Highly sensitive and fast responsive fluorescence turn-on chemodosimeter for Cu2+ and its application in live cell imaging

Article abstract of DOI:10.1002/chem.200800005

A rhodamine B derivative 4 containing a highly electron-rich S atom has been synthesized as a fluorescence turn-on chemodosimeter for Cu²⁺. Following Cu²⁺-promoted ring-opening, redox and hydrolysis reactions, comparable amplifications of absorption and fluorescence signals were observed upon addition of Cu²⁺; this suggests that chemodosimeter 4 effectively avoided the fluorescence quenching caused by the paramagnetic nature of Cu²⁺. Importantly, 4 can selectively recognize Cu²⁺ in aqueous media in the presence of other trace metal ions in organisms (such as Fe³⁺, Fe²⁺, Cu⁺, Zn²⁺, Cr ¹⁺, Mn²⁺, Co²⁺, and Ni²⁺), abundant cellular cations (such as Na⁺, K⁺, Mg²⁺, and Ca²⁺), and the prevalent toxic metal ions in the environment (such as Pb²⁺ and Cd²⁺) with high sensitivity (detection limit ≤10ppb) and a rapid response time (≤1 min). Moreover, by virtue of the chemodosimeter as fluorescent probe for Cu²⁺, confocal and two-photon microscopy experiments revealed a significant increase of intracellular Cu ²⁺ concentration and the subcellular distribution of Cu²⁺, which was internalized into the living HeLa cells upon incubation in growth medium supplemented with 50 μM CuCl₂ for 20 h.

Full text of DOI:10.1002/chem.200800005

DOI: 10.1002/chem.200800005
Highly Sensitive and Fast Responsive Fluorescence Turn-On
Chemodosimeter for Cu2+ and Its Application in Live Cell Imaging
Mengxiao Yu,[a] Mei Shi,[a] Zhigang Chen,[a] Fuyou Li,*[a] Xinxin Li,[b] Yanhong Gao,[c]
Jia Xu,[a] Hong Yang,[a] Zhiguo Zhou,[a] Tao Yi,[a] and Chunhui Huang*[a]
Abstract: A rhodamine B derivative 4
containing highly electron-rich
recognize Cu2+ in aqueous media in
the presence of other trace metal ions
in organisms (such as Fe3+, Fe2+, Cu+,
Zn2+, Cr3+, Mn2+, Co2+, and Ni2+),
abundant cellular cations (such as Na+,
K+, Mg2+, and Ca2+), and the preva-
lent toxic metal ions in the environ-
ment (such as Pb2+ and Cd2+) with
high
sensitivity
(detection
limit
a
S
ꢀ10 ppb) and a rapid response time
(ꢀ1 min). Moreover, by virtue of the
chemodosimeter as fluorescent probe
for Cu2+, confocal and two-photon mi-
croscopy experiments revealed a signif-
icant increase of intracellular Cu2+
concentration and the subcellular dis-
tribution of Cu2+, which was internal-
ized into the living HeLa cells upon in-
cubation in growth medium supple-
mented with 50 mm CuCl2 for 20 h.
atom has been synthesized as a fluores-
cence turn-on chemodosimeter for
Cu2+. Following Cu2+-promoted ring-
opening, redox and hydrolysis reac-
tions, comparable amplifications of ab-
sorption and fluorescence signals were
observed upon addition of Cu2+; this
suggests that chemodosimeter 4 effec-
tively avoided the fluorescence quench-
ing caused by the paramagnetic nature
of Cu2+. Importantly, 4 can selectively
Keywords: chemodosimeter · cop-
per · fluorescent probes · imaging
agents · rhodamine
Introduction
ions is associated with some serious neurodegenerative dis-
eases, including Menkes and Wilson diseases,[2] Alzheimerꢀs
disease,[3] familial amyotropic lateral sclerosis,[4] and prion
diseases.[5] In particular, exposure to a high level of copper
even for a short period of time can cause gastrointestinal
disturbance, and long-term exposure causes liver or kidney
damage,[6] as a result of its ability to displace other metal
ions that act as cofactors in enzyme-catalyzed reactions.[7]
Thus, visualizing the concentration and subcellular distribu-
tion of copper in physiological processes may greatly con-
tribute to understanding its complex physiological functions
and nosogenesis.
Copper, after iron and zinc, is the third most abundant es-
sential trace element in the human body, and plays an im-
portant role in many fundamental physiological processes in
organisms.[1] Alteration in the cellular homeostasis of copper
[a] M. Yu, Dr. M. Shi, Z. Chen, Prof. F. Li, J. Xu, H. Yang, Z. Zhou,
Prof. T. Yi, Prof. C. Huang
Department of Chemistry & Laboratory of Advanced Materials
Fudan University, 220 Handan Road, Shanghai 200433 (PR China)
Fax : (+86)21-5566-4621
E-mail: fyli@fudan.edu.cn
By virtue of its highly sensitive and high-speed spatial
analysis of cells, fluorescence bioimaging has provided a
facile and less cell-damaging means of visualizing analytes
of biological interest in living cells.[8,9] To image intracellular
metal ions, highly sensitive and selective probes that exhibit
an enhanced visible fluorescent emission in aqueous media
need to be developed. A widely used strategy is to closely
linkthe metal recognition portion to a fluorophore as the
signal generation moiety and then a selective metal ion
binding process could induce the fluorescence increase. In
this way, several “turn-on” fluorescent probes in living cells
for sensing main Group II metal ions (such as Ca2+ and
chhuang@fudan.edu.cn
[b] Prof. X. Li
School of Materials Science and Engineering
East China University of Science and Technology
130 Meilong Road, Shanghai 200237 (PR China)
[c] Dr. Y. Gao
Hospital of Xinhua, Medicine School of Shanghai Jiaotong University
1665 Kongjiang Road, Shanghai 200092 (PR China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800005: UV/Vis and fluores-
cence spectra, charge numbers of atoms in 1 and 4, ESI mass spectra,
XPS spectrum, fluorescence images, the result of trypan blue viability
test and the other supplementary materials, including colour versions
of Figure 5, Scheme 3, and Table 1.
[10]
[11,12]
Mg2+
)
and transition-metal ions (such as Zn2+
,
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FULL PAPER
[13]
[15]
[16]
Cd2+
,
Cu+,[14] Pb2+
,
Fe3+
,
and Hg2+[17]) have been
rhodamine B derivative 4 containing a highly electron-rich S
successfully designed and synthesized. However, this
atom has been demonstrated as a chemodosimeter for Cu2+
.
method has been applied to Cu2+ with only limited suc-
Furthermore, confocal and two-photon fluorescence micro-
scopy experiments have demonstrated that 4 can be used to
image Cu2+ in living cells.
cess.[18,19] Due to the intrinsic fluorescence quenching prop-
erty that stems from the paramagnetic nature of Cu2+
,
[20]
most hitherto reported Cu2+ sensors have shown decreased
emission upon Cu2+ binding.[18] Such fluorescence “turn-off”
sensors may give false positive results caused by other
quenchers in real samples and are undesirable for analytical
purposes. In addition, although few fluorescence “turn-on”
Cu2+ sensors[19] with nanomolar sensitivity,[19a–c,g] high selecti-
vity,[19a,g] rapid response, or good water solubility[19g] have
been reported, sensors which combine all these features
have not been obtained up until now. Based on the above
reasons, to the best of our knowledge, no work on fluores-
cence bioimaging of Cu2+ has previously been reported.
Thus, the development of highly sensitive turn-on fluores-
cent probes for monitoring Cu2+ in living cells still remains
a significant challenge.
Results and Discussion
Synthesis and characterization of 4: Probe 4 was easily syn-
thesized in 68% yield by a condensation reaction of rhoda-
hydrazide (3) and n-butyl isothiocyanate
(Scheme 2). The structure of 4 was confirmed by H NMR,
mine
B
1
To date, the chemodosimeter has attracted a tremendous
amount of attention as an alternative to sensors based on
metal ion binding mechanism mentioned above due to its
high sensitivity and a rapid response.[21,22] Chemodosimeter
indicates the presence of an analyte through the generation
of a fluorescent or colored product resulting from a specific
chemical reaction between the dosimeter molecule and the
target species. In particular, the chemodosimeter provides
an ideal way to design fluorescence “turn-on” probes for
paramagnetic metal ions such as Cu2+, since the fluorescent
product does not coordinate to metal ions. However, the
lackof highly selective reactions induced by Cu 2+, which
occur fast in aqueous solution at room temperature, remain
Scheme 2. Chemical structure and synthestic routine of 4.
13C NMR spectroscopic, and MS data and by single-crystal
X-ray diffraction analysis. A single crystal of 4 was obtained
by slow evaporation of the solvent from a solution in
CH3CN. The single-crystal X-ray diffraction study reveals
that the molecules of 4 adopt a spirocyclic form in the crys-
tal (Figure 1). The butyl group is split between two locations
with equal occupancies, reflecting its propensity to swing in
the crystal.
A solution of 4 in CH3CN or CH3CN/HEPES (50 mm, pH
7.2, 3:7, v/v) solution was colorless and weakly fluorescent,
indicating that the spirocyclic form was retained in solution.
The characteristic peakat d=67.0 ppm in the 13C NMR
spectrum of 4 also supported this conclusion.
a big hurdle for obtaining such probes. Based on a Cu2+
promoted hydrolysis reaction of rhodamine hydrazide, Czar-
-
2+
niket al. designed a chemodosimeter for Cu
in water,[21]
although its ability to sense Cu2+ in cells was not investigat-
ed. Recently, Tae et al. reported that a rhodamine 6G deriv-
ative 1 was capable of serving as a chemodosimeter for
[23]
Hg2+
.
This derivative responds to Hg2+ stoichiometrically,
Hg2+/Cu2+ response of 4: Based on the well-known equilib-
rium between non-fluorescent spirolactam and the fluores-
cent ring-opened amide of rhodamine,[16,17d,25] it is reasoned
that 4 with a thiourea group can be used as a fluorescent
probe for soft metal ions. Since 4 is similar in chemical
structure to 1, the response of 4 to Hg2+ was investigated by
UV/Vis absorption and fluorescence spectra. Interestingly,
the behavior of 4 in sensing Hg2+ was distinct from that of 1
in response to Hg2+. Tae et al.[23] described that the fluores-
cence intensity of 1 was nearly proportional to the Hg2+
concentration when up to one equivalent of Hg2+ was
added. In our case, a weakincrease in the absorption and
emission of 4 was observed upon addition of less than one
equivalent of Hg2+, as shown in Figure 2. Further addition
of Hg2+ caused a significant increase in the absorption band
centered at 566 nm and fluorescent emission peaked at
591 nm (see Figures S1 and S2 in the Supporting Informa-
tion), corresponding to the appearance of a purple-red color
and an intense orange-red fluorescence. These facts imply
rapidly, and irreversibly at room temperature through desul-
furization and cyclization reactions which produce a strongly
fluorescent product 2 (Scheme 1).
Inspired by such a chemodosimeter for Hg2+, we surmised
that a marked increase of the electronegativity of the S
atom could lead to similar reactions being induced by other
sulfophilic elements, such as copper. In the present work, a
Scheme 1. Conversion of nonfluorescent 1 to strongly fluorescent 2 by
Hg2+
.
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F. Li, C. Huang et al.
ence of Cu2+ was distinct from that of 4 upon addition of
Hg2+ (the inset of Figure 2a). The emission maximum of the
mixture of 4 and Hg2+ (lmaxem =591 nm) was red-shifted by
ꢁ10 nm in comparison with that of 4 in response to Cu2+
(lmaxem =580 nm), possibly indicating different products of 4
in the sensing of Cu2+ and Hg2+. Moreover, the intense
fluorescence of 4 induced by Hg2+ or Cu2+ could not be
suppressed by further addition of excess chelating agent,
such as EDTA (see Figures S4 and S5 in the Supporting In-
formation), which suggests irreversible sensing processes of
4 in response to these metal ions.
Recognition mechanism of 4 towards Hg2+/Cu2+: To under-
stand the different recognition abilities of 1 and 4 towards
Cu2+, the HOMO and LUMO distributions of 1 and 4 were
determined by density functional theory (DFT) calculations.
As shown in Table 1, the HOMO distribution of 4 is concen-
trated exclusively on the S atom of the thiourea group,
while that of 1 is located diffusely over the thiourea and
xanthene moieties. This result reveals that the introduction
of the n-butyl group significantly increased the HOMO elec-
tron density on the S atom. Furthermore, the charge on the
S atom in 4 is À0.20, whereas in 1 it is À0.16 (see Figures S6
and S7 in the Supporting Information). In the metal ion pro-
moted desulfurization reaction, the S atom of 4 becomes an
electron-rich center and exhibits a higher affinity for the
metal ion. Thus, the HOMO distribution exclusively on the
S atom of 4 and the increased electronegativity would con-
Figure 1. ORTEP drawing of 4 (30% ellipsoid). H atoms are omitted for
clarity.[24]
stitute a reason for its unique response to Cu2+
.
We further investigated the final reaction products of 4
with Cu2+ and Hg2+ by ESI mass spectrometry. The ESI
mass spectrum of 4 with two equiv Hg2+ showed a unique
peakat m/z 538.3 (calcd for 538.3) (see Figure S8 in the
Supporting Information), corresponding to a desulfurized
and cyclized product [5]+ (Scheme 3). In the case of 4 with
two equiv Cu2+, a peakat m/z 443.2 (calcd for 443.3) as-
signed to [6]+ (namely rhodamine B, see Scheme 3) was
clearly observed (see Figure S9 in the Supporting Informa-
tion). Corroborating evidence was provided by independent
syntheses of 5 and 6 from the direct reactions of 4 with Hg-
Figure 2. Distinct difference in Cu2+ and Hg2+ sensing behavior of 4 in
CH3CN/HEPES (50 mm, pH 7.2, 3:7, v/v) solution. a) Time course of
20 mm 4 upon gradual addition of Cu2+ and Hg2+. Absorbance at 555 nm
(for Cu2+) and 566 nm (for Hg2+) was recorded respectively. Inset shows
the fluorescent responses of 4 to 5 equiv of Cu2+ and Hg2+ under 365 nm
UV excitation. b) Color changes of 20 mm 4 in the presence of different
A
N
1
perature; H NMR and MS data confirmed their structures.
These results indicate a distinct recognition mechanism of 4
towards Cu2+ as compared to that of its response to Hg2+
.
concentrations of Cu2+ or Hg2+
.
Recently, Chorev et al.[26] have reported that dialkyl-sub-
stituted thioureas undergo rapid formation of the intermedi-
that 4 might show a distinct recognition of metal ions as
compared to 1.
ate carbodiimide in the presence of Hg(OAc)2 (Scheme 4).
A
Thus, we can deduce a recognition mechanism of 4 towards
Hg2+ as follows (Scheme 3): a) In the presence of less than
one equiv Hg2+, 4 undergoes an irreversible desulfurization
reaction and is thereby converted into an intermediate car-
bodiimide 7, in accordance with the observations of Chorev
et al.[26] This is further confirmed by the appearance of a
peakat m/z 538.2 (calcd for 538.3) assigned to [7+H]+ in
the ESI mass spectrum of a colorless and weakly fluorescent
mixture of 4 with 0.5 equiv of Hg2+ in ethanol (see Figure
S10 in the Supporting Information). The weakincrease in
More interestingly, unlike 1, 4 can also respond to Cu2+
.
When Cu2+ was gradually added to a solution of 4, a new
absorption band with a maximum at 555 nm appeared, cor-
responding to the observation of a new emission band cen-
tered at 580 nm (Figure S3 in the Supporting Information).
Unlike the response of 4 to Hg2+, a proportional increase in
absorption and emission was observed in its sensing of Cu2+
(Figure 2 and Figure S3 in the Supporting Information). It is
worthy of note that the fluorescent color of 4 in the pres-
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Fluorescence Turn-On Chemodosimeter
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Table 1. HOMOÀ1, HOMO, LUMO and LUMO+1 distributions of 1 and 4 calculated by DFT calculations.
tion process similar to that of
its response to Hg2+ occurred
HOMO-1
HOMO
LUMO
LUMO+1
possibly for 4 response to Cu2+
.
Detection of Cu2+ in vitro: In
view of the absence of Hg2+ in
normal living samples, the pos-
sibility of using 4 as a fluores-
cent probe for monitoring intra-
cellular Cu2+ was also investi-
gated. Such a similar treatment
is commonly used in the case of
Zn2+ bioimaging by Zn2+-selec-
1
2
tive fluorescent sensors with in-
[12a,b]
terference from Cd2+
.
The
pH dependence of the Cu2+
sensing by 4 was determined. No obvious fluorescence emis-
sion of 4 was observed between pH 4.8 and 11.8, suggesting
that the compound is stable over a wide range. In the range
pH 5.8–8.8, a marked fluorescence enhancement was mea-
sured upon addition of Cu2+ (see Figure S14 in the Support-
ing Information). These data establish that 4 could act as a
fluorescent probe for Cu2+ under physiological pH condi-
tions.
In absorption and fluorescence titrations of 20 mm 4 in
CH3CN/HEPES (50 mm, pH 7.2, 3:7, v/v) solution, a 430-
fold increase in the absorbance at 555 nm was observed
upon addition of 5 equiv Cu2+ (Figure 3) and a comparable
fluorescence enhancement (470-fold) at 580 nm was also de-
tected (Ff =0.31); this suggests that the chemodosimeter ef-
fectively avoided the fluorescence quenching caused by the
paramagnetic nature of Cu2+. Moreover, to assess the possi-
bility of detecting Cu2+ at a low concentration, fluorescence
titrations were conducted with 1 mm 4. The fluorescence in-
tensity was found to increase linearly with the Cu2+ concen-
tration in the range of 4.5–160 ppb (Figure 4). The detection
limit of this chemodosimeter system was estimated to be
10 ppb (Figure 4b), which is comparable to those of some
previously reported highly sensitive sensors.[19a–c,g] In addi-
absorption and fluorescence indicates that the molecules of
7 adopt a spirolactam form. b) Upon further addition of
more than one equiv Hg2+, the ring-opened product 5 con-
taining a 1,3,4-oxadiazole moiety is formed by an Hg2+-pro-
moted cyclization reaction, resulting in an obvious absorp-
tion and fluorescence enhancement.
In the case of copper, although Cu+ does not induce the
response of 4 (see Figure S11 in the Supporting Informa-
tion), the Cu 2p X-ray photoelectron spectra (XPS) analyses
demonstrate the presence of Cu+. In the mixture of 4 with
0.5 equiv Cu2+, the peaks having binding energies of 932.2
and 951.8 eV, corresponding to Cu 2p3/2 and 2p1/2 of Cu+, re-
spectively, were observed (see Figure S12 in the Supporting
Information). In addition, the S atom of the thiourea group
in 4 shows higher electronegativity as compared to that in 1.
Based on these facts, the mechanism of Cu2+ induced fluo-
rescence increase of
4 can be proposed as follows
(Scheme 3): c) A Cu2+-promoted ring-opening reaction of 4
occurs instantly upon the addition of Cu2+, owning to the
strong binding ability of the O, N, and S atoms towards
Cu2+. d) Due to the rather electron-rich rhodamine–thiour-
ea moiety as electron donor, a redox reaction between 4
and Cu2+ could occur, thus reducing Cu2+ to Cu+. A peak
at m/z 634.2 (calcd for 634.2) assigned to an intermediate 8
including a Cu+ was evident from the ESI mass spectrum of
4 with 0.5 equiv Cu2+ in ethanol. The peaks at m/z 1205.4
(calcd for 1205.5), 1268.3 (calcd for 1268.5), 1331.2 (calcd
for 1331.4), which could be identified as a series of inter-
mediates such as CuL2, Cu2L2, Cu3L2 (L stands for the
ligand 4) were also detected (see Figure S13 in the Support-
ing Information). e) Subsequently, a further hydrolysis
occurs, resulting in the formation of the final product 6,
which exhibits intense photoluminescence. In aqueous
media, the Cu2+-promoted ring-opening, redox and hydroly-
sis reactions described above happen rapidly, therefore, one
can observe a continuous variation in the absorption and
fluorescence spectra when Cu2+ is gradually added
(Figure 2). Naturally, a peakat m/z 538.2 in the ESI mass
spectrum of the mixture of 4 with Cu2+ (see Figure S13 in
the Supporting Information) implies that another recogni-
tion, different copper salts, such as CuCl2, Cu(NO3)2, and
A
CuSO4, gave rise to the same fluorescence profiles in its re-
sponse to Cu2+ (see Figure S15 in the Supporting Informa-
tion), indicating a negligible effect of the counteranions on
the recognition ability and photophysical properties of 4.
Furthermore, the time dependence of the response of 4 to
Cu2+ was monitored by means of absorption spectroscopy
(Figure 5). The results revealed that the reaction of 20 mm 4
and Cu2+ (ꢀ 100 mm) was complete within 1 min. In particu-
lar, an instantaneous response towards Cu2+ at low concen-
trations (ꢀ1 mm) was observed. Therefore, this system could
be used for real-time tracking of Cu2+ in cells and organ-
isms.
Achieving high selectivity for the analyte of interest over
other potentially competing species is a necessity for bio-
imaging probes. Thus, the selectivity and competition experi-
ments were extended to various metal ions, such as abun-
dant cellular cations (Na+, K+, Mg2+, and Ca2+), trace
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F. Li, C. Huang et al.
Figure 3. Absorption spectra of 20 mm
4
upon addition of Cu2+ in
CH3CN/HEPES (50 mm, pH 7.2, 3:7, v/v) solution. Inset shows the ab-
sorbance at 555 nm of 4 as a function of Cu2+ concentration.
Scheme 3. Proposed mechanisms for Hg2+ and Cu2+-promoted fluores-
cence enhancement of 4. a) Hg2+-triggered desulfurization reaction to
produce an intermediate carbodiimide 7 with weakly fluorescence. b)
Hg2+-induced ring-opening of the spirolactam and cyclization reaction of
7, resulting in the formation of fluorescent product 5. c) Cu2+-promoted
ring-opening reaction of 4, owning to the strong binding ability of the O,
N, and S atoms towards Cu2+. d) Redox reaction between Cu2+ and 4,
due to the rather electron-rich rhodamine-thiourea moiety as electron
donor. An intermediate 8 including a Cu+ is demonstrated by the ESI
mass spectrum analyses. e) Hydrolysis reaction in aqueous media, pro-
ducing the final product 6 (namely rhodamine B).
Figure 4. Fluorescence responses of 1 mm 4 upon addition of 4.5–160 ppb
Cu2+ in CH3CN/HEPES (50 mm, pH 7.2, 3:7, v/v) solution. Inset and b)
show the fluorescence intensity at 580 nm of 4 as a function of Cu2+ con-
centration (inset of a) 0–160 ppb; b) 0–21 ppb).
only Cu2+ induced a prominent fluorescence enhancement,
whereas very weakfluorescence variations were observed
for the other metal ions. Additionally, these co-existent ions
had negligible interfering effect on Cu2+ sensing by 4, even
when Na+, K+, Mg2+, and Ca2+ were present at micromolar
levels. Therefore, the excellent selectivity of 4 for Cu2+ over
these physiological metal ions in aqueous media indicates its
utility for a wide range of biological applications. Important-
ly, its high selectivity for Cu2+ over Cu+ suggests that 4
might be used in tracking copper-related redox processes.
Scheme 4. Formation of intermediate carbodiimide from a 1,3-dialkyl-
thiourea.[26]
metals in organisms (Fe3+, Fe2+, Cu2+, Cu+, Zn2+, Cr3+
,
Mn2+, Co2+, and Ni2+), and the prevalent toxic metals in
the environment (Pb2+ and Cd2+). As shown in Figure 6,
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Fluorescence Turn-On Chemodosimeter
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Figure 5. Time course of the response of 4 to different concentrations of
Cu2+. Absorbance at 555 nm was recorded in 20 mm 4 in CH3CN/HEPES
(50 mm, pH 7.2, 3:7, v/v) solution.
Figure 6. Fluorescence intensity changes (F/F0) of 20 mm 4 upon the addi-
tion of various metal ions in CH3CN/HEPES (50 mm, pH 7.2, 3:7, v/v) so-
lution. Darkbars represent the fluorescence response of 4 to the metal
ion of interest (10 mm for K+, Na+, Ca2+ and Mg2+; 25 mm for Fe2+ and
Cu+; and 100 mm for other metal ions; X is a mixture of K+, Na+, Mg2+
and Ca2+). Gray bars represent the subsequent addition of 100 mm Cu2+
to above solutions. Excitation and emission was at 510 and 580 nm, re-
spectively.
Figure 7. Confocal fluorescence and brightfield images of HeLa cells. a)
Cells stained with 5 mm 4 for 10 min at 258C. b) Cells supplemented with
50 mm CuCl2 in the growth media for 20 h at 378C and then incubated
with 5 mm 4 for 10 min at 258C. c) Brightfield image of cells shown in
panel b. The overlay image of b) and c) is shown in d) (lex =543 nm).
e) Two-photon excited fluorescence image of Cu2+ supplemented HeLa
cells stained with 5 mm 4 for 10 min at 258C (lex =880 nm). f) Fluores-
cence intensity profile across a HeLa cell shown in a).
Fluorescence imaging of intercellular Cu2+: We proceeded
to investigate the practical applicability of 4 as a Cu2+ probe
in the fluorescence imaging of living cells. As determined by
laser scanning confocal microscopy, staining HeLa cells with
5 mm 4 for 10 min at 258C gave no intracellular fluorescence
(Figure 7a). When the cells were supplemented with 50 mm
CuCl2 in the growth medium for 20 h at 378C and then incu-
bated with 4 under the same conditions, a significant fluores-
cence increase from the intracellular region was observed
(Figure 7b). Control experiments on Cu2+-supplemented
cells without staining with 4 exhibited negligible background
fluorescence (data not shown). Brightfield measurements
after treatment with Cu2+ and 4 confirmed that the cells
were viable throughout the imaging experiments (Figure 7c).
The overlay of fluorescence and Brightfield images revealed
that the fluorescence signals were localized in the perinu-
clear region of the cytosol (Figure 7d), indicating the subcel-
lular distribution of Cu2+ which was internalized into the
living cells from the growth medium.
We further explored the dose-dependent response of 4 to
Cu2+ in live cells by fluorescence imaging of MCF-7 cells
supplemented with different concentrations of Cu2+. After
incubation with 50 mm 4 for 30 min at 378C, cells pretreated
with 50 mm Cu2+ (for 20 h at 378C) showed fluorescence ex-
clusively in the perinuclear region of the cytosol, and cells
supplemented with 100 or 200 mm Cu2+ exhibited more in-
tense fluorescent signals that were located diffusely over the
cytosol (see Figure S19 in the Supporting Information). In
the control experiments, 50 mm 4 incubated cells without
supplement with Cu2+ exhibited negligible background fluo-
rescence (see Figure S19 in the Supporting Information).
These results indicate that the presence of Cu2+ causes the
fluorescence increase observed in cells. Furthermore, the cy-
totoxicity of the probe 4 was determined by trypan blue via-
bility test (see Figure S20 in the Supporting Information).
The viabilities of MCF-7 cells still retained 94.3 and 90.2%
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F. Li, C. Huang et al.
after the incubation with 50 or 200 mm Cu2+, respectively,
and then staining with 4; this suggests that most of the
MCF-7 cells were viable in the fluorescence imaging experi-
ments mentioned above. In the control experiments without
staining with 4, the cellular viabilities were 98.2 and 93.6%
corresponding to the 50 or 200 mm Cu2+-supplemented cells,
respectively, further indicating that this Cu2+ fluorescent
probe 4 can be considered to have low cytotoxicity. These
results demonstrate that 4 can be used for monitoring Cu2+
within biological samples.
Compared with the single-photon related confocal fluo-
rescent bioimaging technique, two-photon laser scanning mi-
croscopy (TPLSM) imaging has the advantages of high
transmission at low incident intensity and reduced back-
ground cellular autofluorescence,[27] thus has been widely
used for the in vivo imaging in neuroscience.[28] Because
rhodamine B is a classical two-photon active dye,[29] it is rea-
soned that chemodosimeter 4 may be used in two-photon
imaging. Cu2+-supplemented HeLa cells were stained with 4
under the same loading conditions; then, when excitation
was provided at 880 nm, intense intracellular fluorescence
signals at 550–650 nm were detected (Figure 7e). As shown
in Figure 7f, quantization by line plots shows a signal-to-
noise ratio (I2/I1) of 4.3 between cytoplasm (region 2) and
background (region 1) and a ratio (I2/I3) of 3.5 between cy-
toplasm (region 2) and nucleus (region 3), further confirm-
ing the cytoplasmic distribution of intracellular Cu2+. These
facts reveal the potential utility of 4 as a Cu2+-sensitive two-
photon excited fluorescent probe for the in vivo bioimaging
by TPLSM.
Experimental Section
General:
N-(2-Hydroxyethyl)piperazine-N’-2-ethanesulfonic
acid
(HEPES) was purchased from Acros Organics. All other chemicals were
purchased from Sigma-Aldrich and were used as received. TLC analyses
were performed on silica gel 60 F254. Column chromatographic purifica-
tions were carried out on silica gel (HG/T2354-92). NMR spectra were
recorded on a Mercury Plus 400 MHz spectrometer (Varian Gemini-400).
All chemical shifts are reported in the standard d notation of parts per
million. Mass spectra (EI) were measured on an MA1212 instrument
under standard conditions. Electrospray ionization mass spectra (ESI-
MS) were measured on a Micromass LCTTM system. Elemental analyses
were performed on a VarioEL III O-Element Analyzer system. UV/Vis
absorption spectra were recorded on a Shimadzu 3000 spectrophotome-
ter. Fluorescence emission spectra were measured on an Edinburgh
LFS920 luminescence spectrometer with a 1000 W xenon lamp. Lumines-
cence quantum yields in solution were measured by using rhodamine B
(FF =0.69 in ethanol)[30] Samples for absorption and emission measure-
ments were contained in 1 cm”1 cm quartz cuvettes.as a reference. De-
ionized water was used to prepare all aqueous solutions. Solutions of
Cu+, Hg2+, Fe2+, Mn2+, Na+, K+, and Ca2+ were prepared from their
chloride salts; solutions of Zn2+, Cd2+, Fe3+, Pb2+, Co2+, Ni2+, Cr3+
,
Ag+, and Mg2+ were prepared from their nitrate salts. Solutions of Cu2+
were prepared from its chloride, nitrate, and sulfate salts. HEPES buffer
solutions (pH 7.2) were prepared using 50 mm HEPES and the appropri-
ate amount of NaOH. All spectroscopic measurements were performed
in CH3CN/HEPES (50 mm, pH 7.2, 3:7, v/v) solution.
Synthesis of 4: According to previous literature reports,[16,21] rhodamine
B hydrazide (3) was prepared and then characterized by its NMR and
mass spectra. n-Butyl isothiocyanate (2.5 g, 22 mmol) was added to a so-
lution of 3 (1.0 g, 2.2 mmol) in chloroform (10 mL). The mixture was
then heated under reflux for 3 d. Thereafter, the solvent was evaporated
under reduced pressure, and the crude product was purified by column
chromatography (petroleum ether/ethyl acetate 4:1) to give 4 as a color-
less solid (0.85 g, 68%). M.p. 106–1098C; 1H NMR (400 MHz, CDCl3,
258C, TMS): d=0.786 (t, J=7.0 Hz, 3H, C(=S)NHCH2CH2CH2CH3),
1.15 (m, 16H, NCH2CH3, C(=S)NHCH2CH2CH2CH3), 3.23 (q, J=6.4 Hz,
2H, C(=S)NHCH2CH2CH2CH3), 3.31 (q, J=7.1 Hz, 8H, NCH2CH3), 5.88
(t, J=4.8 Hz, 1H, C(=S)NHCH2CH2CH2CH3), 6.28 (d, J=8.4 Hz, 2H,
xanthene-H), 6.40 (s, 3H, xanthene-H), 6.42 (s, 1H, xanthene-H), 6.75 (s,
1H, NHC(=S)NHCH2CH2CH2CH3), 7.23 (d, J=7.6 Hz, 1H, phenyl-H),
7.54 (m, 2H, phenyl-H), 7.98 ppm (d, J=7.6 Hz, 1H, phenyl-H);
13C NMR (400 MHz, CDCl3, 258C, TMS): d=12.8, 13.9, 20.1, 30.9, 44.6,
44.9, 67.0, 98.5, 104.6, 108.3, 124.0, 125.0, 127.8, 129.1, 129.3, 134.4, 149.4,
150.6, 154.4, 167.4, 183.1 ppm; MS (EI): m/z (%): 571.5 (27) [M+]; ele-
mental analysis calcd (%) for C33H41N5O2S: C 69.32, H 7.23, N 12.25;
found: C 69.25, H 7.48, N 12.50.
Conclusion
In conclusion, we have developed a rhodamine B derivative
4 and demonstrated its utility as a fluorescence turn-on che-
modosimeter that responds stoichiometrically, rapidly, and
highly sensitively to Cu2+ in aqueous media. The recognition
process involves Cu2+-promoted ring-opening, redox and
hydrolysis reactions, which may be attributed to the highly
electron-rich S atom in 4. Comparable amplifications of the
absorption and fluorescence signals were observed in its re-
sponse towards Cu2+, suggesting that chemodosimeter 4 ef-
fectively avoided the fluorescence quenching caused by the
paramagnetic nature of Cu2+. This chemodosimeter dis-
played very high sensitivity (detection limit ꢀ10 ppb), a
rapid response time (ꢀ1 min), and high selectivity for Cu2+
over other trace transition metal ions and abundant cellular
cations. Moreover, confocal and two-photon fluorescence
microscopy experiments have established the utility of 4 in
monitoring Cu2+ within living cells and mapping its subcel-
lular distribution. We anticipate that this probe will be of
great benefit to biomedical researchers for studying the ef-
fects of Cu2+ in biological systems.
Synthesis of 5 from 4 upon addition of Hg2+: Mercury(II) perchlorate hy-
drate (0.18 g, 0.36 mmol) was added to a solution of 4 (0.10 g, 0.18 mmol)
in CH3CN (2 mL). The mixture was stirred at room temperature for 1 d.
Thereafter, the solvent was removed under reduced pressure, and the
crude product was purified by column chromatography (CHCl3/MeOH
6:1) to give 5 as a dark-purple solid (0.032 g, 33%). 1H NMR (400 MHz,
CDCl3, 258C, TMS): d=0.697 (t, J=7.2 Hz, 3H, oxadiazole-N-
CH2CH2CH2CH3), 1.07 (m, 2H, oxadiazole-N-CH2CH2CH2CH3), 1.25
(m, 14H, NCH2CH3, oxadiazole-N-CH2CH2CH2CH3), 2.84 (s, 2H, oxa-
diazole-N-CH2CH2CH2CH3), 3.55 (q, J=7.1 Hz, 8H, NCH2CH3), 6.78 (s,
2H, xanthene-H), 6.82 (d, J=9.6 Hz, 2H, xanthene-H), 7.05 (d, J=
9.2 Hz, 2H, xanthene-H), 7.27 (m, 1H, Ar-H), 7.67 (m, 2H, Ar-H),
8.14 ppm (m, 1H, Ar-H); MS (EI): m/z (%): calcd for C33H40N5O2: 537.3
[MÀH]+; found: 537.4.
Synthesis of 6 from 4 upon addition of Cu2+: Copper(II) nitrate hydrate
(0.086 g, 0.36 mmol) was added to a solution of 4 (0.10 g, 0.18 mmol) in
CH3CN (2 mL). The mixture was stirred at room temperature for 10 min.
Thereafter, the solvent was removed under reduced pressure, and the
crude product was purified by column chromatography (CHCl3/MeOH
6:1) to give 6 as a dark-purple solid (0.021 g, 26%). 1H NMR (400 MHz,
CDCl3, 258C, TMS): d=1.29 (t, J=7.0 Hz, 12H, NCH2CH3), 3.53 (q, J=
6898
www.chemeurj.org
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 6892 – 6900

Fluorescence Turn-On Chemodosimeter
FULL PAPER
7.2 Hz, 8H, NCH2CH3), 6.70 (d, J=2.4 Hz, 2H, xanthene-H), 6.75 (dd,
J1 =9.6 Hz, J2 =2.4 Hz, 2H, xanthene-H), 7.10 (d, J=9.2 Hz, 2H, xan-
thene-H), 7.19 (m, 1H, Ar-H), 7.67(m, 2H, Ar-H), 8.47 ppm (m, 1H, Ar-
H); MS (EI): m/z (%): calcd for C28H31N2O3: 442.2 [MÀH]+; found:
442.3.
Trypan blue viability test: MCF-7 cells (1”108 L) were plated on 14 mm
glass coverslips and allowed to adhere for 24 h. Subsequently, cells were
supplemented with 50 or 200 mm CuCl2 in the growth medium for 20 h at
378C, and were incubated with 50 mm 4 for 30 min at 378C. After washing
with PBS, 0.1% trypan blue solution was dropped onto the cells that ad-
hered to the coverslips. Finally, viable (unstained by trypan blue) and
non-viable (stained by trypan blue) cells were counted under a micro-
scope. The cellular viability was calculated by the following formula: Via-
bility (%) = total viable cells/total cells (viable and non-viable)”100.
X-ray crystallographic analyses: A single crystal of 4 was mounted on a
glass fiber and transferred to a Bruker SMART CCD area detector.
Crystallographic measurements were made on a Bruker SMART CCD
diffractometer, with s scans and graphite-monochromated MoKa radia-
tion (l=0.71073 Š) at room temperature. The structure was solved by
direct methods and refined by full-matrix least-squares on F2 values using
the program SHELXS-97.[31] All non-hydrogen atoms were refined aniso-
tropically. Hydrogen atoms were calculated in ideal geometries. For the
full-matrix least-squares refinements [I > 2s(I)], the unweighted and
Acknowledgements
weighted agreement factors of R1=ꢀ
A
[ꢀw(Fo2ÀFc2)2/ꢀwFo4]1/2 were used.
The authors are thankful for the financial support from National Natural
Science Foundation of China (20490210, 20501006 and 20775017), Na-
tional High Technology Program of China (2006 AA03Z318), NCET-06-
0353, Shanghai Science and Technology Community (06QH14002), Huo
Yingdong Education Foundation (104012) and Shanghai Leading Aca-
demic Discipline Project (B108). We also thankProfessor Cong-Jian Xu
and Dr. Xiao-Yan Zhang for their helpful discussion.
Theoretical calculations: The structures of 1 and 4 were optimized using
density functional theory (DFT) by the B3LYP method with the 3-21G**
basis set. The contours of the HOMO and LUMO were plotted. The
DFT calculations were performed using the Gaussian 03 program.[32]
X-ray photoelectron spectroscopy analyses: An aqueous solution of cop-
per(II) chloride (20 mL, 0.44 molLÀ1; 0.0015 g, 0.009 mmol) was added to
a solution of 4 (0.010 g, 0.018 mmol) in ethanol (1 mL). The mixture was
stirred at room temperature for 10 min. Thereafter, the solvent was re-
moved under reduced pressure, and the dark-purple product was used for
XPS experiments. The measurements were carried out on a RBD up-
graded PHI-5000C ESCA system (Perkin Elmer) with MgKa radiation
(hn=1253.6 eV).
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Procedures for metal-ion sensing: Stocksolutions of the metal ions
(2.5 mm) were prepared in deionized water, except for Cu+, which was
dissolved in CH3CN. A stocksolution of 4 (1 mm) was also prepared in
CH3CN, and was then diluted to 20 mm or 1 mm with CH3CN/HEPES
(50 mm, pH 7.2, 3:7, v/v) solution. Titration experiments were performed
by placing 2.5 mL of a solution of 4 (20 or 1 mm) in a quartz cuvette of
1 cm optical path length, and then adding the Cu2+ or Hg2+ stocksolu-
tion incrementally by means of a micro-pipette. Spectra were recorded
3 min after the addition. Test samples for selectivity experiments were
prepared by adding appropriate amounts of metal ion stocksolutions to
2.5 mL of a solution of 4 (20 mm). In competition experiments, Cu2+ was
added to solutions containing 4 and the other metal ions of interest. All
test solutions were stirred for 1 min and then allowed to stand at room
temperature for 30 min. For fluorescence measurements, excitation was
provided at 510 nm, and emission was collected from 520 to 700 nm.
Cell culture: The cell lines HeLa and MCF-7 were provided by the Insti-
tute of Biochemistry and Cell Biology, SIBS, CAS (China). The HeLa
cells were grown in MEM (modified Eagleꢀs medium) supplemented with
10% FBS (fetal bovine serum) at 378C and 5% CO2. The MCF-7 cells
were grown in MEM (modified Eagleꢀs medium) supplemented with
10% FBS (fetal bovine serum) and 1% insulin (10 mL/400 U) at 378C
and 5% CO2. Cells (5”108 L) were plated on 14 mm glass coverslips and
allowed to adhere for 24 h. Experiments to assess Cu2+ uptake were per-
formed over 20 h in the same medium supplemented with 50, 100, or
200 mm CuCl2.
Fluorescence imaging: Immediately before the experiments, cells were
washed with PBS buffer, and then HeLa cells were incubated with 5 mm 4
in PBS for 10 min at 258C, while MCF-7 cells were incubated with 50 mm
4 in PBS for 30 min at 378C. Cell imaging was then carried out after
washing the cells with PBS. Confocal fluorescence imaging was per-
formed with an OLYMPUS IX81 laser scanning microscope and a 60”
oil-immersion objective lens. Cells loaded with 4 were excited at 543 nm
using an HeNe laser. Emission was collected from 550 to 650 nm. Two-
photon fluorescence imaging was performed using an OLYMPUS
BX61W1 laser scanning microscope and a 40” water-immersion objec-
tive lens. Two-photon fluorescence was excited by a Ti/sapphire femto-
second laser source (Coherent Chameleon Ultra) set at 880 nm and an
output power of 2 W, which corresponded to an average power of ap-
proximately 50 mW in the focal plane. Emission was collected from 550
to 650 nm.
Chem. Eur. J. 2008, 14, 6892 – 6900
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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Received: January 2, 2008
Revised: April 3, 2008
Published online: July 7, 2008
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