A highly selective and reversible fluorescent Cu2+ and S2- probe under physiological conditions and in live cells

Article abstract of DOI:10.1039/c4ra03916e

A new spiropyran functionalized rhodamine derivative RB-SP2 has been synthesized and applied to detect Cu²⁺ and S^2-. RB-SP2 was then used as an imaging probe for detection of these ions in HeLa cells at the physiological pH. This jou

Full text of DOI:10.1039/c4ra03916e

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A Highly Selective and Reversible Fluorescent Cu2+
and S2ꢀ Probe in Physiological Conditions and in Live
Cells
Cite this: DOI: 10.1039/x0xx00000x
Quan Zhou, Yong Zhu, Pengtao Sheng, Zeming Wu, Qingyun Cai*
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
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A new spiropyran functionalized rhodamine derivative
RBꢀSP2 has been synthesized and applied to detect Cu2+
and S2ꢀ. RBꢀSP2 was then used as an imaging probe for
detection of these ions in HeLa cells at the physiological
pH.
powder under the acidic conditions to give the target compound
RBꢀSP2 (Scheme 1). Details about the synthesis and
characterization of RBꢀSP2 are presented in the ESI
†
5
The fluorescent chemosensor capable of sensing specific
analytes has potential applications in chemistry and biology,1 as it
generally allows detection of analytes present in ultratrace
quantity. Such detection of heavy metal ions is of high
10 importance due to the high toxicity of these metal ions toward
human health.2 Fluorescent sensors for the detection of Cu2+ are
actively investigated, as it is a significant environmental pollutant
and also an essential element for humans.3 Meanwhile,
development of selective and efficient signaling units for
15 detection of various chemically and biologically important anions
has also attained significant interest.4 Being one of the
biologically and environmentally important anions, sulfide is
largely used in industrial processes.5 Consequently, there are
high risks for the sulfide ions to be exposed to drinking water.
20 Sulfide can damage mucous membranes and can cause
unconsciousness and respiratory problems.6
Therefore,
40
development of a quick and sensitive fluorescence probes for the
detection of S2ꢀ and Cu2+ in aqueous media and in biological
systems is of high interest.
Scheme 1 Synthesis of compound RBꢀSP2
The compound RBꢀSP2 is synthesized by reducing RBꢀSP1
25
Although a significant number of rhodamineꢀbased fluorescent
probes have been developed for different metal ions (Zn2+, Fe3+,
Hg2+),7 a few ones have been reported for copper.8 In this work, a
new fluorescent probe RBꢀSP2 (Scheme 1) is proposed. RBꢀSP2
is capable of detection both Cu2+ and S2ꢀ at physiological pH.
Compound RBꢀSP2 was readily synthesized in six steps.
Condensation of 1 with 2 afforded the intermediate 3; which was
then treated with intermediate 5 under the basic conditions to
give the intermediate compound 6; and 7 was treated with
hydrazine hydrate affording the intermediate 8, then compound 6
which is composed of spiropyran and rhodamine. The N and O
45 atoms of rhodamine can coordinate with metal ions inducing the
ringꢀopen of spiropyran, and consequently an increase in
fluorescence. Considering that the Schiff base of RBꢀSP1 may
respond to many transition metal ions, and in order to enhance
the selectivity of the fluorescent probe, RBꢀSP1 was reduced to
50 RBꢀSP2, and the next experiment has proved this point.
30
35 with 8 afforded 9 (RBꢀSP1), which was then treated with znic
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binding interactions among the metal center, hydrazide and
phenolate hydroxy group (Fig. S4, see ESI†). Furthermore, in
order to verify Cu2+ꢀtriggered spiro ringꢀopening process, we
55 studied mass spectrum of the RBꢀSP2−Cu system, which shows
a molecularꢀion peak at m/z 898.3, corresponding to [RBꢀSP2
+
Cu + Cl] (Fig. S5, see ESI†). Taken these results together, a
likely sensing mechanism based on the Cu2+ꢀtriggered spiro ringꢀ
opening process is proposed in Scheme 2. Based on the
60 relationship between the fluorescence intensity and concentration
of Cu2+, the binding constant was calculated to be logK = 6.11
(Fig. S6, see ESI†).
Fig. 1. Changes in the (a) fluorescence (λex = 500 nm) and (b) UVꢀvis
absorption spectra of RBꢀSP2 (10 µM) in the presence of increasing
concentrations of Cu2+ (0ꢀ10 µM) in C2H5OH/aqueous PBS (1 mM, pH 7.4;
4:6 v/v; 1% DMSO) solutions. Inset: the Cu2+ concentrationꢀdependent
responses at room temperature.
5
As evident from Fig. 1a, excitation of the initial solution of
RBꢀSP2 at 500 nm wavelength did not show any significant
emission over the range from 560 to 680 nm (orange line). This
10 supports the fact that in absence of Cu2+, RBꢀSP2 remains in the
ringꢀclose form. Addition of Cu2+ to RBꢀSP2 solution induces a
significant switch ON fluorescence response near 590 nm, with a
visual display of reddish fluorescence. The switch ON responses
for the emission band at ∼590 nm on binding to Cu2+ suggest the
15 opening of the spirolactam ring in RBꢀSP2 through the
coordination with Cu2+. The fluorescence intensity at ∼590 nm
increases with increasing the Cu2+ concentration, with about
300 % increase of its fluorescence intensity upon addition of only
10 µM Cu2+ ions. The minimum detectable concentration was 1 ×
20 10ꢀ7 M. We have measured the liner equation detecting the Cu2+
65
Scheme 2 Proposed mechanism for the fluorescent changes of sensor RBꢀ
SP2 upon the addition of Cu2+ and S2ꢀ
As other metal ions may also coordinate with RBꢀSP2, the
responses to other metal ions (Au3+, K+, Mg2+, Hg2+, Fe2+, Ni2+,
Cd2+, Ba2+, Co2+, Mn2+, Na+, Fe3+, Cu+, Zn2+ and Ag+) were
70 investigated (Fig. S7, see ESI†). There are not any noticeable
changes in fluorescence spectrum with addition of these tested
ions, suggesting that the generation of xanthene moiety is highly
selective toward Cu2+ ions. This conclusion was further
confirmed with the UVꢀvis absorption spectra analysis, no
75 noticeable changes in UVꢀvis absorption were observed with the
addition of these tested ions in RBꢀSP2 solution (Fig. S8, see
(Figure S1, see ESI†) which was y=1.25x+1.24 with a liner range
from 0.1 µM to 1.0 µM, the limit of detection was calculated as
0.05 µM from the 3s method.
ESI†).
UVꢀvis spectra recorded for RBꢀSP2 in C2H5OH/aqueous PBS
25 indicated the maximum absorption at 324 nm (Fig. 1b), which
may be attributed to the intraꢀmolecular π−π* charge transfer
(CT) transition. According to previous studies some transitionꢀ
metal ions can selectively bind with suitable derivatives of
rhodamine,9 inducing the opening of the spirolactam ring and
30 generation of the xanthene form. This structural change is
manifested in the electronic and fluorescence spectral patterns.
Significant change in UVꢀvis spectrum was observed in the
presence of Cu2+ (Fig. 1b). The absorption band appeared around
550 nm increases with increasing Cu2+ concentration from 0 µM
35 to 10 µM (Fig. 1b), and the solution turned from colorless to pink
Fig. 2. Changes in the (a) fluorescence (λex = 500 nm) and (b) UVꢀvis
80 absorption spectra of RBꢀSP2ꢀCu complex (Adding 10 µM Cu2+ to RBꢀSP2
10 µM) in the presence of different concentrations of S2ꢀ (0ꢀ20 µM) in
C2H5OH/aqueous PBS (1 mM, pH 7.4; 4:6 v/v; 1% DMSO) solution. Inset: the
S2ꢀ concentrationꢀdependent response at room temperature.
(Fig. S2, see ESI†). It should be noted that a small adsorption
band between 400 and 450 nm slightly increased with the
increase of the band around 550 nm, which clearly indicated that
Cu2+ caused the ringꢀopen of not only RB but also SP.
40
We have examined mechanism using FTIR techniques. The IR
spectra of RBꢀSP2 revealed that the peak of C=O amide bond at
1687 cm−1, the characteristic stretching frequency for the C=O
amide bond of the rhodamine unit, shifted to 1636 cm−1 in
The aboveꢀmentioned studies show that RBꢀSP2 selectively
85 binds with Cu2+ to form RBꢀSP2ꢀCu complex resulting in
considerable changes in the spectral properties. One may think
that if the RBꢀSP2-Cu complex dissociates, the fluorescence
would be quenched. Herein sulfide was introduced to quench the
fluorescence by forming CuS. Fig. 2a shows that the fluorescence
90 intensity of 10 µM RBꢀSP2ꢀCu decreases with increasing the
presence of 2 equiv of the Cu2+ ion (Figure S3, see ESI
†). Such
45 shift in the stretching frequency of C=O amide bond of the
rhodamine unit on binding to a metal ion has been reported
earlier9c, indicating that the amide carbonyl group is involved in
the interactions with Cu2+. This is the key to the spiro ringꢀ
opening and fluorescence turnꢀon of the rhodamine dye.
50 Meanwhile, the pronounced down field shifts of the methylene
protons of RBꢀSP2 in the 1H NMR spectra indicate that the
concentration of S2ꢀ. In the presence of 20 µM S2 anion the
‑
intensity of RBꢀSP2ꢀCu decreases by about 280%. Similar
results are observed on the UV−vis spectral pattern (Fig. 2b)
which is in a reverse direction to the spectral pattern as shown in
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Fig. 1b. These results further confirmed the conclusion that the 50 was conspicuous in the perinuclear region of HeLa cells.
RBꢀSP2-Cu complex results in the switch ON of fluorescence
response (Scheme 2). Their corresponding fluorescence changes
were also shown in Figure S9 (see ESI†), implying the
reproducibility and reversibility of this experiment.
Interestingly, sulfide sensing inside HeLa cells by RBꢀSP2ꢀCu
complex could also be pursued as evident from the remarkable
switchꢀOFF of the red fluorescence inside cells following
incubation with Na2S solution (Fig. 3b. 3). Essentially, the
55 fluorescence microscopic analysis strongly suggested that probe
RBꢀSP2 could readily cross the membrane barrier, permeate into
5
The spectral responses of the RBꢀSP2ꢀCu complex were also
investigated in the presence of other anions including F−, Cl−,
HeLa cells, and rapidly sense intracellular Cu2+ and S . It is
significant to mention here that brightfield images of treated cells
did not reveal any gross morphological perturbations, which
60 suggested that HeLa cells were viable. This finding is
encouraging for future in vivo biomedical applications of the
probe.
2
‑
−
−
−
2−
−
Br−, I−, CN−, H2PO4 , NO3 , ClO−, ClO4 , SO4 and HSO3 .
These investigated anions show little fluorescence quenching
10 effect except the S2 (Fig. S10, see ESI
†
). The reason is that
−
these anions cannot form stable precipitate/complex or the
formed precipitate/complex is with less stability than RBꢀSP2ꢀ
Cu. As expected, same results were observed on the UVꢀvis
absorption spectral analysis, which also demonstrate high
Conclusions
15 selectivity toward S2‑ ions (Fig. S11, see ESI
†).
A highly selective and reversible fluorescent probe RBꢀSP2
65 was designed for the detection of Cu2+ and S2ꢀ by incorporating
both spiropyran and rhodamine. RBꢀSP2 can be employed to
image Cu2+ and S2ꢀ in living cells. Notably, the reduction of RBꢀ
SP1 to RBꢀSP2 guaranteed the reversible as well as selective
response to Cu2+. This work opens an avenue for development of
70 reversible fluorescent probe from irreversible chemo dosimeters
with high selectivity.
The fluorescence intensity of RBꢀSP2 probe is independent on
pH in the range of pH 5.0ꢀ9.0 either in the absence or presence of
Cu2+ (Fig. S12 see ESI
†), indicating that the probe was suitable
for the detection of Cu2+ at physiological pH.
20
Based on the above experiment, it was conceived that
compound RBꢀSP2 could be exploited for fluorescence imaging
of live cells, particularly for sensitive detection of intracellular
Cu2+. To pursue this goal, it was pertinent to assess the cytotoxic
effect of compound RBꢀSP2 on live cells. Various
25 concentrations of compound RBꢀSP2 and RBꢀSP2ꢀCu complex
were thus chosen, and their cytotoxic effects on HeLa cells were
ascertained following an exposure period of 24 h. The wellꢀ
established MTT assay, which is based on mitochondrial
dehydrogenase activity of viable cells, was adopted. It is quite
30 evident from Fig. 3a that compound RBꢀSP2 does not exert any
effect on the viability of HeLa cells. However, exposure of HeLa
We are grateful for the financial support from the NSFC
(2009CB421601)
Notes and references
75 State Key Laboratory of Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering, Hunan University, Changsha, 410082,
China; Tex/Fax: +86 731 88822170; E-mail: qycai0001@hnu.edu.cn
Electronic Supplementary Information (ESI) available:
Experimental details, synthesis and characterization of RBꢀSP2,
80 optimized procedure. See DOI: 10.1039/c000000x/
cells to 100 µM RBꢀSP2ꢀCu complex resulted in a decline in cell
viability. In the presence of higher concentrations of RBꢀSP2ꢀCu
complex, the cytotoxic effect was more prominent as a result of
35 cytotoxic and antiproliferative effects of Cu2+ complex on cancer
cells.10
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Fig. 3. (a) MTT assay to determine the cytotoxic effect of compound RBꢀSP2
and RBꢀSP2ꢀCu complex in HeLa cells. (b) Fluorescence microscopic images
40 of HeLa cells: (1) after treating with 10 µM RBꢀSP2 (under green light); (2)
after adding 10 µM of Cu2+ (under green light) to the RBꢀSP2 treated cells,
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and (3) after adding 20 µM S2ꢀ (under green light) to the (RBꢀSP2ꢀCu2+
)
treated cells.
100
Fluorescence microscopic studies reveals that HeLa cells
45 treated with probe RBꢀSP2 alone show no fluorescence (Fig. 3b.
1). Upon incubation with 10 µM Cu(ClO4)2 for 1 h, a striking
switchꢀON fluorescence is observed inside HeLa cells, indicating
the formation of RBꢀSP2ꢀCu complex (Fig. 3b. 2), as observed
earlier in solution studies. Further, an intense red fluorescence
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