A mitochondrial-targeted two-photon probe for zinc ion

Article abstract of DOI:10.1021/ja200444t

We report a two-photon probe (SZn-Mito) for mitochondrial zinc ions ([Zn²⁺]_m). This probe shows a 7-fold enhancement of two-photon-excited fluorescence in response to Zn²⁺ with a dissociation constant (KdTP) of 3.1 ± 0.1 n

Full text of DOI:10.1021/ja200444t

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A Mitochondrial-Targeted Two-Photon Probe for Zinc Ion
Goutam Masanta,† Chang Su Lim,§ Hyung Joong Kim,‡ Ji Hee Han,§ Hwan Myung Kim,*,†,‡ and
Bong Rae Cho*,§
†Molecular Science and Technology Research Center, ‡Division of Energy Systems Research, Ajou University, Suwon 443-749, Korea
§Department of Chemistry, Korea University, 1-Anamdong, Seoul 136-701, Korea
S Supporting Information
b
Scheme 1. Synthesis of SZn-Mitoa
ABSTRACT: We report a two-photon probe (SZn-Mito)
for mitochondrial zinc ions ([Zn2þ]m). This probe shows a
7-fold enhancement of two-photon-excited fluorescence in
response to Zn2þ with a dissociation constant (KTdP) of 3.1 (
0.1 nM and pH insensitivity in the biologically relevant range,
allowing the detection of [Zn2þ]m in a rat hippocampal slice at
a depth of 100ꢀ200 μm without interference from other metal
ions through the use of two-photon microscopy.
inc is the second most abundant transition-metal ion in the
Z
human body.1 In the brain, 5ꢀ20% of the total Zn2þ is stored
a Conditions (a): p-toluenesulfonic acid, CHCl3.
in presynaptic vesicles, and the highest intracellular free Zn2þ
([Zn2þ]i) is found in the hippocampus.2 Zn2þ modulates brain
excitability and plays a key role in synaptic plasticity.3 For proper
brain function, maintaining [Zn2þ]i homeostasis is vital. Imbalance
in [Zn2þ]i has been linked to neurological disorders such as
Alzheimer’s and Parkinson’s diseases.4 Recent studies suggest that
mitochondria take up evoked [Zn2þ]i increases, thereby controlling
the [Zn2þ]i homeostasis.5,6 On the other hand, a strong elevation of
intramitochondrial Zn2þ ([Zn2þ]m) concentration can promote
mitochondrial dysfunction and generation of reactive oxygen species
(ROS).5,6 To understand the physiology of Zn2þ in the brain, it is
crucial to monitor [Zn2þ]m in intact brain tissues.
for Zn2þ,13 and TPP is an effective mitochondrial-targeting site.14,15
DPEN and TPP are separated as far as possible to minimize the
interactions between them. Herein we report that SZn-Mito can be
used for selective detection of [Zn2þ]m in live cells and intact tissues
at >100 μm depth by TPM.
The preparation of SZn-Mito is described in the Supporting
Information (SI). The solubility of SZn-Mito in 3-(N-morpholi-
no)propanesulfonic acid (MOPS) buffer solution (30 mM,
100 mM KCl, pH 7.2) as determined by the fluorescence method17
is ∼2 μM, which is sufficient to stain the cells (Figure S2 in the SI).
The photophysical properties of SZn-Mito were studied in
MOPS buffer solution. In metal-free buffer (30 mM MOPS,
100 mM KCl, 10 mM EGTA, pH 7.2), SZn-Mito has absorption
and emission maxima at 388 nm (ε = 1.87 ꢁ 104 Mꢀ1 cmꢀ1) and
500 nm (Φ = 0.15), respectively. Both spectra showed gradual red
shifts with increasing solvent polarity (Figure S1 and Table S1). The
shifts were greater for the fluorescence spectrum (45 nm) than for
the absorption spectrum (5 nm). Upon addition of Zn2þ, the
fluorescence intensity increased dramatically (Φ = 0.92) and
exhibited a slight blue shift in both its absorption (375 nm, ε =
2.31 ꢁ 104 Mꢀ1 cmꢀ1) and emission maxima (493 nm) (Figure
S3a,b). This outcome can be attributed to the blocking of the
photoinduced electron transfer process and the decrease in the
electron-donating ability of the amino group by the complexation of
SZn-Mito with Zn2þ, of which the former appears to be predomi-
nant. A nearly identical result was observed in the TP process
(Figure 1a). The fluorescence enhancement factors [FEF = (F ꢀ
Fmin)/Fmin] of SZn-Mito determined for the OP and TP processes
were both equal to 7 in the presence of excess Zn2þ (Table S2).
To detect [Zn2þ]m in cells, a one-photon (OP) fluorescent probe
(RhodZin-3) has been developed and commercialized.7,8 This small-
molecule probe does not require transfection, as does its protein
counterparts,9,10 but it suffers from the lack of selectivity for mito-
chondria and/or Zn .
2þ 8,9b,10 Moreover, use of this probe with one-
photon microscopy (OPM) requires a rather short excitation
wavelength (∼550 nm) that limits its use in tissue imaging because
of the shallow penetration depth (<100 μm), photobleaching, and
cellular autofluorescence. Overcoming these problems requires a
two-photon (TP) probe that can be used for detection of [Zn2þ]m in
intact tissues through two-photon microscopy (TPM). TPM, which
utilizes two photons of lower energy for the excitation, is a new
technique that can visualize biological events deep inside intact
tissues (>500 μm) for extended periods of time.11,12 We therefore
designed a new TP probe for [Zn2þ]m, SZn-Mito (Scheme 1),
which is derived from 6-(benzo[d]thiazol-20-yl)-2-(N,N-dimethyl-
amino)naphthalene (BTDAN) as the reporter, N,N-di-(2-picolyl)-
ethylenediamine (DPEN) as the Zn2þ chelator, and triphenyl-
phosphonium salt (TPP) as the mitochondrial-targeting site.13ꢀ15
BTDAN is closely related to 6-(benzo[d]oxazol-20-yl)-2-(N,N-di-
methylamino)naphthalene, which has been successfully utilized in
the TP probe for Ca2þ (BCaM);16 DPEN is a well-known receptor
Received: January 17, 2011
Published: March 30, 2011
r
2011 American Chemical Society
5698
dx.doi.org/10.1021/ja200444t J. Am. Chem. Soc. 2011, 133, 5698–5700
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Figure 1. (a) Two-photon fluorescence spectra of 1 μM SZn-Mito in
the presence of free Zn2þ (0ꢀ127 nM). The excitation wavelength was
760 nm. (b) Two-photon action spectrum of SZn-Mito in the presence of
47 nM free Zn2þ. These data were measured in 30 mM MOPS buffer
(100 mM KCl, 10 mM EGTA, pH 7.2).
Figure 3. (aꢀc) TPM images of 0.5 μM SZn-Mito-labeled HeLa cells
(a) before and (b) after addition of 150 μM DTDP to the imaging
solution and (c) after addition of 10 μM CCCP to (b). (d) Relative
TPEF intensity of SZn-Mito-labeled HeLa cells as a function of time.
The TPEF intensities were collected at 425ꢀ575 nm upon excitation at
760 nm with femtosecond pulses. Scale bar: 10 μm. Cells shown are
representative images from replicate experiments (n = 5).
rather homogeneous and slightly more hydrophobic than that of
MOPS buffer. Therefore, we detected [Zn2þ]m by using the detec-
tion window at 425ꢀ575 nm.
Figure 2. (a) TPM and (b) OPM images of HeLa cells colabeled with
(a) SZn-Mito (0.5 μM) and (b) Mitotracker Red FM (1 μM) for 30 min at
37 °C. (c) Colocalized image. The wavelengths for TP and OP excitation
were 760 and 514 nm, respectively, and the corresponding emissions were
collected at 450ꢀ550 (SZn-Mito) and 600ꢀ700 nm (Mitotracker Red
FM). Scale bar: 20 μm. Cells shown are representative images from replicate
experiments (n = 5).
To assess whether SZn-Mito can detect [Zn2þ]m in cells, the
HeLa cells were colabeled with SZn-Mito and Mitotracker Red FM,
a well-known OP probe for mitochondria.8 The TPM and OPM
images (Figure 2a,b) were obtained by using the detection windows
at 450ꢀ550 and 600ꢀ700 nm, respectively, to selectively detect the
two signals with equal intensities (Figure S8). The two images
overlapped well (Figure 2c), and the Pearson’s colocalization
coefficient, A, for SZn-Mito and Mitotracker Red FM was 0.89, as
calculated using Autoquant X2 software. In addition, the TPEF
intensity of SZn-Mito-labeled HeLa cells decreased dramatically
upon addition of N,N,N0,N0-tetrakis(2-pyridyl)ethylenediamine
(TPEN), a membrane-permeable Zn2þ chelator that can effectively
remove [Zn2þ]m (Figure S6c,d).5b These results confirmed that the
bright regions in the TPM image reflect the presence of [Zn2þ]m.
Moreover, SZn-Mito showed negligible toxicity as measured by
using a CCK-8 kit (Figure S9). Further, the TPEF intensity at a
given spot on the SZn-Mito-labeled HeLa cells remained nearly the
same after continuous irradiation with the femtosecond pulses for
60 min, indicating its high photostability (Figure S7d).
The dissociation constants (KOd P and KTdP) ofSZn-MitofortheOP
and TP processes were calculated from the fluorescence titration
curves (Figure S3c).16,18 The titration curves were fitted well with a
1:1 binding model (Figure S3c); the Hill plots were linear with a slope
of 1.0 (Figure S3d), and the Job’s plot exhibited a maximum point at a
mole fraction of 0.50 (Figure S4), indicating 1:1 complexation
between the probe and Zn2þ. TheKdOP and KTdP values for Zn2þ were
3.1 nM; thus, this probe can detect Zn2þ in the nanomolar range.
SZn-Mito showed high selectivity for 1 μM Zn2þ over 1 mM
Naþ, Kþ, Ca2þ, and Mg2þ and 1 μM Mn2þ, Fe2þ, and Fe3þ but
only modest selectivity over 1 μM Cd2þ (Figure S5b). In the
presence of 1 μM Co2þ, Ni2þ, and Cu2þ, the fluorescence was
quenched as a result of metal-to-ligand electron transfer upon
excitation.19 Since Co2þ, Ni2þ, and Cu2þ ions rarely exist in the
cells,20 this probe can detect [Zn2þ]m with minimum interference
from other competing metal ions. Moreover, SZn-Mito is pH-
insensitive in the biologically relevant pH range (Figure S5a).
The TP action spectra of SZn-Mito in MOPS buffer contain-
ing excess Zn2þ indicated a Φδ value of 75 GM at 750 nm
(Figure 1b), which is comparable to those of existing TP probes.12
This predicts a bright TPM image of the cells stained with SZn-Mito,
as observed (Figure 2a). In comparison with Figure 2a, the TPM
image of HeLa cells labeled with 0.5 μM RhodZin-3 AM was hardly
visible and that labeled with 5 μM RhodZin-3 AM and 5 μMPluronic
F-127, a surfactant that enhances the cell loading,8 was much dimmer
(Figure S6). This result underlines the advantage of SZn-Mito over
commercial probes in TPM imaging. Further, the TP-excited fluore-
scence (TPEF) spectrum of SZn-Mito in the cells was symmetrical
and slightly blue-shifted relative to that measured in the MOPS
buffer, with an emission maximum at 475 nm (Figure S7b),
presumably because the polarity of the probe environment was
We then tested whether SZn-Mito could be used to monitor
changes in [Zn2þ
] in live cells. The TPEF intensity increased
m
when 2,20-dithiodipyridine (DTDP; 150 μM), a reagent that
promotes the release of Zn2þ from Zn2þ-binding proteins,21
was added to HeLa cells labeled with SZn-Mito (Figure 3a,b),
and it decreased upon addition of carbonyl cyanide m-chlorophen-
ylhydrazone (CCCP; 10 μM), a compound that promotes the
release of intramitochondrial cations by collapsing the mitochon-
drial membrane potential (Figure 3c).10 Moreover, the TPEF
intensity increased when the cells were treated with 100 μM Zn2þ
and 100 μM pyrithione (2-mercaptopyridine N-oxide), a reagent
that can bring Zn2þ into the cytoplasm,22 and decreased upon
treatment with CCCP (Figure S10). These results established the
capability of SZn-Mito for detecting [Zn2þ]m in live cellsfor a long
period of time with minimum interference from other competing
metal ions, pH, cytotoxicity, and photostability.
We further investigated the utility of this probe in tissue
imaging. TPM images were obtained from a slice of 14-day-old
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dx.doi.org/10.1021/ja200444t |J. Am. Chem. Soc. 2011, 133, 5698–5700

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’ ACKNOWLEDGMENT
This work was supported by the National Research Founda-
tion (NRF) through grants funded by the Korean Government
(2010-0016997 and 2010-0018921), the Priority Research Cen-
ters Program through the NRF funded by the Ministry of
Education, Science, and Technology (2010-0028294 and 2010-
0020209), and the Ajou University Research Fellowship of 2010.
C.S.L., H.J.K., and J.H.H. were supported by BK21 Scholarships.
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m
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’ AUTHOR INFORMATION
Corresponding Author
kimhm@ajou.ac.kr; chobr@korea.ac.kr
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dx.doi.org/10.1021/ja200444t |J. Am. Chem. Soc. 2011, 133, 5698–5700