
results led us to exclude the possibility that ꢀOH participated in the
fluorescence H2O2 response of ZP1Fe2.
As shown in Fig. 2, addition of an excess of H2O2 to a 10 mM
ZP1Fe2 solution provoked fluorescence turn-on at pH 7.0 and
25 1C, with a 22-fold increase in the fluorescence quantum yield
(F = 0.38). This turn-on ratio is smaller than those of the
boronate-based fluorescent H2O2 probes,12 but is still applicable
in typical fluorescence microscope experiments. The ZP1Fe2
fluorescence turn-on response was analyzed by fitting to a
pseudo-first order kinetics model, giving a rate constant k =
0.49 ꢂ 0.03 Mꢁ1 sꢁ1 (ESIw, Fig. S5). The limit of detection
(LOD) for H2O2 was determined to be 29 mM using the three-
sigma method. The H2O2 probe exhibited exceptional selectivity
over other ROSs. Asꢁsummarized in Fig. 3, almost no response
The reaction between ZP1Fe2 and H2O2 produces 2,7-dichloro-
fluorescein through dissociation of the Fe ionophores, as
evidenced by the identical UV-vis absorption and fluorescence
spectra of the product solution and the authentic 2,7-dichloro-
fluorescein compound (ESIw, Fig. S8). Since the dinuclear iron
centers in ZP1Fe2 complicated the precise analyses, we synthesized
a mononuclear iron complex, 2-((di(2-picolyl)amino)methyl)-
phenol ([Fe(DPAPhOH)]), as a model compound of ZP1Fe2
(Fig. 4a). [Fe(DPAPhOH)] displayed an absorption maximum
at 512 nm (e = 688 Mꢁ1 cmꢁ1) due to a ligand-to-metal
charge-transfer (LMCT) transition between the phenolate and
the Fe center.22 Upon addition of 50 equiv. of H2O2, this band
disappeared with a rate constant k = 0.11 Mꢁ1 sꢁ1 (Fig. 4b),
indicating liberation of the phenolate moiety. Cleavage of
the phenolate moiety was also observed by 1H NMR
ꢀ
was observed for O2
,
ꢁOCl, t-BuOOH, NO, and 1O2, and only
a small increase in the fluorescence intensity was observed in the
ꢀ
cases of t-BuOꢀ and OH. In addition, chemical oxidants, such
as (NH4)2CeIV(NO3)6 (CAN) and 2,3-dichloro-5,6-dicyano-p-
benzoquinone (DDQ), did not produce fluorescence turn on
even at concentrations as high as 1 mM. The improved H2O2
selectivity suggested that the fluorescence response of ZP1Fe2
was a consequence of a reaction involving H2O2 bound at
the iron center rather than other oxidants derived from H2O2
(e.g., ꢀOH). In fact, the fluorescence intensity of 10 mM ZP1Fe2
Fig. 2 (a) Fluorescence spectral changes of ZP1Fe2 (10 mM, PIPES
Fig. 4 (a) Crystal structurey (drawn at the 30% probability level) and
H2O2-induced cleavage of [Fe(DPAPhOH)]. Hydrogen atoms are omitted
for clarity. (b) UV-vis absorption spectra of [Fe(DPAPhOH)] (0.5 mM,
pH 7.0 PIPES buffer, 25 1C) after the addition of H2O2 (50 equiv.): black,
before the addition of H2O2; red, 10 min after the addition of H2O2.
buffer at pH 7.0, 25 1C) after the addition of H2O2 (100 equiv.). lex
=
512 nm. (b) Time traces of the fluorescence intensity of a ZP1Fe2
solution observed at 524 nm in the presence of various concentrations
of H2O2 (0–1000 equiv.).
c
5450 Chem. Commun., 2012, 48, 5449–5451
This journal is The Royal Society of Chemistry 2012

View Online
from the fluorophore. The unique mechanism affords exceptional
selectivity for H2O2 over other ROSs. This H2O2 probe is
applicable to live cell imaging and has the ability to detect
intracellular H2O2 at the lysosome.
This research was supported by CRI, GRL (2010-00353),
and WCU programs (R31-2008-000-10010-0) (W.N.), Basic
Science Research Program (2011-0010514) (D.K.), and Ewha
Womans University (RP-Grant 2010) (Y.Y.).
Notes and references
z Refer to ESI for a description of the ROSs preparation.
Fig. 5 (a) Fluorescent detection of intracellular H2O2 in live HeLa
cells: Bright field (top) and fluorescence (bottom) micrographs of
HeLa cells incubated with ZP1Fe2 (10 mM, 30 min). Cells shown
in the right panels were treated with H2O2 (200 mM, 10 min). Scale
bar = 50 mm. (b) Subcellular localized fluorescence signals from the
lysosomes. HeLa cells were pretreated with H2O2 (200 mM, 10 min)
and incubated with ZP1Fe2 (10 mM, 30 min) and LysoTracker-Red
(50 nM, 1 h): (i) ZP1Fe2 signals; (ii) LysoTracker-Red signals;
(iii) merged images (i) and (ii); (iv) bright field image. Scale bar =
10 mm.
y Crystal
C21H24Cl2FeN4O10, monoclinic, Cc, Z = 4, a = 11.3781(2), b =
data
for
[Fe(DPAPhOH)(H2O)(CH3CN)](ClO4)2:
18.5161(3), c = 12.4123(2) A, b = 91.2170(10)1, V = 2614.41(8) A3,
r
calcd = 1.573 g cmꢁ3, m = 0.842 mmꢁ1, R1 = 0.0304, wR2 = 0.0802,
GOF = 0.913 for 5695 unique reflections and 356 variables. Flack
parameter
=
ꢁ0.001(11). The crystallographic data for
[Fe(DPAPhOH)(H2O)(CH3CN)](ClO4)2 are listed in ESI, Table S1,
and Table S2 lists the selected bond distances and angles. CCDC
865223 for [Fe(DPAPhOH)(H2O)(CH3CN)](ClO4)2 contains the
supplementary crystallographic data associated with this paper.
1 R. S. Balaban, S. Nemoto and T. Finkel, Cell, 2005, 120, 483–495.
2 S. G. Rhee, Science, 2006, 312, 1882–1883.
3 H. A. Woo, S. H. Yim, D. H. Shin, D. Kang, D.-Y. Yu and
S. G. Rhee, Cell, 2010, 140, 517–528.
4 J. D. Lambeth, Nat. Rev. Immunol., 2004, 4, 181–189.
5 N. Soh, Anal. Bioanal. Chem., 2006, 386, 532–543.
6 X. Chen, X. Tian, I. Shin and J. Yoon, Chem. Soc. Rev., 2011, 40,
4783–4804.
¨ ¨
7 M. Schaferling, D. B. M. Grogel and S. Schreml, Microchim. Acta,
2011, 174, 1–18.
(D2O solution containing 2.5 vol% d6-DMSO) and electro-
spray ionization mass spectrometry (ESIw, Fig. S9 and S10). It
is likely that the dissociation proceeded via a reactive H2O2
adduct of the iron center, such as FeIII–OOH or FeIVQO,
similar to the N-dealkylation mediated by cytochrome P-450
enzymes.23 In fact, the formation of a reactive high-spin FeIII
species was supported by the observation of a strong EPR
signal (g = 4.30) in aqueous solutions containing H2O2 and
[Fe(DPAPhOH)] (ESIw, Fig. S11).
8 N. Soh, O. Sakawaki, K. Makihara, Y. Odo, T. Fukaminato,
T. Kawai, M. Irie and T. Imato, Bioorg. Med. Chem., 2005, 13,
1131–1139.
With an understanding of the fluorescence turn-on response,
we evaluated the biological utility of the H2O2 probe. ZP1Fe2 is
cell-permeable and nontoxic to live mammalian cells. In fact,
MTT assays revealed that the cell viabilities of HeLa cells and
COS7 cells were not affected by incubation with 0–20 mM
ZP1Fe2 for 24 h. In contrast, significant cell death was observed
when the cells were incubated with 1 mM staurosporine under
identical conditions (ESIw, Fig. S12). Live HeLa cells were
therefore incubated with 10 mM ZP1Fe2 for 30 min, and
fluorescence micrographs were taken before and after treatment
with 200 mM H2O2. Turn-on fluorescence signals were observed
for the HeLa cells treated with H2O2 (Fig. 5a). Interestingly, the
punctate spots were scattered near the perinuclear regions
rather than being evenly distributed in the cytosol. The signal
localization was examined by performing colocalization experi-
ments using the live HeLa cells pretreated with 200 mM H2O2,
and then incubated with 10 mM ZP1Fe2 (30 min) and 50 nM
LysoTracker-Red (1 h), a lysosome-specific stain. As shown
in Fig. 5b, the fluorescence patterns of ZP1Fe2 and the
LysoTracker-Red signals overlapped perfectly, indicating
unambiguously that the fluorescence response was localized
at the lysosome. Because intracellular H2O2 metabolism by
lysosomal enzymes is closely linked to oxidative stress,24 the
subcellular detection utility of ZP1Fe2 will be useful in the studies
of lysosomal H2O2.
9 S. L. Hempel, G. R. Buettner, Y. Q. O’Malley, D. A. Wessels and
D. M. Flaherty, Free Radical Biol. Med., 1999, 27, 146–159.
10 A. Negre-Salvayre, N. Auge, C. Duval, F. Robbesyn, J.-C. Thiers,
´
D. Nazzal, H. Benoist and R. Salvayre, Methods Enzymol., 2002,
352, 62–71.
11 M. Zhou, Z. Diwu, N. Panchuk-Voloshina and R. P. Haugland,
Anal. Biochem., 1997, 253, 162–168.
12 A. R. Lippert, G. C. Van de Bittner and C. J. Chang, Acc. Chem.
Res., 2011, 44, 793–804.
13 H. Maeda, Y. Fukuyasu, S. Yoshida, M. Fukuda, K. Saeki,
H. Matsuno, Y. Yamauchi, K. Yoshida, K. Hirata and
K. Miyamoto, Angew. Chem., Int. Ed., 2004, 43, 2389–2391.
14 K. Xu, B. Tang, H. Huang, G. Yang, Z. Chen, P. Li and L. An,
Chem. Commun., 2005, 5974–5976.
15 M. Abo, Y. Urano, K. Hanaoka, T. Terai, T. Komatsu and
T. Nagano, J. Am. Chem. Soc., 2011, 133, 10629–10637.
16 K.-i. Setsukinai, Y. Urano, K. Kakinuma, H. J. Majima and
T. Nagano, J. Biol. Chem., 2003, 278, 3170–3175.
17 O. S. Wolfbeis, A. Duerkop, M. Wu and Z. Lin, Angew. Chem.,
Int. Ed., 2002, 41, 4495–4498.
18 G. Battistuzzi, M. Bellei, C. A. Bortolotti and M. Sola, Arch.
Biochem. Biophys., 2010, 500, 21–36.
19 T. Zhang, H. Fan, G. Liu, J. Jiang, J. Zhou and Q. Jin, Chem.
Commun., 2008, 5414–5416.
20 Y. Hitomi, T. Takeyasu, T. Funabiki and M. Kodera, Anal.
Chem., 2011, 83, 9213–9316.
21 G. K. Walkup, S. C. Burdette, S. J. Lippard and R. Y. Tsien,
J. Am. Chem. Soc., 2000, 122, 5644–5645.
22 Y. Nishida, H. Shimo and S. Kida, J. Chem.Soc., Chem. Commun.,
1984, 1611–1612.
23 F. P. Guengerich and T. L. Macdonald, Acc. Chem. Res., 1984, 17,
9–16.
24 T. Kurz, A. Terman, B. Gustafsson and U. T. Brunk, Biochim.
Biophys. Acta, Gen. Subj., 2008, 1780, 1291–1303.
In summary, we developed a new fluorescent probe for
H2O2 based on the cleavage of the paramagnetic Fe ionophore
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 5449–5451 5451