A novel near-infrared turn-on and ratiometric fluorescent probe capable of copper(ii) ion determination in living cells

Article abstract of DOI:10.1039/d0cc01481h

A near-infrared ratiometric fluorescent probe CR-Ac based on a coumarin-benzopyrylium platform has been developed for selective detection of Cu²⁺. The cell imaging data revealed the capabilities of CR-Ac in monitoring the dynamic changes of sub

Full text of DOI:10.1039/d0cc01481h

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A novel NIR ratiometric fluorescent probe has been developed for monitoring the dynamic changes of subcellular Cu and
DOI: 10.1039/D0CC01481H
quantifying its concentration at nanomolar level in live-cells.
Fred / Fblue
ws1 cells + CR-Ac
O
O
N
N
N
O
O
O
N
Ex = 425 nm
+Cu2+
Em = 520 nm
-Cu2+
ws1 cells + CR-Ac + Cu2+
O
X
O
Cu
N
N
N
O
O
O
N
Ex = 650 nm
Em = 696 nm

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DOI: 10.1039/D0CC01481H
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A novel near-infrared turn-on and ratiometric fluorescent probe
capable of copper(II) ion determination in living cells
Ziya Aydin,*a,b Bing Yan,c Yibin Wei,c and Maolin Guo*b,c
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
recently been reported for Cu2+ imaging5,7. These sensors offer
A near-infrared ratiometric fluorescent probe CR-Ac based on a
coumarin-benzopyrylium platform has been developed for
selective detection of Cu2+. Cell imaging data revealed the
capabilities of CR-Ac in monitoring the dynamic changes of
subcellular Cu2+ and quantification of Cu2+ levels in live cells.
effective tools for Cu2+ detection in living systems, however,
quantitative measurement of free copper ion concentration in
live-cells is still very challenging5. A carbonic anhydrase-dye
conjugation has recently been applied for quantification of free Cu2+
5c
levels in cytoplasm via fluorescence lifetime imaging microscopy
.
However, the cytoplasm localization of the conjugate limits its ability
in Cu2+ detecting in cellular organelles where most of the free Cu2+
are located1. Ratiometric probes may provide better tools for
quantification as the ratio between two intensities can be used
to measure analyte concentration and they also provide built-in
correction for environmental effects. So far, only a few
ratiometric probes5,8 have been reported for Cu2+ and only one
(ACCu2)9 has achieved quantification of Cu2+ concentration in live
cells or tissues. However, the ACCu2 probe is a turn-off type
with emission in the visible region, requiring two-photon
excitation and the fluorescent ratio of the ACCu2-copper
complex is sensitive to pH in physiological pH range9, impeding
its broad application in biological settings. Herein, we report a
new turn-on NIR ratiometric fluorescent probe that overcomes
the pH and two-photon issues and is capable of quantifying Cu2+
concentration in live cells in real time.
Introduction
It is well known that copper is one of the most abundant
transition metals in living systems, third after iron and zinc, and
a crucial micronutrient for animals and plants1. Copper (Cu+ and
Cu2+) functions as a key factor in many physiological processes
such as signal transduction, redox reactions, the central nervous
system, and energy generation1. On the other hand, it is highly
toxic to living systems under unhealthy levels1. The toxicity of
copper has been associated with various diseases, including
Menkes and Wilson diseases, Alzheimer’s and Parkinson’s
diseases2. Moreover, enormous quantity of copper pollution
was generated to our living environment due to its widely usage
in industrial sectors3. It is thus important to develop effective
detection methods to efficiently evaluate Cu2+ levels in
environments and biological systems.
Spirocyclization of xanthene dyes has become a powerful
technique for developing fluorescent probes10. Recently, this
unique fluorescence switching mechanism has been extended
to near-infrared dyes with a coumarin-benzopyrylium platform
which displays coumarin emission even in its spirocylic closing
form10. This enables the dye to exhibit visible and near-infrared
emissions in its spirocylic ring-closing and opening forms,
respectively. Taking this advantage, we designed the NIR
fluorescent probe CR-Ac for turn-on and ratiometric sensing of
Cu2+ by employing the coumarin-benzopyrylium platform as the
dye scaffold and a group consisting of O/N/O receptor moiety
for Cu2+. The probe, CR-Ac, was synthesized via a 4-step
procedure as outlined in Scheme 1 and detailed in supporting
information, with an overall yield of 34%.
Over the past few decades, optical imaging and small molecule
fluorescent probes have gained growing interest in detecting
metal ions in living systems4. Probes for both Cu+ and Cu2+ have
been reported1e,5 but developing Cu2+-probes is more
challenging due to its paramagnetic quenching nature and
selectivity issues5. It is thus most of the current Cu2+-probes are
limited to “turn-off” type, providing useful information but
suffering from poor sensitivity, or interference from other metal
ions6. Recently, “turn-on” copper sensors have been reported
but most of them require excitation using short-wavelength UV−vis
light (350−500 nm) and emit lights in the visible range5,6. Near
-
infrared (NIR) probes are highly desirable due to better tissue
penetration, less photo damage, minimum fluorescence background,
and less light scattering5. Several interesting NIR probes have
We first evaluated the spectroscopic properties of CR-Ac and its
interactions with various metal ions. The yellow compound CR-
Ac (20 µM) in ACN/MOPS buffer (10 mM, pH 7.04, v/v 1:1)
displays a maximum absorption at 424 nm (corresponding to
the coumarin moiety) and almost no absorption above 500 nm,
indicating that CR-Ac was dominantly in the spirocylic form10.
The metal ions such as Ni2+, Mn2+, Hg2+, Na+, Ca2+, Zn2+, Ag+,
Mg2+, Pb2+, K+, Fe3+, Co2+, Fe2+, Cu+ and Cr3+ gave little response
a. Vocational School of Technical Sciences, Karamanoğlu Mehmetbey University,
70100 Karaman, Turkey, email: ziyaaydin@kmu.edu.tr
b. Department of Chemistry, University of Massachusetts Amherst, 710 North
Pleasant Street, Amherst, MA 01003 (USA). Email: mguo@umassd.edu
c. Department of Chemistry and Biochemistry, University of Massachusetts
Dartmouth, 285 Old Westport Road, Dartmouth, MA 2747 (USA).
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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to CR-Ac (Figure 1a). In contrast, with increasing in Cu2+ as a result of Cu2+ binding. Meanwhile, an isosbestic point was
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concentration, the absorbance at 424 nm (ε = 5.1 × 104 M-1 cm- observed at 448 nm, indicating the cleanly conversion of the
DOI: 10.1039/D0CC01481H
1, CR-Ac only) decreased, while the absorbance at 650 nm (ε = free sensor into its Cu2+-complex. The immediate and dramatic
2.52 × 104 M-1 cm-1, CR-Ac:Cu2+, 1:1) increased concomitantly change in colour from yellow to greenish-blue allows a ‘‘naked-
(Figure 1b), suggesting that the spirocylic ring-opening of CR-Ac eye’’ detection of Cu2+ (Figure 1a inset).
O
O
O
O
O
OH
N
N
N
NH2.NH2.H2O
BOP, DCM
O
NH2
EtOH, Overnight
75 o
C
N
O
N
O
N
O
O
O
N
O
O
N
O
O
N
ClO4
CR-Ac
CR-NH2
CR
Scheme 1. Synthesis of CR-Ac
ratiometric fluorescence response (F650/F520) of CR-Ac to
determine its selectivity to metal ions. As shown in Figure 3a,
the solution of CR-Ac exhibits a very low fluorescent ratiometric
value (F650/F520) and it remains very low in the presence of the
various metal ions (gray bars); but upon the addition of 1 equiv.
of Cu2+, there is a strong enhancement of this value, even in the
presence of other metal ions tested (black bars). These data
demonstrate that CR-Ac is highly selective to Cu2+ and the
fluorescence response is not influenced by the other metal ions.
Figure 1. (a) Absorption responses of 20 µM CR-Ac to various metal ions
(20 µM for Cu2+, Ni2+, Mn2+, Hg2+, Zn2+, Ag+, Mg2+, Pb2+, Fe3+, Co2+, Fe2+,
Cu+ and Cr3+; 100 µM for Na+, K+, Mg2+ and Ca2+) in ACN/MOPS buffer (10
mM, pH 7.04, v/v 1:1). (b) UV–vis spectra of CR-Ac with the addition of
various concentrations of CuCl2 in ACN/MOPS buffer (10 mM, pH 7.04).
To examine the fluorescent response to Cu2+, a solution of CR-
Ac in ACN/MOPS buffer (10 mM, pH 7.04, v/v 1:1) was titrated
with various concentrations of Cu2+ and monitored with a
fluorometer by excitation at 425 or 650 nm, individually. When
excited at 650 nm, CR-Ac itself is non-fluorescent (φF = 0.02).
When Cu2+ was added to the CR-Ac solution, it displayed an
emission peak with the maximum at 696 nm (φF = 0.24) (Figure
2a). When it was excited at 425 nm, the CR-Ac solution
displayed one strong emission peak at 473 nm and a very weak
one at 696 nm (Figure 2b). Upon Cu2+ was added to CR-Ac
solution, a significant decrease in intensity of the 473 nm peak
which gradually shifted to 520 nm and a marked concomitantly
increase in intensity of the emission peak at 696 nm were
observed (Figure 2b). The quenching of the ~473-520 nm
emission can be attributed to the binding of the paramagnetic
Cu2+ moiety while the “turn-on” to the 696 nm emission is due
to the coordination-induced ring-opening of the spirocylic
moiety3d. These Cu2+-induced spectral features on CR-Ac laid
the foundation for Cu2+ detection and its concentration
measurement via ratiometric fluorescent techniques.
Figure 2. (a) Fluorescence response of 20 µM CR-Ac to increasing
concentration of Cu2+ (bottom to top, 0.0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.5,
0.7, 0.8, 0.9, 1.0, 1.5 and 2.0 eq. in ACN/MOPS buffer (10 mM, pH 7.04,
v/v 1:1), (λEx 650 nm). (b) Fluorescence response of 20 µM CR-Ac to
increasing concentration of Cu2+ in ACN/MOPS buffer (10 mM, pH 7.04,
v/v 1:1) (λEx 420 nm)
Moreover, the effects of pH on the stability of the probe and its
Cu2+-complex were investigated and monitored by both
absorption and fluorescent spectroscopies (Figure 3b and
Figure S2). In contrast to the dramatic pH-dependent profile of
the ACCu2-copper complex9, we found that the spectra of our
probe CR-Ac and its Cu2+-complex are both nicely stable over
the biologically relevant pH range 5-9, which is key to its biological
application. The fluorescence goes up a bit at low or high pH.
We next tested the changes in fluorescence properties of CR-
Ac as a result of the addition of various metal ions including
Cu2+, Ni2+, Mn2+, Hg2+, Na+, Ca2+, Zn2+, Ag+, Mg2+, Pb2+, K+, Fe3+,
Co2+, Fe2+, Cu+ and Cr3+. As shown in Figure S1, significant 696
nm fluorescence “turn-on” (excitation at 650 nm) was only
induced by Cu2+ (>65-fold enhancement with 1 eq. of Cu2+) with
Fe3+ showing very minor response. The presence of any of the
other metal ions tested (Figure 3a) does not affect the
fluorescent intensity of CR-Ac with Cu2+, suggesting little
interferences from the other metal ions. We also examined the
Figure 3. (a) Fluorescent responses F696/F520 of 20 µM CR-PK to the
presence of various metal ions (gray bars) and the subsequent addition
of Cu2+ (black bar) in ACN/MOPS buffer (10 mM, pH 7.04, v/v 1:1). (b)
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Variation of fluorescent response (696 nm) of CR-Ac and CR-Ac + Cu2+ (20 µM
each) at various pH values in ACN/H2O (1/1, v/v) solution.
to the solution of CR-Ac-Cu2+ caused the disappearance of the
absorption signals of CR-Ac-Cu2+ (Figure S5), suggeVsiteiwngArttichleaOtntlhinee
DOI: 10.1039/D0CC01481H
Cu2+-binding process is reversible. The possible structures of
this reversible binding process are shown in Scheme 2.
The binding stoichiometry of CR-Ac and Cu2+ was investigated
by UV-vis titration which gives a 1:1 ratio (see Figure 1b inset)
and was confirmed by Job's plot (Figure S3). The binding
X
O
O
O
Cu
constant was calculated following
a method reported
O
N
+Cu2+
-Cu2+
N
N
previously11, using absorption values at 650 nm, and was
determined to be 1.92 × 107 M−1 (log K = 7.28) which is over 2
orders of magnitude higher than that of Cu2+ to ACCu29.
Moreover, the fluorescent ratiometric change (F696/F520) was
linearly dependent on the concentration of Cu2+ over the range
from 0 to 10 µM (R2 = 0.997). The fluorescent detection limit in
solution was determined to be 0.20 µM (based on 3σ/k, Figure
S4) which is 4 times lower than that of the ACCu2 sensor 9
N
N
O
N
O
O
O
N
O
O
N
Ex = 425 nm
Em = 696 nm
Ex = 650 nm
Em = 520 nm
X = Cl- or solvent molecule
Scheme 2. The proposed reversible 1:1 binding mode between CR-Ac and
Cu2+ (X represents a possible ligand from the solvent)
Reversibility is also key to monitoring the dynamics of Cu2+ levels in
intracellular store. The addition of metal chelator EDTA (5.0 eq.)
Figure 4. Confocal microscopy images (with DIC) of fibroblast cells (ws1) treated with (b,c) 10 µM CR-Ac sensor after 30 min incubation; (f,g) the cells were
preincubated with Cu2+ (20 µM) for 1 h before incubation with the sensor for 30 min; (j,k) the Cu2+-loaded cells were incubation with 100 μM chelator SIH for
7 h. Excitation wavelength was 458 nm for the blue channel (b,f,j) and 633 nm for the red channel (c,g,k). (d,h,l) are ratio images of c/b, g/f, k/j, respectively.
The fluorescent intensities are shown in the bar chart at the bottom (n =6). Confocal ratiometric images are the average ratio in regions of interest.
Encouraged by the above promising results, we next tested the 1,13. When the cells were preloaded with Cu2+ (20 µM) for 1 h,
usefulness of the sensor CR-Ac in detecting Cu2+ ions in living and then incubated with CR-Ac (10 µM) for 30 min, a marked
cells. Primary human fibroblast cells (ws1) were treated with 10 enhancement in the red emission (Figure 4g and bar chart) was
o
-400 µM CR-Ac for 30-60 min at 37 C and cell viability was observed, matching those Cu2+-induced fluorescence changes
monitored by confocal microscopy with Hoechst 33258 (Figure 2), suggesting detectable level of free Cu2+ in the cells.
staining4d. Little cell death was observed even at 400 µM CR-Ac, When the Cu2+-loaded cells were incubated with
suggesting negligible cytotoxicity. The cells exhibited a strong salicylaldehyde isonicotinoyl hydrazone (SIH, 100 μM), a
fluorescence in the blue channel (Figure 4b), indicating that the membrane-permeable metal ion chelator that effectively
sensor CR-Ac is cell permeable. However, barely any fluorescent removes Cu2+ from cells12, a marked decrease in the red
signals were detected in the red channel (Figure. 4c), emission (Figure 4k and bar chart) and an increase in the blue
presumably due to the very low basal level of free Cu2+ in cells emission (Figure 4j and bar chart) were observed, indicating a
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significant reduction in cellular free Cu2+ level and that the The conflicts of interest issue is being processed by the OTCV
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binding of CR-Ac to Cu2+ is readily reversible in live-cells. Thus, office of University of Massachusetts Dartmouth.
DOI: 10.1039/D0CC01481H
CR-Ac exhibits facile fluorescent responses to Cu2+ ions in living
cells and is capable of imaging the presence of Cu2+ ions as well
as its dynamic changes in cells.
The dynamic changes of free Cu2+ levels in the Cu2+-loaded cells
and SIH-treated cells are also readily revealed by ratiometric
imaging (Figure 4d,h,l and bar chart).
To quantify free Cu2+ levels in cells, in situ cell calibrations12 were
performed via ratiometric imaging using pyrithione as a Cu2+
ionophore5c in FBS-free media. The calibration curve gives a linear
response to cellular free [Cu2+] up to 400 nM with an in situ detection
limit ca. 7 nM (Figure S6). Evident cell death at higher [Cu2+],
presumably due to copper toxicity, prevents meaningful [Cu2+]
analysis. Analysis of the ratiometric images in Figure 4 gives free
[Cu2+] 262 nM in Cu2+-loaded ws1 cells (Figure 4h) and 89 nM in
the subsequently SIH-treated cells (Figure 4l). The free [Cu2+] in
untreated ws1 cells (Figure 4d) is below the detection limit of
the sensor. This is reasonable because free copper ions are
known to be very low in cells1,5c,14. The nanomolar level of free
Cu2+ in ws1 cells after Cu2+-loading is lower than those in a
previous report9 which in situ cell calibration was not used for
[Cu2+] determination and different cell lines were used.
Notes and references
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The images of the Cu2+-loaded cells from the red and ratiometric
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cells (ws1) may be localized in certain subcellular compartments
(organelles) and that CR-Ac may be capable of imaging Cu2+ ions
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o
colocalization between the Cu2+-induced fluorescence (red) and
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8
detected free Cu2+ is not located in lysosomes. In contrast,
a
complete colocalization between the red fluorescence and the
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cells. This mitochondria location of the Cu2+ pool is reasonable
as it is where Cu2+ is needed in cells for Cu-relying enzymes such
as cytochrome c oxidase.1
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Collectively, the results show excellent sensor characteristics of
CR-Ac in reporting the dynamic changes in cytoplasmic labile
Cu2+ at subcellular resolution as well as concentration
estimation at nanomolar level. The one-photon, turn-on,
ratiometric and NIR photo properties, high solubility in culture
medium and selectivity, reversible response, resistant to pH
change and fast response time make it an excellent tool to study
the cell biology of copper as well as its related diseases.
We thank the National Science Foundation (CHE-1213838, CHE-
1229339) and NIH (1R15GM126576-01) for funding.
Conflicts of interest
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