A highly sensitive luminescent probe based on Ru(II)-bipyridine complex for Cu2+, L-Histidine detection and cellular imaging

Article abstract of DOI:10.1016/j.saa.2018.05.001

A ruthenium(II) bipyridyl complex conjugated with functionalized Schiff base (RuA) has been synthesized and functioned as a luminescent probe. The luminescence of RuA was greatly quenched by Cu²⁺ due to its molecular coordination with paramagnetic Cu²⁺. Subsequently, the addition of L-Histidine can turn on the luminescence of the RuA-Cu(II) ensemble, which can be attributed to the replacement of RuA in RuA-Cu(II) ensemble by L-Histidine. On the basis of the quenching and recovery of the luminescence of RuA, we proposed a rapid and highly sensitive on-off-on luminescent assay for sensing Cu²⁺ and L-Histidine in aqueous solution. Under the optimal conditions, Cu²⁺ and L-Histidine can be detected in the concentration range of 5 nM–9.0 μM and 50 nM–30 μM, respectively, and the corresponding detection limits were calculated to be 0.35 and 0.44 nM (S/N=3), separately. The proposed luminescent probe has been successfully utilized for the analysis of Cu²⁺ and L-Histidine in real samples (drinking water and biological fluids). Furthermore, the probe revealed good photostability, low cytotoxicity and excellent permeability, making it a suitable candidate for cell imaging and labeling in vitro.

Full text of DOI:10.1016/j.saa.2018.05.001

Accepted Manuscript
A highly sensitive luminescent probe based on Ru(II)-bipyridine
complex for Cu2+, l-histidine detection and cellular imaging
Shi-Ting Zhang, Panpan Li, Caiyun Liao, Tingting Luo,
Xingming Kou, Dan Xiao
PII:
DOI:
Reference:
S1386-1425(18)30391-3
doi:10.1016/j.saa.2018.05.001
SAA 16024
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 16 August 2017
Revised date:
Accepted
date:
29 April 2018
1 May 2018
Please cite this article as: Shi-Ting Zhang, Panpan Li, Caiyun Liao, Tingting Luo,
Xingming Kou, Dan Xiao , A highly sensitive luminescent probe based on Ru(II)-
bipyridine complex for Cu2+, l-histidine detection and cellular imaging. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Saa(2017), doi:10.1016/j.saa.2018.05.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
A highly sensitive luminescent probe based on Ru(II)-bipyridine
complex for Cu2+, L-Histidine detection and cellular imaging
Shi-Ting Zhanga, Panpan Lib, Caiyun Liaoa, Tingting Luoa, Xingming Kou*,a, Dan
Xiao*,a,c
aCollege of Chemistry, Sichuan University, Chengdu 610064, China.
bDepartment of Architecture and Environment, Sichuan University, Chengdu
610065, China
cCollege of Chemical Engineering, Sichuan University, Chengdu 610065, China.
*Corresponding Author: Xingming Kou E-mail: kouxm@scu.edu.cn; Dan Xiao E-mail:
xiaodan@scu.edu.cn.
1

ACCEPTED MANUSCRIPT
Abstract
A ruthenium(II) bipyridyl complex conjugated with functionalized Schiff base (RuA)
has been synthesized and functioned as a luminescent probe. The luminescence of RuA
was greatly quenched by Cu2+ due to its molecular coordination with paramagnetic Cu2+.
Subsequently, the addition of L-Histidine can turn on the luminescence of the RuA-Cu(II)
ensemble, which can be attributed to the replacement of RuA in RuA-Cu(II) ensemble by
L-Histidine. On the basis of the quenching and recovery of the luminescence of RuA, we
proposed a rapid and highly sensitive on-off-on luminescent assay for sensing Cu2+ and
L-Histidine in aqueous solution. Under the optimal conditions, Cu2+ and L-Histidine can
be detected in the concentration range of 5 nM−9.0 M and 50 nM−30 M, respectively,
and the corresponding detection limits were calculated to be 0.35 and 0.44 nM (S/N=3),
separately. The proposed luminescent probe has been successfully utilized for the analysis
of Cu2+ and L-Histidine in real samples (drinking water and biological fluids). Furthermore,
the probe revealed good photostability, low cytotoxicity and excellent permeability,
making it a suitable candidate for cell imaging and labeling in vitro.
Keywords
Luminescent detection; Cu2+; L-Histidine; Ruthenium(II) complex; Cell imaging
1. Introduction
As an essential element of the organism, copper is the third largest essential trace
element in the human body after iron and zinc [1,2]. Specially, Cu2+ plays a key role in
signal transduction, cellular energy generation, together with oxygen transport and
activation [3,4], because copper ions are the redoxcofactor of many mitochondria, cytosol
and vesicle oxidase coenzyme [5]. However, due to the uncontrolled reaction presented in
Cu2+ with oxygen and reactive oxygen species (ROS), it may also cause oxidative damage
to nucleic acids, proteins and lipids [6-9]. For example, some grievous neurodegenerative
2

ACCEPTED MANUSCRIPT
diseases, such as Wilson, Menkes, prion and Alzheimer's disease, are associated with the
change of homeostasis Cu2+ in intracellular [3,10-12].
In addition to the transition metal, we are also concerned about the physiological role
of amino acids. L-Histidine (L-His) is involved in numerous key biological activities in
the human body, including minimizing minimally invasive internal bleeding and
controlling the transport of metals in vital genes [13,14]. For example, L-His, as an
important neurotransmitter, is the metabolic disorder indicator of many diseases and
psychological disorders, such as the failure of normal erythropoietin and Parkinson's,
epilepsy disease, etc [15-18].
Therefore, the design and synthesis of probes for Cu2+ and L-His detection have
attracted much attention. As one of the important methods for testing Cu2+, luminescent
probe displays much clear superiority in selectivity and biological imaging due to its
sensitivity, specificity, easy operation and real-time detection with fast response [19-21].
As for L-His, luminiferous detection has received great attention for its temporal and
spatial resolution and high sensitivity [22,23]. Some metal indicator complexes (called
ensembles) have been considered as molecular luminescent probes for L-His [24-26].
In recent years, many probes have been reported for the detection of Cu2+. However,
many of them performed in acetonitrile, ethanol, methanol or other organic solvents
[27-30] and required prolonged reaction time [31,32], which limits their practical
applications especially in biological systems and further lead to lower detection efficiency.
To the best of our knowledge, due to their satisfied water solubility, intense polarized
luminescence, high chemical and optical stability, large Stokes shifts, relatively long
lifetimes and low-energy absorption [33,34], ruthenium(II) polypyridyl complexes have
turned into prospective probe for environmental monitoring and biological probing
[35-38].
Therefore, in this work, we have designed a Ru(II) polypyridyl complex-based probe
that can selectively detect Cu2+, and then the probe-Cu(II) complex acted as an ensemble
3

ACCEPTED MANUSCRIPT
to identify L-His. Specifically, the design strategy was based on the imino N and phenol O
atoms of RuA (the abbreviation of Ru(bpy)2(A)Cl2, where bpy = 2,2′-bipyridine, A
=N,N′-5,6-(1,10-phenanthroline)-bis(5-methoxylsalicylidene)diamine) can efficiently
combine Cu2+ centre (d9) resulting in the luminescence quenching [39-41]. In order to
further validate the sensing mechanism, we synthesized a series of compounds (RuB,
RuC and RuD) with the similar molecular structure and conducted comparative studies.
Moreover, the luminescent intensity of RuA-Cu(II) can be obviously enhanced by adding
L-His, which indicated that RuA-Cu(II) ensemble can be used as the luminescent probe
for L-His detection. Besides, the obtained probe exhibited many favorable properties,
such as red emission, 100% water solubility and biocompatibility, implying its potential
to be used in the bio-detection in vivo and in vitro. Consequently, RuA showed a
possibility to serve as an on-off-on luminescent switch probe to selectively, sensitively
and quickly detect Cu2+ and L-His in aqueous solution and biological fluids. Furthermore,
cell imaging experiments showed that RuA exhibited fine membrane permeability, very
low toxicity, and the capability of visualizing Cu2+ and L-His in living cells.
2. Experimental Section
2.1. Materials
1,10-phenanthroline-5,6-diamine was synthesized and purified according to our
previous literature [42]. 2-hydroxyl-5-methoxybenzaldehyde was purchased from Aladdin
Industrial Corporation (Shanghai, China). Cis-Bis-(2,2’-bipyridine) dichlororuthenium(II)
dihydrate (Ru(bpy)2Cl2·2H2O, 99%) was gained from Alfa Aesar (Johnson Matthey
Company, U.S.A.). Mice B16 melanoma cells were purchased from the Shanghai Institute
of Biochemistry and Cell Biology, Chinese Academy of Sciences. Roswell Park
Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) were obtained
from Life Technologies Corporation (Gibco, USA). A Cell-Counting Kit-8 (CCK-8) was
purchased from Dojindo Laboratories (Tokyo, Japan). Lyophilized human serum samples
were purchased from Boer Xi Science and Technology (China) Co., Ltd. Other chemical
4

ACCEPTED MANUSCRIPT
reagents and solvents were purchased from commercial chemical suppliers and used
without further purification.
2.2. Apparatus
Fluorescence spectra (FL) were measured on a Hitachi F-7000 fluorescence
spectrophotometer (Japan). UV-Vis absorption spectra were recorded on U-2900
1
spectrophotometer (HITACHI, Japan). H NMR was measured on a Bruker AV II-400
MHz spectrometer. FT-IR was performed on a Thermo Scientific Nicolet 6700 FT-IR
spectrometer (Sugar Land, TX, USA). Mass spectra of A was obtained from the MAT-261
spectrometer (Thermo Fisher Scientific, San Jose, CA). Matrix-assisted laser
desorption/ionization time of flight mass spectrums (MALDI-TOF-MS) of RuA was
obtained from a Bruker Ultrafle Xtreme matrix-assisted laser desorption/ionization time
of flight mass spectrometer (Germany). Fluorescence lifetime measurements were
acquired using a Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon) with a NanoLED
(454 nm, Horiba Scientific) as the excitation source and a picosecond photon detection
module (PPD-850, Horiba Scientific) as the detector. Confocal luminescence imaging
was performed by a confocal laser scanning microscopy (Nikon, N-SIM).
2.3. Synthesis
N,N’-5,6-(1,10-phenanthroline)-bis(5-methoxyl-salicylidene)diamine (A), RuA, RuB,
RuC and RuD were prepared by the following steps (Scheme 1, Scheme S1). Products
were characterized by 1H NMR, FT-IR, HRMS and elemental analysis (Fig. S15-23†).
2.3.1. Synthesis of A
A mixture of 1,10-phenanthroline-5,6-diamine (200 mg, 0.95 mmol) and
2-hydroxy-5-methoxybenzaldehyde (475 L, 3.8 mmol) was dissolved in ethanol (20 mL)
and a few drops of triethyl orthoformate were added. Then the mixture was stirred at
76 °C for 4 h under reflux in a nitrogen atmosphere. After cooling to room temperature,
the product was obtained via filtration, washed with diethyl ether and dried under vacuum.
1
Yield: 53.7%. H NMR (400 MHz, DMSO-d6) δ(ppm): δ 12.28 (s, 2H), 9.08 (s, 3H),
5

ACCEPTED MANUSCRIPT
8.98–8.87 (m, 3H), 7.95–7.74 (m, 5H), 7.05 (s, 4H), 3.86 (s, 5H). FT-IR (KBr): 3430.36
-1
-1
-1
cm−1 (OH), 1565.38 cm (C=N imines), 1492.49 cm (OCH3), 1223.26 cm and 1039.94
-1
-1
cm (=C-O-C-), 737.22 cm (benzene ring substitution). ESI-MS: calcd for C28H22O4N4
([M+Na]+): 501.502, found: 501.155. Elemental analysis: calcd (%) for C28H22O4N4
(478.512): C 70.28, H 4.63, N 11.71; found: C 69.55, H 4.92, N 12.25.
2.3.2. Synthesis of RuA
Ru(bpy)2Cl2·2H2O (30 mg, 0.057 mmol) and A (27.3 mg, 0.057 mmol) were dissolved
in dry ethanol (15 mL) under nitrogen. The solution was refluxed at 80 °C for 12 h under
nitrogen atmosphere. Then the deep red solution was cooled and the solvent was removed
by vacuum distillation. The crude residue was purified by column chromatography (SiO2,
CH2Cl2/CH3OH 4:1, v/v) and a red solid was obtained. Yield: 79.4%. 1H NMR (400 MHz,
DMSO-d6) δ(ppm): δ 8.90 (dd, J = 15.4, 8.2 Hz, 6H), 8.23 (td, J = 8.0, 1.3 Hz, 3H), 8.11
(td, J = 8.0, 1.3 Hz, 3H), 8.02 (d, J = 4.7 Hz, 3H), 7.92–7.83 (m, 5H), 7.67–7.56 (m, 5H),
7.40–7.33 (m, 3H), 7.19–7.12 (m, 1H), 6.98 (s, 2H), 6.76 (d, J = 7.5 Hz, 1H), 3.87 (s, 4H).
-1
-1
FT-IR (KBr): 3392.67 cm−1 (OH), 1598.59 cm (C=N imines), 1439.10 cm (OCH3),
-1
-1
-1
1239.25 cm and 1028.71 cm (=C-O-C-), 764.50 cm (benzene ring substitution).
ESI-MS: calcd for C48H38O4N8RuCl2 ([M-2Cl+H]3+): 890.950, found: 890.938.
([M-Cl+Na+2H]2+): 952.417, found: 952.895. Elemental analysis: calcd (%) for
C48H38O4N8RuCl2･6H2O (1070.960): C 53.83, H 4.71, N 10.46; found: C 54.17, H 4.42,
N 13.17.
2.3.3. Synthesis of RuB, RuC, RuD
The synthesis methods of RuB, RuC and RuD were similar to the RuA.
Compound RuB has been fully characterized and the results were shown in our
1
previous report [42]. Compound RuC H NMR (400 MHz, DMSO) δ(ppm): δ 9.29 (s,
2H), 8.90 (dd, J = 15.7, 8.2 Hz, 5H), 8.52 (d, J = 7.3 Hz, 2H), 8.22 (td, J = 8.0, 1.3 Hz,
3H), 8.11 (td, J = 8.0, 1.3 Hz, 3H), 7.95 (d, J = 4.8 Hz, 2H), 7.85 (dd, J = 9.4, 5.4 Hz, 5H),
7.66–7.51 (m, 8H), 7.47 (t, J = 7.3 Hz, 1H), 7.40–7.32 (m, 2H), 7.20–7.11 (m, 1H),
6

ACCEPTED MANUSCRIPT
6.79–6.72 (m, 1H). Compound RuD 1H NMR (400 MHz, DMSO) δ(ppm): δ 9.29 (s, 2H),
8.90 (dd, J = 15.6, 8.2 Hz, 5H), 8.22 (t, J = 7.3 Hz, 3H), 8.11 (t, J = 7.2 Hz, 5H), 7.94 (s,
2H), 7.87 (d, J = 4.8 Hz, 5H), 7.60 (d, J = 12.5 Hz, 5H), 7.45 (s, 1H), 7.36 (t, J = 6.7 Hz,
3H), 7.16 (s, 1H), 7.02 (d, J = 6.4 Hz, 1H), 6.76 (d, J = 8.5 Hz, 1H), 3.91 (s, 4H).
2.4. Solution preparation and metal ion titration
RuA stock solution (0.5 mM) was prepared in HEPES buffer (10 mM, pH=7.4) and
further diluted to 15 M for the assay. Stock solutions of various metal ions of nitrate
salts (Cu2+, Pb2+, Na+, Co2+, Fe3+, K+, Ba2+, Al3+, Cr3+, Ca2+, Mg2+, Ag+, Fe2+, Cd2+, Mn2+,
Ni2+ and Li+), amino acids (L-histidine, L-cysteine, glutathione, L-methionine,
L-threonine, L-glutamic acid, L-leucine, L-isoleucine, L-aspartic acid, L-arginine,
L-proline, L-proline, L-serine, L-tryptophan, L-lysine, D-phenylalanine, DL-alanine and
glycine) with the concentration of 15 mM were prepared in distilled water and then
diluted to appropriate concentrations. NEM (60 mM) stock solution was prepared in
distilled water and kept at 4 ℃ before used. RuA (1.00 mL, 15 M) and suitable amounts
of ions solution were added into the cuvette. As for the selective and competitive
detection of various metal cations and luminescence titration of Cu2+, the luminescence
spectra were recorded after incubation for 1 min at room temperature. Similarly, the
incubation time was 4 min for the detection of L-His. All the luminescence spectra were
collected under excitation at 460 nm. Slit: 10/10 nm.
2.5. Cell culture and cell imaging
Mice B16 melanoma cells were cultured in 1640 Medium with 1% antibiotics
(penicillin-streptomycin, 10 kU mL-1) and 10% FBS (fetal bovine serum) under a 5%
CO2 humid atmosphere (37 ºC). Firstly, the cells were seeded into a 48-well plate and
incubated for 24 h. Then the cells were washed three times with phosphate buffered
saline (PBS, pH=7.4). Next, after incubating with 30 μM RuA for 2 h, the cells were
washed thrice again with PBS to remove the free RuA. Then 1640 culture medium (1 mL)
was added and the cellular uptake was observed by confocal laser scanning microscopy
7

ACCEPTED MANUSCRIPT
with 460 nm as excitation wavelength. For the second experiment, cells were
pre-incubated with N-ethylmaleimide (400 μM) for 15 min and washed with PBS three
times, then the cells were treated with RuA (30 μM) for 2 h, and then treated with Cu2+
(40 μM). For the third experiment, cells were pre-incubated with N-ethylmaleimide (400
μM) for 15 min, and then treated with RuA (30 μM), Cu2+ (40 μM) for 2 h. Prior to
imaging, cells were incubated with L-His (100 μM).
3. Results and Discussion
Scheme 1. The synthesis of RuA and the structure of RuB, RuC and RuD.
3.1. Optical properties of RuA
The optical properties of probe RuA were studied by UV−Vis absorption and
luminescence spectroscopy. As shown in Figure 1A, the UV−Vis absorption spectrum of
RuA showed a typical absorption peak around 290 nm due to the π−π* transition of
2,2’-bipyridine moieties; the peaks at 340 nm and 460nm were ascribed to the charge
transfer (CT) transition of phenanthroline-based units (Schiff base cavity) and a metal to
ligand charge transfer (MLCT) absorption band, respectively [34,43]. Moreover, it can be
seen that RuA exhibited the maximum emission at 604 nm and the maximum excitation
at 460 nm, and no excitation dependence was observed (Fig. 1B). Therefore, in the
8

ACCEPTED MANUSCRIPT
following experiments, the excitation wavelength of probe RuA was fixed at 460 nm,
which corresponded to the UV−Vis peak of MLCT.
Fig. 1. (A) The UV-Vis absorption spectrum, excitation and emission luminescence
spectra of RuA (15 M). (B) Luminescence emission spectra of RuA (15 M) at the
different excitation wavelength. All spectra were measured in HEPES buffer (10 mM,
pH=7.4).
3.2. Investigation of the analytical conditions
At first, we studied the effect of the concentration of probe RuA. As shown in Fig. 2A,
when the concentration of RuA was more than 12 M, the quenching efficiency ((I0-I)/I0)
changed moderately by adding the same concentration of Cu2+ into the solution.
Considering the gain of low detection limit and good luminescence spectrum, 15 M was
served as the appropriate concentration of RuA in this experiment. pH was another vital
factor to affect the luminescent detecting performance. So the effect of pH towards the
luminescence quenching efficiency of RuA was also investigated in the solution of Cu2+
(15 M) and RuA (15 M). The result as shown in Fig. 2B illustrated that a high
quenching efficiency could be obtained at pH=7.2-9.0. This can be attributed to that the
deprotonation of phenolic hydroxyl in mildly basic condition would induce the oxygen
atom and nitrogen atom to bind to Cu2+ better [44]. Considering that the normal
physiological conditions is pH=7.4, therefore, this value was selected as the operating pH
condition in our experiment. Furthermore, the influence of reaction time was shown in
9

ACCEPTED MANUSCRIPT
Fig. S1A. It can be seen that the luminescence intensity of RuA decreased by 90% in less
than 1 min with the addition of Cu2+, revealing a fast reaction rate. However, the
luminescence intensity increased gradually and achieved a stable state after 4 min when
L-His was added into the RuA–Cu(II) solution (Fig. S1B†). Therefore, the reaction time
of 1 min and 4 min were selected for the detection of Cu2+ and L-His, respectively.
Additionally, the stability of RuA-Cu (II) and RuA-Cu (II) + L-His was investigated in
different concentrations of NaCl solution, and only mild luminescence intensity changes
can be observed even at the high ionic strength condition (Fig. 2C). The photostability of
RuA was also studied, and the luminous intensity remained almost constant under 2 h
continuous irradiation (460 nm) with a Xe lamp (Fig. 2D). Based on above results, the
probe can be applied to biologic imaging and biomarkers.
Fig. 2. (A) The effect of concentration of RuA on the luminescence quenching efficiency.
(B) The effect of pH on the luminescence quenching efficiency of RuA. (C)
Luminescence intensity of RuA-Cu(II) (15 M) and RuA-Cu(II) (15 M) + L-His (30
10

ACCEPTED MANUSCRIPT
M) containing different concentrations of NaCl (error bars, SD, n=3). (D) The
photostability of RuA (15 M) under continuous UV irradiation. The excitation/emission
wavelengths were 460/604 nm. All spectra were tested in HEPES buffer (10 mM,
pH=7.4).
3.3. Detection of Cu2+
The selectivity of RuA for Cu2+ sensing was evaluated by adding various metal ions
with a certain concentration. As shown in Fig. 3A and Fig. S2, other metal cations (Pb2+,
Na+, Co2+, Fe3+, K+, Ba2+, Al3+, Cr3+, Ca2+, Mg2+, Ag+, Fe2+, Cd2+, Mn2+, Ni2+ and Li+)
marginally affected the luminescence intensity of RuA, while Zn2+ caused a slight
luminescence quenching. This may be ascribed to the chelation effect between RuA and
Zn2+, which can lead to the electron transfer and the change in the order of energy levels
of the excited states [45]. Besides, other divalent transition metal ions including Fe2+,
Ni2+ performed nearly no effect on the luminescence of RuA. Thus, the probe RuA
showed high selectivity for Cu2+. Moreover, the binding behavior of the probe towards a
variety of metal ions was studied by the UV-Vis spectra (Fig. 3B). A bathochromic shift
of 23 nm was observed in RuA+Cu2+, in comparison with the RuA, while other metal
ions had nearly no interference. The UV-Vis absorption titration of RuA with Cu2+ was
also completed, and we can clearly find that the peak at 340 nm moved slowly toward the
long wavelength direction with the increase of Cu2+ (Fig. S3†). This was due to the
coordination of the Cu2+ to the phenol oxygen atom and imino nitrogen atom in Schiff
base cavity, which reduced the ability of the imino group to participate in the charge
transfer (CT) transition [46]. These results indicated that RuA possessed a strong binding
with Cu2+ and therefore, can be applied to detect Cu2+ quickly, selectively and sensitively
in aqueous solution.
11

ACCEPTED MANUSCRIPT
Fig. 3. (A) Selectivity of the RuA (15 M) sensing system for Cu2+ (15 M) to other
competing metal ions (15 M) (error bars, SD, n=3), where I0 and I refer to the
luminescence intensities of the system (λem=604 nm) in the absence and presence of
metal ions, respectively. (B) Absorption spectra of RuA (15 M) upon addition of
different metal ions (15 M). All spectra were detected in HEPES buffer (10 mM,
pH=7.4).
The linear range and detection limit of Cu2+ detection was assessed by changing the
concentration of Cu2+ at the best condition discussed above. The luminescence of RuA
was quenched gradually by adding Cu2+ (Fig. 4A). The corresponding detection limit and
linear range have been calculated to be 0.35 nM (S/N=3) limited from 5 nM to 9.0 M
(Fig. 4B), which is superior to the other organic probes reported previously (Table S1†).
Besides, the Job's plot for the binding activity of RuA to Cu2+ was researched according
to the luminescence titration (Fig. 4C), and a 1:1 stoichiometry was obtained.
Furthermore, the association constant (K) was calculated to be 4.437*103 M-1 based on
the Benesi–Hildebrand plot (Fig. 4D) [47].
12

ACCEPTED MANUSCRIPT
Fig. 4. (A) Luminescence emission spectra of RuA (15 M) in the presence of different
concentrations of Cu2+. Insert: photograph of RuA (a), and RuA+Cu2+ (b) under UV
illumination (365 nm). (B) The plots of relative luminescence intensity (I0−I)/I0 versus
the concentration of Cu2+ (error bars, SD, n=3). (C) Job's plot of the complex formed by
RuA and Cu2+ at a constant total concentration of 15 M. (D) Benesi-Hildebrand analysis
of RuA (15 M) at different Cu2+ concentration. I0 and I are the luminescence intensities
of the probe at 604 nm in the absence and presence of Cu2+, respectively. All spectra were
detected in HEPES buffer (10 mM, pH=7.4).
3.4. Detection of L-His
As expected, the RuA-Cu(II) ensemble was almost non-luminescent excited at 460 nm.
However, all the L-His, cysteine (Cys) and glutathione (GSH) can induce a great increase
of luminescent intensity at around 604 nm (Fig. S4†), which brought the interference
toward L-His detection. Thus, we introduced N-ethylmaleimide (NEM) as a masking
13

ACCEPTED MANUSCRIPT
agent to eliminate the effect of thiol groups in the detecting system [48,49] (the reaction
process was shown in Scheme S2†).
Fig. 5. (A) Luminescence responses of RuA-Cu(II) (15 M) to amino acids (30 M) and
common biological species. (B) Luminescence responses of RuA-Cu(II) (15 M) to
L-His (30 μM) in the presence of various amino acids (30 M) and common biological
species. (C) Luminescence spectra of the RuA–Cu(II) complex (15 M) towards various
concentrations of L-His. Insert: photograph of RuA-Cu(II) complex (a), and
RuA-Cu(II)+L-His (b) under UV illumination (365nm). (D) Plots of the luminescence
value as a function of the L-His concentration (error bars, SD, n=3). All spectra were
measured in HEPES buffer (10 mM, pH=7.4).
Then we inspected the selectivity of the probe towards other amino acids (30 M) and
common biological species (KCl 4 mM, NaCl 4 mM, MgCl2 400 M, CaCl2 400 M,
FeCl3 20 M, glucose 300 M, ascorbic acid 50 M, urea 500 M). As shown in Figure
14

ACCEPTED MANUSCRIPT
5A, the luminescence of the system initiated by L-His was much more pronounced than
those by other substances. We also conducted a competitive determination to further
assess the selectivity, and the results showed that the probe can still particularly respond
to the L-His in the presence of other related species (Fig. 5B). Therefore, the RuA-Cu(II)
ensemble possessed good selectivity towards L-His and can be used as a good probe to
detect L-His in biological fluids with subtle interference.
Fig. 5C showed the titration results of L-His to RuA–Cu(II) ensemble. As indicated in
Fig. 5C, the luminescent intensity of RuA–Cu(II) ensemble increased significantly with
adding L-His. For example, when 30 M of L-His was added, a 10-fold of increase of
luminescence intensity was obtained compared with the original intensity. In addition, as
indicated in the titration curve (Fig. 5D), the emission intensity at 604 nm increased
linearly with adding L-His from 50 nM to 30 μM (R2=0.9955), and a 0.42 nM of the
detection limit (S/N=3) can be obtained, which means that our work presented better
linear range and detection limit than other previous reports (Table S2†) and have a
potential to be used in practical biological fluids [50]. Furthermore, the 2: 1 binding
model of L-His to Cu2+ has been demonstrated by Job’s plot (Fig. S5A†) and a 1.025*104
M-1 of association constant (K) was estimated on the grounds of a Benesi–Hildebrand
plot (Fig. S5B†). Moreover, Cys and GSH also displayed similar luminescence
enhancement, and the detection limits were calculated to be 2.21 nM and 2.49 nM,
respectively (Fig. S6† and Fig. S7†).
3.5. Mechanism for the detection of Cu2+ and L-His
Due to the paramagnetic nature of Cu2+, the sensing mechanism of RuA for Cu2+ may
be attributed to the luminescence quenching induced by copper-binding [51], which
ultimately allows the Cu2+ to exhibit recognizable luminescence quenching through
electron and/or energy transfer processes [52]. It is well-known that the outer layer
electron configuration for Cu2+ is 3s23p63d9, while the electron sub-layer 3d, 4s and 4p
with the relatively low level of the empty track, which can accept lone electron pair
15

ACCEPTED MANUSCRIPT
provided by N, O atoms to form coordination compounds. Thus, the proposed specific
recognition site of RuA for Cu2+ was Schiff base cavity composed of the phenol oxygen
atom and imino nitrogen atom. Therefore, this dramatic quenching of the initial
luminescence of RuA induced by Cu2+ is due to the reverse photoinduced electron
transfer (PET) from the 2,2’-bipyridine ruthenium(II) moieties to the phenol oxygen atom
and imino nitrogen atom because of the decrease in the electron density upon Cu2+
complexation [53,54]. Besides, in order to further prove the response mechanism of RuA,
complexes of RuB, RuC and RuD were prepared, which own the similar structure as
shown in Scheme 1. Specially, the position of the amino and hydroxyl groups of the RuB
complex is consistent with the RuA, and it should also respond to the Cu2+. However, the
methoxy group in the RuB complex is designed to be located next to the hydroxyl group
so as to explore the steric hindrance in the process of the combination of L-His and Cu2+.
In addition, for the purpose of further investigating the role of O atom in the binding of N,
O hole in Schiff base with Cu2+, no hydroxyl exists in the complexes of RuC and RuD,
which should be no response to copper ions. Besides, we also explored the selectivity of
RuB, RuC and RuD toward different metal ions (Fig. S8†, Fig. S9† and Fig. S10†). Then
the responses about these four complexes toward Cu2+ were compared in Fig. 6A. As
expected, RuB showed an obvious luminescence quenching toward Cu2+, while RuC and
RuD exhibited marginal change. The reason may be that both the phenolic hydroxyls and
imino group exist in RuB and the location is adjacent to each other, while no phenolic
hydroxyls exists in RuC and RuD. These results demonstrated that the adjacent oxygen
atom plays an indispensable role in the Cu2+ coordination. It is well known, the general
coordination number of Cu2+ is four, so the adjacent amido and hydroxyl of the probe
provide four coordination groups to combine with Cu2+ to form a complex. Moreover, we
noticed that the luminescence can be enhanced by introducing L-His into the RuA-Cu(II)
and RuB-Cu(II) systems (Fig. 6B), implying that the strong binding capacity of the
imidazole group from L-His with Cu2+ and freeing Ru(II) compounds from the
16

ACCEPTED MANUSCRIPT
complexes and thus recovering their luminescence [55]. However, RuA had better
luminescence recovery than RuB, thus RuA was selected as the representative probe in
this work.
Fig. 6. The luminescence response of Ru compounds (15 M) toward Cu2+ (15 M) (A)
and L-His (30 M) (B) in HEPES buffer (10 mM, pH=7.4), where I0 and I refer to the
luminescence intensities of the system (λem=604 nm) in the absence and presence of Cu2+
or L-His, respectively.
Above hypothesis also has been confirmed by MALDI-TOF-MS spectrum of RuA
solution in the presence of Cu2+ from Fig. 7A, which two peaks related to RuA–Cu(II)
ensemble have been observed. Then the ESI-MS spectra of RuA+Cu(II)+L-His solution
were also obtained (Fig. 7B), in which the peaks at m/z ∼ 890.33 and 984.17 were related
to RuA, and the appearance of a peak at 374.10 evidenced the formation of the complex
1
[2L-His+Cu2+]. Additionally, H NMR titration of the RuA can further support the
proposed mechanism (Fig. 7C). It can be seen from Fig. 7C that the peaks about RuA in
1
the H NMR spectrum were broadened and even almost vanished when the Cu2+ was
added, which may be due to the coordination of paramagnetic Cu2+ and RuA [56]. And
1
the addition of L-His into the RuA-Cu (II) solution resulted in a recovery of the fine H
NMR spectrum of RuA in the mixture to some extent, which indicated that L-His was
able to remove Cu2+ from the RuA-Cu (II) complex. What’s more, the displacement
mechanism was further testified by UV spectra, in which the formation of free RuA was
17

ACCEPTED MANUSCRIPT
clearly observed when adding L-His into RuA−Cu(II) solution (Fig. S11†). The quantum
yield of RuA and RuA-Cu(II)+L-His were 0.084 and 0.081, respectively (reference was
fluorescein in 0.1 N NaOH, Φ = 0.85) (Fig. S12†) [57]. Besides, a luminescence decay
experiment was also conducted, and the lifetimes of RuA, RuA-Cu(II) and
RuA-Cu(II)+L-His were determined to be 258.7, 108.8 and 250.8 ns, respectively, by
fitting the data into a single exponential decay function (Fig. S13†). These results were
all consistent with the proposed mechanism, as indicated in Scheme 2.
18

ACCEPTED MANUSCRIPT
Fig. 7. (A) MALDI-TOF-MS of RuA+Cu2+ in methanol. (B) ESI-MS spectrum of
1
RuA-Cu(II)+L-His in HEPES buffer solution. (C) H NMR titration spectrum of RuA
with Cu2+ followed by L-Histidine in D2O.
19

ACCEPTED MANUSCRIPT
Scheme 2. The proposed mechanism of Cu2+ and L-His detection.
3.6. Real sample analysis
3.6.1. Cu2+ detection in water samples
According to the water quality standard of US Environmental Protection Agency
(EPA), 20 μM is the highest acceptable level of Cu2+ in drinking water [58]. RuA probe
was water-soluble with the luminescent detection limit of 0.35 nM and this suggested that
it can be applied to detecting Cu2+ in drinking water samples. In fact, the good results
have been gained from the detection of Cu2+ in tap water (Table S3).
3.6.2. L-Histidine detection in biological fluids
Since L-His is an essential component of many proteins and associated with various
diseases, it is important to detect L-His in biological fluids such as human serum and
urine. Before the detection, the blood and urine samples were treated according to the
method described in the supporting information. In order to evaluate the accuracy of the
proposed method, the spiked recovery test of L-His in the actual samples was carried out.
The determination of L-His for blood samples was shown in Table S4. The original
content of L-His in human serum was in the range of 15-75 M [50]. And the average
contents of L-His in two urine samples were measured to be 470 and 543 M,
respectively (Table S5), which were included in the normal level (130–2100 M) [59].
All of these results manifested that the proposed method for sensing L-His in biological
fluids was accurate and reliable.
20

ACCEPTED MANUSCRIPT
3.7. Application of probe in living cellular imaging
It is widely known that bioimaging probes for analytical diagnostics in vitro should be
non-toxic. Actually, because of much smaller scattering, lower background signals and
almost no damage to viable cells, the emission spectrum of the red or near-infrared (NIR)
region are favorable for their use as biological probes in biological samples [60,61]. To
further explore the potential applications of the RuA in the biological system, the
cytotoxicity of RuA towards mice B16 melanoma cells was firstly assessed by CCK-8
assay after a 24 h treatment (Fig. S14†). The percentage of cell survival was more than
97%, which revealed that the RuA was non-cytotoxic towards B16 cells. Subsequently,
luminescence imaging experiments of RuA for living cells were performed. As shown in
Fig. 8, a bright red luminescence was observed when B16 cells were treated with the
RuA probe only. When cells were pre-treated with an appropriate amount of
thiol-scavenger (NEM) to eliminate all free thiols present in the cells, and then incubated
with RuA and Cu2+, the observed luminescence signal in cells was significantly reduced.
However, a remarkable luminescence signal was investigated when cells were incubated
with RuA, Cu2+ and L-His. These results indicated that the probe RuA can be used for
imaging Cu2+ and L-His in living cells.
21

ACCEPTED MANUSCRIPT
Fig. 8. Luminescence images of the living cells. (A) B16 cells were incubated with RuA
(30 μM) for 2 h. Cells pre-incubated with 0.12 mM NEM for 15 min, washed thrice,
treated with RuA (30 μM) for 2 h, then treated with Cu2+ (40 μM) (B) and then treated
with L-His (100 μM) (C). Luminescence images were acquired by confocal microscopy
(excitation 460 nm, emission 550−680 nm). Scale bar: 25 μm.
4. Conclusion
In summary, the water-soluble luminescent RuA was used as an on-off-on probe for
sensitively and selectively detecting Cu2+ and L-Histidine, revealing the low detection
limits of 0.35 nM and 0.44 nM for Cu2+ and L-Histidine, respectively. Cu2+ can
significantly quench the luminescence of RuA by the coordination of Cu2+ with the N, O
22

ACCEPTED MANUSCRIPT
atoms of the probe molecule. Furthermore, the sensing mechanism was demonstrated by
luminescent behaviour of similar structure compounds (RuB, RuC and RuD) and
MALDI-TOF-MS spectrum of co-existence solution of RuA and Cu2+. Upon adding
L-Histidine into RuA-Cu(II) solution, the luminescence can be almost recovered to the
original intensity. The possible cause of the luminescence enhancement was L-Histidine
being able to remove Cu2+ from RuA-Cu(II) due to the strong binding ability towards
Cu2+ of the amino and imidazole group of L-Histidine and RuA released from the
1
ensemble. In addition, this replace mechanism was also testified by MS, H NMR,
UV-Vis and other characterizations. Besides, the probe can practically be applied to the
analysis of real samples. Importantly, the low-toxicity probe can be used for imaging
Cu2+ and L-His in living cells. Finally, the excellent performance of the probe indicated
its potential application in the field of environmental and clinical diagnostics.
Acknowledgements
We would like to thank the National Natural Science Foundation of China (Nos.
21407109) for its financial support to this research.
Appendix A. Supplementary Data
Supplementary data to this article can be found online at
Reference
[1] M. Debabrata, T. Govindaraju, Chem - Eur. J. 17 (2011) 1410-1414.
[2] N. Narayanaswamy, T. Govindaraju, Sens. Actuators B 161 (2012) 304-310.
[3] E. Gaggelli, H. Kozlowski, D. Valensin, G. Valensin, Chem. Rev. 37 (2006)
1995-2044.
[4] R. Uauy, M. Olivares, M. Gonzalez, Nutr. Rev. 45 (1987) 176–180.
[5] M. C. Linder, M. Hazeghazam, Am. J. Clin. Nutr. 63 (1996) 797S-811S.
[6] J. S. Valentine, P. J. Hart, P. Natl. Acad. Sci. 100 (2003) 3617-3622.
[7] L. I. Bruijn, T. M. Miller, D. W. Cleveland, Annu. Rev. Neurosci. 27(2004) 723-749.
23

ACCEPTED MANUSCRIPT
[8] T. Chen, Y. Hu, Y. Cen, X. Chu, Y. Lu, J. Am. Chem. Soc. 135 (2013) 11595-11602.
[9] D. R. Brown, H. Kozlowski, Dalton. T. 13 (2004) 1907-1917.
[10] S. G. Kaler, Nat. Rev. Neurol. 7 (2011) 15-29.
[11] S. Lutsenko, A. Gupta, J. L. Burkhead, V. Zuzel, Arch. Biochem. Biophys. 476 (2008)
22-32.
[12] G. L. Millhauser, Accounts Chem. Res. 37 (2004) 79-85.
[13] S. V. Staden, L. Holo, Sens. Actuators B 120 (2007) 399-402.
[14] L. D. Li, Z. B. Chen, H. T. Zhao, L. Guo, Biosens. Bioelectron. 26 (2011)
2781-2785.
[15] W. M, S. ME, Q. AR, G.-L. E, B. P, H. O, S. P, L. B, Am. J. Clin. Nutr. 87 (2008)
1860-1866.
[16] G. N. Chen, X. P. Wu, J. P. Duan, H. Q. Chen, Talanta 49 (1999) 319-330.
[17] M. L. Rao, H. Stefan, C. Scheid, A. D. Kuttler, W. Froscher, Epilepsia 34 (1993)
347-354.
[18] A.L. Jones, M.D. Hulett, C.R. Parish, Immunol. Cell. Biol. 83 (2005) 106-118.
[19] E.L. Que, D.W. Domaille, C.J. Chang, Chem. Rev. 39 (2008) 1517-1549.
[20] X. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon, Chem. Rev. 112 (2012) 1910-1956.
[21] J. Du, M. Hu, J. Fan, X. Peng, Chem. Soc. Rev. 43 (2012) 4511-4535.
[22] L. Prodi, F. Bolletta, M. Montalti, N. Zaccheroni, Coordin. Chem. Rev. 205 (2000)
59-83.
[23] C. Bargossi, M.C. Fiorini, M. Montalti, L. Prodi, N. Zaccheroni, Cheminform 208
(2000) 17-32.
[24] Z. Huang, J. Du, J. Zhang, X.Q. Yu, L. Pu, Chem. Commun. 48 (2012) 3412-3414.
[25] F. Pu, Z. Huang, J. Ren, X. Qu, Anal. Chem. 82 (2010) 8211-8216.
[26] S. K. Sun, K. X. Tu, X. P. Yan, Analyst 137 (2012) 2124-2128.
[27] P. N. Basa, A. G. Sykes, J. Org. Chem. 77 (2012) 8428-8434.
24

ACCEPTED MANUSCRIPT
[28] X. Li, Y. Chen, J. Meng, K. Lü, A. Zhang, B. Zhang, Chinese J. Chem. 29 (2011)
1947-1950.
[29] J. Xie, M. Ménand, S. Maisonneuve, R. Métivier, J. Org. Chem. 72 (2007)
5980-5985.
[30] B. Khan, M.R. Shah, D. Ahmed, M. Rabnawaz, I. Anis, S. Afridi, T. Makhmoor,
M.N. Tahir, J. Hazard. Mater. 309 (2016) 97-106.
[31] M.H. Kim, H.H. Jang, S. Yi, S.K. Chang, M.S. Han, Chem. Commun. 32 (2009)
4838-4840.
[32] N. Li, Y. Xiang, A. Tong, Chem. Commun. 46 (2010) 3363-3365.
[33] V. Balzani, G. Bergamini, F. Marchioni, P. Ceroni, Coordin. Chem. Rev. 250 (2006)
1254-1266.
[34] M. Hardy, D. Konetski, C. Bowman, N. Devaraj, Org. Biomol. Chem. 14 (2016)
5555-5558.
[35] P. Zhou, Z. Zheng, W. Lu, F. Zhang, Z. Zhang, D. Pang, B. Hu, Z. He, H. Wang,
Angew. Chem. Int. Edit. 51 (2012) 670-674.
[36] Y. Yang, Q. Zhao, W. Feng, F. Li, Chem. Rev. 113 (2013) 192-270.
[37] N. P. Cook, K. Kilpatrick, L. Segatori, A. A. Martí, J. Am. Chem. Soc. 134 (2012)
20776-20782.
[38] H. Komatsu, K. Yoshihara, H. Yamada, Y. Kimura, A. Son, K. Tanabe, Chem - Eur. J.
19 (2013) 1971–1977.
[39] W. Hao, A. Mcbride, S. Mcbride, P. G. Jian, Y. W. Zhi, J. Mater. Chem. 21 (2010)
1040-1048.
[40] M. Suresh, A. Ghosh, A. Das, Chem. Commun. 2008 (2008) 3906-3908.
[41] G. U. Reddy, P. Das, S. Saha, M. Baidya, S. K. Ghosh, A. Das, Chem. Commun. 49
(2013) 255-257.
[42] P. P. Li, Z. Y. Jin, M. L. Zhao, Y. X. Xu, Y. Guo, D. Xiao, Dalton T. 44 (2015)
2208-2216.
25

ACCEPTED MANUSCRIPT
[43] R. Lo, S. Roy, T. Banerjee, B. Ganguly, A. Das, Chem. Commun. 49 (2013)
9818-9820.
[44] T. Chen, L. Yin, C. Huang, Y. Qin, W. Zhu, Y. Xu, X. Qian, Biosens. Bioelectron. 66
(2015) 259-265.
[45] T. Kowalczyk, Z. Lin, T.V. Voorhis, J. Phys. Chem. A 114 (2010) 10427-10434.
[46] T. Gunnlaugsson, J. P. Leonard, N. S. Murray, Org. Lett. 6 (2004) 1557-1560.
[47] M. Hariharan, S. C. K. And, D. Ramaiah, Org. Lett. 9 (2007) 417-420.
[48] H. S. Jung, J. H. Han, Y. Habata, C. Kang, J.S. Kim, Chem. Commun. 47 (2011)
5142-5144.
[49] H. Li, J. Liu, Y. Fang, Y. Qin, S. Xu, Y. Liu, E. Wang, Biosens. Bioelectron. 41 (2013)
563-568.
[50] H. Agarwalla, N. Taye, S. Ghorai, S. Chattopadhyay, A. Das, Chem. Commun. 50
(2014) 9899-9902.
[51] Z. Zheng, L. Wang, W. Tang, P. Chen, H. Zhu, Y. Yuan, G. Li, H. Zhang, G. Liang,
Biosens. Bioelectron. 83 (2016) 200-204.
[52] S. Anbu, S. Shanmugaraju, R. Ravishankaran, A.A. Karande, P.S. Mukherjee, Inorg.
Chem. Commun. 25 (2012) 26-29.
[53] S. Anbu, S. Shanmugaraju, R. Ravishankaran, A.A. Karande, P.S. Mukherjee, Dalton
T. 41 (2012) 13330-13337.
[54] S. Anbu, R. Ravishankaran, G.D.S. Mf, A.A. Karande, A.J. Pombeiro, Inorg. Chem.
53 (2014) 6655-6664.
[55] F. Pu, Z. Huang, J. Ren, X. Qu, Anal. Chem. 82 (2010) 8211-8216.
[56] L. Xu, Y. Xu, W. Zhu, B. Zeng, C. Yang, B. Wu, X. Qian, Org. Biomol. Chem. 9
(2011) 8284-8287.
[57] T. Miura, Y. Urano, K. Tanaka, T. Nagano, K. Ohkubo, S. Fukuzumi, J. Am. Chem.
Soc. 125 (2003) 8666-8671.
26

ACCEPTED MANUSCRIPT
[58] H. S. Jung, P. S. Kwon, J. W. Lee, J. I. Kim, C. S. Hong, J. W. Kim, S. Yan, J. Y. Lee,
J. H. Lee, T. Joo, J. Am. Chem. Soc. 131 (2009) 2008-2012.
[59] P. M. Kovach, M. E. Anal. Chem. 54 (1982) 217-220.
[60] B. Tang, H. Huang, K. Xu, L. Tong, G. Yang, X. Liu, L. An, Chem.Commun. 0
(2006) 3609-3611.
[61] M. Li, X. Wu, Y. Wang, Y. Li, W. Zhu, T.D. James, Chem.Commun. 50 (2014)
1751-1753.
27

ACCEPTED MANUSCRIPT
Graphical abstract
28

ACCEPTED MANUSCRIPT
Highlights
 A novel near-infrared luminescent probe for the highly sensitive detection of
Cu2+ and L-Histidine in aqueous solution has been established.
 Low detection limits and favorable linear ranges for Cu2+ and L-Histidine were
obtained.
 The mechanism for detecting Cu2+ and L-Histidine has been comprehensively and cl
early proposed via various methods like MS, 1H NMR, UV-Vis etc.
 The probe that exhibited many satisfied properties has been successfully adopted to
many practical applications including imaging cancer cells.
29