Fluorescence turn-on detection of Cu2+ in water samples and living cells based on the unprecedented copper-mediated dihydrorosamine oxidation reaction

Article abstract of DOI:10.1039/b919531a

A fluorescence turn-on probe for Cu²⁺ based on the novel copper-mediated dihydrorosamine oxidation reaction has been constructed and employed in the detection of Cu²⁺ in water, new born calf serum, and living cells, and the new coppe

Full text of DOI:10.1039/b919531a

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Fluorescence turn-on detection of Cu2+ in water samples and living cells
based on the unprecedented copper-mediated dihydrorosamine oxidation
reactionw
Weiying Lin,* Lingliang Long, Bingbing Chen, Wen Tan and Wensha Gao
Received (in Cambridge, UK) 18th September 2009, Accepted 17th December 2009
First published as an Advance Article on the web 15th January 2010
DOI: 10.1039/b919531a
A fluorescence turn-on probe for Cu2+ based on the novel
copper-mediated dihydrorosamine oxidation reaction has been
constructed and employed in the detection of Cu2+ in water, new
born calf serum, and living cells, and the new copper-mediated
dihydrorosamine oxidation reaction likely proceeds by a copper
redox mechanism.
oxidation reactions mediated by Cu2+ which are clean and
complete.
The rosamine dyes have excellent photophysical properties,
such as long wavelength absorption and emission, large
extinction coefficients, and high fluorescence quantum yields.4
In the reduced form, the xanthene rings of dihydrorosamines
are deconjugated and thus non-fluorescent. Nevertheless,
upon oxidation, the xanthene rings transform into their con-
jugated forms, and the fluorescence is switched on. Some
fluorescent probes for reactive oxygen species have been
constructed by the oxidation of dihydrorosamines, and they
tend to have poor stability due to auto-oxidation in air.5
However, to the best of our knowledge, dihydrorosamine
oxidation by metal ions has been previously unknown, and
thus the metal-mediated dihydrorosamine oxidation has not
been exploited in the design of fluorescent probes for metal
ions. Herein, we present dihydrorosamine 1 (Fig. 1), a new
type of fluorescence turn-on copper probe, as the first metal-
oxidation based fluorescence turn-on probe that can operate in
biological conditions. In addition, we also proposed that the
unprecedented copper-mediated dihydrorosamine oxidation
reaction employed for the probe development likely proceeds
by a copper redox mechanism.
Spectacular successes have been achieved in the organic
synthesis field in the preparation of a wide variety of species.
However, the central themes in organic synthesis are carbon–
carbon bond formation and functional group transformation.
In most cases, functional group transformation may lead
to pronounced changes in electronic properties. Thus, if a
functional group transformation occurs in close proximity to a
fluorescent dye, this will have a significant effect on the
photophysical states of the dye resulting in a visible variation
in the dye emission profile. This constitutes the basis of
the organic reaction-based fluorescent probe development
approach.1
Oxidation reactions are an important class of organic
reactions. Transition metal ions are known to be able to facilitate
the oxidation reactions. Thus, transition metal-based oxida-
tions may be exploited to design fluorescent probes for transi-
tion metal ions of interest. Recently, the groups of Burdette,2a
The synthesis of probe 1 is outlined in Scheme S1.w The
structure of compound 1 was fully characterized by 1H NMR,
13C NMR, HRMS, and single crystal X-ray diffraction
analysis.z The single-crystal X-ray crystallographic analysis
revealed that compound 1 adopts a deconjugated form and
that carbon 9 (labeled as C7 in the ORTEP drawing in Fig. 2)
is in the sp3 hybrid state. The fact that the crystal structure of
compound 1 was successfully acquired indicates that it is
relatively stable. Notably, this is the first report of a dihydro-
rosamine crystal structure.
Gopidas,2b and Bruckner2c have elegantly constructed fluores-
¨
cent probes by metal-based oxidations. However, these probes
have not been utilized in sensing metal ions in biological
samples likely due to limited biocompatibility. To examine
the feasibility of the construction of fluorescent probes based
on the metal-mediated oxidation strategy that can function
under biocompatible conditions, we were interested in creating
Cu2+ fluorescent probes by exploiting the oxidation ability of
copper, as Cu2+ plays a critical role in living systems.3 To
realize this goal, the major challenges include: (1) selection of
the oxidation reactions mediated by Cu2+ which can proceed
readily in biocompatible conditions; (2) transducing the
changes in the electronic properties of the probes in light of
the oxidation to a marked fluorescence turn-on signal; (3) the
stability of the probes in the open air; (4) choice of the
Probe 1 exhibited essentially no emission (Fig. 3) due to its
deconjugated structure. However, when probe 1 was titrated
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering, Hunan University,
Changsha, Hunan 410082, P. R. China. E-mail: weiyinglin@hnu.cn;
Fax: +86-731-88821464
w Electronic supplementary information (ESI) available: Experimental
procedures, characterization data, and some spectra for probe 1.
CCDC 748393. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/b919531a
Fig. 1 Structures of probe 1 and its conjugated compound 2; visible
color and visual fluorescence color of probe 1 and compound 2 on
excitation at 365 nm using a handheld UV lamp. The oxidation
reaction proceeded in 20 mM HEPES buffer, at pH 7.4, containing
40% CH3CN as a cosolvent.
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Fig. 2 ORTEP diagram of compound 1. Notably, carbon 9 is labeled
as C7.
with increasing concentrations of Cu2+, a drastic increase in
the featured emission of conjugated rosamine around 580 nm
was observed, and the fluorescence intensity enhancement was
up to 100-fold. Consistently, the visual emission color of the
probe 1 solution turned from dark to bright orange (Fig. 1).
Moreover, the probe showed high sensitivity toward Cu2+
with a detection limit of 2.61 ꢁ 10ꢂ7 M (S/N = 3), which is
superior or comparable to most of the reported fluorescent
Cu2+ probes2b,6,7 and is much lower than the typical con-
centration of blood copper (11.8–23.6 mM) in normal individuals8
and the limit of copper in drinking water (B20 mM) set by the
U.S. Environmental Protection Agency.
Fig. 4 The fluorescence intensity of probe 1 (10 mM) at 580 nm in the
absence and presence of different species (5 equiv.) in 20 mM HEPES
buffer, pH 7.4, containing 40% CH3CN as a cosolvent.
Thus, probe 1 has much less auto-oxidation and is significantly
more stable than other dihydrorosamine-based reactive oxygen
probes.5 Presumably, the bulky isopropyl silane group may
contribute to the stability.
The conjugated product of probe 1 + Cu2+ was isolated by
column chromatography, and its identity was confirmed to be
conjugated rosamine 2 (fluorescence quantum yield Ff = 0.22)
based on the 1H NMR, 13C NMR, mass spectrometry,
absorption, emission, and excitation spectroscopic studies
(Fig. S6–S11w). The oxidation conversion of probe 1 to
compound 2 essentially reached 100% in the presence of
5 equiv. Cu2+ at 30 min (Fig. S12w), indicating that the
oxidation reaction is clean and complete. This is highly desirable
for reaction-based probes, as the fluorimetric assay should be
much simpler and more reliable. As the oxidation reaction was
essentially complete in 30 min (Fig. S13w), in this work, an
assay time of 30 min was selected in the evaluation of the
We then proceeded to examine the selectivity of the probe.
As shown in Fig. 4, introduction of Cu2+ to the probe 1
solution resulted in a significant enhancement in the fluores-
cent intensity at 580 nm. By contrast, no marked changes in
the emission were noted upon addition of the representative
species such as Ag+, Al3+, Ca2+, Cd2+, Co2+, Fe2+, Fe3+
,
Hg2+, Mn2+, Ni2+, Pb2+, Zn2ꢃ+, Fꢂ, NO2ꢂ, H2O2 + horse-
radish peroxidase (HRP), and OH, indicating that the probe
has a high selectivity for Cu2+. This is also supported by the
observation that other species have negligible interference
with the fluorescence response. The absorption spectra also
showed the similar selectivity to the species (Fig. S2w).
Furthermore, the visual response of probe 1 to various species
(Fig. S3w) demonstrates that the probe can be used conveniently
for Cu2+ detection by simple visual inspection.
sensitivity and selectivity of probe 1 toward Cu2+
.
Although copper ions are known to facilitate the oxidation
of phenolic compounds,9 the Cu2+-mediated oxidation of
dihydrorosamine has not been revealed in the literature and
the oxidation mechanism is unknown. To gain insight into the
likely mechanism of the novel Cu2+-mediated oxidation of
dihydrorosamine, we investigated the kinetic profile of the
formation of conjugated compound 2 from probe 1 in the
presence of different equiv. of Cu2+ by absorption spectro-
scopy. When probe 1 (10 mM) was treated with various equiv.
of copper ions, the maximal absorption at 558 nm could be
reached after 30 min and 215 h in the presence of 5 and
0.1 equiv. Cu2+, respectively (Fig. 5). This preliminary kinetic
study implies that the Cu2+-mediated oxidation plays an
important role in the oxidation of non-fluorescent probe 1 to
fluorescent rosamine 2.9 The presence of Cu+ in the process
was detected by the Cu+ indicator neocuproine in light of the
formation of an absorption band around 460 nm (Fig. S14w),
attributed to the [Cu(neocuproine)2]+ complex.10 These
findings suggest that the oxidation process likely involves a
copper redox mechanism.9
Probe 1 could be employed to detect Cu2+ in the pH range
3.0–8.0 (Fig. S4w). This indicates that the probe functions
properly at physiological pH. The solid powder of probe 1 was
stable at ꢂ20 1C for at least one year, and there were no
obvious changes in the fluorescence intensity of probe 1 in the
assay solution for three days at room temperature (Fig. S5w).
The linear calibration curve (the inset in Fig. 3) indicates
that the probe may be employed to determine copper con-
centration in water. Indeed, probe 1 was able to quantitatively
detect various concentrations of Cu2+ spiked in water samples
from Yuelu spring with excellent recovery (Table S1w). Thus,
Fig. 3 Fluorescence spectral changes of probe 1 (10 mM) upon addition
of increasing concentrations (0–5 equiv.) of Cu2+ (lex = 554 nm) in
20 mM HEPES buffer, pH 7.4, containing 40% CH3CN as a cosolvent.
The inset shows the correlation between the fluorescence intensity and
copper concentration.
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broadly extended to develop biocompatible fluorescent probes
for various metal ions of interest. Our research is now in
progress along this line.
This research was supported by the NSFC (20872032,
20972044), NCET (08–0175), and the Key Project of Chinese
Ministry of Education (No:108167).
Notes and references
Fig. 5 The variations of absorbance of probe 1 (10 mM) in 20 mM
HEPES buffer, pH 7.4, containing 40% CH3CN as a cosolvent in the
z Crystal data for probe 1: C36H52N2O2Si, M = 572.89, monoclinic,
space group P21/c, a = 19.8209(19), b = 13.1032(12), c = 13.9853(13) A,
a = 90.00, b = 108.316(2), g = 90.001, V = 3448.2(6) A3,
T = 293(2) K, Z = 4, Dc = 1.104 Mg mꢂ3, F000 = 1248, m(MoKa) =
0.100 mmꢂ1, l = 0.71073 A, 2ymax = 51.01. 17 917 reflections
measured, 6406 unique (Rint = 0.1102). The structures were solved
by direct methods and refined by a full-matrix least-squares technique
presence of 0 equiv. (’), 0.1 equiv. ( ), 0.5 equiv. ( ) and 5 equiv. (
)
Cu2+ as a function of time. (a) The variations of the absorbance in the
initial 30 min (notably, it took roughly 0.5 min to mix the solution and
record the spectra); (b) the variations of the absorbance from the
subsequent 30 min to 215 h. The absorbance was recorded around
558 nm at room temperature.
on F2 using the SHELXL97 program. Final GooF = 0.859, R1
0.0769, wR2 = 0.1948, R indices based on 2743 reflections and 375
refined parameters, with I 4 2s(I). CCDC 748393.
=
probe 1 is satisfactory for monitoring copper in environmental
water samples. Furthermore, probe 1 could also sense Cu2+ in
new born calf serum by simple visual inspection (Fig. S15w).
The utility of probe 1 for fluorescence imaging of Cu2+ in
living cells was investigated. Staining of nasopharyngeal
carcinoma cells with only probe 1 provided no significant
fluorescence (Fig. 6a). In Fig. 6b, the cells were pre-treated
with Cu2+ in the growth medium for 30 min. The cells were
then washed with PBS to remove the remaining Cu2+ and
further incubated with probe 1 for 30 min. The resulting
bright fluorescence image demonstrates that probe 1 with
suitable amphipathicity is cell membrane permeable and able
to display a fluorescence turn-on response to Cu2+ in the
living cells.
1 Some recent examples of reaction-based probes: (a) A. L. Garner,
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K. R. Gopidas, Dalton Trans., 2009, 1180; (c) N. C. Lim,
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The desirable features of probe 1 include high sensitivity and
selectivity for Cu2+, working well in biocompatible conditions,
the clean and complete oxidation reaction mediated by Cu2+
,
6 For some recent examples of fluorescence ‘‘turn-off’’ Cu2+ probes,
see: (a) H. S. Jung, P. S. Kwon, J. W. Lee, J. I. Kim, C. S. Hong,
J. W. Kim, S. Yan, J. Y. Lee, J. H. Lee, T. Joo and J. S. Kim,
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and satisfactory stability in the open air. Notably, one striking
character of probe 1 is a fluorescence turn-on response by
circumventing the intrinsic fluorescence quenching nature of
paramagnetic Cu2+. This may be considered as the advantage
of the metal-oxidation based probe development strategy.
In conclusion, we have created a novel type of fluorescent
copper probe 1 based on the new copper-mediated dihydro-
rosamine oxidation reaction, and the probe has been employed
to sense Cu2+ in water, new born calf serum, and living cells.
Notably, probe 1 represents the first fluorescence turn-on
probe on the basis of metal-mediated oxidation that can work
in biological conditions. Furthermore, we also proposed that
the novel copper-mediated dihydrorosamine oxidation reac-
tion likely proceeds by a copper redox mechanism. We expect
that the general metal-mediated oxidation approach could be
7 For some recent examples of fluorescence ‘‘turn-on’’ Cu2+ probes,
see: (a) M. Yu, M. Shi, Z. Chen, F. Li, X. Li, Y. Gao, J. Xu,
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Fig. 6 Fluorescence and brightfield images of cells. (a) Cells stained
with probe 1 (10 mM) for 30 min at 37 1C; (b) cells pre-treated with
Cu2+ (5 equiv.) for 30 min at 37 1C and then further incubated with
probe 1 (10 mM) for 30 min at 37 1C; (c) brightfield image of cells
shown in pane b; (d) an overlay image of (b) and (c). The cell culture
and imaging conditions are in accordance with those in ref. 6a,7a,b,g.
8 J. Popko, S. Olszewski, K. Huka"owicz, R. Markiewicz,
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Chem. Commun., 2010, 46, 1311–1313 | 1313