Near-infrared fluorescent probe for selective detection of Cu2+ in living cells and in Vivo

Article abstract of DOI:10.1016/j.saa.2019.03.062

A NIR-rhodamine fluorescent probe was designed and successfully synthesized. The structure of the probe NRh-Cu was characterized by ¹H NMR, ¹³C NMR and HRMS. The probe was found to show high sensitivity and high selectivity. The dete

Full text of DOI:10.1016/j.saa.2019.03.062

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 216 (2019) 404–410
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Near-infrared fluorescent probe for selective detection of Cu2+ in living
cells and in Vivo
⁎
Zhiyang Xu, Hong Wang, Zhen Chen, Hongli Jiang, Yanqing Ge
School of Chemistry and Pharmaceutical Engineering, Taishan Medical University, Taian, Shandong 271016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A NIR-rhodamine fluorescent probe was designed and successfully synthesized. The structure of the probe NRh-
Cu was characterized by 1H NMR, 13C NMR and HRMS. The probe was found to show high sensitivity and high
selectivity. The detection limit was calculated to be as low as 0.95 ppb. The sensing mechanism was proposed
and confirmed by HRMS spectra. Furthermore, it could be used for imaging Cu2+ in living cells and in vivo.
© 2019 Elsevier B.V. All rights reserved.
Received 16 November 2018
Received in revised form 19 February 2019
Accepted 18 March 2019
Available online 19 March 2019
Keywords:
NIR
Bioimaging
Fluorescent probe
Cu2+
1. Introduction
2. Experimental section
Copper, as an important trace element in the human body, plays a
major role in a variety of fundamental physiological processes in or-
ganisms ranging from bacteria to mammals [1–3]. However, exces-
sive amount of copper results in liver disease, psychiatric or
neurologic symptoms by damaging the liver and nervous system
while hematological and neurological disorder such as Menkes dis-
ease is reported to be resulted from copper deficiency [4–6]. Hence,
it is very important to develop various methods to efficiently detect
copper in vitro and in vivo.
Luminescence bioimaging has attracted great attention in the
medical diagnosis and biological analysis [7,8]. Up to now, a large
number of fluorescent probes for detection of Cu2+ have been re-
ported [9]. However, most of them have relatively short absorption
and emission wavelength (UV/Vis), which renders them difficult to
be used for sensing and imaging in living animals due to their poor
tissue penetration ability [10]. On the other hand, the near-infrared
(NIR) fluorophores at around 650–900 nm have many obvious ad-
vantages such as the reduction of background absorption, fluores-
cence, and light scattering. Furthermore, they show good tissue
penetration ability with less damage [11–13]. Therefore, it is signifi-
cant and appealing to develop NIR probes to detect Cu2+ in vivo.
Herein, we report the synthesis, spectroscopic properties, and
in vivo biological imaging applications of NIR Cu2+ probes.
2.1. Materials and equipment
UV–vis spectra and fluorescence spectra were recorded on a U-3900
UV–Vis spectrometer and FS 5 luminescence spectrophotometer at
room temperature, respectively. 1H NMR and 13C NMR spectra were
measured on a Bruker 400 (400 MHz) instrument (CDCl3 as solvent
and tetramethylsilane as an internal standard). The mass spectra were
recorded on an AB SCIEX 5800 matrix-assisted laser desorption ioniza-
tion time-of flight mass spectrometer. All reagents and solvents were
purchased from commercial sources and used without further purifica-
tion. The solutions of metal ions were prepared from chlorizated salts
which were dissolved in deionized water, and the latter was used
throughout the process of absorption and fluorescence determination.
The limit of detection (LOD) for the Cu(II) was calculated as 3 times
the standard deviation for the average measurements of 10 blank sam-
ples by slope (LOD = 3σ/K).
2.2. Cell culture and imaging
The HeLa cell lines were provided by the Institute of Biochemistry
and Cell Biology, SIBS, CAS (China). The HeLa cells were cultured in
MEM (modified Eagle's medium) supplemented with 10% FBS (fetal
bovine serum) at 37 °C and 5% CO2. Cell images were obtained via a
confocal microscope from FV1000 (Olympus) at excitation of
630 nm.
⁎
Corresponding author.
E-mail address: geyanqing2016@126.com (Y. Ge).
https://doi.org/10.1016/j.saa.2019.03.062
1386-1425/© 2019 Elsevier B.V. All rights reserved.

Z. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 216 (2019) 404–410
405
2.3. Fluorescence in vivo imaging
2.5. Synthesis of compound 7
Animal procedures were in agreement with the guidelines of the
Institutional Animal Care and Use Committee. In this small animal
in vivo fluorescence imaging system, a 635 nm continuous wave-
length laser (Connet Fiber Optics, China) was used as the excitation
source, and the fluorescence signal was collected by Andor DU897
EMCCD.
Compound 6 (0.67 g, 1 mmol), hydrazine hydrate (1.00 g, 20 mmol)
and BOP Reagent (0.45 g, 1 mmol) were dissolved in CH2Cl2 (10 mL),
and the reaction mixture was stirred at room temperature for 2 h.
Then, the solvent was removed under reduced pressure to give the
crude product, which was purified by silica gel flash chromatography
using hexane/ethyl acetate (5:1) as eluent to afford compound 7 as a
yellow solid in 87% yield. 1H NMR (400 MHz, CD3COCD3): δ 7.76 (m,
1H), 7.53 (m, 3H), 7.25 (m, 1H), 7.17 (m, 2H), 6.82 (m, 1H), 6.74 (m,
1H), 6.41 (d, J = 3.2 Hz, 1H), 6.35 (m, 1H), 6.31 (d, J = 16.0 Hz, 1H),
5.55 (d, J = 12.0 Hz, 1H), 4.14 (s, 2H), 3.77 (q, J = 7.2 Hz, 2H), 3.40
(q, J = 7.2 Hz, 4H), 2.56 (m, 2H), 1.70 (s, 6H), 1.56 (m, 2H), 1.30 (m,
2H), 1.21(t, J = 7.2 Hz, 3H), 1.15 (t, J = 7.2 Hz, 6H).
Compounds 1, 2, 3 and 5 were synthesized according to the litera-
ture [4,14].
2.4. Synthesis of compound 6
Compound 3 (0.42 g, 1 mmol) and compound 5 (0.48 g, 1 mmol)
were dissolved in acetic anhydride (6 mL), and the reaction mixture
was heated to 50 °C and further stirred for 60 min. Then, water
(20 mL) was added to the reaction mixture to quench the reaction.
The solvent was removed under reduced pressure to give the crude
product, which was purified by silica gel flash chromatography
using CH2Cl2 to CH2Cl2/ethanol (0 to 6:1) as eluent to afford com-
pound 6 as a green solid in 65% yield. 1H NMR (400 MHz, CDCl3): δ
8.62 (s, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.73 (m, 1H), 7.63 (m, 1H),
7.43 (m, 2H), 7.30 (m, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.74 (d, J =
8.0 Hz, 1H), 6.58 (m, 1H), 6.10 (m, 1H), 4.19 (s, 2H), 3.56 (s, 4H),
2.91 (s, 3H), 2.69 (s, 2H), 2.31 (s, 2H), 1.83 (s, 6H), 1.50 (t, J =
7.2 Hz, 3H), 1.28 (t, J = 7.2 Hz, 6H).
2.6. Synthesis of sensor NRh-Cu
Compound
7 (0.59 g, 1 mmol) and 4‑(diethylamino)‑2‑
hydroxybenzaldehyde (0.23 g, 1.2 mmol) were dissolved in ethanol
(10 mL) and heated to reflux for 6 h. After removal of ethanol under vac-
uum, the residue was purified by silica gel flash chromatography using
hexane/ethyl acetate (10:1) as eluent to afford compound NRh-Cu as a
yellow solid in 85% yield. 1H NMR (400 MHz, CDCl3): δ 11.25 (s, 1H),
9.06 (s, 1H), 7.90 (d, J = 4.0 Hz, 1H), 7.51–7.42 (m, 3H), 7.21–7.15 (m,
3H), 6.98 (d, J = 8.0 Hz, 1H), 6.84 (m, 1H), 6.60 (d, J = 8.0 Hz, 1H), 6.46
(d, J = 8.0 Hz, 1H), 6.36 (s, 1H), 6.25 (d, J = 8.0 Hz, 1H), 6.13 (m, 2H),
5.43 (d, J = 12.0 Hz, 1H), 3.67 (q, J = 8.0 Hz, 2H), 3.39 (m, 8H), 2.52
Scheme 1. Synthetic route of NRh-Cu.

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Z. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 216 (2019) 404–410
Fig. 1. Changes in the absorption (a) and emission (b) spectra of probe NRh-Cu (10 μM) upon the addition of Cu2+ from 0 to 15 μM in ethanol/H2O (1:1 v/v) and the analysis of the
absorption (c) and emission (d) detection limit of the probe.
(m, 2H), 1.74 (d, J = 8.0 Hz, 4H), 1.59 (m, 8H), 1.23 (t, J = 7.2 Hz, 3H),
1.15 (m, 12H). 13C NMR (100 MHz, CDCl3): 164.22, 160.79, 155.89,
154.69, 152.54, 150.50, 149.73, 148.74, 148.00, 144.55, 139.12,
132.90, 128.35, 127.76, 127.68, 123.45, 122.99, 121.61, 120.17,
119.60, 119.11, 108.33, 107.41, 105.58, 103.83, 103.34, 98.22,
97.94, 91.83, 68.13, 445.56, 44.50, 44.28, 36.72, 28.42, 28.33, 25.32,
22.90, 22.22, 12.66, 11.04. MS: m/z calcd for C49H56N5O3 [M + H]+
762.4383, found 762.4272.
3. Results and discussion
3.1. Synthesis
The rhodamine hydrazide 7 was prepared according to the previously
reported methods with minor modifications (Scheme 1) [14]. Compound
2, which was easily obtained by alkylation of 2,3,3‑trimethyl‑3H‑indole 1
and ethyl iodide, was converted to the desired intermediate 3 by using N,
Scheme 2. Proposed mechanism for the off-on sensing of NRh-Cu for Cu2+
.

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407
Fig. 2. Absorption and fluorescence spectra of probe (10 μM) in the presence and absence of different metal ions (10 μM) in EtOH-H2O (1:1, v/v).
N′‑bis‑phenylformamidine in 89% yield. Intermediate 5 was obtained
from cyclohexanone and 2‑(4‑diethylamino‑2‑hydroxybenzoyl)benzoic
acid by a condensation reaction in 90% yield. Compound 7 was conve-
niently synthesized by condensation of acid 6 with hydrazine hydrate
where (benzotriazol‑1‑yloxy)tripyrrolidino‑phosphonium hexafluo‑
rophosphate (PyBOP) was used as the coupling reagent. The yield in-
creased to 70%, in comparison with a low yield (≈20%) in the literature
method [14]. The probe NRh-Cu was synthesized by condensation of rho-
damine hydrazide 7 and 4‑(diethylamino)‑2‑hydroxybenzaldehyde. The
structure of NRh-Cu was characterized by 1H NMR, 13C NMR and HRMS
(see the Experimental Section and Supporting Information for details).
solution became green and strongly fluorescent, indicating that the
ring-opened amide form of NRh-1 was formed (Scheme 2, Fig. S7, S8).
3.4. Selectivity
A wide range of metal ions such as Li+, Na+, K+, Mg2+, Ca2+, Al3+
Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Zn2+, Cd2+, Ag+, Pb2+, Sn4+, and
Hg2+ were selected to investigate the selectivity toward Cu2+ (Fig. 2).
These results suggest that probe NRh-Cu has excellent selectivity to-
ward Cu2+ over other metal ions. Moreover, a kinetic study was exam-
ined over a 20-min time period. As shown in Fig. 3, Cu2+ (2 equiv.)
exhibited a rapid turn-on response that reached the maximum emission
intensity at 740 nm within 10 min. In contrast, other metal ions were al-
most nonemissive and stable.
,
3.2. Optical Response of NRh-Cu to Cu2+
The spectral response of probe NRh-Cu toward Cu2+ was investi-
gated in ethanol/H2O (1:1 v/v) solution. As shown in Fig. S1, NRh-Cu
displays significant enhancement of fluorescent emission after
interacting with Cu2+. To achieve more accurate results, we chose 50%
of ethanol as cosolvent. With increasing amounts of Cu2+, the intensity
of maximum absorption band at 717 nm was steadily enhanced causing
a significant color change in solution (Fig. 1a). These results suggest that
NRh-Cu could be used as a “naked-eye” probe for Cu2+. Moreover, in
the absence of Cu2+, NRh-Cu displayed a weak fluorescence emission
at 740 nm when excitated at 680 nm. In contrast, NRh-Cu showed grad-
ual fluorescence emission enhancement following the addition of in-
creasing concentrations of Cu2+ due to the Cu2+-induced ring opening
of the spirolactam form (Fig. 1b). The fluorescence intensity at 740 nm
has a good linear relationship with Cu2+ concentration ranging from
0.1 to 0.6 μM as shown in Fig. 1d and an ultralow detection limit is de-
termined to be 0.95 ppb. In addition, a detection limit of 6.2 ppb is
also obtained by absorption spectroscopy (Fig. 1c). The above results in-
dicate that the NRh-Cu could serve as an excellent NIR probe for the Cu2
+ detection.
3.5. Practical Applications
In order to check the cytotoxicity of probe NRh-Cu, the HeLa cells
were incubated for 48 h with varied concentrations of NRh-Cu (2–80
3.3. Sensing Mechanism
Phenol O, imino N, and carbonyl O atoms were designed to chelate
with copper ions. The rhodamine was selected as a signal switcher
due to its outstanding properties and a structural change from the
spirocyclic state (non-fuorescent) to the ring-open state (fuorescent)
induced by metal ions. The probe solution is weakly fluorescent and col-
orless, indicating that NRh-Cu exists in the spirolactam form, which was
also supported by the characteristic peak of the 9‑carbon of near 68 ppm
in the 13C NMR spectrum (Fig. S6). Upon the addition of 1.5 equiv. of Cu2
+, the absorbance at 717 nm was obviously enhanced and the probe
Fig. 3. Time-dependent fluorescence intensity change of probe (λem = 735 nm, 10 μM) in
the presence (blank) and absence of Cu2+ (2 equiv) in ethanol/H2O (v/v = 1:1).

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Z. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 216 (2019) 404–410
Fig. 4. Fluorescence microscope images of NRh-Cu in living HeLa cells. (a) HeLa cells incubated with NRh-Cu (10 μM) for 1 h. (b) HeLa cells pretreated with Cu2+ (20 μM) for 1 h and
followed by incubation with NRh-Cu (10 μM) for 30 min.
Fig. 5. Fluorescent images of living mice (a) negative control; treated with 0.2 mL of saline (b) negative control; injected with probe NRh-Cu (c) preinjected with NRh-Cu, and then
injected with Cu2+
.

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