A ratiometric luminescence probe for highly reactive oxygen species based on lanthanide complexes

Article abstract of DOI:10.1021/ic202195a

Reactive oxygen species (ROS) are important mediators in a variety of pathological events, but the oxidative stress owing to excessive generation of ROS is implicated in many human diseases. In this work, we designed and synthesized a novel dual-functional chelating ligand, [4′-(p-aminophenoxy) methylene-2,2′:6′,2″-terpyridine-6,6″-diyl] bis(methylenenitrilo)tetrakis(acetic acid) (AMTTA), that can strongly coordinate with both Eu³⁺ and Tb³⁺ in aqueous solutions for the recognition and time-gated luminescence detection of highly ROS (hROS), hydroxyl radical (?OH), and hypochlorite (ClO^-). The complexes AMTTA-Ln³⁺ (Ln = Eu and Tb) are almost nonluminescent because of the photoinduced electron transfer from the electron-rich aminophenyl group to the terpyridine-Ln³⁺ moiety but can rapidly react with hROS to afford highly luminescent complexes (4′-hydroxymethyl-2,2′:6′, 2″-terpyridine-6,6″-diyl)bis(methylenenitrilo)tetrakis(acetate) -Ln³⁺ (HTTA-Ln³⁺). Interestingly, when the AMTTA-Eu ³⁺/Tb³⁺ mixture (AMTTA/Eu³⁺/Tb³⁺ = 2/1/1) was reacted with hROS, the intensity ratio of its Tb³⁺ emission at 540 nm to its Eu³⁺ emission at 610 nm, I ₅₄₀/I₆₁₀, showed a ratiometric response toward hROS, and the dose-dependent increase of the ratio displayed a double-exponential correlation to the concentration of hROS. This unique luminescence response allowed the AMTTA-Eu³⁺/Tb³⁺ mixture to be used as a ratiometric probe for the time-gated luminescence detection of hROS.

Full text of DOI:10.1021/ic202195a

Article
pubs.acs.org/IC
A Ratiometric Luminescence Probe for Highly Reactive Oxygen
Species Based on Lanthanide Complexes
Yunna Xiao, Zhiqiang Ye,* Guilan Wang, and Jingli Yuan*
State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, People's Republic of
China
S
* Supporting Information
ABSTRACT: Reactive oxygen species (ROS) are important mediators
in a variety of pathological events, but the oxidative stress owing to
excessive generation of ROS is implicated in many human diseases. In
this work, we designed and synthesized a novel dual-functional chelating
ligand, [4′-(p-aminophenoxy)methylene-2,2′:6′,2″-terpyridine-6,6″-diyl]-
bis(methylenenitrilo)tetrakis(acetic acid) (AMTTA), that can strongly
coordinate with both Eu3+ and Tb3+ in aqueous solutions for the
recognition and time-gated luminescence detection of highly ROS
(hROS), hydroxyl radical (•OH), and hypochlorite (ClO−). The
complexes AMTTA-Ln3+ (Ln = Eu and Tb) are almost nonluminescent
because of the photoinduced electron transfer from the electron-rich
aminophenyl group to the terpyridine-Ln3+ moiety but can rapidly react
with hROS to afford highly luminescent complexes (4′-hydroxymethyl-
2,2′:6′,2″-terpyridine-6,6″-diyl)bis(methylenenitrilo)tetrakis(acetate)-
Ln3+ (HTTA-Ln3+). Interestingly, when the AMTTA-Eu3+/Tb3+ mixture (AMTTA/Eu3+/Tb3+ = 2/1/1) was reacted with hROS,
the intensity ratio of its Tb3+ emission at 540 nm to its Eu3+ emission at 610 nm, I540/I610, showed a ratiometric response toward
hROS, and the dose-dependent increase of the ratio displayed a double-exponential correlation to the concentration of hROS.
This unique luminescence response allowed the AMTTA-Eu3+/Tb3+ mixture to be used as a ratiometric probe for the time-gated
luminescence detection of hROS.
distinguish a specific ROS. To solve this problem, many efforts
have been motivated to develop fluorescent probes that can
distinguish an individual species of ROS in recent years.14
INTRODUCTION
■
Reactive oxygen species (ROS), including alkylperoxyl radicals
(ROO•), superoxide anion radical (O2•−), hydrogen peroxide
(H2O2), singlet oxygen (1O2), hypochlorite (ClO−), hydroxyl
radical (•OH), and so on, are collective terms representing the
chemical species that are formed from the incomplete reduction
of oxygen.1−3 It has been known that ROS are important
signaling molecules in biological systems to regulate a wide
range of physiological functions, but their overproduction can
result in oxidative stresses, which are involved in the
pathogenesis of many diseases, such as carcinogenesis,4
inflammation,5 ischemia-reperfusion injury,6 cardiovascular
disease, and neurological disorders.7−10 Therefore, detection
methods for ROS could be powerful tools to elucidate the
molecular mechanisms that underlie such physiological and
pathological conditions.11 In various methods, fluorescence
analysis in combination with a ROS-responsive luminescence
probe is generally considered to be one of the most promising
methods because of its high sensitivity, selectivity, experimental
feasibility, and convenience in data collection.12
•
Among various ROS, ClO− and OH are considered to be
highly ROS (hROS) because of their strong oxidant property
that can directly oxidize DNA-duplex, proteins, and lipids.15
Thus, the development of fluorescent probes for detecting
hROS in vivo and in vitro remains an active and challenging
work. To this end, several organic dye-based fluorescence
probes that can selectively respond to hROS, such as 2-[6-(4′-
hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid and 2-[6-
(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid15 and
MitoAR and MitoHR,16 have been developed and used for
imaging hROS production in living cells.
In most cases, the responses of fluorescent probes to analytes
are based on the increase or decrease of the fluorescence
intensities of the probes, which have an unavoidable critical
problem that the signal intensity could be influenced by many
factors, e.g., excitation intensity, probe concentration, sample
environment, etc. In contrast to the intensity-based probes, a
ratiometric fluorescence probe can get rid of most or all of
these impacts by the self-calibration of two emission bands at
To date, a variety of fluorescent probes for the detection of
ROS have been developed. The generally used probes, such as
2,7-dichlorodihydrofluorescein13a and dihydrorhodamine
123,13b can be oxidized by various ROS to afford strong
luminescence signals, but they lack the recognition selectivity to
Received: October 11, 2011
Published: February 22, 2012
© 2012 American Chemical Society
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a
Scheme 1. Luminescence Response Reaction of the AMTTA-Eu3+/Tb3+ Mixture toward hROS
a
The photographs show the luminescence colors of the complex solutions under a 365 nm UV lamp.
system from ferrous ammonium sulfate and H2O2.22 Peroxynitrite
(NaONOO) was synthesized from sodium nitrite (0.6 M) and H2O2
(0.65 M) in a quenched-flow reactor (excess H2O2 was used to
minimize nitrite contamination).23 After the reaction, the solution was
treated with MnO2 to eliminate the excess H2O2. The concentration of
the NaONOO stock solution was determined by measuring the
absorbance at 302 nm with a molar extinction coefficient of 1670 cm−1
M−1.24 Singlet oxygen (1O2) was generated from the Na2MoO4/H2O2
system in 0.05 M carbonate buffer of pH 10.5.25 Superoxide anion
radical (O2•−) was generated from the xanthine−xanthine oxidase
system.26 A stock solution of ClO− was prepared from a commercial
sodium hypochlorite solution. Unless otherwise specified, all chemical
materials were purchased from commercial sources and used without
further purification.
different wavelengths to increase the accuracy of the
fluorescence measurement.17 Furthermore, the fluorescence
measurement for a complicated biological sample is also
profoundly interfered by the strong autofluorescence from the
sample. On the basis of the above opinions, we have attempted
to develop ratiometric luminescence probes based on the
luminescent lanthanide complexes because their sharp and
long-lived emissions enable them to be ideal probes for time-
gated luminescence detection to discriminate the background
noises, and the dilanthanide ensemble consisting of a mixture of
Eu3+/Tb3+ complexes can be easily used to generate ratiometric
luminescence probes.18
In this work, a novel dual-functional chelating ligand, [4′-(p-
aminophenoxy)methylene-2,2′:6′,2″-terpyridine-6,6″-diyl]bis-
(methylenenitrilo)tetrakis(acetic acid) (AMTTA), that can
strongly coordinate with both Eu3+ and Tb3+ and specifically
react with hROS in aqueous solutions was designed and
synthesized. Its complexes with Ln3+ ions, AMTTA-Ln3+ (Ln =
Eu and Tb), are highly stable and water-soluble and almost
nonluminescent because of photoinduced electron transfer
(PET) from the electron-rich p-aminophenyl group to the
terpyridine-Ln3+ moiety. However, upon reaction with hROS,
due to cleavage of the p-aminophenyl ether in the complex, the
highly luminescent complexes (4′-hydroxymethyl-2,2′:6′,2″-
terpyridine-6,6″-diyl)bis(methylenenitrilo)tetrakis(acetate)-
Ln3+ (HTTA-Ln3+) can be generated. It was found that the
AMTTA-Eu3+/Tb3+ mixture (AMTTA/Eu3+/Tb3+ = 2/1/1)
showed a ratiometric luminescence response to hROS. The
intensity ratio of its Tb3+ emission at 540 nm to its Eu3+
emission at 610 nm, I540/I610, could be remarkably augmented
upon reaction with hROS. Thus, a ratiometric and time-gated
luminescence detection method for hROS using the AMTTA-
Eu3+/Tb3+ mixture as a probe was developed. Scheme 1 shows
the structures of AMTTA-Eu3+/Tb3+ and HTTA-Eu3+/Tb3+
and the luminescence response reaction of AMTTA-Eu3+/Tb3+
toward hROS.
1H and 13C NMR spectra were recorded on a Bruker Avance
1
spectrometer (400 MHz for H and 100 MHz for 13C). Electrospray
ionization mass spectrometry (ESI-MS) spectra were measured on a
HP1100LC/MSD electrospray ionization mass spectrometer. Ele-
mental analysis was carried out on a Vario-EL CHN analyzer.
Absorption spectra were measured on a Perkin-Elmer Lambda 35
UV−vis spectrometer. All time-gated luminescence spectra and
luminescence properties were measured on a Perkin-Elmer LS 50B
luminescence spectrometer with the following settings: delay time, 0.2
ms; gate time, 0.4 ms; cycle time, 20 ms; excitation slit, 10 nm;
emission slit, 5 nm. The luminescence quantum yields of AMTTA-
Ln3+ and HTTA-Ln3+ were measured by using the previous
methods.18c,27
Syntheses of AMTTA and HTTA. The reaction pathways for the
synthesis of AMTTA and HTTA are shown in Scheme S1 in the
Supporting Information, and the details are described as follows.
4′-Hydroxymethyl-6,6″-bis(bromomethyl)-2,2′:6′,2″-terpyri-
dine (2). LiAlH4 (183.1 mg, 4.82 mmol) was added slowly into a
solution of compound 1 (1.05 g, 2.21 mmol) in dry THF (80 mL) at 0
°C, and the suspension was stirred for 2.5 h in an ice−water bath. After
the precipitate was removed by filtration and washed three times with
dry THF, the filtrate was evaporated. The residue was purified by silica
gel column chromatography with CH2Cl2/CH3OH (100/1, v/v) as
the eluent to afford compound 2 as a white solid (246 mg, 25% yield).
1H NMR (400 MHz, CDCl3): δ 4.66 (s, 4H), 4.97 (s, 2H), 7.51 (d, J =
8.0 Hz, 2H), 7.86 (t, J = 7.8 Hz, 2H), 8.49 (d, J = 7.8 Hz, 2H), 8.56 (s,
2H). 13C NMR (100 MHz, CDCl3): δ 34.02, 63.85, 118.95, 120.59,
123.74, 137.98, 153.72, 156.29, 156.82, 158.76. ESI-MS (m/z): 472.0
([M + Na]+, 100%), 450.0 ([M + H]+, 20%).
Tetraethyl (4′-Hydroxymethyl-2,2′:6′,2″-terpyridine-6,6″-
diyl)bis(methylenenitrilo)tetrakis(acetate) (3). Diethyl iminodia-
cetate (356.8 mg, 3.49 mmol) and NaH (110 mg, 4.58 mmol) were
added into dry acetonitrile (20 mL) under an argon atmosphere. After
the suspension was stirred for 1 h, a solution of compound 2 (680 mg,
1.52 mmol) in dry THF (15 mL) was poured into the above
suspension. The mixture was further stirred overnight under an argon
atmosphere. After the precipitate was removed by filtration and
washed three times with dry THF, the filtrate was evaporated, and the
residue was purified by silica gel column chromatography with
EXPERIMENTAL SECTION
■
Materials and Physical Measurements. Ethyl 6,6″-bis-
(bromomethyl)-2,2′:6′,2″-terpyridine-4′-carboxylate (compound 1)
was synthesized according to a previously reported method.19
Tetrahydrofuran (THF) and acetonitrile were used after appropriate
distillation and purification. 1-Hydroxy-2-oxo-3-(3-aminopropyl)-3-
methyl-1-triazene (NOC-13, a NO donor with a half-life of 13.7 min)
was synthesized using a reported method.20 Hydrogen peroxide
(H2O2) was diluted immediately from a stabilized 30% solution and
was assayed by using its molar absorption coefficient of 43.6 cm−1 M−1
at 240 nm.21 Hydroxyl radical (•OH) was generated in the Fenton
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petroleum ether/ethyl acetate (1/1, v/v) as the eluent to afford
compound 3 as yellowish crystals (560 mg, 56% yield). 1H NMR (400
MHz, CDCl3): δ 1.24 (t, J = 6.0 Hz,12H), 3.68 (s, 8H), 4.14−4.21 (m,
12H), 4.87 (s, 2H), 7.60 (d, J = 8.0 Hz, 2H), 7.82 (t, J = 7.8 Hz, 2H),
8.41 (s, 2H), 8.43 (d, J = 7.8 Hz, 2H). 13C NMR (100 MHz, CDCl3):
δ 14.21, 55.09, 59.46, 60.72, 61.56, 119.47, 120.46, 123.87, 138.80,
153.52, 153.61, 157.84, 169.42, 171.15. ESI-MS (m/z): 666.3 ([M +
H]+, 100%), 688.3 ([M + Na]+, 45%).
8.51 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 54.80,
59.75, 62.42, 118.52, 119.86, 123.68, 138.44, 154.51, 154.91, 155.11,
158.92, 172.78. Elem anal. Calcd for C26H27N5O9·H2O (HTTA·H2O):
C, 54.64; H, 5.11; N, 12.25. Found: C, 54.93; H, 4.86; N, 11.90. ESI-
MS (m/z): 552.2 ([M − H]−, 100%).
Syntheses of the Ln3+ Complexes. Stock solutions of AMTTA-
Ln3+ and HTTA-Ln3+ (Ln = Eu and Tb) complexes were prepared by
in situ mixing equivalent molar of the ligand (0.01 mmol) and
LnCl3·6H2O (0.01 mmol) in 5.0 mL of 50 mM HEPES buffer of pH
7.2. Stock solutions of the AMTTA-Eu3+/Tb3+ and HTTA-Eu3+/Tb3+
mixtures (ligand/Eu3+/Tb3+ = 2/1/1) were prepared by adding the
ligand (0.01 mmol), TbCl3·6H2O (0.005 mmol), and EuCl3·6H2O
(0.005 mmol) into 5.0 mL of 50 mM HEPES buffer of pH 7.2. All
stock solutions were stored at room temperature and suitably diluted
with aqueous buffers before use. ESI-MS (m/z) for AMTTA-Eu3+:
817.0 ([M + Na]+, 100%), 795.1 ([M + H]+, 70%). ESI-MS (m/z) for
AMTTA-Tb3+: 823.3 ([M + Na]+, 100%), 801.2 ([M + H]+, 70%).
ESI-MS (m/z) for HTTA-Eu3+: 724.6 ([M − 2H + Na]−, 100%),
702.6 ([M − H]−, 70%). ESI-MS (m/z) for HTTA-Tb3+: 730.6 ([M −
2H + Na]−, 100%), 708.6 ([M − H]−, 70%).
Tetraethyl (4′-Bromomethyl-2,2′:6′,2″-terpyridine-6,6″-
diyl)bis(methylenenitrilo)tetrakis(acetate) (4). To a solution of
compound 3 (550 mg, 0.83 mmol) in 15 mL of dry THF was added
dropwise PBr3 (268.2 mg, 1.00 mmol) with stirring. After the solution
was stirred for 2 h at room temperature, 150 mL of CHCl3 was added.
The solution was washed three times with 100 mL of water and dried
with Na2SO4. The solvent was evaporated, and then the residue was
purified by silica gel column chromatography with petroleum ether/
ethyl acetate (2/1, v/v) as the eluent to afford compound 4 as a white
1
oil (390 mg, 65% yield). H NMR (400 MHz, CDCl3): δ 1.26 (t, J =
6.0 Hz, 12H), 3.71 (s, 8H), 4.17−4.22 (m, 12H), 4.59 (s, 2H), 7.66 (d,
J = 8.0 Hz, 2H), 7.85 (t, J = 7.6 Hz, 2H), 8.49 (s, 2H), 8.51 (s, 2H).
13C NMR (100 MHz, CDCl3): δ 14.24, 55.03, 59.67, 60.73, 61.71,
119.89, 120.50, 123.69, 138.26, 155.47, 155.29, 157.79, 167.68, 171.00.
ESI-MS (m/z): 728.3/730.3 ([M + H]+, 100%), 750.3/752.3 ([M +
Na]+, 90%).
Ratiometric Luminescence Detection of hROS in Aqueous
Media. The reactions of the AMTTA-Eu3+/Tb3+ mixture (total
concentration of 20 μM; Eu3+/Tb3+ = 1/1) with different
concentrations of ClO− and •OH were carried out in 50 mM
HEPES buffer of pH 7.2. The ClO− solutions with different
concentrations were prepared by dilution of a stock solution of
sodium hypochlorite and directly added into the AMTTA-Eu3+/Tb3+
solution. As for •OH luminescence detection, different concentrations
Tetraethyl [4′-(p-Aminophenoxy)methylene-2,2′:6′,2″-ter-
pyridine-6,6″-diyl]bis(methylenenitrilo)tetrakis(acetate) (5).
After a mixture of p-aminophenol (197.9 mg, 1.82 mmol) and NaH
(44 mg, 1.82 mmol) in 20 mL of dry acetonitrile was stirred at room
temperature for 15 min under an argon atmosphere, compound 4 (440
mg, 0.61 mmol) was added. The suspension was further stirred
overnight under an argon atmosphere. After filtration, the solvent was
evaporated, and the residue was purified by silica gel column
chromatography with CH2Cl2/CH3OH (40/1, v/v) as the eluent to
afford compound 5 as a yellow oil (230 mg, 50% yield). 1H NMR (400
MHz, CDCl3): δ 1.23 (t, J = 6.0 Hz, 12H,), 3.69 (s, 8H), 4.15−4.20
(m, 12H), 5.15 (s, 2H), 6.65 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.8 Hz,
2H), 7.62 (d, J = 8.0 Hz, 2H), 7.82 (t, J = 7.6 Hz, 2H), 8.47 (s, 2H),
8.50 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 14.08, 55.01, 59.40,
60.70, 61.89, 116.19, 116.40, 118.89, 120.04, 120.95, 124.10, 138.89,
149.01, 154.56, 155.16, 159.85, 168.96, 172.85. ESI-MS (m/z): 757.3
([M + H]+, 100%), 779.2 ([M + Na]+, 80%).
•
of OH were in situ generated by adding Fe2+ (60 μM) and different
concentrations of H2O2 into the AMTTA-Eu3+/Tb3+ solution. All of
the above reaction solutions were stirred at room temperature for 30
min and then subjected to time-gated luminescence measurement on a
Perkin-Elmer LS 50B luminescence spectrometer.
Reactions of AMTTA-Ln3+ with Different ROS. The reactions of
AMTTA-Ln3+ (10 μM AMTTA-Eu3+, 10 μM AMTTA-Tb3+, or a
mixture of 10 μM AMTTA-Eu3+ and 10 μM AMTTA-Tb3+) with
ClO− (15 μM NaClO), •OH (80 μM Fe2+ + 80 μM H2O2), NO (100
−
μM NOC-13), H2O2 (100 μM), NO2 (100 μM NaNO2), ONOO−
(100 μM NaONOO), and O2·− (100 μM xanthine + 100 μM xanthine
oxidase) were carried out in 50 mM HEPES buffer of pH 7.2. The
1
reaction with O2 (1.0 mM Na2MoO4 + 100 μM H2O2) was carried
out in 50 mM carbonate buffer of pH 10.5. All of the solutions were
stirred at room temperature for 30 min and then subjected to time-
gated luminescence measurement on a Perkin-Elmer LS 50B
luminescence spectrometer.
AMTTA. A mixture of compound 5 (230 mg, 0.31 mmol), KOH
(0.45 g, 8.04 mmol), 1.13 mL of water, and 13 mL of ethanol was
stirred at room temperature for 20 h. After evaporation, the residue
was dissolved in 3 mL of water, and the pH of the solution was
adjusted to ∼3 with HCl (3 M). The suspension was stirred for a
further 20 h at room temperature, and then the precipitate was
collected by filtration. After drying, the precipitate was added to 30 mL
of dry acetonitrile, and the mixture was refluxed for 30 min. The
precipitate was filtered and dried to afford AMTTA as a light-brown
solid (122 mg, 61% yield). 1H NMR (400 MHz, DMSO-d6): δ 3.56 (s,
8H), 4.10 (s, 4H), 5.30 (s, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.98 (d, J =
8.8 Hz, 2H), 7.65 (d, J = 7.6 Hz, 2H), 7.99 (t, J = 7.6 Hz, 2H), 8.47 (s,
2H), 8.50 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ
54.82, 59.72, 68.92, 116.21, 116.41, 118.95, 119.82, 120.45, 123.80,
138.44, 149.32, 154.07, 154.63, 155.55, 159.25, 172.85. Elem anal.
Calcd for C32H32N6O9·2.5H2O (AMTTA·2.5H2O): C, 55.73; H, 5.41;
N, 12.19. Found: C, 55.50; H, 5.05; N, 11.94. ESI-MS (m/z): 643.2
([M − H]−, 100%), 321.1 ([M − 2H]2−, 80%).
RESULTS AND DISCUSSION
■
Design and Photophysical Properties of the Ln3+
Complexes. To design a ratiometric fluorescence probe, it is
vital that two emissions of the probe at different wavelengths
should originate from the same excited state, allowing the
emission intensity ratio to be independent of the concentration,
photobleaching, and power fluctuation of the excitation source.
Recently, a robust approach using a heterometallic bis-
(lanthanide) ensemble for the design of ratiometric lumines-
cence probes has been reported, wherein a mixture of Eu3+ and
Tb3+ complexes outfitted with the same antenna ligand to
display both the Eu3+ and Tb3+ emissions having different
responses to the analyte could be combined.28 In this work, we
designed and synthesized a novel dual-functional ligand,
AMTTA, by incorporating an electron-rich group, p-amino-
phenyl, into a strongly coordinating antenna, (2,2′:6′,2″-
terpyridine-6,6″-diyl)bis(methylenenitrilo)tetrakis(acetic acid),
capable of sensitizing the luminescence of both Eu3+ and Tb3+
ions.29 The p-aminophenyl group in the AMTTA-Ln3+ complex
has two functions. One is to quench the Ln3+ luminescence via
a PET process to make the Ln3+ luminescence be turned-off,
HTTA. A mixture of compound 3 (99 mg, 0.15 mmol), KOH (0.19
g, 3.39 mmol), 0.52 mL of water, and 6.3 mL of ethanol was stirred at
room temperature for 20 h. After evaporation, the residue was
dissolved in 2 mL of water, and the pH of the solution was adjusted to
∼3 with HCl (3 M). The suspension was stirred for a further 20 h at
room temperature, and then the precipitate was collected by filtration.
After drying, the precipitate was added to 30 mL of dry acetonitrile,
and the mixture was refluxed for 30 min. The precipitate was filtered
1
and dried to afford HTTA as a white solid (43 mg, 51% yield). H
NMR (400 MHz, DMSO-d6): δ 3.63 (s, 8H), 4.16 (s, 4H), 4.73 (s,
2H), 7.65 (d, J = 7.6 Hz, 2H), 8.00 (t, J = 7.6 Hz, 2H), 8.40 (s, 2H),
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Figure 1. (A) Time-gated excitation and emission spectra of AMTTA-Ln3+ (3.5 μM, red line for AMTTA-Eu3+, green line for AMTTA-Tb3+) and
HTTA-Ln3+ (3.5 μM, blue line for HTTA-Eu3+, purple line for HTTA-Tb3+) in 50 mM borate buffer of pH 9.1. (B) Emission spectra of the
AMTTA-Eu3+/Tb3+ mixture (total concentration of 7.0 μM; Eu3+/Tb3+ = 1/1; red line) and HTTA-Eu3+/Tb3+ mixture (total concentration of 7.0
μM; Eu3+/Tb3+ = 1/1; black line) in 50 mM borate buffer of pH 9.1.
and the other is to enable the complex to selectively react with
hROS to cleave the p-aminophenyl ether from the ligand. Thus,
the weakly luminescent AMTTA-Eu3+/Tb3+ mixture is
expected to serve as a ratiometric luminescence probe for
hROS because the mixture could be considered to display
different responses at Eu3+ and Tb3+ emissions toward hROS
based on the different emission efficiencies of the cleavage
reaction products, HTTA-Eu3+ and HTTA-Tb3+.29
HTTA-Eu3+/Tb3+ mixture. These results indicate that the
luminescence response sensitivity of AMTTA-Tb3+ at 540 nm
is remarkably higher than that of AMTTA-Eu3+ at 610 nm
toward hROS, suggesting that the AMTTA-Eu3+/Tb3+ mixture
could be used as a ratiometric probe for the time-gated
luminescence detection of hROS with the I540/I610 ratio as a
signal.
Time-Gated Luminescence Detection of hROS Using
AMTTA-Eu3+/Tb3+ as a Ratiometric Probe. Quantitative
The photophysical properties of AMTTA-Eu3+, AMTTA-
Tb3+, HTTA-Eu3+, and HTTA-Tb3+ were measured in 50 mM
borate buffer of pH 9.1. As shown in Figure 1A, the two
AMTTA-Ln3+ complexes are weakly luminescent with very low
quantum yields (ε331 nm = 1.37 × 104 cm−1 M−1, ϕ = 0.59%, and
τ = 1.02 ms for AMTTA-Eu3+; ε331 nm = 1.35 × 104 cm−1 M−1,
ϕ = 0.19%, and τ = 0.39 ms for AMTTA-Tb3+). However, after
reacting with hROS to afford HTTA-Ln3+, the complexes
become highly luminescent with remarkable increases of their
quantum yields and luminescence lifetimes (ε331 nm = 1.36 ×
104 cm−1 M−1, ϕ = 16.3%, and τ = 1.38 ms for HTTA-Eu3+;
ε331 nm = 1.38 × 104 cm−1 M−1, ϕ = 11.5%, and τ = 1.22 ms for
HTTA-Tb3+). The product of AMTTA-Tb3+ reacting with
ClO− was confirmed by time-of-flight ESI-MS spectrum
(Figure S1 in the Supporting Information), in which the base
peak at m/z 708.0745 could be assigned to be the molecular ion
peak of HTTA-Tb3+. All of the Ln3+ complexes are highly stable
in aqueous media because of the nonadentate terpyridine−
polyacid structure of the ligand.18c,27
•
time-gated luminescence detections of OH and ClO− using
the AMTTA-Eu3+/Tb3+ mixture (10 μM AMTTA-Eu3+ + 10
μM AMTTA-Tb3+) as a ratiometric probe were carried out in
•
50 mM HEPES buffer of pH 7.2. Because OH was in situ
generated by the Fenton reaction, before •OH detection, it was
confirmed that the probe could not react with H2O2 or Fe2+ by
reacting the AMTTA-Eu3+/Tb3+ mixture with H2O2 or Fe2+
alone (no time-gated luminescence response was observed).
However, in the presence of Fe2+, the intensities of both Eu3+
and Tb3+ emissions of the probe were significantly increased
upon the addition of H2O2. These results clearly indicate that
the luminescence enhancement of the probe was caused by
•OH and not by H2O2 or Fe2+ species.
Figure 2 shows the time-gated emission spectra of the probe
in the presence of Fe2+ (60 μM) and different concentrations of
H2O2 in the buffer. Although the intensities of the probe at
Eu3+ and Tb3+ emissions were simultaneously increased because
of the different luminescence response behaviors of AMTTA-
•
Eu3+ and AMTTA-Tb3+ to OH, the reaction between the
The time-gated emission spectra of mixtures of AMTTA-
Eu3+/Tb3+ and HTTA-Eu3+/Tb3+ (ligand/Eu3+/Tb3+ = 2/1/1)
in 50 mM borate buffer of pH 9.1 are shown in Figure 1B.
Because both of the Eu3+ and Tb3+ complexes were outfitted
with the same antenna chromophore, the “cocktails” of the
complexes showed simultaneously Eu3+ emission at 610 nm
•
probe and OH gave rise to an increase of the I540/I610 ratio
from 0.18 to 1.4 to provide a comfortable 7.8-fold contrast
•
window in which to detect OH radicals. The inset curve in
Figure 2, fitted to a double-exponential correlation, shows the
variation of the I540/I610 ratio as a function of the H2O2
concentration, in which a good linear correlation between the
I540/I610 ratio and H2O2 concentration (Figure S2 in the
Supporting Information) can be fitted in the lower concen-
tration range of H2O2 (5.0−30 μM, which can be considered to
7
7
(5D0 → F2) and Tb3+ emission at 540 nm (5D4 → F5) when
excited at the same wavelength (maximum at 331 nm). In fact,
when the AMTTA-Eu3+/Tb3+ mixture was reacted with hROS,
the emission color of the weakly luminescent solution turned
strongly yellow (Scheme 1), the mixed color of green emission
from HTTA-Tb3+ and red emission from HTTA-Eu3+. In
addition, Figure 1B also shows that the intensity of Eu3+
emission (610 nm) is ∼5-fold higher than that of Tb3+
emission (540 nm) in the spectrum of the AMTTA-Eu3+/
Tb3+ mixture, while the intensity of Eu3+ emission (610 nm) is
only half that of Tb3+ emission (540 nm) in the spectrum of the
•
correspond to the concentration range of OH because one
H2O2 molecule can generate one •OH in the presence of excess
Fe2+).30 This result demonstrated the feasibility of the
ratiometric probe for the quantitative time-gated luminescence
•
detection of OH radicals.
Figure 3 shows the time-gated emission spectra of the probe
in the presence of different concentrations of ClO− in the
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shown in Figure 4, the luminescence intensity of AMTTA-Tb3+
itself was very weak and stable but could be increased
Figure 2. Time-gated emission spectra (λex = 331 nm) of the
AMTTA-Eu3+/Tb3+ mixture (total concentration of 20 μM, Tb3+/Eu3+
= 1/1) in the presence of Fe2+ (60 μM) and different concentrations
of H2O2 (0.0, 2.0, 4.0, 6.0, 8.0, 10, 12, 14, 16, 18, 20, 30, 40, 60, 80,
100, and 200 μM) in 50 mM HEPES buffer of pH 7.2. The inset
shows the intensity ratio of I540/I610 as a function of the H2O2
concentration.
Figure 4. Time course of the luminescence response of AMTTA-Tb3+
(10 μM) to the addition of ClO− in 50 mM HEPES buffer of pH 7.2
at room temperature (the 3.0 μM ClO− solution was added six times
at a, b, c, d, e, and f, respectively). The luminescence intensity at 540
nm was determined (λex = 331 nm).
immediately to reach the maximum value and then kept at a
steady level for a long time upon the addition of ClO−. This
result indicates that the reaction between AMTTA-Ln3+ and
ClO− is very fast, which is favorable for the detection of ClO−
in complicated samples to avoid the effects of other undesirable
reactions.
Effects of the pH and ROS. The effects of the pH on the
I
540/I610 ratios of the AMTTA-Eu3+/Tb3+ (5.0 μM AMTTA-
Eu3+ + 5.0 μM AMTTA-Tb3+) and HTTA-Eu3+/Tb3+ (5.0 μM
HTTA-Eu3+ + 5.0 μM HTTA-Tb3+) mixtures were investigated
in 50 mM Tris-HCl buffers with different pHs ranging from 4
to 10. As shown in Figure 5, the I540/I610 ratios of the AMTTA-
Figure 3. Time-gated emission spectra (λex = 331 nm) of the
AMTTA-Eu3+/Tb3+ mixture (total concentration of 20 μM; Eu3+/Tb3+
= 1/1) in the presence of different concentrations of ClO− (0.0, 0.25,
0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 15, 20, 30, and 40 μM)
in 50 mM HEPES buffer of pH 7.2. The inset shows the intensity ratio
of I540/I610 as a function of the ClO− concentration.
buffer. Compared to the response to •OH, the probe showed a
higher sensitivity to respond to ClO− with a faster increase of
its I540/I610 ratio at low concentrations of ClO− (the I540/I610
ratio reached to the maximum value upon the addition of 10
μM ClO−), and the variation of the I540/I610 ratio against the
ClO− concentration (the inset in Figure 3), fitted also to a
double-exponential correlation, provided a broader contrast
window from 0.18 to 2.23 (12.3-fold) for the detection of
ClO−. We also investigated the luminescent responses of
AMTTA-Eu3+ (10 μM) and AMTTA-Tb3+ (10 μM) toward
ClO−, respectively. As shown in Figure S3B in the Supporting
Information, after reaction with ClO−, the ratio of the Tb3+ and
Eu3+ emissions (I540/I610) increased from 0.19 to 2.16 (11.4-
fold), which is almost the same as that of the AMTTA-Eu3+/
Tb3+ mixture and indicates that the effect of the differential
metal displacement of the lanthanide complexes caused by
hROS on the ratiometric detection is small and can be
negligible. To examine the response kinetics of the probe
toward ClO−, the real-time luminescence responses of
AMTTA-Tb3+ (10 μM) to the repeat additions of ClO− (3.0
μM) in 50 mM HEPES buffer of pH 7.2 were determined. As
Figure 5. Effects of the pH on the I540/I610 ratios of AMTTA-Eu3+/
Tb3+ (a, total concentration of 10 μM, Eu3+/Tb3+ = 1/1) and HTTA-
Eu3+/Tb3+ (b, total concentration of 10 μM, Eu3+/Tb3+ = 1/1) in 50
mM Tris-HCl buffers with different pHs.
Eu3+/Tb3+ and HTTA-Eu3+/Tb3+ mixtures are not remarkably
affected by the pH value, which suggests that the AMTTA-
Eu3+/Tb3+ mixture could work well as a ratiometric probe for
the time-gated luminescence detection of hROS in weakly
acidic, neutral, and weakly basic buffers.
To evaluate the response specificity of the probe to hROS,
the reactions of AMTTA-Eu3+ (10 μM), AMTTA-Tb3+ (10
μM), and the AMTTA-Eu3+/Tb3+ mixture (10 μM AMTTA-
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Figure 6. (A) Luminescence intensities of the products of AMTTA-Tb3+ (10 μM, black column) and AMTTA-Eu3+ (10 μM, gray column) reacted
with various ROS in 50 mM HEPES buffer of pH 7.2. (B) I540/I610 ratios of the products of the AMTTA-Eu3+/Tb3+ mixture (total concentration of
20 μM, Tb3+/Eu3+ = 1/1) reacted with various ROS in 50 mM HEPES buffer of pH 7.2.
Eu3+ + 10 μM AMTTA-Tb3+) with various ROS were carried
out in 50 mM HEPES buffer of pH 7.2. As shown in Figure 6A,
both AMTTA-Eu3+ and AMTTA-Tb3+ almost did not give
ASSOCIATED CONTENT
* Supporting Information
Schematic pathways for the synthesis of AMTTA and HTTA
■
S
and three supplementary figures. This material is available free
of charge via the Internet at http://pubs.acs.org.
observable luminescence responses to the addition of excess
(100 μM) NO, H2O2, NO2 , ONOO−, O2•−, and 1O2, whereas
−
the luminescence intensities of AMTTA-Tb3+ and AMTTA-
Eu3+ were 233- and 19-fold increased upon the addition of
ClO− (15 μM) and 23- and 3.2-fold increased upon the
addition of •OH (80 μM). These results indicate that AMTTA-
AUTHOR INFORMATION
Corresponding Author
*E-mail: zhiqiangye2001@yahoo.com.cn (Z.Y.), jingliyuan@
yahoo.com.cn (J.Y.). Phone/Fax: +86-411-84986041.
■
•
Ln3+ cannot react with other ROS apart from OH and ClO−,
Notes
so that the AMTTA-Eu3+/Tb3+ mixture can be used as a
ratiometric probe for hROS with high specificity. Indeed, when
the AMTTA-Eu3+/Tb3+ mixture was reacted with various ROS,
the I540/I610 ratio of the mixture did not show remarkable
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the National Natural Science
Foundation of China (Grant 20835001) is gratefully acknowl-
edged.
responses to NO, H2O2, NO2 , ONOO−, O2•−, and O2 (the
change <0.1), while that was 7.8- and 12.3-fold increased after
−
1
reaction with OH and ClO−, respectively (Figure 6B).
•
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■
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CONCLUSION
■
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