A rhodamine-appended water-soluble conjugated polymer: An efficient ratiometric fluorescence sensing platform for intracellular metal-ion probing

Article abstract of DOI:10.1039/c3cc48649d

The water-soluble CP was conjugated with a rhodamine spirolactam for the first time to develop a new FRET-based ratiometric fluorescence sensing platform (CP 1) for intracellular metal-ion probing. CP 1 exhibits excellent water-solubility with two well-resolved emission peaks, which benefit ratiometric intracellular imaging applications.

Full text of DOI:10.1039/c3cc48649d

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A rhodamine-appended water-soluble conjugated
polymer: an efficient ratiometric fluorescence
sensing platform for intracellular metal-ion probing†
Yong-Xiang Wu,a Jun-Bin Li,a Li-Hui Liang,b Dan-Qing Lu,a Jing Zhang,a
Guo-Jiang Mao,a Li-Yi Zhou,a Xiao-Bing Zhang,*a Weihong Tan,*a Guo-Li Shena
and Ru-Qin Yua
Cite this: Chem. Commun., 2014,
50, 2040
Received 13th November 2013,
Accepted 19th December 2013
DOI: 10.1039/c3cc48649d
www.rsc.org/chemcomm
The water-soluble CP was conjugated with a rhodamine spirolactam for carboxylate, phosphate and quaternary ammonium) as building
the first time to develop a new FRET-based ratiometric fluorescence blocks. Such a structural modification could obviously improve the
sensing platform (CP 1) for intracellular metal-ion probing. CP 1 exhibits solubility of CPs in aqueous media, which is favorable for their
excellent water-solubility with two well-resolved emission peaks, which applications in biological samples.4e,5,6
benefit ratiometric intracellular imaging applications.
Rhodamine derivatives with the spirolactam form are colorless
(weak absorption) and non-fluorescent, whereas the ring-opened
Monitoring of the distribution of intracellular metal ions is critical amide form induced by analytes gives rise to an appearance of pink
for understanding their physiological or pathological functions.1 color (strong absorption) and a strong fluorescence emission at a
Fluorescence bio-imaging through staining with a molecular probe relatively long wavelength.7,8 So the rhodamine framework is an ideal
might be one of the most attractive techniques in this field.2 To platform to construct a turn-on type fluorescent probe, and much
achieve this goal, quite a few of fluorescent small organic molecular research effort has been devoted to develop such probes for various
probes based on target-triggered single emission enhancement,2 or metal ions in the past decades.9 Ratiometric probes based on the
ratiometric intensity changes of two emissions,3 have been designed fluorescence resonance energy transfer (FRET) mechanism can theore-
and developed for sensing intracellular metal ions. Among these, the tically afford two well-resolved emission peaks, which benefits observa-
ratiometric probes involve the observation of target-triggered ratio- tion of the fluorescence signal change at two different emission
metric changes of the fluorescence intensities at two wavelengths, wavelengths at high resolution.3 The rhodamine framework has also
which could provide built-in correction for eliminating the environ- been proved to be a perfect acceptor and a trigger for constructing
mental effects, and are beneficial for increasing the dynamic FRET based ratiometric probes for various metal ions,10 although these
response concentration range.3 They seem to be more suitable for small molecule-based probes usually exhibit poor water solubility.
monitoring bio-related species in complex biological systems. How-
In this work, by combining a rhodamine spirolactam with water-
ever, most of the previously reported small molecular ratiometric soluble ionic CPs, we have designed a new ratiometric fluorescence
probes suffer from poor water-solubility, and the necessary addition sensing platform CP 1 for the detection of metal ions in living cells
of organic cosolvents might cause non-negligible bio-toxicity and (Scheme 1). Fe3+ was chosen as a model metal ion, since iron is a vital
dissatisfactory sensitivity in case less cosolvent is involved.
constituent of plant and animal life.11 However, under conditions that
In the past decade, conjugated polymer (CP)-based fluorescent exceed the level at which the organisms safely use it, iron can be toxic
probes have attracted increasing attention, and usually exhibit remark- because of its ability to promote oxidation of lipids, proteins and other
ably higher sensitivity than that of small molecule-based probes, due cellular components.12 Moreover, although several small molecular
to the so-called ‘‘molecular wire’’ effect, which could afford fast energy fluorescent probes have been developed for Fe3+ with satisfactory
migration along the conjugated backbone.4 Water-soluble ionic
CP-based probes have been recently developed by employing different
ionic aromatic monomers (involving ions including sulfonate,
a Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/
Biosensing and Chemometrics, College of Chemistry and Chemical Engineering,
College of Biology, Hunan University, Changsha 410082, China.
E-mail: xbzhang@hnu.edu.cn, tan@chem.ufl.edu
b Hunan Provincial People’s Hospital, Changsha, 410002, P. R. China
† Electronic supplementary information (ESI) available: Experimental details and
Scheme 1 Structure and response mechanism of the probe CP 1 toward Fe3+
.
supplementary figures. See DOI: 10.1039/c3cc48649d
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sensitivity and selectivity, most of these probes still exhibit poor water-
solubility.13 Poly[p(phenylene ethynylene)-alt-(thienylene ethyn-ylene)]
(PPETE) was chosen as the energy donor, because its fluorescence is
insensitive to environmental factors, and its fluorescence emission
spectrum matches well with the absorption spectrum of rhodamine
6G (see Fig. S1, ESI†). To improve its solubility, we modified its
monomer with two trimethylamine groups, and the as-prepared
FRET-based ratiometric CP 1 exhibits excellent solubility in pure
aqueous solution, with no requirement of any organic cosolvent.
The synthetic routes of the monomers and polymers are shown in
Schemes S1 and S2 (see ESI†). The copolymerizations were carried out
using compound 5 and compound 9 in a mixture of dry toluene and
anhydrous triethylamine (Et3N) at 60 1C under an atmosphere of N2,
using Pd(PPh3)4 and CuI as the catalysts, which affords CP 2 with a
molecular weight (Mn) of 2084 with a polydispersity index (PDI) of 1.17.
The water soluble polymer CP 1 is prepared by treatment of CP 2 with
condensed trimethylamine in a THF–water mixture for 24 h (see ESI†).
The changes in the UV-vis spectra of CP 1 in buffered solution upon
the addition of Fe3+ are first investigated (see Fig. S2, ESI†). It can be
observed that the free CP 1 shows a maximum absorption of the donor
(CP backbones) chromophore at 400 nm, with no characteristic
absorption peak of the rhodamine 6G moiety observed, demonstrating
its existence in the spirolactam form. However, when Fe3+ was added
into the buffered sensing system, in addition to the absorption peak at
400 nm, a new characteristic absorption peak at 530 nm is observed,
Fig. 1 (a) The fluorescence spectra of CP 1 ([RU] = 10 mM) in the presence
of different concentrations of Fe3+ (0, 1, 8, 20, 50, 80, 90, 100, 150, 200,
250, 300, 350, and 500 mM) in buffer solution (Tris-HCl, pH = 7.2). lex
=
400 nm. Inset shows change in fluorescence of CP 1 ([RU] = 10 mM) upon
which accords with the characteristic absorption peak of the ring- addition of Fe3+ (50 Â 10À5 M) in buffer solution (Tris-HCl, pH = 7.2),
excitation at 365 nm under UV lamp. (b) The calibration curve of the probe
opened form of the rhodamine 6G moiety in CP 1. The obvious
CP 1. The inset shows the linear response at low Fe3+ concentrations.
changes indicate that the addition of Fe3+ ions can induce the transfer
of CP 1 from the spirolactam form to the ring-opened form.
The fluorescence-sensing ability of CP 1 toward Fe3+ was evaluated
toward Fe3+ ions in the neutral pH range (7.0–8.0) was observed.
in pure aqueous buffer solution. Fig. 1a shows the emission changes
of CP 1 upon the addition of different concentrations of Fe3+. The
The results showed that CP 1 is suitable for application under
introduction of Fe3+ induced a clear see-saw-type dual-emission
physiological conditions.
The selectivity and competition experiments of CP 1 were then
extended to various metal ions. Fig. 2 (the black bar portion) shows
the relative fluorescence intensity ratio (I538/I442) of CP 1 in the
presence of different metal ions in aqueous buffer solution. The
results indicated that our proposed probe exhibits high selectivity
toward Fe3+ over other competing metal ions. It is worthy to note
change, suggesting the recovery of FRET from the CP backbones
(donor) (Imax at 442 nm) to the rhodamine 6G (Imax at 538 nm). The
ratio R of the emissions at the two wavelengths (R = I538/I442) increased
from 0.18 to 3.7 with the dynamic response concentration range for
Fe3+ covering from 0 to 500 mM (Fig. 1b), with a linear range up to
1.0 Â 10À4 M (inset of Fig. 1b), and a detection limit (LOD) of
3.0 Â 10À7 M (3s/slope). Moreover, a remarkable fluorescence change
from blue to yellow (excitation at 365 nm under UV lamp) could be
observed easily by the naked eye (inset of Fig. 1a). The fluorescence
quantum yield (QY) of PPETE backbones (donor) is calculated to be
0.34 with quinine bisulfate in 0.1 M H2SO4 as the standard.14 The
¨
Forster energy transfer efficiency between CPs and rhodamine 6G
in CP 1 was calculated to be 61.8%, with an estimated r value (the
distance between the acceptor and the donor) of 4.06 nm,15 indicating
the occurrence of an efficient FRET (see ESI†).
To study the practical applicability of the probe in the physiolo-
gical environment, the effects of pH on the fluorescence response of
CP 1 toward Fe3+ were also investigated (see Fig. S3, ESI†). Because
the fluorescence of CP backbones was not influenced by the pH,16
the acidity of the system mainly affects the fluorescence intensity of
the rhodamine 6G moiety in CP 1. Upon the addition of Fe3+, the
emission intensity ratio at I538/I442 of CP 1 shows obvious changes at
different pH values from 5.0 to 11, and a good fluorescence response
Fig. 2 Relative fluorescence intensity (I538/I442) response of CP 1 ([RU] =
10 mM) to 2 Â 10À4 M of Fe3+ or 2 Â 10À4 M of other metal ions (the
black bar portion); and to the mixture of 2 Â 10À4 M of other metal ions
with 2 Â 10À4 of Fe3+ (the gray bar portion). lex = 400 nm.
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that CP 1 could distinguish Fe3+ from Fe2+. The competition experiment (Fig. 3g) were observed. Moreover, the treatment of Hela cells with
showed that the response of the probe CP 1 toward Fe3+ is unaffected probe CP 1 or Fe(NO3)3 did not result in any critical change in the cell
by the presence of the other possible contaminating metal ions (the morphology or cell death (Fig. 3a and e), indicating that the cellular
gray bar portion in Fig. 2), demonstrating that our new designed probe toxicity of probe CP 1 is negligible. These preliminary experimental
CP 1 could meet the selective requirements for biomedical and results demonstrate that probe CP 1 could be applied to ratiometric
environmental applications. The high selectivity of probe CP 1 might imaging of Fe3+ in biological samples at high resolution.
be ascribed to the high specificity of the Fe3+-induced ring-opening
In summary, we have designed and synthesized a rhodamine-
reaction of the spirolactam structure of CP 1 through the chelation of appended water-soluble conjugated polymer CP 1 as a FRET-based
Fe3+ with the two oxygen atoms and two nitrogen atoms of the ratiometric probe for intracellular Fe3+ probing. With no requirement
rhodamine 6G moiety (Scheme 1), which triggered the FRET of CP 1 of any organic cosolvent, CP 1 exhibits excellent solubility in pure
to afford ratiometric fluorescence response toward Fe3+.10c,13b–f
aqueous solutions. It shows two well-resolved emission peaks, which
The chemical reversibility behavior of the binding of CP 1 with benefits observation of double-channel fluorescence signal changes at
Fe3+ was then studied in the buffered aqueous solution (see Fig. S4, high resolution. In addition, probe CP 1 shows high selectivity and
ESI†), which indicated that the Fe3+ binding of CP 1 in buffered good reversibility toward Fe3+. The excellent water-solubility, the high
solution is chemically reversible. The response time of the probe sensitivity and selectivity, and the good reversibility of the CP probe
toward Fe3+ is concentration-dependent, and the reaction of the may provide a new platform for intracellular metal-ion probing.
probe and 8 mM of Fe3+ was completed within 5 min (see Fig. S5,
This work was supported by the National Key Scientific Program
ESI†), which is favorable for real-time tracking of the Fe3+ levels in of China (2011CB911000), NSFC (Grants 21325520, 21327009,
biological samples. The practical applications of the designed probe 21221003, J1210040, 21177036, 21135001), the National Instrumen-
were further evaluated by the detection of the recovery of spiked Fe3+ tation Program (2011YQ030124), the Ministry of Education of China
in river water (see Table S1, ESI†). The results obtained in real water (20100161110011), and the Hunan Provincial Natural Science
samples show good recovery values, which confirmed that the Foundation (Grant 11JJ1002).
proposed probe was applicable for practical Fe3+ detection in real
samples with other co-existing potentially competing species.
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CP 1, should benefit ratiometric imaging of Fe3+ in biological
samples at high resolution. Consequently, we performed the ratio-
metric confocal fluorescence imaging experiment for Fe3+ in living
cells. Fig. 3 shows the results of double-channel fluorescence
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Fig. 3 Images of Hela cells treated with probe CP 1 in the absence or
presence of Fe3+. (a) Bright field image of Hela cells incubated with CP 1
([RU] = 10 mM) for 1 h; (b) fluorescence image of (a) from channel 1;
(c) fluorescence image of (a) from channel 2; (d) merge image of frames
(a), (b), and (c); (e) bright field image of Hela cells incubated with CP 1
([RU] = 10 mM) for 1 h, then treated with Fe3+ (5 Â 10À4 M) for another 1 h;
(f) fluorescence image of (e) from channel 1; (g) fluorescence image of
(e) from channel 2; (h) merge image of frames (e), (f), and (g). Excitation
was set at 405 nm.
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2042 | Chem. Commun., 2014, 50, 2040--2042
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