New Dual Fluorescent Probe for Simultaneous Biothiol and Phosphate Bioimaging

Article abstract of DOI:10.1002/chem.201502799

The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS-GG by biological thiols at near-neutral pHvalues, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence-decay time. The new dual probe was tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using fluorescence-lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.

Full text of DOI:10.1002/chem.201502799

DOI: 10.1002/chem.201502799
Full Paper
&
Imaging Agents
New Dual Fluorescent Probe for Simultaneous Biothiol and
Phosphate Bioimaging
Sandra Resa,[a] Angel Orte,[b] Delia Miguel,[a] Jose M. Paredes,[b] Virginia Puente-MuÇoz,[b]
Rafael Salto,[c] Maria D. Giron,[c] Maria J. Ruedas-Rama,[b] Juan M. Cuerva,*[a] Jose M. Alvarez-
Pez,[b] and Luis Crovetto*[b]
Dedicated to the memory of Professor Carlos Orte
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Abstract: The simultaneous detection of relevant metabo-
lites in living organisms by using one molecule introduces
an approach to understanding the relationships between
these metabolites in healthy and deregulated cells. Fluores-
cent probes of low toxicity are remarkable tools for this type
of analysis of biological systems in vivo. As a proof of con-
cept, different naturally occurring compounds, such as bio-
thiols and phosphate anions, were the focus for this work.
The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-
tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one
(Granada Green; GG) were designed and synthesized. This
new sulfinyl xanthene derivative can act as a dual sensor for
the aforementioned analytes simultaneously. The mechanism
of action of this derivative implies thiolysis of the sulfinyl
group of the weakly fluorescent DNBS-GG by biological
thiols at near-neutral pH values, thus releasing the fluores-
cent GG moiety, which simultaneously responds to phos-
phate anions through its fluorescence-decay time. The new
dual probe was tested in solution by using steady-state and
time-resolved fluorescence and intracellularly by using fluo-
rescence-lifetime imaging microscopy (FLIM) in human epi-
thelioid cervix carcinoma (HeLa) cells.
Introduction
processes that combine alterations of bone metabolism with
increased oxidative stress, such as obesity and diabetes.[4]
To our knowledge, no fluorescent probes with the ability to
measure these parameters simultaneously have been de-
scribed to date. Therefore, we believe that a fluorescence-
based multiplexed probe that can jointly sense biological
thiols and inorganic phosphate ions inside and outside the cell
would be of broad interest and utility.
Fluorescent probes of low toxicity for multiplex sensing are re-
markable tools for the analysis of biological systems.[1] To this
end, different properties of the sensor must be related to unre-
lated bioanalytes, which is a difficult task. Within this context,
we focused on very different naturally occurring compounds,
such as biothiols and phosphate anions. Both of these com-
pounds are widely present in living organisms and are relevant
because their deregulation is related to cellular dysfunction.
Biological thiols play crucial roles in biological systems; there-
fore, there has been a great effort to develop new methods to
detect them over the last two decades. Among the methods
for the detection and quantification of biological thiols, fluo-
rescence approaches have proved to be the most suitable due
to the advantages derived from their high sensitivity, simplicity,
and low cost.[2] Phosphate ions play important roles in signal
transduction and energy storage in biological systems; there-
fore, the detection and quantification of these ions inside the
cell and extracellular media are relevant parameters in biologi-
cal studies.[3] The measurement of intracellular phosphate ions
as a marker of osteoblast differentiation and bone deposition
combined with the assay of biothiol levels as a marker of oxi-
dative stress would be a useful tool to address pathological
The key point in this study was the careful selection of the
fluorophore. In recent years, fluorescein-based compounds
have evolved to yield simple structures that can retain their
striking photophysical properties. Thus, for example, some
aryl-[5] and alkyl-substituted xanthenones[6] have simplified the
original prototropic equilibrium in fluorescein, thereby improv-
ing the analysis of the photophysical data (Figure 1). Therefore,
these molecules have been used as fluorescent probes for
many biologically relevant analytes, such as b-galactosidase,[5,7]
hydrogen peroxide,[8] nucleotide pyrophosphatases/phospho-
diesterases,[9] uridine diphosphate glucuronosyltransferase
(UDPGT),[10] and BlaC hydrolase of Mycobacterium tuberculosis[11]
as well as in Western blot analysis.[12] Within this context,
a new methodology based on the dependence of the fluores-
cence-decay time of these xanthene-based dyes on the phos-
phate-buffer concentration at near physiological pH values has
recently been developed in our laboratories.[13] The highest
sensitivity was obtained with a customized compound, called
Granada Green (GG), with a fine-tuned pKa value of 7.3.[14]
[a] S. Resa, Dr. D. Miguel, Dr. J. M. Cuerva
Department of Organic Chemistry
Faculty of Sciences, University of Granada
C. U. Fuentenueva s/n, 18071 Granada (Spain)
Fax: (+34)958248437
E-mail: jmcuerva@ugr.es
[b] Dr. A. Orte, Dr. J. M. Paredes, V. Puente-MuÇoz, Dr. M. J. Ruedas-Rama,
Prof. J. M. Alvarez-Pez, Dr. L. Crovetto
Department of Physical Chemistry
Faculty of Pharmacy, University of Granada
Cartuja Campus, 18071 Granada (Spain)
Fax: (+34)958244090
E-mail: luiscrovetto@ugr.es
[c] Dr. R. Salto, Dr. M. D. Giron
Department of Biochemistry and Molecular Biology
Faculty of Pharmacy, University of Granada
Cartuja Campus, 18071 Granada (Spain)
Supporting information and ORCID(s) from the author(s) for this article is
available on the WWW under http://dx.doi.org/10.1002/chem.201502799.
Figure 1. Xanthenone-based fluorescent compounds.
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Considering the remarkable properties of these compounds,
we decided to demonstrate their selective ON–OFF fluorogenic
behavior toward biological thiols. In this manner, the liberated
fluorophore could be used for dual purposes: 1) for the detec-
tion of the presence of biothiols and 2) for providing informa-
tion on the local phosphate-ion concentration (Scheme 1).
Scheme 2. Working hypothesis and structure of DNBS-GG.
Scheme 1. Xanthenone-based fluorescent compounds and proposed func-
tions of the simultaneous probe.
as a dual probe for biothiols and phosphate anions at near-
neutral pH values in vitro and in an extracellular medium.
In 2005, Maeda et al. reported that 2,4-dinitrobenzenesulfon-
yl fluorescein derivatives can act as ON–OFF fluorescent
probes toward thiols because of the highly selective aromatic
nucleophilic addition of thiols to the highly electron-deficient
aromatic ring.[15] These seminal studies allowed the extension
of the reactive 2,4-dinitrobenzenesulfonyl derivatives to new
fluorescent compounds, such as cyanine,[16] merocyanine,[17]
boron-dipyrromethene (BODIPY),[18] benzothiazol,[19] and cou-
marin.[20] Remarkably, despite the excellent properties of xan-
thene-based dyes, the corresponding 2,4-dinitrobenzenesulfon-
yl derivatives have not been described to date. We then pre-
pared corresponding 2,4-dinitrobenzenesulfonate derivatives
of 9-[1-(2-methyl-4-methoxy-phenyl)]-6-hydroxy-3H-xanthen-3-
one (TGII), 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-
xanthen-3-one (Granada Green, GG; compound 1 in the Sup-
porting Information), and 6-hydroxy-9-isopropyl-3H-xanthen-3-
one (iso-Prop-Xanth), which unfortunately presented a fast and
unselective hydrolysis reaction in aqueous or methanolic solu-
tions. Moreover, the poor solubility in pure water was also lim-
iting for bioimaging in vivo. One possible reason for this unde-
sirable solvolysis reaction is the low pKa values of the starting
hydroxyxanthenones, which enhances their properties as nu-
cleofuges in a direct substitution reaction at the electron-defi-
cient sulfur center. After obtaining these unfruitful results, we
thought that the undesirable hydrolysis reaction (Scheme 2,
path a) could be changed to the desired reaction if the reac-
Results and Discussion
For our purposes, the fluorescent probe should fulfill three pri-
mary conditions: 1) the nucleophilic aromatic substitution reac-
tion should be turned off, 2) the water-promoted hydrolysis re-
action should be diminished, and 3) the thiolysis reaction
should be selective. To this end, less-deactivated aromatic sul-
fonates were prepared, but they were unreactive toward any
of the previously mentioned reactions. One solution would be
the use of other less-electrophilic functional groups, such as
sulfinates (R1-SO-O-R2). In addition, the dipolar moment of the
resulting molecule could increase its water solubility. The re-
quired sulfinate moiety must also be in a carefully balanced
equilibrium between the undesirable hydrolysis and the desira-
ble thiolysis reactions. Thus, the probe must be sufficiently
robust to resist hydrolysis, but must also react with sulfur nu-
cleophiles.
After some experimentation, we found that both the xan-
thene dye and the substitution of the sulfinate group are key
factors for the success of the approach. Only the combination
of GG as a xanthene dye and the 2,4-dinitro substitution of the
benzene sulfinate in DNBS-GG was shown to be appropriate
for this application because they are stable and soluble in
water and present the expected selective reactivity toward bio-
thiols. However, the corresponding DNBS derivatives of TGII
and iso-Prop-Xanth were labile toward water. The reason for
this different reactivity is unclear, but could be related to the
higher pKa value of GG.
tion was transformed in
a
selective thiolysis[21] reaction
(Scheme 2, path b).[22] Therefore, a new biothiol-sensitive group
could be developed if the thiolysis reaction took place in aque-
ous solution without the interference of oxygenated nucleo-
philes.
The primary synthetic problem in this study is that the re-
quired 2,4-dinitrobenzenesulfinyl chloride has not been de-
scribed to date.[24] Therefore, we used an unusual reaction of
electron-deficient sulfonyl chlorides, which allows the reduc-
tion of the sulfur atom in situ from +6 to +4, with triethyla-
mine as a reductant (Scheme 3).[25]
In this study, we present the synthesis of the 2,4-dinitroben-
zenesulfinate (DNBS) derivative of GG, that is, DNBS-GG, a new
sulfinate-based[23] biothiol-sensitive group based on such a se-
lective thiolysis reaction. This compound presents an excellent
performance in terms of avoiding undesirable hydrolysis reac-
tions, thus increasing the specificity of the fluorogenic probe
toward biothiols. Moreover, DNBS-GG shows excellent behavior
To explore the use of the new fluorescent probe to detect
biologically relevant substances containing a thiol group (e.g.,
cysteine, homocysteine, and glutathione), the temporal de-
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the probe concentration and re-
action time in the temporal gen-
eration of the fluorescent signal.
First, we studied the limit in the
linearity of the fluorescence
signal with the concentration of
the free dye (see Figures S3 and
S5 in the Supporting Informa-
tion). The maximum suitable
concentration of the probe was
selected as 6.5 mm. Biological ex-
perimental conditions were es-
tablished for the reaction at
378C and pH 7.35 because we
intended to perform the detec-
tion inside living cells. Figure 2B
shows the fluorescence intensity
Scheme 3. Synthesis of DNBS-GG.
of DNBS-GG in the presence of
Cys, Hcy, or GSH at l=519 nm,
pendence of the DNBS-GG fluorescence emission in the pres-
ence of these biothiols was investigated at pH values of 9 (at
which DNBS-GG shows the highest sensitivity to biothiols due
to the influence of the pKa value of the aliphatic thiols on the
reaction), 4.80 (to check there are not undesirable hydrolysis
reactions under acidic conditions, thus ruling out any potential
interference of lysosomal actions in the use of the dye in cells),
and 7.35 (which is interesting for physiological applications).
The fluorescence emission of DNBS-GG in the absence of bio-
thiols was very weak. However, an emission band centered at
l=520 nm, characteristic of GG, gradually increased over time
in the presence of biothiols. This fluorescence increase indi-
cates the cleavage of DNBS-GG and its separation into two
moieties, which releases the fluorescent compound GG from
the electron-withdrawing 2,4-dinitrobenzene-sulfinate moiety.
Figure 2A shows representative fluorescence-emission spectra
at different reaction times of DNBS-GG in the presence of glu-
tathione (GSH) at pH 7.35. Similar reactions were found upon
reaction with cysteine (Cys) and homocysteine (Hcy; see Fig-
ure 2B and Figure S1 in the Supporting Information). The ab-
sorption spectra of DNBS-GG (see Figure S2 in the Supporting
Information) exhibit a clear change upon reaction with thiols,
which give a dual absorption band with maxima at l=436
and 461 nm; furthermore, the reaction gave rise to the spectral
profile characteristic of free GG (absorption maximum at l=
495 nm).
thus increasing sharply with the reaction time from 0 to 3 h
and reaching a nearly constant value at approximately 3 h. Fig-
ure S4 (see the Supporting Information) shows the relative re-
sponse of DNBS-GG toward Cys, Hcy, and GSH after 2 hours of
reaction under physiological experimental conditions. These re-
sults confirm a highly similar sensitivity of the probe toward
the three biothiols.
We then attempted to analyze the fate of the DNBS-based
fragment. Control experiments that used sodium propiolate as
a nucleophile showed that the nucleophilic attack does not
occur at the aromatic ring, as expected. The sulfur nucleophile
attacks the sulfur electron-deficient center, thus initially leading
to the thiolysis of DNBS-GG. After the hydrolysis of the result-
ing propyl 2,4-dinitrobenzenethiosulfinate[26] we could isolate
2,4-dinitrobenzenesulfinic acid.[27,28] This result is relevant be-
cause the sulfur nucleophile can be slowly regenerated over
time and can influence the overall kinetics of the process.
To optimize the experimental conditions to use the DNBS-
GG in biothiol detection, we further investigated the effects of
Figure 2. A) Fluorescence emission spectra (lex =485 nm) of DNBS-GG
(6.5 mm) in the presence of 6.5 mm GSH at pH 7.35 in 10 mm Tris solution, re-
corded every five minutes until reaching 1300 minutes. B) Fluorescence in-
tensity at lex =485 and lem =519 nm versus time for DNBS-GG (6.5 mm) in
&
the presence of 6.5 mm GSH ( ), Cys (~), or Hcy (*) at pH 9 and 408C.
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We also investigated potential interference from the pres-
ence of other amino acids (which lack a thiol group) or by re-
active oxygen species (ROS) to study the specific response of
the probe to biothiols. The increase in the fluorescence-emis-
sion intensity of DNBS-GG in the presence of l-alanine, l-
serine, H2O2, or FeII ions was low (see Figure S4 in the Support-
ing Information). The low response to ROS indicates that the
probe would be a good candidate for thiol detection in cases
of cellular stress due to high levels of ROS in which the thiols
are the first to respond to oxidative stress.
ered lifetimes show a decrease with the phosphate-ion con-
centration, thus revealing that the product of the DNBS-GG
cleavage is the free GG, which is sensitive toward phosphate
ions through an excited-state proton-transfer (ESPT) reaction
(see Scheme S1 in the Supporting Information).
The recovered fluorescence lifetimes after the reaction with
the thiols are in excellent agreement with those previously re-
ported for free GG in the presence of phosphate ions
(Figure 3).[14]
The innovative dual probe for the detection of thiols and
phosphate ions was also tested intracellularly by means of
fluorescence-lifetime imaging microscopy (FLIM). DNBS-GG was
added to the extracellular medium of human epithelioid cervix
carcinoma (HeLa) cell cultures, and FLIM images were collected
at different times. The evaluation of the fluorescence-intensity
changes displayed with time due to biological thiols shows
that the probe is cell-membrane permeable and can selectively
detect thiols in living cells (Figure 4A,C). To verify this conclu-
sion, an excess of N-methylmaleimide (NMM) was added to the
extracellular medium to block the cellular thiols. The fluores-
cence signal of the probe did not appreciably change with
time (Figure 4B,C). More examples of fluorescence images at
different reaction times can be seen in videos in the Support-
ing Information.
In previous studies, we have demonstrated the fluorescence-
decay time of GG to be a good estimator of the phosphate-ion
concentration.[14] Given that the product of the reaction be-
tween DNBS-GG and thiols is free GG, the measurements of
the fluorescence-decay time must provide the basis for a dual
sensor. To test the capability of the DNBS-GG dye to jointly es-
timate the concentrations of phosphate ions and biothiols, we
studied the reaction between 6.5 mm DNBS-GG and GSH in the
presence of phosphate buffer at different concentrations in
the range 0–0.4m and at pH 7.35. Experimental measurements
were completed in the time-resolved regime, and Figure 3
shows the recovered results. As can be observed, the recov-
To verify the phosphate sensitivity by the changes in the
fluorescence lifetime, the cells were incubated with a-toxin,
a nonspecific ionophore that opens membrane pores to allow
a free flow of small ions, including the phosphate buffer added
Figure 3. A) Fluorescence-decay time (open symbols, left y axis) and normal-
ized emission intensity (closed symbols, right y axis) of DNBS-GG
(6.5”10À6 m) in the presence of GSH (6.5”10À6 m) at pH 7.35 and total phos-
phate-ion concentration of 0 (black squares), 200 (blue triangles), and
400 mm (red circles). B) Average of the fluorescence-decay time of DNBS-GG
(6.5”10À6 m) in the presence of GSH (6.5”10À6 m) at pH 7.35, collected every
10 min for 1 h of reaction in the presence of different amounts of total
phosphate ions between 0 and 400 mm.
Figure 4. Fluorescence-intensity images of cell cultures with DNBS-GG
(1”10À7 m) at different reaction times (in minutes). A) Physiological condi-
tions with natural thiols present. B) Cells incubated with an excess of NMM.
Scale bars represent 10 mm. C) Average number of photons per pixel con-
taining the fluorescence of the probe (cell cytoplasm) from cells under phys-
iological conditions (black squares) or incubated with an excess of NMM
(open circles). Error bars represent standard deviations from the measure-
ments of at least five different cells.
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Conclusion
We have designed and synthesized a new sulfinyl xanthene de-
rivative that can act as a simultaneous dual sensor of different
analytes, such as biological biothiols and phosphate anions.
The mechanism of action of this derivative implies thiolysis of
the sulfinyl group of the weakly fluorescent DNBS-GG by bio-
logical biothiols, thus releasing a fluorescent GG moiety that si-
multaneously responds to phosphate anions through its fluo-
rescence-decay time. The efficiency of this new dye as a dual
sensor was probed in experiments at two significant pH values
in vitro; that is, pH 9, at which the probe responds faster and
is more sensitive to biothiols, and pH 7.35, which is required
for biological applications. In both cases, we could detect the
presence of cysteine, homocysteine, or glutathione and deter-
mine the phosphate-ion concentration at the same time. Note
that this probe is suitable for thiol detection in cases of cellular
stress due to its low response to ROS. Moreover, this new dye
was tested intracellularly by using FLIM in HeLa cells and
showed permeability through the cell membrane. Again, the
increase in the fluorescence intensity confirmed the ability of
this dye to detect thiols, and changes in the fluorescence life-
time enabled its behavior as a phosphate-ion concentration
sensor.
Figure 5. A) FLIM images of cells incubated with a-toxin and with DNBS-GG
(1”10À7 m) at pH 7.35 with phosphate-ion concentrations of 0, 100, and
200 mm (arbitrary color scale). The FLIM images were collected after 30 mi-
nutes to allow the reaction of DNBS-GG with the intrinsic thiols. Scale bar
represents 10 mm. B) Recovered lifetime of DNBS-GG in a-toxin-treated cells
in the presence of different phosphate-ion concentrations. Error bars repre-
sent standard deviations from the measurements of at least five different
images containing multiple cells.
to the extracellular media. Along with the increase in the fluo-
rescence emission due to a reaction with the internal thiols,
the FLIM images show changes in the fluorescence lifetime of
the probe because of the changes in the total phosphate-ion
concentration (Figure 5A,B). Previously,[24] we have reported
the kinetic rate constant of the buffer-mediated proton-trans-
fer reaction in the ground state in different buffers by using
fluorescence correlation spectroscopy (FCS) and showed that
only a suitable buffer, such as phosphate, makes the reaction
fast enough to compete with fluorescence emission; therefore,
we could assign these changes in lifetime to phosphate ESPT
among other cell species. Although Dt shows a large error bar,
this is intrinsic to cell-to-cell variation. Nevertheless, we have
shown the excellent feasibility and specificity of this method in
previous reports for following variations of the phosphate-ion
concentration in living cells between 10 and 600 mm.[13;14,29]
We show that this dye can detect thiols and phosphate ions
at the same time. The combined sensing of inorganic phos-
phate ions and GSH has a clear biological relevance. In bone
tissue, the regulation of bone synthesis and degradation is
modulated by oxidative stress. The molecular bases of these
interactions have been proposed, and the role of specific
genes that promote oxidative stress in the development of os-
teoporosis has been described (e.g., the NOX4 gene promotes
oxidative stress and osteoporosis,[30] whereas FoxO1 gene ex-
pression exerts a protective effect).[31] In addition, sensing of
phosphate-based energy-rich compounds and oxidative stress
is relevant in biology in pathological processes that include al-
teration of the energetic metabolism combined with an in-
crease in oxidative stress. Examples of these situations are
cancer,[32] obesity, diabetes,[4] and hypoxia[33]. Studies that use
our dye for some of these biological diseases and sensibility
against phosphate compounds are in progress.
Experimental Section
General
All reactions were performed in dry glassware in an air atmos-
phere. All of the commercially available reagents (2,4-dinitrobenze-
nesulfonyl chloride and 2,4-dinitrobenzenesulfonic acid) and sol-
vents (triethylamine, dichloromethane, and methanol) were used
without further purification. TLC analysis was performed on alumi-
num-backed plates coated with silica gel 60 (230–240 mesh) with
F254 indicator. The spots were visualized with UV light (l=
254 nm) and/or staining with Ce/Mo reagent or phosphomolybdic
acid solution and subsequent heating. NMR spectra were mea-
1
sured at room temperature and the H NMR spectra were recorded
at 500 or 600 MHz.
Synthesis and spectroscopic data of DNBS-GG
2,4-Dinitrobenzenesulfonyl chloride (29 mg, 0.11 mmol) was added
to Granada Green (GG, 20 mg, 0.056 mmol) dissolved in Et3N and
CH2Cl2 (4 mL, 1:1 v/v; Scheme 3). The reaction was monitored by
TLC analysis and 2,4-dinitrobenzenesulfonyl chloride (29 mg,
0.11 mmol) was added every 60 min until no starting material was
observed. The solvent was removed by evaporation, and the resi-
due was purified by flash chromatography with CH2Cl2/MeOH as
the eluent to give the corresponding DNBS-GG derivative as
1
a dark-orange solid (16 mg, 50%). H NMR (600 MHz, [D6]acetone):
d=8.95 (d, J=2.7 Hz, 1H), 8.60 (dd, J=9.2, 2.8 Hz, 1H), 7.66 (d, J=
9.2 Hz, 1H), 7.35 (s, 1H), 7.34 (d, J=2.4 Hz, 1H), 7.29–7.25 (m, 3H),
7.17 (dd, J=8.8, 2.4 Hz, 1H), 7.08 (d, J=9.8 Hz, 1H), 6.44 (dd, J=
9.8, 1.7 Hz, 1H), 6.16 (d, J=1.7 Hz, 1H), 3.82 (s, 3H), 1.44 ppm (s,
9H); 13C NMR (151 MHz, [D6]acetone): d=187.1 (C), 161.1 (C), 160.9
(C), 159.5 (C), 157.9 (C), 156.2 (C), 148.1 (C), 146.1 (C), 143.7 (C),
133.7 (CH), 133.2 (CH), 133.0 (CH), 132.8 (CH), 132.1 (CH), 130.2 (C),
124.8 (CH), 124.6 (CH), 123.2 (C), 121.0 (C), 120.9 (C), 120.5 (CH),
118.3 (CH), 111.7 (CH), 109.4 (CH), 107.9 (CH), 57.8 (CH3), 37.7 (C),
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33.3 (CH3); LSIMS (FAB): m/z calcd for C30H24N2O9S: 588.12 [M+];
found: 588.12.
bled the reconstruction of the lifetime histogram. Raw images
were recorded by raster scanning of an area of 80”80 mm, with
a resolution of 512”512 pixels. The photons of each pixel were
temporally sorted with respect to the excitation pulse in the histo-
Preparation of solutions
grams with a time resolution of 29 pschannelÀ1
.
Tris(hydroxymethyl)aminomethane (Tris) buffer solutions were pre-
pared by mixing the required amounts of Trizma base and Tris–HCl
(both from Sigma-Aldrich) to obtain the desired pH value. For the
preparation of phosphate buffer (pH 6.8) solutions, NaH2PO4·H2O
and Na2HPO4·7H2O (both from Fluka, puriss. p.a.) were used. All of
the solutions were prepared with Milli-Q water as the solvent. All
of the chemicals were used as received without further purifica-
tion. A stock solution of DNBS-GG (10À4 m) in ethanol was pre-
pared. From this stock solution, aqueous solutions with a final con-
centration of 1”10À5 m of dye and pH 7.35 in Tris buffer were pre-
pared. The required amounts of this solution and thiol stock solu-
tion were added to obtain the desired concentrations of DNBS-GG
and thiol in Tris buffer (20 mm) at pH 7.35.
Cell culture
The human epithelioid cervix carcinoma (HeLa; ATCC No. CCL-2)
cell line was provided by the Cell Culture Facility, University of
Granada. The HeLa cells were grown in the Dulbecco modified
Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum (FBS), 2 mm glutamine, 100 UmLÀ1 penicillin, and
0.1 mgmLÀ1 streptomycin in a humidified 5% CO2 incubator.
For the FLIM microscopy experiments, the HeLa cells were seeded
onto coverslips (25 mm) in six-well plates at
a density of
11 250 cellscmÀ2. On the day of the experiment, the cells were
washed twice with Krebs–Ringer phosphate medium (118 mm
NaCl, 5 mm KCl, 1.2 mm MgSO4, 1.3 mm CaCl2, 1.2 mm KH2PO4, 30
mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
pH 7.4) and incubated in the same medium with effectors. When
N-methylmaleimide (NMM) was used to block the intracellular
thiols, the cells were preincubated with 1 mm NMM for 20 min.
NMM was maintained in the medium during the FLIM experiments.
Solutions of all of the compounds for time-resolved fluorescence
measurements were prepared so that the absorbance of the final
solutions at lex was lower than 0.1. Solutions were kept cool in the
dark when not in use to avoid possible deterioration by exposure
to light and heat. The solutions were not degassed.
Instrumentation
Cell permeabilization
Absorption spectra were recorded on a Perkin–Elmer Lambda 650
Cells were permeabilized as reported.[14] Briefly, HeLa cells were
seeded onto coverslips in 12-well plates. On the day of the experi-
ment, the cells were washed twice with phosphate-buffered saline
(PBS) and were perforated by incubation for 15 min at 378C with
2 mgmLÀ1 a-toxin in permeabilization buffer (20 mm potassium 3-
(N-morpholino)propanesulfonate (pH 7.0), 250 mm mannitol, 1 mm
potassium adenosine triphosphate (ATP), 3 mm MgCl2, and 5 mm
potassium glutathione). Subsequently, the cells were washed twice
with PBS and were analyzed by using FLIM in the presence of dif-
ferent concentrations of phosphate ions in the assay medium.
UV/Vis spectrophotometer with
a temperature-controlled cell.
Steady-state fluorescence emission spectra were performed on
a JASCO FP-6500 spectrofluorometer equipped with a 450 W
xenon lamp for excitation and an ETC-273T temperature controller
at 208C. The pH values of the solutions were measured immediate-
ly before the fluorescence measurements at the same temperature.
Fluorescence-decay traces of solutions were recorded by the
single-photon timing method on a FluoTime 200 fluorometer (Pi-
coQuant, Inc.). The excitation was achieved with a LDH-485 laser
head (PicoQuant, Inc.), and the observation was performed
through a monochromator at l=515 nm. The pulse repetition rate
was 20 MHz. Fluorescence-decay histograms were collected in
1320 channels using cuvettes of 10”10 mm. The time increment
per channel was 36 ps. Histograms of the instrument-response
functions (using a LUDOX scatterer) and sample decays were re-
corded until they typically reached 2”104 counts in the peak chan-
nel. Three fluorescence decays were recorded for all of the sam-
ples. The fluorescence-decay traces were individually analyzed by
using an iterative deconvolution method with exponential models
that employed FluoFit software (PicoQuant).
Acknowledgements
This research was funded by MINECO (project CTQ2014-53598)
and CEI-BioTic (UGR).
Keywords: anions
·
fluorescence
lifetime
imaging
microscopy · fluorescent probes · intracellular imaging agents ·
thiols
Fluorescence-lifetime images were recorded on a MicroTime 200
fluorescence-lifetime microscope system (PicoQuant, Inc.) and the
time-tagged time-resolved (TTTR) methodology, which permits
fluorescence-decay histograms to be reconstructed from molecules
in the confocal volume. The excitation source consisted of a laser
diode head (LDH) pulsed laser at l=470 nm (PicoQuant) that oper-
ates at a repetition rate of 20 MHz. The light beam was directed
onto a dichroic mirror (500DCXR) to the oil-immersion objective
(numerical aperture=1.4, 100”) of an inverted microscope system
IX-71 (Olympus). The collected fluorescence light was filtered by an
HP500LP long-pass filter (AHF/Chroma) and focused onto a confo-
cal aperture of 75 mm. After the aperture, the transmitted light was
refocused onto a single-photon avalanche diode SPCM-AQR 14
(Perkin–Elmer) after the signal passed through a 520/35 filter
(Chroma). The data acquisition was performed with a TimeHarp
200 TCSPT module (PicoQuant, Inc.) in the TTTR mode, which ena-
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Received: July 16, 2015
Published online on September 16, 2015
Chem. Eur. J. 2015, 21, 14772 – 14779
www.chemeurj.org
14779
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim