A ruthenium(ii) complex-cyanine energy transfer scaffold based luminescence probe for ratiometric detection and imaging of mitochondrial peroxynitrite

Article abstract of DOI:10.1039/c8cc08061e

A novel ruthenium(ii) complex-cyanine energy transfer scaffold has been established for the development of a ratiometric luminescence probe for ONOO^- detection. The probe, Ru-Cy5, is localized in mitochondria of live cells, allowing ratiometric sensing and imaging of ONOO^- therein.

Full text of DOI:10.1039/c8cc08061e

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A ruthenium(II) complex–cyanine energy transfer
scaffold based luminescence probe for ratiometric
detection and imaging of mitochondrial peroxynitrite†
Cite this: Chem. Commun., 2018,
54, 13698
Received 9th October 2018,
Accepted 12th November 2018
a
Wenzhu Zhang,*a Yi Liu,a Quankun Gao,a Chaolong Liu,a Bo Song,
a
Run Zhang *b and Jingli Yuan
*
DOI: 10.1039/c8cc08061e
rsc.li/chemcomm
A novel ruthenium(II) complex–cyanine energy transfer scaffold has intensity on/off approach,17–20 ratiometric probes that have a
been established for the development of a ratiometric luminescence self-calibration of external interference on emission intensity
probe for ONOOÀ detection. The probe, Ru–Cy5, is localized in are desirable for effective detection of ONOOÀ in mitochondria
mitochondria of live cells, allowing ratiometric sensing and imaging of live cells.21–23
of ONOOÀ therein.
The ratiometric changes in luminescence intensities at two
¨
emission wavelengths mainly arise from the factor of Forster
As a member of reactive nitrogen species (RNS), peroxynitrite resonance energy transfer (FRET) where generally organic dyes act
(ONOOÀ) is endogenously generated by the diffusion-controlled as energy donors and acceptors. Energy transfer (ET) scaffolds
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reaction of endogenous nitric oxide (NO) and superoxide (O2
)
established by exploiting luminescent metal complexes, especially
mainly in mitochondria.1 In living organisms, ONOOÀ at a ruthenium(II) complexes, are scarcely reported,24,25 but such a
reasonable concentration acts as a signalling transduction scaffold is important due to the abundant photophysics and
molecule to modulate a series of cellular functions, such as photochemistry properties of these luminophores, such as strong
selective nitration of tyrosine residues.2 On the other hand, an visible absorptions and emissions, large Stokes shifts, and good
elevated level of ONOOÀ can directly oxidize or decompose water solubility.26–33 Furthermore, previous research of metal
lipids, proteins, and nucleic acids, leading to the apoptosis and complex probes is mainly focused on the modulation of their
death of cells.2 Growing evidence has established that the level excited states through photoinduced electron transfer (PET) and
of ONOOÀ is highly correlated with the progress of various intramolecular charge transfer (ICT), leaving the ET scaffold
diseases and disorders, such as cardiovascular and metabolic strategy yet to be well demonstrated.
diseases, shock, arteriosclerosis, neurodegenerative diseases
In this contribution, we report a Ru(II) complex–cyanine
and even cancers.3 Therefore, an effective method for ONOOÀ based ET scaffold and the development of a luminescence probe
detection is required that might benefit disease early diagnosis for ratiometric detection of ONOOÀ. Based on this ET system,
and treatment monitoring.
the ratiometric luminescence probe, Ru–Cy5, was engineered by
Towards this end, the development of molecular probes that are employing a Ru(II) complex as the energy donor and Cy5 as the
capable of sensing and imaging ONOOÀ has attracted considerable energy acceptor. The absorption of Cy5 (labs = 630 nm) is
attention due to the advantages of high sensitivity, selectivity, and diminished due to the ONOOÀ-mediated oxidative cleavage of
simplicity in data collection of sensing technology.4–7 In the past few the polymethine bridge (Scheme 1A).22 The ET from the Ru(II)
years, a number of probes have been engineered, allowing ONOOÀ complex to Cy5 is thus significantly interrupted (Scheme 1B),
detection to be performed by recording their emission intensity leading to the decrease of Cy5 emission (lem = 660 nm) and
changes during the sensing process.8–16 In comparison with the the increase of Ru(II) emission (lem = 610 nm) for ratiometric
(I610/I660) detection of ONOOÀ. Furthermore, owing to the mito-
chondria targeting feature of Cy5 after cellular internalization,20
a State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of
Ru–Cy5 is expected to be localized in mitochondria, to enable
ONOOÀ in this organelle to be visualized (Scheme 1C) under a
ratiometric mode. To the best of our knowledge, this is the first
report on constructing a Ru(II) complex–Cy5 ET probe for ratio-
metric luminescent sensing and imaging of biomolecules.
To synthesize Ru–Cy5, a Cy5 derivative, compound 8, was
first synthesized (Scheme S1, ESI†). Then, Ru–Cy5 was obtained
Technology, Dalian 116024, China. E-mail: wzhzhang@dlut.edu.cn,
jlyuan@dlut.edu.cn
b Australian Institute for Bioengineering and Nanotechnology, The University of
Queensland, St. Lucia, QLD 4072, Australia. E-mail: r.zhang@uq.edu.au
† Electronic supplementary information (ESI) available: General information,
experimental section, synthesis and characterisation of Ru–Cy5, spectrometric
response of Ru–Cy5 towards ONOOÀ, and MTT assay. See DOI: 10.1039/
c8cc08061e
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Fig. 1 (A) Absorption spectrum changes of Ru–Cy5 (10 mM) in the presence
of different concentrations of ONOOÀ (0, 30, 80, 100, 150 and 200 mM) in
25 mM PBS buffer of pH 7.4 (inset: color changes of the Ru–Cy5 solution
upon reaction with different concentrations of ONOOÀ); (B) emission spectra
(lex = 450 nm) of Ru–Cy5 (10 mM) upon reaction with different concentrations
of ONOOÀ (0–220 mM); (C) the I610/I660 ratio as a function of the ONOOÀ
concentration; (D) calibration curve for the ratiometric luminescence detection
of ONOOÀ. The absorption and emission spectra were recorded after
incubating Ru–Cy5 with ONOOÀ for 30 min.
Scheme 1 (A) ONOOÀ-mediated oxidative cleavage of the polymethine
bridge of Cy5; (B) FRET scaffold of Ru–Cy5 and its response mechanism to
ONOOÀ; (C) Ru–Cy5 in the sensing and imaging of ONOOÀ in mitochon-
dria of live cells.
450 nm, Ru–Cy5 exhibited a strong emission band centred
at 660 nm, the characteristic emission of Cy5, which suggested
the effective ET from the Ru(II) complex to the Cy5 moiety. In
by reacting compound 8 with a Ru(II)-bipyridine complex derivative the presence of ONOOÀ, owing to the oxidation of Cy5 and the
(complex 10) that was synthesized through chloridization of the corruption of ET (Scheme 1B), the emission band at 660 nm
carboxyl acid in complex 9 (Scheme S1, ESI†). The chemical was remarkably decreased with the increase of the emission
structures of all intermediates and Ru–Cy5 were confirmed by intensity at 610 nm. The ratio of the intensity at 610 nm to that
NMR, ESI-MS, HRMS, and elemental analysis (Fig. S1–S17, ESI†). at 660 nm (I610/I660) was found to be more than 46-fold enhanced
To validate the reaction mechanism of Ru–Cy5 with ONOOÀ, their in the presence of 200 mM ONOOÀ (Fig. 1C). Furthermore, the
reaction products were investigated by ESI-MS analysis. As shown enhancement in the I610/I660 ratio showed an excellent linear correla-
in Fig. S18 (ESI†), the peak at m/z 632.20 was absent, and a new tion to the concentration of ONOOÀ in the range from 0–30 mM.
peak at m/z 448.70 that can be assigned to the species of [Ru–Cy]2+ Based on the concentration corresponding to three standard devia-
was found, which confirmed the reaction of Ru–Cy5 with ONOOÀ, tions of the background signal, the detection limit (3s/k) was
with the formation of [Ru–Cy]2+ as proposed in Scheme 1B.
calculated to be 0.28 mM. This result suggests that Ru–Cy5 could
In the absence and presence of ONOOÀ, changes of UV-vis be used as a ratiometric luminescence probe for highly sensitive
spectra in Ru–Cy5 were initially examined in 25 mM PBS buffer of quantification of ONOOÀ in aqueous media.
pH 7.4. As shown in Fig. 1A, the absorption spectrum of Ru–Cy5
The specificity of Ru–Cy5 for responding to ONOOÀ over
showed two intense absorption bands centred at 450 nm and other interfering species, including ROS, RNS, amino acids and
630 nm, which can be ascribed to the metal-to-ligand charge metal ions, was investigated. As shown in Fig. 2A, more than
transfer (MLCT) absorption of the Ru(II) complex and the typical 46-fold enhancement in the I610/I660 ratio was noticed after
absorption of Cy5. Upon the reaction with ONOOÀ, the absorption Ru–Cy5 was reacted with 20 equiv. of ONOOÀ, while the
band at 630 nm was remarkably decreased due to the oxidization of reactions of Ru–Cy5 with 100 equiv. of other species, including
Cy5, while no obvious changes at 450 nm were noticed, indicating HOCl (20 equiv.), H2O2, OH, O2 À, O2, NO, NO2À, HSÀ, Cys,
the tolerance of the Ru(II) complex to ONOOÀ. On increasing the Hcy, GSH (5 mM), Ca2+, Zn2+, K+, Na+, Mg2+, Fe2+ and Fe3+,
concentration of ONOOÀ, the color of the solution was clearly could not induce the change in the I610/I660 ratio. These results
changed from blue to yellow (Fig. 1A inset). These facts indicate reveal that the luminescence response of Ru–Cy5 towards
that Ru–Cy5 can act as a ‘‘naked-eye’’ probe for ONOOÀ detection. ONOOÀ is highly specific. The effect of pH on the ratiometric
Significant changes in the emission spectrum of Ru–Cy5 were luminescence response of Ru–Cy5 towards ONOOÀ was then
also observed in the presence of different concentrations of ONOOÀ evaluated in 25 mM PBS buffer with different pH values (pH 4–10).
in 25 mM PBS buffer of pH 7.4 (Fig. 1B). Under excitation at As shown in Fig. 2B, in the absence of ONOOÀ, Ru–Cy5 exhibited
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Fig. 2 (A) The I610/I660 ratios of Ru–Cy5 (10 mM) upon reactions with
various ROS, RNS, amino acids and ions (a, control; b, ONOOÀ; c, HOCl; d,
NO2 ; e, NO; f, HS ; g, H2O2; h, ꢀOH; i, O2 À; j, 1O2; k, Cys; l, Hcy; m, GSH
(5 mM); n, Ca2+; o, Zn2+; p, K+; q, Na+; r, Mg2+; s, Fe2+; and t, Fe3+) in
25 mM PBS buffer of pH 7.4. (B) Effects of pH on the I610/I660 ratios of
Ru–Cy5 (10 mM) before and after the reaction with SIN-1 (200 mM).
Excitation was performed at 450 nm.
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Fig. 3 Colocalization analysis of internalized Ru–Cy5 and MitoTrackert
Green FM in HeLa cells. (A) Green channel of MitoTrackert Green FM;
(B) red channel of Ru–Cy5; (C) merged image of A, B, and a bright-field
image; (D) intensity correlation plot of Ru–Cy5 and MitoTrackert Green
FM; (E) intensity profiles of the linear regions of interest (ROIs) across
HeLa cells. Excitation at 488 nm was used for both the red channel
(640–680 nm) and the green channel (500–540 nm).
small and stable I610/I660 ratios in the pH range from 4 to 10. Upon
reaction with 3-morpholinosydnonimine (SIN-1, a ONOOÀ donor,
200 mM), the I610/I660 ratio remained low at pH below 6.5, and then
rapidly increased to the maximum level at pH 7.5. Considering the
weakly basic pH in mitochondria (pH B8),34,35 the above result luminescence imaging approach. As shown in Fig. 4, under
indicates that Ru–Cy5 is favorable to be used for the detection of laser excitation at 488 nm, the control group of HeLa cells
ONOOÀ in the mitochondria of live cells.
(incubated with 25 mM Ru–Cy5 for 2 h only) showed a weak signal in
Fig. S19 (ESI†) illustrated the time profile changes of the the Ru(II) channel (590–630 nm) and an intense signal in the Cy5
I610/I660 ratio of Ru–Cy5 in the absence and presence of 200 mM channel (640–680 nm). After supplying with 500 mM SIN-1 for
ONOOÀ. The I610/I660 ratio of Ru–Cy5 kept at a low and stable value 30 min, significant decrease of the Cy5 channel signal and increase
during the period of 25 min. However, upon the addition of ONOOÀ, of the Ru(II) channel signal were observed, with a more than 7-fold
the I610/I660 ratio was gradually increased, and reached a maximum change of the luminescence intensity ratio. These results
after 20 min incubation, which reveals that the reaction between demonstrated the feasibility of Ru–Cy5 for the visualization of
Ru–Cy5 and ONOOÀ could be completed within 20 min. Upon the mitochondrial ONOOÀ in live cells under ratiometric mode.
addition of ONOOÀ, a good linearity between ONOOÀ concentration
In summary, a Ru(II) complex–cyanine energy transfer based
and the corresponding initial reaction rate is obtained (Fig. S20, luminescence probe, Ru–Cy5, has been developed for the ratiometric
ESI†). According to the reported methods,36,37 the total reaction rate
constant (ktot) and reaction half-life time (t1/2) were estimated to be
6.48 Â 104 MÀ1 minÀ1 and 1.07 Â 10À5 min, respectively.
On the basis of the above results, the feasibility of Ru–Cy5 for
sensing and imaging of mitochondrial ONOOÀ was evaluated. MTT
assay showed that Ru–Cy5 has low toxicity towards live cells. As
shown in Fig. S21 (ESI†), no obvious changes in cell proliferation
were noticed when HeLa cells were incubated with increased
concentrations of Ru–Cy5. The cell viability remained more than
84% even after incubating HeLa cells with 50 mM of Ru–Cy5 for
24 h, suggesting the low cytotoxicity of Ru–Cy5 for loading live cells.
The distribution of Ru–Cy5 in mitochondria of live cells was
confirmed by colocalization analysis with a commercially available
mitochondria labeling agent, MitoTrackert Green FM. HeLa cells
were incubated with 50 mM Ru–Cy5 for 2 h, and then stained with
MitoTrackert Green FM for the colocalization analysis. As shown
in Fig. 3, the mitochondrial distribution was supported by the
synchronous luminescence signals in the linear region of interest
across the cells. The excellent overlapping of red (Ru–Cy5) and
Fig. 4 Confocal luminescence images of ONOOÀ in HeLa cells using
Ru–Cy5 as a probe. (A) HeLa cells were incubated with Ru–Cy5 (25 mM) for
2 h at 37 1C and then treated with 500 mM SIN-1 for 30 min (control group:
HeLa cells were stained with Ru–Cy5 only). Image analysis: luminescence
green (MitoTrackert Green FM) channels was observed. The
Pearson’s correlation coefficient (0.94) is close to 1, revealing the
mitochondrial localization of the internalized Ru–Cy5 molecules.
With the mitochondria-localization feature of Ru–Cy5, mito-
chondrial ONOOÀ in live cells was visualized via a ratiometric
intensity analysis (B) and changes of the luminescence intensity ratio (C).
Scale bar: 10 mm.
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detection of ONOOÀ. This probe showed high response specificity
and sensitivity to ONOOÀ, as well as low cytotoxicity, good cell
membrane permeability and mitochondria-localization feature,
which enabled it to be successfully used for the ratiometric sensing
and imaging of ONOOÀ in the mitochondria of live cells, to provide
a useful tool for investigating the functions of ONOOÀ in live cells. In
addition, the ET scaffold strategy described in this work also
contributes to the future development of luminescent transition
metal complex-based ratiometric probes.
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We gratefully acknowledge the financial support from the
National Natural Science Foundation of China (Grant No.
21475015, and 21775015), and Australian Research Council
(DE170100092). The facilities and assistance of the Queensland
Node of the Australian National Fabrication Facility (ANFF-Q),
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Conflicts of interest
There are no conflicts to declare.
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