Single molecular multianalyte (Ca2+, Mg2+) fluorescent probe and applications to bioimaging

Article abstract of DOI:10.1021/ja0528228

Intracellular signal transduction relies on spatial and temporal signal transmitter dynamics. To clarify the correlations of these transmitter molecules, multicolor-imaging has been widely used. However, in the case of applying multiple indicators in a ce

Full text of DOI:10.1021/ja0528228

Published on Web 07/14/2005
Single Molecular Multianalyte (Ca2+, Mg2+) Fluorescent Probe and
Applications to Bioimaging
Hirokazu Komatsu, Takahiro Miki, Daniel Citterio, Takeshi Kubota, Yutaka Shindo,
Yoshiichiro Kitamura, Kotaro Oka, and Koji Suzuki*
Departments of Applied Chemistry and of Bioscience and Informatics, Faculty of Science and Technology, Keio
UniVersity, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
Received April 30, 2005; E-mail: suzuki@applc.keio.ac.jp
Fluorescent imaging based on fluorescent probes offers a highly
sensitive, high-speed spatial and temporal analysis of cells1 and is
widely used in the field of biology and physiology. The develop-
ment of excellent fluorescent probes is required for better fluorescent
imaging, thus highly selective fluorescent probes have already been
developed, including fura-22 and fluo-33 for Ca2+, KMG-204 and
1045 for Mg2+, DAFs6 for NO, etc. With these molecular fluorescent
probes, selective imaging of a particular analyte has been investi-
gated. Since intracellular signal transduction results from various
signal transmitters, the simultaneous monitoring of multiple analytes
has been attempted with simultaneous loading of several indicators
in a cell.7 However, the combination of several indicators produces
cross-talk, a larger invasive effect, and the different localization,
metabolisms, and photobleaching rates of individual indicators make
the situation complicated and unsuitable for quantitative analysis.
In this study, we demonstrate that the use of a single-molecular
multianalyte sensor, namely, a single probe that allows the sensing
of multiple analytes with different spectral responses, is a promising
way to overcome the difficulties encountered when loading multiple
indicators.
Ca2+ is the intracellular divalent cation with the largest concen-
tration variations and plays a critical role as a signal transmitter,8
while Mg2+ is the most abundant divalent cation and acts as a
cofactor in many situations.9 However, the correlation of the Ca2+
and Mg2+ concentrations is not clear and has sometimes been
reported to behave proportionally10 and at other times inverse
proportionally.11 Thus, to clarify the situation, a highly sensitive
spatially and temporally resolved monitoring of both cations is
required.
KCM-1 was successfully synthesized starting from the reported
fluorine-substituted BAPTA derivative13b (details in Supporting
Information).
Figure 1 shows the absorbance and fluorescence spectra of
KCM-1 under simulated biological conditions (pH 7.20, 50 mM
HEPES, 130 mM KCl, 20 mM NaCl) for different Ca2+ or Mg2+
concentrations. Upon complexation to Ca2+, KCM-1 shows a 45
nm blue shift in absorbance and a 5 nm blue shift in the fluorescence
spectrum. For Mg2+, a 21 nm red shift in absorbance and a 5 nm
red shift in fluorescence occurred.
The binding characteristics of fluorescent probes are often
compared based on their dissociation constant, Kd. The dissociation
constants of KCM-1 with Ca2+ or Mg2+ were calculated using the
double logarithm plot method2 and found to be 14 ( 1 µM in the
2+
2+
case of Kd(Ca ) and 26 ( 3 mM for Kd(Mg ). While the intracellular
Ca2+ concentration is on the order of 0.1-1 µM and the Mg2+
concentration is 1 mM, the experimentally determined dissociation
constants indicate relatively low affinities. However, low-affinity
indicators are reported to be useful for quantification of high
concentrations and of transient responses.15 The basic optical
properties and the dissociation constants are summarized in Table
1 and are consistent with the molecular design strategy.
To investigate the performance of KCM-1 in the simultaneous
presence of Ca2+ and Mg2+, the absorbance and fluorescence spectra
of the probe were measured under simulated biological conditions
(pH 7.20, 50 mM HEPES, 130 mM KCl, 20 mM NaCl) with
different mixed Ca2+ and Mg2+ concentrations.
On the basis of the assumption of independent complexation of
Ca2+ and Mg2+ to KCM-1, fluorescence intensity at certain
excitation/emission wavelength was calculated as following.
We now report on the simultaneous imaging of intracellular Ca2+
and Mg2+ with a novel single-molecular multianalyte sensor, the
Ca2+-Mg2+ multifluorescent probe.
fluorescence intensity (λex, λem) ) [I]0{fI + fICa2+KCa2+[Ca2+] +
fIMg2+KMg2+[Mg2+]}(1 + KCa2+[Ca2+] + KMg2+[Mg2+])-1 (1)
We designed the first Ca2+-Mg2+ multifluorescent probe, KCM-
1, by combining in a single molecule a coumarin moiety as a stable
fluorophore4 excitable with visible light, BAPTA (O,O′-bis(2-
aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid) as the Ca2+
selective binding site1-3,12 at the electron-donor site of the chro-
mophore, and a charged â-diketone as the Mg2+ selective binding
site4,5 at the electron-accepting site. The dissociation constants of
the BAPTA derivative are typically on the order of 0.1-1 µM for
Ca2+ and 1 mM for Mg2+, while those of the charged â-diketone
are on the order of 10 mM for Ca2+ and 1-10 mM for Mg2+. A
fluorine-substituted BAPTA13 was chosen as the Ca2+ selective
binding site due to its low Mg2+ affinity. In the event of Ca2+
binding to BAPTA, the cation binding to the electron-donor site
of the chromophore leads to a blue shift in the absorbance and
fluorescence spectra based on an ICT-type mechanism.14 On the
contrary, Mg2+ binding to the â-diketone electron-acceptor site
induces a red shift of the spectral bands.
where K represents the association constant; I stands for the
indicator (fluorescent probe) and ICa2+ and IMg2+ for the indica-
tor-ion complex; f is a proportionality factor for the individual
fluorescent compounds.
Inserting the ratio of the fluorescence intensities at different
wavelengths results in eq 1 becoming independent of the initial
indicator concentration, and using data obtained from a combination
of three different excitation/emission wavelengths, the Ca2+ and
Mg2+ concentrations can be determined. (For further details, refer
to the Supporting Information.) A curve fitting based on eq 1 to
the experimental data was in good agreement for concentrations
lower than 10 µM Ca2+ and 10 mM Mg2+(R > 0.999).
At very high Ca2+ and Mg2+ levels, a slight deviation from the
fit according to eq 1 is observed, although such situations are not
likely to occur in normal cells. The deviation is assumed to result
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J. AM. CHEM. SOC. 2005, 127, 10798-10799
10.1021/ja0528228 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. FCCP responses of Ca2+ and Mg2+ in cytosol of a PC12 cell
loaded with KCM-1AM; time course of concentrations of Ca2+ (a) and
Mg2+ (b) in cytosol, fluorescent image of Ca2+ change (c, between 0 and
21 s) and of Mg2+ change (d, between 0 and 38 s). FCCP was applied at
5 s. Fluorescent image of the PC12 cell excited at 420 nm (e).
Figure 1. (a) Structure of Ca2+-Mg2+ multifluorescent probe KCM-1.
(b-d) Absorbance and fluorescence spectra of 10 mM KCM-1 in the
absence of Ca2+ and Mg2+ (black line), in the presence of 1 mM Ca2+
(blue line), 500 mM Mg2+ (red line), and coexistence of 10 mM Ca2+ and
10 mM Mg2+ (green line); (b) absorbance, (c) fluorescence (excited at 358
nm), (d) fluorescence (excited at 424 nm). All spectra were measured at
pH 7.20 (50 mM HEPES, 130 mM KCl, 20 mM NaCl).
the measurement of the intracellular local concentrations will
become possible. This is the first reported example of the
simultaneous determination of multiple analytes in a cell with a
single-molecular multianalyte sensor. Soon, observations of multiple
signal transmitter dynamics are going to clarify the central problems
in cellular biology.
Table 1. Optical Properties and Binding Characteristics of KCM-1a
λ
nm
max
ꢀ
Fλ
nm
1
compound
M-1 cm-
Kd
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
KCM-1
KCM-1 (Ca2+
KCM-1 (Mg2+
403
358
424
10400
7100
12900
480
475
485
)
)
14 µM
26 mM
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a All data were taken in buffer simulating biological conditions (50 mM
HEPES, pH 7.2, 130 mM KCl, 20 mM NaCl); ꢀ stands for the extinction
coefficient, and Kd for the dissociation constants. The probe and cation
concentrations during the measurements were 10 µM for KCM-1, 1 mM
Ca2+ for KCM-1 (Ca2+) and 500 mM Mg2+ for KCM-1 (Mg2+).
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