A turn-on fluorescent probe based on hydroxylamine oxidation for detecting ferric ion selectively in living cells

Article abstract of DOI:10.1039/c2cc31426f

We have described a turn on fluorescent probe BOD-NHOH based on hydroxylamine oxidation for detecting intracellular ferric ions. The probe comprises a signal transducer of BODIPY dye and a Fe³⁺-response modulator of hydroxylamine. It is readily employed for assessing intracellular ferric ion levels, and confocal imaging is achieved successfully.

Full text of DOI:10.1039/c2cc31426f

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A turn-on fluorescent probe based on hydroxylamine oxidation for
detecting ferric ion selectively in living cellsw
Rui Wang,a Fabiao Yu,b Ping Liua and Lingxin Chen*a
Received 27th February 2012, Accepted 22nd March 2012
DOI: 10.1039/c2cc31426f
We have described a turn on fluorescent probe BOD-NHOH
based on hydroxylamine oxidation for detecting intracellular
ferric ions. The probe comprises a signal transducer of BODIPY
dye and a Fe3+-response modulator of hydroxylamine. It is
readily employed for assessing intracellular ferric ion levels, and
confocal imaging is achieved successfully.
much time and effort into dicscovering a new chelator-independent
method of detecting Fe3+ ions with a turned on chemical switch.
It is noteworthy that hydroxylamine can be easily oxidized
by Fe3+ while other metal ions have almost no interference.13
As far as we know, this reaction has never been used to
develop a selective fluorescent probe for Fe3+. We anticipated
that the lone pair electrons in the ammonia group could
quench the fluorescence of an appropriate fluorophore
through a fast photoinduced electron transfer (PET) process.
With this in mind, we designed and synthesized a turn-on
fluorescence probe of 8-hydroxyethanam-ine-4,4-difluoro-1,7-
diphenyl-3,5-di(1-bromo-phen-yl)-4-bora-3a,4a-diaza-s-indacene
(BOD-NHOH) for the detection of Fe3+ in living cells, as shown
in Scheme 1.
The highly sensitive and selective detection of the ferric ion
(Fe3+) is of great significance for the study of its physiological
functions in organisms.1,2 Iron is the most abundant transition
metal in cellular systems and is of outstanding importance due
to its biological functions, including essential roles in oxygen
uptake, oxygen metabolism, electron transfer,3 and transcriptional
regulation.4 Ferric ion deficiency and overload can induce various
diseases, such as Parkinson’s disease, Alzheimer’s disease and
cancer.5 Therefore, much effort has been focused on the
investigation of the biological functions and deleterious effects
of Fe3+ in vivo. Recently, a number of sensitive and selective
methods have been reported for conducting such research.6 In
comparison with other technologies, fluorescence method provides
greater sensitivity, less invasiveness, and more convenience.7 There
has been an explosive increase in the number of fluorescent
metal ion probes, which are usually developed by combining a
fluorophore with a known metal ion chelator.8 However, Fe3+
is well-known as a fluorescence quencher due to its paramagnetic
nature. As a result, many of the available Fe3+ probes are based
on fluorescence quenching mechanisms.9 Only a few ‘‘turn-on’’
probes that work with a selective response to Fe3+ have been
reported.10 It is generally recognized that one probe with a
fluorescence turn-on signal for specific events is more efficient.7,11
Compared with ‘‘turn-off’’ probes, the greatest advantage of
‘‘turn-on’’ probes is the ease of measuring low-concentration
contrast relative to a ‘‘dark’’ background, which reduces the
likelihood of false positive signals and increases sensitivity, as
demonstrated by numerous studies.12 Therefore, we have invested
As an overall strategy, the probe is composed of two moieties.
One is a fluorophore, for which a BODIPY (4,4-difluoro-4-3a,4a-
diza-s-indacene) platform is selected as the signal transducer,
because the dyes have high molar absorption coefficients and
fluorescence quantum yields, sharp absorption and fluorescence
peaks in the visible region.14 The other moiety is the Fe3+-response
switch, for which hydroxylamine is chosen as the modulator.11
After being equipped with hydroxylamine, the fluorescence of the
BODIPY platform is quenched by PET between the modulator
and the transducer.15 However, excitingly, the hydroxylamine
oxidation can eliminate the PET, triggering an increase of
the fluorescence emission, thereby allowing the formation of
a ‘‘turn-on’’ fluorescent probe (Scheme 1). As expected, the
probe exhibits a selective fluorescence response to Fe3+
,
instead of other physiological relevant metal ions. We next
described the synthesis, spectroscopy, and application of the
Fe3+-selective fluorescent probe.
We examined the effect of pH on the fluorescence. The
fluorescence of the probe began to quench at pH above 5.8. On
the other hand, we explained the phenomenon of the fluorescence
a Key Laboratory of Coastal Zone Environmental Processes,
Yantai Institute of Coastal Zone Research, Chinese Academy of
Sciences, Yantai 264003, P. R. China. E-mail: lxchen@yic.ac.cn;
Fax: (+86) 535-2109130; Tel: (+86) 535-2109130
b School of Chemical Engineering, Dalian University of Technology,
Dalian 116024, P. R. China. E-mail: yufabiao@163.com
w Electronic supplementary information (ESI) available: Synthesis;
experimental details and spectrocopical data; additional cell images.
See DOI: 10.1039/c2cc31426f
Scheme 1 Structure of BOD-NHOH and proposed mechanism of
fluorescence enhancement.
c
5310 Chem. Commun., 2012, 48, 5310–5312
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decrease under alkaline conditions in terms of deprotonation of
the ammonia group which increased the electron-donating
ability of the lone pair electrons in the ammonia group and
raised the PET. The pKa value of protonated hydroxylamine is
5.97.16 These spectral properties suggest that PET is regulated
in the molecule successfully.12 As shown in Fig. S1w, the pH of
the medium had hardly any effect on the oxidized probe
(BOD-CH3) over the pH range from 6.8 to 7.8. Thus, the probe
was expected to work well under physiological conditions.17
Simultaneously, considering the pKa of the probe and the
hydrolysis property of Fe3+, pH 7.0 was chosen as part of the
optimal test conditions,18 i.e. pH 7.0, 40 mM HEPES.
Fig. 2 (a) Fluorescence responses (lex = 580 nm, lem = 610 nm)
of 1.0 mM probe to diverse metal ions in 40 mM HEPES (pH 7.0): 1,
Fe3+ (50.0 mM); 2, Cu2+ (0.2 mM); 3, Ag+ (0.3 mM); 4, Na+
(120 mM); 5, Ca2+ (0.5 mM); 6, Mg2+ (0.5 mM); 7, K+ (120 mM); 8,
Zn2+; 9, Pb2+; 10, Mn2+; 11, Hg2+; 12, Co2+; 13, Ni2+; 14, Cd2+
(above ions were all 0.3 mM); 15, Fe2+ (0.2 mM). Bars present
fluorescence responses to various metal ions. In each group, the black
bar represents the fluorescence intensity after addition of analytes,
and the gray bar represents that after the subsequent addition of
50.0 mM Fe3+. (b) Response of BOD-NHOH to Fe3+ and various other
cations in (a) over 1 h.
The fluorescence and absorption spectra of the probe (1.0 mM)
were examined in the HEPES aqueous buffer (pH 7.0, 40 mM).
Under these simulated physiological conditions, BOD-NHOH
exhibits an absorption maximum at lmax = 580 nm (e = 1.7 Â
106 MÀ1 cmÀ1). There is no significant change in the absorption
spectrum upon changing the state from ‘‘off’’ to ‘‘on’’ (Fig. 1a).
The fluorescence titration of the probe in the presence of different
concentrations of Fe3+ was then performed. As shown in
Fig. 1b, the fluorescence intensity (lem = 615 nm) increases
significantly upon addition of Fe3+. When increasing the
concentration of Fe3+ up to 50 mM, there is an impressive
fluorescence enhancement from the background level of the
probe (from F = 0.01 to F = 0.35). These spectral properties
indicate that the PET process is successfully regulated in the
molecule.19 We tested the ability of the probe to quantify
Fe3+ in water solution. Fig. 1b inset demonstrates that there
is a good linear dependence of fluorescence intensity on Fe3+
concentration (0–50 mM), and the regression equation was
F615 nm = 2674.9 [Fe3+] (mM) + 9457.7, with r = 0.997.
Upon increasing the Fe3+ concentration to 60 mM, the
fluorescent emission intensity reaches the saturation threshold
(Fig. S2w).
Ag+ levels (50 mM) will minimize this interference. The results
indicate that the present probe shows excellent selectivity for
Fe3+ over other abundant cellular cations. To check whether
the chemoselective hydroxylamine switch might turn on upon
incubation with other cations over time, the probe’s response to
various cations was measured for 1 h. As Fig. 2b demonstrates,
BOD-NHOH selectively responded to Fe3+ by a turn-on fluores-
cence switch and avoided a host of other ions.
We next established the ability of BOD-NHOH to track
Fe3+ levels in living cells by using a MCF-7 cell model exposed
to Fe3+. The MCF-7 cells were incubated with 0.01, 0.1, 1, 10,
and 100 mM Fe3+ solutions for 30 min in RPMI 1640 Medium
at 37 1C, and then washed with physiological saline to remove
excess Fe3+ ions. The treated cells were incubated with BOD-
NHOH (10.0 mM) in RPMI 1640 Medium for 15 min. After
being washed with physiological saline, the cells were imaged
by a confocal fluorescence microscope. As a control, the cells not
treated with Fe3+ were also imaged. The control experiments
showed faint fluorescence (Fig. 3a), but those treated with
various concentrations of Fe3+ displayed different fluorescence
intensities. The confocal fluorescence images grew brighter as the
concentration of Fe3+ increased from 0.1 to 100 mM (Fig. 3b–f).
We selected the whole region in the visual field (Fig. 3b–f) as the
BOD-NHOH exhibits a selective turn-on fluorescence response
to Fe3+ in water. Responses of 1.0 mM BOD-NHOH to the
presence of various biologically relevant metal ions are shown in
Fig. 2. The fluorescence profiles of BOD-NHOH are unchanged
in the presence of Zn2+, Pb2+, Mn2+, Hg2+, Co2+, Ni2+, Cd2+
and Fe2+ (all in 0.3 mM), Na+ and K+ (both in 120 mM), Ca2+
and Mg2+ (both in 0.5 mM). Of all the tested metal ions, only the
addition of Cu2+ (in 0.2 mM) and Ag+ (in 0.3 mM) gave a
limited increase in fluorescence intensity. But lower Cu2+ and
Fig. 1 (a) Absorption spectra of probe (1.0 mM) treated with various
concentrations of Fe3+ in 40 mM, pH 7.0 HEPES aqueous buffer.
(b) Fluorescence responses of 1.0 mM probe to Fe3+ concentrations of
0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mM. Spectra were acquired in
40 mM, pH 7.0 HEPES buffer with excitation at 585 nm and emission
ranging from 590 to 650 nm. Inset: Relationship between the fluores-
cence intensity at 615 nm of probe and [Fe3+].
Fig. 3 Confocal fluorescence images of living MCF-7 cells incubated
with various concentrations of Fe3+. MCF-7 cells loaded with
10.0 mM BOD-NHOH and Fe3+ for 15 min of (a) Control,
(b) 0.01 mM, (c) 0.1 mM, (d) 1 mM, (e) 10 mM and (f) 100 mM. Scale
bar is 10 mm.
c
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Chem. Commun., 2012, 48, 5310–5312 5311

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grant No. 20833008), NKBRSF (grant No. 2007CB815202),
the Innovation Projects of the Chinese Academy of Sciences
(grant No. KZCX2-EW-206), and the 100 Talents Program of
the Chinese Academy of Sciences.
Notes and references
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Fig. 5 Confocal fluorescence images of MCF-7 cells incubated with
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In summary, we have developed a new fluorescent probe
that exhibits high sensitivity and selectivity for monitoring
Fe3+ both in aqueous solution and living cells. The probe
exhibits a turn-on fluorescent response upon detecting Fe3+
compared to other biologically relevant metal ions. Confocal
microscopy images indicate that BOD-NHOH can be used for
detecting changes in Fe3+ levels within living cells. To the best of
our knowledge, this is the first example of a fluorescence probe
that can be used to successfully detect Fe3+ based on hydroxyl-
amine oxidation both in aqueous solution and living cells. We
anticipate that the new probe will lead to many new opportunities
for studying the biological effect of Fe3+ in living cells.
This work was financially supported by the National
Natural Science Foundation of China (grant No. 20975089,
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5312 Chem. Commun., 2012, 48, 5310–5312
This journal is The Royal Society of Chemistry 2012