Synthesis, characterization and photophysical properties of a new Cu2+ selective phosphorescent sensor

Article abstract of DOI:10.1016/j.jorganchem.2017.10.032

A new Cu(II) selective phosphorescent sensor based on a cationic iridium(III) complex was synthesized and characterized. Compared with other commonly coexisting metal ions, the Ir(III) complex displayed significant phosphorescence quenching for detecting Cu²⁺ with a 1:1 stoichiometry in MeOH/H₂O (1:1, v/v) solution. Upon addition of Cu²⁺, the rapid quenching of emission intensity provided a sensitive method for detection of Cu²⁺. The corresponding detection limit was determined to be 7.6 × 10^?8 M and the association constant between the Ir(III) complex and Cu²⁺ was calculated to be 0.31 × 10⁵ M^?1. Luminescence imaging experiments demonstrated that this Ir(III) complex was cell permeable and suitable for monitoring intracellular Cu²⁺ in living Hela cells by confocal microscopy.

Full text of DOI:10.1016/j.jorganchem.2017.10.032

Accepted Manuscript
2+
Synthesis, characterization and photophysical properties of a new Cu selective
phosphorescent sensor
Tianzhi Yu, Qianguang Yang, Zeyang Zhu, Yuling Zhao, Xiaoxiao Liu, Chengjin Wei,
Hui Zhang
PII:
S0022-328X(17)30619-8
10.1016/j.jorganchem.2017.10.032
JOM 20152
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 11 September 2017
Revised Date: 20 October 2017
Accepted Date: 22 October 2017
Please cite this article as: T. Yu, Q. Yang, Z. Zhu, Y. Zhao, X. Liu, C. Wei, H. Zhang, Synthesis,
2+
characterization and photophysical properties of a new Cu selective phosphorescent sensor, Journal
of Organometallic Chemistry (2017), doi: 10.1016/j.jorganchem.2017.10.032.
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ACCEPTED MANUSCRIPT
Synthesis, characterization and photophysical properties of a new
Cu2+ selective phosphorescent sensor
Tianzhi Yu1,*, Qianguang Yang1, Zeyang Zhu1, Yuling Zhao2, Xiaoxiao Liu3, Chengjin Wei1,
Hui Zhang1
1Key Laboratory of Opto-Electronic Technology and Intelligent Control (Ministry of Education),
Lanzhou Jiaotong University, Lanzhou 730070, China
2School of Chemical and Biological Engineering, Lanzhou Jiaotong University, Lanzhou 730070,
China
3School of Mechatronic Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China
Abstract: A new Cu(II) selective phosphorescent sensor based on a cationic iridium(III) complex
was synthesized and characterized. Compared with other commonly coexisting metal ions, the
Ir(III) complex displayed significant phosphorescence quenching for detecting Cu2+ with a 1:1
stoichiometry in MeOH/H2O (1:1, v/v) solution. Upon addition of Cu2+, the rapid quenching of
emission intensity provided a sensitive method for detection of Cu2+. The corresponding detection
limit was determined to be 7.6 × 10-8 M and the association constant between the Ir(III) complex
and Cu2+ was calculated to be 0.31 × 105 M−1. Luminescence imaging experiments demonstrated
that this Ir(III) complex was cell permeable and suitable for monitoring intracellular Cu2+ in living
Hela cells by confocal microscopy.
Keywords: Cationic iridium(III) complex; Phosphorescence quenching; Phosphorescent
*Corresponding Author. Tel. +86-931-4956935; Fax: +86-931-4938756; e-mail: yutzh@mail.lzjtu.cn (T.Z. Yu).
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chemosensor; Cu2+ detection; Bioimaging
1. Introduction
Recently, phosphorescent heavy-metal complexes have attracted tremendous attentions due to
their efficient luminescent properties and potential applications in organic light-emitting diodes
[11-14]
(OLEDs) [1-6], light-emitting electrochemical cells [7-10], chemosensors
and biological
labeling [15,16]. Compared with the fluorescent chemosensors, the phosphorescent heavy-metal
complexes have attracted more attentions and increasingly used as colorimetric or luminescent
probes for biomolecules and metal ions because their relatively long phosphorescence emission is
unaffected by short-lived background noise such as fluorescence and scattered light [17-20]. Among
phosphorescent heavy-metal complexes, the phosphorescent Ir(III) complexes show some
advantageous photophysical properties such as short excited-state lifetime, high quantum
efficiency, color diversity and high photostability, and some Ir(III) complexes have been used as
luminescent probes.
As trace element and nutrient in living organisms, the copper( ) is essential and plays a
significant role in a basic physiological process [13]. However, the copper( ) is also one of
significant metal pollutants and toxic to living cells [21]. Aberrant levels of copper( ) ions can
result in neurodegenerative diseases such as Alzheimer's disease, prion disease, Menkes disease,
Parkinson’s disease and Wilson disease [22-24]. As a result, to seek a rapid sensitive sensor to detect
and quantify copper( ) ion in environment and in living systems is very importance for biologic
research and medical diagnosis. A number of fluorescent chemosensors for the highly sensitive
and selective detection of Cu (II) ion have been reported in the literatures [25-31]. Recently, the
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phosphorescent Ir(III)-based chemosensors have been successfully applied for the detection of
Cu2+ ions [13,14,19,32-35]. Moreover, the phosphorescent Ir(III) complexes as luminescent probes
present important advantages as their simplicity, easy visualization and high sensitivity. The
incorporation of a Lewis base as a cation receptor on the ancillary ligands of the luminescent Ir(III)
complexes is a very used strategy for the recognition of cations. The coordination interaction of
the Cu2+ with an ion recognition group on the Ir(III) complex causes variations in the
luminescence intensity that may be used to detect Cu2+. For example, You et al. [19] synthesized the
Ir(III) complex having two cyclometalating 2-phenylpyridine (ppy) ligands and one
2-(2′-benzo[b]thienyl)pyridine (btp) tethered a di(2-picolyl)amine (DPA) copper ion receptor by a
methylene linker, which was used as a Cu2+-sensitive probe. This Ir(III) complex has a 1:2
stoichiometry (complex : Cu2+) that was demonstrated by the binding titration data, and the limit
of detection for Cu2+ was measured to be range of 5.8 – 8.1 µM.
The di(2-picolyl)amine moiety (DPA) always exhibits high affinity and specificity for Zn2+
and Cu2+ ions over the other metal ions, which has been frequently used as turn-on sensors for
Zn2+ and Cu2+ ions. In this paper we used 2-(1-naphthyl)benzothiazole (nbt) as cyclometalated
ligand and 2,2′-dipyridine tethered DPA moiety by a methylene linker (bp-DPA) as the neutral
auxiliary ligand to prepare a new cationic Ir(III) complex, [Ir(nbt)2(bp-DPA)]PF6. The
photophysical properties and ion-recognition behaviors of the cationic Ir(III) complex with some
heavy-metal ions are investigated. This cationic Ir(III) complex exhibited good sensitivity and
selective on–off type response toward Cu2+ over other metal ions including Zn2+ and Hg2+.
Furthermore, the confocal luminescence imaging was performed to validate the ability of the Ir(III)
complex to monitor intracellular Cu2+ ions.
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The synthetic routes of the neutral auxiliary ligand bp-DPA and the cationic Ir(III) complex
were shown in Scheme 1.
(nbt)2Ir(µ-Cl)2Ir(nbt)2
[Ir(nbt)2(bp-DPA)]PF6
Scheme 1. Synthetic routes of bp-DPA and [Ir(nbt)2(bp-DPA)]PF6.
2. Experimental
2.1. Materials and instruments
2-Aminothiophenol, 1-naphthaldehyde and 4,4′-dimethyl-2,2′-bipyridine were bought from
Alfa Aesar. 2,2'-Dipicolylamine was obtained from Energy Chemical (China). IrCl3•nH2O (iridium
content > 60.0%) was bought from Shanxi Kaida Chemical Co. Ltd. (China) and used without
further purification. All the other chemicals were analytical grade reagent. All of the organic
solvents used in this study were dried over appropriate drying agents and freshly distilled prior in
the related reaction.
4-Methyl-2,2′-bipyridine-4′-carboxaldehyde was synthesized as the procedure described in
literature [36]. The cyclometalated Ir(III) µ-chlorobridged dimer of (nbt)2Ir(µ-Cl)2Ir(nbt)2 was
prepared as previously described [37]
.
1H NMR spectra were recorded on Agilent Technologies DDZ 600 MHz. Mass spectrum was
obtained from a Thermo Scientific Orbitrap Elite mass spectrometer. Thermogravimetric analysis
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(TGA) was performed on a Perkin-Elmer Pyris system. UV-vis absorption and photoluminescent
spectra were recorded on a Shimadzu UV-2550 spectrometer and on a Perkin-Elmer LS-55
spectrometer, respectively. All pH measurements were made with apH-10C digital pH meter.
2.2. Synthesis and characterization of bp-DPA and [Ir(nbt)2(bp-DPA)]PF6
4-Methyl-4′-(di(2-picolyl)aminomethyl)-2,2′-bipyridine (bp-DPA): Di(2-picolyl)amine
(0.729 g, 3.66 mmol) and 4-Methyl-2,2′-bipyridine-4′-carboxaldehyde (0.725 g, 3.66 mmol) were
dissolved in fresh MeOH (70 mL). A catalytic amount (two drops) of glacial acetic acid was added
to the solution, which was refluxed for 0.5 h. The reaction mixture was cooled by using an ice bath,
and then NaBH3CN (0.460 g, 7.32 mmol) was slowly delivered to the solution. The ice bath was
removed and the reaction mixture was stirred overnight at room temperature. Basic work-up using
saturated aqueous sodium carbonate solution and subsequent extraction using CH2Cl2 was
performed. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuum. The
crude was purified by chromatography on silica gel using acetone/petroleum ether (1:10, v/v) to
1
obtain a sticky brown oily-liquid. Yield (1.185 g, 84.9%). H NMR (600 MHz, CDCl3, δ, ppm):
8.50 (d, J = 7.2 Hz, 1H), 8.40 (t, J = 9.6 Hz, 3H), 8.29 (s, 1H), 8.09 (s, 1H), 7.53 (t, J = 9.0 Hz,
2H), 7.47 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 7.2 Hz, 1H), 7.02-6.97 (dd, J = 9.6, 8.4 Hz, 3H), 3.74 (s,
4H), 3.69 (s, 2H), 2.26 (s, 3H).
[Ir(nbt)2(bp-DPA)]PF6: The chloro-bridged dimer complex (nbt)2Ir(µ-Cl)2Ir(nbt)2 (0.799 g,
0.534 mmol) and bp-DPA (0.5111 g, 1.340 mmol) were refluxed in 60 mL of
dichloromethane/methanol (1:1, v/v) under nitrogen atmosphere at 75 for 4 h with keeping in
dark place. After cooling, an excess of KPF6 (15 equiv) was slowly added to the reaction mixture
and the solution was stirred for additional 12 h. After completion of the reaction, the solids in the
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solution were removed by filtration. The residue was purified through chromatography on silica
1
gel using CH2Cl2/MeOH (9:1, v/v) as the eluent to obtain a red powder (0.98 g, 74%). H NMR
(600 MHz, CDCl3, δ, ppm): δ 8.66 (dd, J = 9.0, 8.4 Hz, 3H), 8.44 (t, J = 6.8 Hz, 3H), 7.95 (dd, J =
8.4, 7.8 Hz, 2H), 7.87 (d, J = 6.0 Hz, 1H), 7.82 (d, J = 6.0 Hz, 1H), 7.76-7.68 (m, 4H), 7.57 (t, J =
7.8 Hz, 2H), 7.51-7.44 (m, 5H), 7.41 (t, J = 7.2 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.25 (t, J = 7.2
Hz, 2H), 7.20 (dd, J = 8.4, 6.0 Hz, 2H), 7.03 (t, J = 6.6 Hz, 2H), 6.98 (t, J = 7.8 Hz, 1H), 6.56 (dd,
J = 8.4, 8.4 Hz, 2H), 6.37 (d, J = 8.4 Hz, 1H), 6.15 (d, J = 8.4 Hz, 1H), 4.03 (s, 2H), 3.85 (s, 4H),
2.63 (s, 3H). HR EI-MS: Calcd. for [C58H43IrN7S2]+, 1094.27; Found, 1094.2637.
2.3. Spectroscopic experiments
Spectrophotometric titrations of the Ir(III) complex were performed in air-equilibrated
CH3OH solution. The Ir(III) complex stock solution (1.0 × 10−3 M) was prepared in CH3OH. The
fresh metal stock solutions (typically 1.0 × 10−2 M) were prepared in Milli-Q water using the
corresponding chloride salts (Na+, K+, Ca2+, Mg2+, Mn2+, Cu2+, Zn2+, Cd2+, Ni2+, Co2+, Pb2+, Al3+
and Fe3+), nitrate salt (Ag+) and perchlorate salt (Hg2+). For emission titration experiment, 10µL of
methanol solution of the Ir(III) complex (1.0 × 10−3 M) was taken in a quartz optical cell of 1.0 cm
optical path length in 2000 µL and then stock solutions of metal ions were added gradually to it by
using micropipette. Spectral data were recorded at 1 min after the addition of the metal ions for
both titrations.
2.4. Cell cytotoxicity
The cell cytotoxicity of [Ir(nbt)2(bp-DPA)]PF6 was determined by MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assays. Hela cells were
o
placed in microassay culture plates (5 × 104 cells per milliliter) and grown for 24 h at 37 C in a
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5% CO2 incubator. Then [Ir(nbt)2(bp-DPA)]PF6 was added to the wells to achieve final
o
concentrations of 1.25, 2.5, 5, 10 and 20 µM. The plates were incubated at 37 C in a 5% CO2
incubator for another 24 h. Upon completion of the incubation, stock MTT dye solution (20 µL, 1
mg/mL) was added to each well. After 4 h, dimethyl sulfoxide (150 µL) was added to solubilize
the MTT formazan.
2.5. Cell culture and confocal luminescence imaging
Vitro experiments were performed by using Hela cells. Hela cells were cultured in
RPMI-1640 medium supplemented with 10% FBS (fetal bovine serum), 100 units per mL
penicillin, and 100 µg/mL streptomycin at 37 oC in a humid atmosphere containing 5% CO2. Cells
(5 × 108 L−1) were plated on 18 mm glass coverslips and allowed to adhere for 24 h. Then the cells
were treated with [Ir(nbt)2(bp-DPA)]PF6 (20 µM) and incubated for 30 min. Subsequently, the
cells were treated with 100 µM CuCl2·2H2O. The cells were incubated for 30 min and washed
with PBS three times to remove free compound and ions before analysis. Hela cells cells only
incubated with 20 µM [Ir(nbt)2(bp-DPA)]PF6 for 30 min acted as a control. Confocal
luminescence images of Hela cells cells were carried out on an Olympus FV1000 laser scanning
confocal microscope and a 100x oil-immersion objective lens. Emission was collected at 550–630
nm for the Hela cells.
3. Results and discussion
3.1. Synthesis and characterization
The synthesis of the cationic Ir(III) complexes from Scheme 1 began first with preparing the
ancillary ligand bp-DPA. The ligand bp-DPA was successfully obtained through two step
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reactions.
First,
4,4′-dimethyl-2,2′-bipyridine
was
selectively
oxidized
to
be
4-Methyl-2,2′-bipyridine-4′-carboxaldehyde by SeO2 catalyst. Subsequently, this intermediate was
subjected to reductive amination with DPA. The ligand bp-DPA was purified by column
chromatography to give the pure product. The Ir(III) complex [Ir(nbt)2(bp-DPA)]PF6 was
synthesized by reacting the Ir(III) chloro-bridged dimer with the ancillary ligand bp-DPA, and
–
followed by a counter ion-exchange reaction from Cl– to PF6 . The Ir(III) complex was structurally
1
characterized by H NMR and the high resolution electro spray ionization mass spectra (HR
ESI-MS).
3.2. Photophysical and thermal properties of [Ir(nbt)2(bp-DPA)]PF6
The UV-vis absorption and photoluminescence (PL) spectra of [Ir(nbt)2(bp-DPA)]PF6 in
MeOH solution is shown in Fig. 1. As shown in Fig.1, the intense absorption bands before 300 nm
1
in the absorption spectrum are assigned to the spin-allowed (π–π*) ligand-centered transition of
the cyclometalated primary ligand and the ancillary ligand in the complex. The following
relatively weak absorption band at 345 nm could be attributed to both spin-allowed
metal-to-ligand charge-transfer (1MLCT) and ligand-to-ligand charge-transfer (1LLCT) transitions.
Moreover, the weaker absorption band that found at 468 nm is ascribed to the spin-forbidden
3MLCT and 3LLCT [38-40]
.
Under the excitation of 468 nm, the complex [Ir(nbt)2(bp-DPA)]PF6 exhibits red emission
with a maximum peak at 594 nm and a shoulder peak at 642 nm in MeOH solution at room
temperature. The CIE coordinates of [Ir(nbt)2(bp-DPA)]PF6 is (0.610, 0.388). In addition, the
photoluminescence quantum yield of [Ir(nbt)2(bp-DPA)]PF6 at room temperature was measured
to be 5.05% in MeOH solution (ca. 10-6 mol/L) through an absolute method using the Edinburgh
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Instruments spectrometer (FLS920) integrating sphere excited at 468 nm with a Xe lamp. The
photoluminescence decay lifetime of [(nbt)2Ir(bp-DPA)](PF6) in MeOH solution was measured
to be 2.21 µs with a time-correlated single photon counting spectrometer using an Edinburgh
Instruments spectrometer (FLS920) with a microsecond flash lamp as the excitation source
(repetition rate 90 Hz) at room temperature. The observed emission lifetime is in the microsecond
time scale, which indicates the phosphorescent nature of the emission. The result indicates that the
Ir(III) complex possesses a relatively long lifetime to differentiate short-lived background
fluorescence and enhances the signal-to-noise ratio of detection through the use of time-resolved
fluorescence analysis.
UV-vis
PL
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
300
400
500
600
700
800
Wavelength (nm)
Fig. 1. The UV-vis absorption and photoluminescence (PL) spectra of
[Ir(nbt)2(bp-DPA)]PF6 in MeOH solution.
The thermal behavior of the cationic Ir(III) complex was investigated using
thermogravimetric analysis (TGA) under a nitrogen atmosphere. The result of its TGA
measurement is shown in Fig. 2. The complex [(nbt)2Ir(bp-DPA)](PF6) exhibited good thermal
o
stability up to 256 C. With increasing temperature, [(nbt)2Ir(bp-DPA)](PF6) undergoes two
large-stage decomposition at 259 and 382 oC accompanied by two sharp weight losses in its TGA
curve. It indicates that the complex meets with the thermal stability requirement of chemical
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sensor application.
100
80
60
40
20
0
0
100 200 300 400 500 600 700
Temperature (
)
Fig. 2. Thermogravimetric analysis (TGA) of [Ir(nbt)2(bp-DPA)]PF6 in nitrogen atmosphere
(heating rate 10 /min).
3.2. Selectivity and cation-competitive experiments
The selectivity of the Ir(III) complex (10 µM) for metal ions investigated by monitoring its
photoluminescence changes upon addition of various metal ions (1 equiv) in MeOH solution is
shown in Fig. 3a. The result indicated that the photoluminescence of the Ir(III) complex was
quenched significantly upon addition of Cu2+ ion and quenched slightly upon addition Ca2+ or Ni2+
ion, whereas very weak variations in the emission spectra of the Ir(III) complex were observed
upon addition of other metal ions, such as Zn2+, Na+, Mg2+, Cd2+, Fe3+, Mn2+, K+, Co2+, Hg2+, Al3+,
Pb2+ and Ag+. Therefore, the Ir(III) complex displayed a turn-off phosphorescence response and
high selectivity in sensing Cu2+ ion.
To further investigate the interference of other metal ions on the detection of Cu2+ ion, the
cation-competitive experiments were also performed by adding Cu2+ ion to solutions of the Ir(III)
complex in the presence of other metal ions (Fig. 3b). As shown in Fig. 3b, whether in the absence
or in the presence of the other metal ions, obvious spectral changes were observed for
[Ir(nbt)2(bp-DPA)]PF6 only upon addition of Cu2+ ion. The results suggests that
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[Ir(nbt)2(bp-DPA)]PF6 possesses good selectivity for Cu2+ ion.
400
probe
Zn2+
Ca2+
Na+
a
Mg2+
Cd2+
Ni2+
Fe3+
Mn2+
K+
300
200
100
0
Co2+
Hg2+
Al3+
Pb2+
Ag+
Cu2+
2+
Cu
550
600
650
700
Wavelength (nm)
2+
with Cu
400
350
300
250
200
150
100
50
b
2+
without Cu
0
Zn Ca Mg Ni Cd Mn K Na Co Fe Ag Pb Al Hg
Fig. 3. (a) Photoluminescence spectra of [Ir(nbt)2(bp-DPA)]PF6 (10 µM) upon addition of
various metal ions (1 equiv) in MeOH solution; (b) Column diagrams of the PL intensity of
[Ir(nbt)2(bp-DPA)]PF6 + Mn+. Red bars represent the addition of 10 equiv of various metal ions
to the blank solution and black bars represent the subsequent addition of Cu2+ (1 equiv) to the
aforementioned solutions. (λex = 468 nm, λex = 594 nm)
3.3. Cu2+ titration analysis
To further evaluate the sensing properties of the Ir(III) complex towards Cu2+ ion, the
emission titration experiments of [Ir(nbt)2(bp-DPA)]PF6 with Cu2+ ion in varying concentrations
were carried out by monitoring the emission intensity changes of [Ir(nbt)2(bp-DPA)]PF6. As
shown in Fig. 4a, the lone Ir(III) complex exhibited strong phosphorescence emission with a
maximum peak at 594 nm and a shoulder peak at 642 nm upon excitation at 468 nm, and the PL
intensity of the Ir(III) complex gradually decreased with continuous addition of Cu2+ (0–2.0 eq.) to
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the solution. The emission intensity of the Ir(III) complex drops by 97.4% as Cu2+ ion
concentration achieves 1.0 equivalents, which indicates that there is a strong association
interaction between Cu2+ and the Ir(III) complex.
The Job’s plot experiment was performed by monitoring the PL intensity change of
[Ir(nbt)2(bp-DPA)]PF6 with the variation of the concentration of both [Ir(nbt)2(bp-DPA)]PF6
and Cu2+ ion (Fig. 4b), indicating a 1:1 stoichiometry between the Ir(III) complex and Cu2+ ions.
400
[Cu2+] (eq.)
a
0.0
0.1
0.2
350
0 eq.
0.3
0.4
300
[Cu2+]
0.5
0.6
0.7
0.8
0.9
1.0
1.2
1.4
1.6
1.8
2.0
250
200
150
100
50
2 eq.
0
550
600
650
700
Wavelength (nm)
300
250
200
150
100
50
b
0
0.0
0.2
0.4
0.6
0.8
1.0
XCu2+
Fig. 4. (a) Photoluminescence spectra of [Ir(nbt)2(bp-DPA)]PF6 (10 µM) upon titration with
Cu2+ (0–2.0 eq.) in MeOH solution; (b) Job’s plot for the complex formation between
2+
[Ir(nbt)2(bp-DPA)]PF6 and Cu2+ ion in MeOH solution. (λex = 468 nm, λex = 594 nm, XCu
[Cu2+]/[[Ir(nbt)2(bp-DPA)]PF6]+[Cu2+])
=
From the titration experiments, plotting emission intensity versus Cu2+ concentration (0–1.0
equiv.) afforded a good linear relationship (R = 0.9897) (Fig. 5a). The detection limit of
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[Ir(nbt)2(bp-DPA)]PF6 was calculated from the equation DL = 3S/k, where S is the standard
deviation of blank measurement, k is the slope between intensity versus sample concentration
[33,41,42]. The limit of detection was calculated to be 7.6 × 10-8 M. The association constant was
determined by the Benesi-Hildebrand equation and calculated to be 0.31 × 105 M−1 (Fig. 5b).
Compared with the similar Ir(III) complex [19], our synthesized Ir(III) complex exhibited lower
detection limit for Cu2+ ion.
300
250
200
150
100
50
a
0
0.0
5.0x10-6
[Cu2+]
1.0x10-5
2
1
0
b
2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6
[Cu2+]
Fig. 5. (a) The linear response of emission intensity at 594 nm of [Ir(nbt)2(bp-DPA)]PF6 (10
µM) to the Cu2+ concentrations changes (0–1.0 equiv.) in MeOH solution; (b) Benesi-Hildebrand
plot of [Ir(nbt)2(bp-DPA)]PF6 at different Cu2+ concentrations in MeOH solution. (λex = 468 nm)
Reversibility of the probe is an important consideration in practical application. The
reversibility of interaction between [Ir(nbt)2(bp-DPA)]PF6 and Cu2+ ion was performed by
addition of an aqueous of EDTA disodium (1 eq.) to the mixture of [Ir(nbt)2(bp-DPA)]PF6 (10
µM) and Cu2+ ion (10 µM) in MeOH/H2O (1:1, v/v) solution (Fig. 6). As shown in Fig. 6, after
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adding of Na2EDTA (1 eq.) to the mixture of the Ir(III) complex and Cu2+ ion, the emission
intensity at 594 nm was almost completely recovered. The result indicated that the interaction
between [Ir(nbt)2(bp-DPA)]PF6 and Cu2+ ion is chemically reversible.
400
probe
probe + Cu2+
probe + Cu2+ + Na2EDTA
300
200
100
0
550
600
650
700
Wavelength (nm)
Fig. 6. The emission spectra of [Ir(nbt)2(bp-DPA)]PF6 (10 µM), [Ir(nbt)2(bp-DPA)]PF6 (10
µM) + Cu2+ (10 µM) and [Ir(nbt)2(bp-DPA)]PF6 (10 µM) + Cu2+ (10 µM) + Na2EDTA (10 µM)
in MeOH/H2O (1:1, v/v) solution. (λex = 468 nm, λex = 594 nm)
800
without Cu2+
with Cu2+
700
600
500
400
300
200
100
0
0
2
4
6
8
10
12
14
pH
Fig. 7. The emission intensity of [Ir(nbt)2(bp-DPA)]PF6 (10 µM) with and without Cu2+ (10
µM) as a function of pH in MeOH/H2O (1:1, v/v) solution. (λex = 468 nm, λex = 594 nm)
The effect of pH on the photoluminescence response of [Ir(nbt)2(bp-DPA)]PF6 to Cu2+ ions
was also investigated in MeOH/H2O (1:1, v/v) solution (Fig. 7). As shown in Fig. 7, the PL
intensity of [Ir(nbt)2(bp-DPA)]PF6 solutions in the presence and absence of Cu2+ were both no
dramatic change over a pH range of 2–10. However, pH＞10，with the coordinated Cu2+ ions
gradually dissociating from the Ir(III) complex–Cu2+ system, the emission of free the Ir(III)
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complex recovered gradually.
From the above results, the possible mechanisms of detection and the structures of
[Ir(nbt)2(bp-DPA)]PF6, both with and without the addition of Cu2+, are shown in Fig. 8. In the
absence of Cu2+, the Ir(III) complex exhibits strong red emission with a maximum peak at 594 nm
when excited at 468 nm. After coordinated with Cu2+, the paramagnetic effect from spin-orbit
coupling of the Cu2+ induces the emission of the Ir(III) complex quenching. Upon the addition of
Na2EDTA into the [Ir(nbt)2(bp-DPA)]PF6–Cu2+ system, the emission of the Ir(III) complex was
restored due to dissociating from the Ir(III) complex–Cu2+ system by EDTA, which indicates the
regeneration of the free [Ir(nbt)2(bp-DPA)]PF6.
Fig. 8. The possible sensing mechanism of [Ir(nbt)2(bp-DPA)]PF6 for Cu2+.
3.4. Cell cytotoxicity and confocal luminescence imaging
The cytotoxicity of [Ir(nbt)2(bp-DPA)]PF6 was investigated in Hela cells using an MTT
assay (Fig. 9). As shown in Fig. 9, the Ir(III) complex had some cytotoxicity to Hela cells. When
the concentration of [Ir(nbt)2(bp-DPA)]PF6 was greater than 5 µM, the viability of the cells kept
changeless and the cell viability remained above 40%. Herein, we selected 20 µM
[Ir(nbt)2(bp-DPA)]PF6 to analyze its recognition ability for Cu2+ ions in living Hela cells by
confocal luminescence imaging (Fig. 10). As shown in Fig. 10a, the Hela cells treated with 20 µM
15

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o
[Ir(nbt)2(bp-DPA)]PF6 alone for 30 min at 37 C exhibited bright red emission. However, when
Cu2+ (100 µM) was introduced into the cells via incubation under the same condition, remarkable
intracellular luminescence quenching was observed (Fig. 10d). Bright-field measurements after
the treatment with [Ir(nbt)2(bp-DPA)]PF6 confirmed that the cells were viable throughout the
imaging experiments (Figs. 10b,e). Overlays of confocal luminescence images also demonstrated
that the luminescence was evident (Figs. 10c,f). Thus, the results indicated that the Ir(III) complex
was cell membrane-permeable and had the ability to detect Cu2+ in living cells.
100
80
60
40
20
0
1.25
2.5
5
10
20
Concentration of the Ir(III) complex (µM)
Fig. 9. Hela cell viability values (%) estimated by MTT proliferation test vs. incubation
concentrations of [Ir(nbt)2(bp-DPA)]PF6. Cells were cultured in the presence of 0–20 µM
[Ir(nbt)2(bp-DPA)]PF6 at 37 oC for 24 h.
Luminescence
Overlay
Bright-field
a
b
e
c
f
d
Fig. 10. Confocal luminescence (a and d), bright field (b and e) and overly images (c and f) of
o
Hela cells. Hela cells were incubated with [Ir(nbt)2(bp-DPA)]PF6 (20 µM) for 30 min at 37 C
(a–c), and subsequently treated with Cu2+ (100 µM) for another 30 min at 37 oC (d–f).
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4. Conclusion
To summarize, we have presented a new cationic Ir(III) complex as phosphorescent sensor
for quantitating Cu2+ ion. The Ir(III) complex exhibited a rapid turn-off phosphorescent response
toward Cu2+ ion and displayed excellent reversibility and selectivity for Cu2+ ion in MeOH/H2O
(1:1) solution with a broad pH range of 2–10. The detection limit and the association constant
were determined to be 7.6 × 10-8 M and 0.31 × 105 M−1 for Cu2+, respectively. Although the Ir(III)
complex had some cytotoxicity to Hela cells, it exhibited well cell-permeable and good imaging
characteristics for the detection of Cu2+ ion in living cells.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant
51563014).
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Highlights:
1. A cationic Ir(III) complex was synthesized for detection of Cu2+ in vitro and cells.
2. The detection limit for Cu2+ was measured to be 10-8 M.
3. The complex has well cell-permeable and imaging characteristics for Cu2+ in cells.