A diaminomaleonitrile-appended BODIPY chemosensor for the selective detection of Cu2+ via oxidative cyclization and imaging in SiHa cells and zebrafish

Article abstract of DOI:10.1016/j.saa.2020.118179

A specific Cu²⁺ sensor, 2-amino-3-(BODIPYmethyleneamino)maleonitrile (BDM), was established by a simple dehydration between BODIPY and diaminomaleonitrile. Cu²⁺ could be recognized by BDM over other competing metal ions in acetonitri

Full text of DOI:10.1016/j.saa.2020.118179

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A diaminomaleonitrile-appended BODIPY chemosensor for the
selective detection of Cu2+ via oxidative cyclization and imaging
in SiHa cells and zebrafish
Xiaochuan Li, Yujie Han, Saisai Sun, Dandan Shan, Xiaoming
Ma, Guangjie He, Naveen Mergu, Jong-Su Park, Cheol-Hee Kim,
Young-A Son
PII:
S1386-1425(20)30157-8
https://doi.org/10.1016/j.saa.2020.118179
SAA 118179
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 5 December 2019
Revised date:
11 February 2020
19 February 2020
Accepted
date:
Please cite this article as: X. Li, Y. Han, S. Sun, et al., A diaminomaleonitrile-appended
BODIPY chemosensor for the selective detection of Cu2+ via oxidative cyclization and
imaging in SiHa cells and zebrafish, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy(2020), https://doi.org/10.1016/j.saa.2020.118179
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A Diaminomaleonitrile-Appended BODIPY Chemosensor for the
Selective Detection of Cu2+ via Oxidative Cyclization and Imaging
in SiHa Cells and Zebrafish
a,
a
a
a
a
b
Xiaochuan Li *, Yujie Han , Saisai Sun , Dandan Shan , Xiaoming Ma , Guangjie He ,
Naveen Mergu c, Jong-Su Park d, Cheol-Hee Kim d, Young-A Son c,*
aHenan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative
Inovation Center of Henan Province for Green Manufacturing of Fine Chemicals, Key
Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of
Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, P.
R. China;
bDepartment of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan 453003, P.
R. China;
cBK21, Department of Advanced Organic Materials Engineering, Chungnam National
University, Daejeon 34134, South Korea.
dDepartment of Biology, Chungnam National University, Daejeon 34134, South Korea.
*Corresponding authors
Xiaochuan Li (E-mail: lixiaochuan@henannu.edu.cn)
Young-A Son (E-mail: yason@cnu.ac.kr)
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Abstract
A specific Cu2+ sensor, 2-amino-3-(BODIPYmethyleneamino)maleonitrile (BDM), was
established by a simple dehydration between BODIPY and diaminomaleonitrile. Cu2+ could
be recognized by BDM over other competing metal ions in acetonitrile with distinct
fluorescence emission signal response. Upon the addition of Cu2+ to BDM in acetonitrile, the
maximum absorption at approximately 530 nm on the longer wavelength side was quenched,
and the emission at 530 nm was ignited simultaneously. The fluorescence intensity
enhancement could reach a maximum of 204 times the intensity of the BDM blank solution.
The fluorescence “off-on” effect is established according to the Cu2+-induced fast
intramolecular oxidative cyclization reaction, which could be deduced from the formation of
an
imidazole
ring
appended
to
the
cyclization
product
(2-BODIPY-1H-imidazole-4,5-dicarbonitrile, BMC). Single-crystal structure analysis of the
sensor BDM and cyclization product BMC further demonstrated this oxidative cyclization.
Finally, the Cu2+ recognition property of BDM was validated in SiHa cells and living
zebrafish. Additionally, the blood-brain barrier of the zebrafish can be penetrated by the BDM
dye and the neuron cells in the brain were stained.
Keywords: Cu2+ sensor, BODIPY, Fluorescence, SiHa cells, Zebrafish, Blood-brain barrier.
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1. Introduction
The configuration of chromophores and/or chromophores that can effectively convert light
signals into fluorescence or absorbance shifts is of great importance to bioimages, molecular
motors, and smart logic operations [1–6]. Among the various chemistries of photosignal
conversion, fluorescence signals are the easiest-to-manipulate tool for the precision
recognition of chemical and biological species due to their unique sensitivity, selectivity, and
simplicity [7–14]. Recognition of a given species must accompany the interactions between
host and guest, including hydrogen bonding, metal-ligand coordination, van der Waals forces,
and efficient chemical reactions [15–22].
Currently, copper-related diseases [23–25], such as prion, Wilson, Menkes, and
Alzheimer’s, are becoming more and more common, which seems unavoidable due to the fast
economic and metallurgical development in postindustrial societies. However, we must
determine where copper is and when we are exposed to it, and offer protection against
cooper-related diseases. The copper intake limitation was set to 1.3 ppm in daily table-water
by the U.S. Environmental Protection Agency (EPA). Therefore, chemically modifying a
molecule to respond specifically to copper ions with fluorescence signal changes is currently
a prime task for medical, environmental, and biological purposes [26]. Ion recognition has
been well documented based on fluorescence signal mechanisms, including signal “on-off”,
“off-on”, and displacement [27, 28]. These last two mechanisms are promising due to the
maneuverability of fluorescence signal enhancement. Unlike most metal ions, copper ions are
paramagnetic. Once the fluorophore coordinates with copper, the fluorescence signal is
always significantly quenched via the electron/energy transfer process [29–35]. Therefore, the
chemistry of copper-induced fluorescence emission enhancement has aroused lasting research
interest [36-38].
Among various well-established fluorescent dyes, BODIPY (4,4'-difluoro-4-bora-3a,4a-
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diaza-s-indacene) and its analogs are promising candidates for modifying compounds for
copper ion recognition due to their unique photophysical properties [38-41]. The high
emission yield and molar extinction coefficient of BODIPY predicts the achievable low
detection limit in recognition. Its chemically tunable framework, together with the robust
photo-/thermoresistance, allows the creation of a perfect fit between structure and desired
spectral character [42]. In this contribution, diaminomaleonitrile is efficiently incorporated
into the BODIPY framework. Its emission is significantly quenched due to its
electron-donating nature and the isomerization of C=N. Most diaminomaleonitrile-containing
sensors that can respond to the existing copper ions are based on coordination complexes.
Theoretically, such complexes cannot circumvent the fluorescence quenching induced by the
paramagnetism of copper [43–49]. Recently, the condensed product of BODIPY and
diaminomaleonitrile has been demonstrated as a hypochlorous acid probe [50]. However, we
found that this condensation product is more sensitive to a Cu(II)-containing environment via
Cu(II)-induced intramolecular cyclization reactions together with the fluorescence signal
enhancement. Without the copper ion involved in fluorescence signal generation, the negative
paramagnetism effect of Cu(II) in fluorescent manipulation can be well circumvented by this
intramolecular cyclization reaction.
2. Experimental
2.1. Instruments and reagents
All chemicals and reagents used in the synthesis of BMC and BDM were analytical-grade
and used with no further purification after receiving from chemical company. Spectroscopic
grade solvents (such as acetonitrile, THF, dioxane, chloroform etc.) were used in the emission
and absorption spectra measurement. Aluminium plate (~0.3 mm) coated with silica gel (5-10
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μm, ~0.25 mm thickness) was used to monitor the reaction progress (Merck DC. Alufolien
Kieselgel 60 GF254). Deionized water was used in glassware pretreatment before drying in
order to avoid unpredictable metal ion contamination. NMR spectra (1H and 13C) were
recorded on a Bruker AM-600 spectrometer with deuterated chloroform or acetone as the
solvents. Singlet (s) and multiplet (m) are used in the description of peak shape. HRMS
(High-resolution mass spectra) was recorded by using a Bruker micrOTOF Focus II
instrument and processed with Bruker Compass Data Analysis 4.4. PERSEE TU-1900 and
Perkin Elmer LS-55 fluorescence spectrophotometers were used to record absorption and
emission spectra, respectively. An FLS 980 fluorometer (Edinburgh Instruments Ltd.),
equipped with an integrating sphere system, was used to recording the absolute quantum yield.
The cuvettes used in the above mentioned spectral measurement were synthetic quartz glass,
which had 1 cm path length and more than 82% transmission of 200~2500 nm light for empty
cell. Leica DMI8 (an inverted fluorescence microscope) was used for the cell imaging
capture.
2.2. X-ray data collection and structure determination
The suitable crystals were selected under a stereoscopic microscope, and then covered with a
layer of hydrocarbon oil (Parabar 10312) to avoid possible efflorescence. Once the selected
crystal was mounted on a CryoLoopTM (Hampton Research), it was moved to the workbench
of four-circle diffractometer (SuperNova, Dual, Cu at home/near, EosS2). The diffraction data
for the crystals were collected on a CCD plate (EosS2) at Cu Kα of micro-focus sealed X-ray
tube (SuperNova Cu X-ray Source, λ = 1.54178 Å). The structures were obtained by
processing the diffraction data with SHELXS-97 as implement in Olex2 and refined against
F2 by full-matrix least-squares. CCDC files 1880861 and 1880862 contain the supplementary
crystallographic data, and a summary of the crystal data can be found in the supplementary
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information. These data can also be obtained free of charge from the Cambridge
Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge, CB2 1EZ, UK
(http://www.ccdc.cam.ac.uk/conts/retrieving.html).
2.3 General procedures
Stock solutions (1.0 × 10−2 M) of the chlorate salts of various metal ions (Hg2+, Ag+, Co2+,
Al3+, Zn2+, Ni2+, Ba2+, Pb2+, Mg2+, Fe3+ Cu2+, Mn2+, Cd2+, and Cr3+) were prepared in
acetonitrile. Probes 2-amino-3-(BODIPYmethyleneamino)maleonitrile (BDM) and
BDM+Cu2+ were dissolved in acetonitrile to prepare the stock solutions (1.0 × 10−5 M). In
titration experiments, 2.5 mL of probe solution in a quartz optical cell was titrated with Cu2+
(40 mM in acetonitrile, each time 10 μL) prepared in advance. After mixing the probe
solutions for several seconds, they were directly used in the spectroscopic measurement. To
guarantee the reproducibility of the experimental phenomena, all experiments were repeated
2~3 times.
2.4 Cell and Zebrafish imaging
Images of Siha cells were captured under a Leica DMI8 inverted fluorescence microscope.
Before digital images acquisition and processing, the SiHa cells were pretreated according to
the standard procedures that we described in previous publication (Details see Supplementary
Information).
In order to get the images of probe and cooper ions interacting in live animal model
organism, zebrafish was chosen as carrier to treat with synthesized probe and cooper ions,
which was approved by Animal Ethics Committee of Chungnam National University (CNU
00393). Zebrafish were obtained from the Zebrafish Center for Disease Modeling (ZCDM).
The obtaining of adult fish was start from the incubation of zebrafish embryos in egg water
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(60 μg/mL sea salt in distilled water, Sigma). Finally, the Sensor and/or cooper ions -treated
zebrafish were used in the image acquisition with a fluorescence microscope (DM 5000B,
Leica). Our animal experimentation was under the restriction of CNU 00393 issued by AEC
(Animal Ethics Committee) of Chungnam National University. More detailed procedure can
be found in Supplementary Information.
2.5. General procedure for the preparation and characterization of BDM and BMC
The synthetic routes for the diaminomaleonitrile and cyclized derivative of BODIPY are
depicted in Scheme 1. The aldehyde unit is introduced under typical Vilsmeier-Haack
conditions [51]. Subsequently, diaminomaleonitrile is condensed with the aldehyde group,
appending to BODIPY. While this manuscript was in preparation, Han’s group reported the
above condensation and its response toward HClO [50]. Our method is slightly different from
that of the literature in that BDM can be obtained under milder conditions. Additionally,
Cu(II)-induced intramolecular oxidative cyclization with γ-aminoimine is more efficient than
that with HClO, yielding 2-BODIPY-1H-imidazole-4,5-dicarbonitrile (BMC).
2.5.1 BDM (2-amino-3-(BODIPYmethyleneamino)maleonitrile)
BY-CHO (600 mg, 1.7 mmol) and diaminomaleonitrile (216 mg, 2.0 mmol) were dissolved in
acetonitrile (40 mL). One drop of trifluoroacetic acid was added, and the mixture was heated
to reflux for 48 hours. Then, it was cooled to 0 °C until the dark-red solid fully precipitated
from the solution. The crystalline BDM was isolated in 82% yield (615 mg).
M.p.: 288-289 ºC; IR (KBr, υ, cm-1): 488, 500, 676, 822, 880, 976, 1022, 1216, 1438, 1460,
1508, 1578, 1601, 1941, 2198, 2225, 2924, 2958, 3370, 3477; 1H NMR (600 MHz, CDCl3) δ:
8.40 (s, 1H), 7.53 (s, 3H), 7.29 (s, 2H), 6.13 (s, 2H), 4.83 (s, 2H), 2.82 (s, 3H), 2.61 (s, 3H),
13
1.58 (s, 3H), 1.41 (s, 3H); C NMR (150 MHz, CDCl3) δ: 160.9, 159.4, 155.7, 153.1, 143.0,
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138.1, 134.5, 133.8, 129.7, 129.6, 128.0, 123.9, 122.6, 117.3, 114.0, 112.3, 110.5, 15.2, 15.0,
14.5, 12.5; HRMS (ESI): C24H21BF2N6Na [M+Na] requires 465.1787; found: 465.1809.
2.5.2 BMC (2-BODIPY-1H-imidazole-4,5-dicarbonitrile)
BDM (60 mg, 0.136 mmol) was dissolved in dichloromethane (40 mL). Cautiously, copper
perchlorate (c = 4.1 × 10−2 M, in acetonitrile) was added dropwise, allowing the solution color
to turn from dark red to yellow-green. Once BDM was fully reacted as confirmed by TLC, the
solvent was removed under vacuum. The residue was loaded onto the column and eluted with
dichloromethane and methanol (100:1), yielding 30 mg of red needles (50% yield).
M.p.: > 300 ºC; IR (KBr, υ, cm-1): 491, 555, 727, 831, 976, 1008, 1050, 1072, 1171, 1190,
1229, 1259, 1302, 1535, 1577, 2226, 2923, 3380; 1H NMR (600 MHz, acetone-d6): δ 12.99 (s,
1H), 7.65 – 7.60 (m, 3H), 7.48 – 7.47 (m, 2H), 6.31 (s, 1H), 2.65 (s, 3H), 2.58 (s, 3H), 1.53 (s,
3H), 1.46 (s, 3H). 13C NMR (150 MHz, acetone-d6) δ 161.2, 152.8, 147.5, 147.2, 144.4, 140.4,
135.3, 134.1, 130.9, 130.5, 130.5, 128.9, 124.3, 119.7, 15.0, 14.9, 13.5, 12.9. HRMS (ESI):
C24H20BF2N6 [M+H] requires 441.1811; found: 441.1826.
3. Results and discussion
3.1 Absorption spectroscopy response toward Cu2+
BDM, the probe we synthesized, is stable both in solid and in common organic solvents. It
exhibits medium to good solubility toward the frequently-used solvents such as toluene,
chloroform, acetonitrile, THF, methanol, DMSO, and DMF. Even though the BDM solutions
are subjected to room light irradiation for several days, there is no observable color fading.
Detailed absorption behavior was investigated in the above mentioned solvents (Fig. S7).
With the variation in solvent polarity, the maximum absorption at longer wavelengths ranged
from 530–540 nm, and the S0 → S1 transition contributed to this absorption. The weaker
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absorption at approximately 300~440 nm, contributed by the S0 → S2 transition, constituted of
0-2, 0-1, and 0-0 vibration transition, respectively. Basically, the variation of polar
environment did not induce significant solvatochromism. It has been reported that the
emission spectral response of BDM to the oxidation of hypochlorous acid (HClO), based on
the double bond (C=N) broken and regeneration of an aldehyde attached BODIPY [50].
Moreover, a high similar analogue of BDM, in which a methoxy group substituted at the
non-function position of BODIPY, fluorescently responded to the oxidation of dichromate,
based on the same mechanism as that of hypochlorous acid [52]. However, the authors, who
configured the analogue of BDM, figured it as an anion probe (dichromate anion) only and
missed out on its response to Cu2+ strangely. In our investigation, we found the more sensitive
response of BDM where Cu2+ is present and report herein a fluorescent probe based on a
Cu2+-induced intramolecular cyclization reaction. Fig. 1 shows the absorption change of
BDM in acetonitrile with Cu2+ addition. Upon the gradual addition of Cu2+ to BDM in
acetonitrile, the absorption bands at longer (530 nm) and shorter (300~430 nm) wavelengths
show a gradual decrease. When totally 4.2 equivalents of Cu2+ was mixed with the BDM
solution, the foregoing maximum absorption at approximately 530 nm decreased to near the
optical transparency. Meanwhile, a new absorption peak, centered around 500 nm, appeared
and increased dramatically. Well-defined isosbestic points appeared at 280 (ε = 1.5 × 104 M-1
cm-1), 434 (ε = 7.0 × 103 M-1 cm-1), and 512 (ε = 5.1 × 104 M-1 cm-1) nm, supporting the
transformation of the two-component system. Furthermore, there exist a perfect linear
dependence between the Cu2+ concentration and absorption intensity with the correlation
coefficients as high as 0.9970 (0-3.75 equivalents). In comparison, the existence of selected
competitive metal ions did not induce a similar absorption blueshift as that of Cu2+ addition
(Fig. 2). This behavior demonstrated the specificity of BDM toward Cu2+.
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3.2 Emission spectroscopic response toward Cu2+
The imine unit, characterized with double bond, came into the state of a rapid isomerization
when excited with light. When it appended to BODIPY, this rapid isomerization in the excited
state is radiationless deactivated and leads to a significant emission quenching of BDM in
solution. The absolute fluorescence quantum yield (AFQY) was measured to be 2.03% in
acetonitrile, which is almost same as that of the relative fluorescence quantum yield (RFQY)
referred to fluorescein in 0.1 M NaOH (2.9%) [50]. However, Cu2+ addition not only
blueshifts the maximum absorption at the longer wavelength but can also significantly
enhance the fluorescence emission (Fig. 3). Upon addition of Cu2+ (0-3.3 equiv.) to BDM
gradually in acetonitrile, the emission centered at 530 nm appeared and the intensity increased
in sync with the Cu2+ addition. Meanwhile, the solution color turned bright yellowish-green
[Supporting video: Fast-response fluorescence of BDM in acetonitrile (1.0 × 10−5 M) toward
Cu2+]. The emission intensity no longer increased when totally 3.15 equiv. of Cu2+ added. At
this point, the ration of the emission intensities centered at 530 nm became as high as 204
times that in the absence of Cu2+. The fluorescence intensity remained constant when more
than 3.15 equivalents of Cu2+ was added. The AFQY of the above solution was determined to
be 51.0%, which further indicates that this solution is more highly emissive than that of BDM
in acetonitrile. The fluorescence lifetime recorded at the emission maximum was estimated to
be 2.8 ns with single-exponential distribution (Fig.3 bottom right). Additionally, there exists
an excellent linear dependence with respect to emission intensity and Cu2+ concentration with
a correlation coefficient of 0.9957 (Fig.3 top right). In terms of this linear relationship, the
detection limits (DL) based on the fluorescence changes were calculated to be 2.92 × 10-7 M
using the expression of 3σ/k (σ: the standard deviation of the blank solution; k: the slope of the
calibration plot), which is low enough and within the limit of maximum acceptable daily
intake limit of Cu2+ ions (1.3 ppm) in table-water set by US EPA. This also demonstrates
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that the emission signal response of BDM induced by Cu2+ is more sensitive than the response
induced by HClO with respect to the equivalent proportion of Cu2+/BDM and fluorescence
signal increment.
To assess the metal-specific fluorescence responses of BDM toward Cu2+, different metal
ions were treated with BDM under identical conditions. The changes in the fluorescence
spectra caused by Cu2+ and other different metal ions (10 μM), containing some alkaline-earth
and transition-metal ions (Hg2+, Ag+, Co2+, Al3+, Zn2+, Ni2+, Ba2+, Pb2+, Mg2+, Fe3+, Mn2+,
Cd2+, and Cr3+), in acetonitrile are shown in Fig. 4. Apart from Cu2+, the metal ions list we
selected cannot induce similar enhancement of the emission intensity as that of Cu2+ induced,
demonstrating the Cu2+-specific response. In addition, selectivity for specially appointed
metal ion over many other competing metal ions is a direct challenge to the chemists in
building chemosensors. The selectivity of BDM to Cu2+ was assessed under competing
conditions. The consequence of competing metal ions was also investigated by the addition of
Cu2+ to the BDM solution with the existence of Hg2+, Ag+, Co2+, Al3+, Zn2+, Ni2+, Ba2+, Pb2+,
Mg2+, Fe3+, Mn2+, Cd2+, or Cr3+ (Fig. 5). The emission intensity enhancement induced by the
addition of Cu2+ did not interfered by the coexistence of other competing ones.
From the point of view of the naked eye, BDM in acetonitrile (10 μM) was also
responsive toward Cu2+. Without the presence of Cu2+, the BDM solution had a pinkish color,
and one can observed fluorescence quenching only (Fig. 6). With the addition of 3 equivalents
of competitive metal ions to the BDM solution (except Cu2+), a change in the color or
fluorescence was unobservable compared to that of the free BDM solution. When 3
equivalents of Cu2+ was dropped into the solution, the pinkish color faded in less than one
second and changed to orange. Under the excitation of a 365 nm handheld UV lamp (~500
mW/cm2), only the solution containing BDM and Cu2+ exhibited bright green fluorescence, as
shown in Fig. 6.
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3.3 X-ray crystal analysis
The crystal structure of BDM and the highly fluorescent product BMC, which was formed by
Cu2+-induced intramolecular cyclization, were obtained by liquid-liquid diffusion of water/
acetonitrile (for BDM) and water/methanol (for BMC). The X-ray molecular structure of
BDM is depicted in Fig. 7a. BDM crystalized in a P1 space group with two molecules per
lattice. The BODIPY ring and diaminomaleonitrile are coplanar and bond to both sides of the
C=N in a trans configuration. It has been demonstrated that the excited dye molecules, when
irradiated with UV-vis light, could release their energy to the ground state by the C=N
isomerization in radiationless way predominantly; thus, most of C=N containing dyes are
often weakly or not fluorescent. Theoretically, inhibition of C=N isomerization or changing
the bonding character of C=N bond could lead to the regeneration of highly efficient
fluorescence emission character, in which the former could be more effective than the latter
due to the easy operation of coordination or “click”-type chemistry. In this paper, Cu2+
induced a fast intramolecular oxidative cyclization of γ-aminoimine, forming a
five-membered ring. When BMC was treated with Cu2+, highly fluorescent BDM was
generated immediately, and the crystal structure of BMC further verified the intramolecular
oxidative cyclization (Fig. 7b). The crystal lattice of BDM is similar to that of BMC with the
same space group and molecule number, in which the solvent molecules of acetonitrile and
methanol provide intermolecular hydrogen bonding, promoting the formation of a single
crystal. The hydrogen bonds can be found in the crystal packing diagrams of BMC (Fig. 7c)
and BDM (Fig. 7d), which is the main driving force for the regular packing of dye molecules.
The crystal structure of BMC also indicated the formation of an imidazole ring, which is on a
plane with the BODIPY ring, and the dihedral angle was calculated to be 4.35. The AFQY of
BMC in acetonitrile was determined to be 51.0%, which is the same as that of BMC+Cu2+ in
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acetonitrile. It is clear that isomerization of C=N is inhibited by the formation of the
imidazole, thereby leading to efficient fluorescence emission. Additionally, this imidazole
avoids the generation of coordination complexes between Cu2+ and BMC due to deficient
coordination sites.
3.4 Fluorescence detection of Cu2+ in SiHa cells
For the biological application of BDM as a probe, the ability of the biosensing molecule to
selectively detect a guest species in living cells is of great importance. SiHa cells were
selected for this end. The SiHa cells were incubated with BDM (2 μM) for not less than 3
hours to guarantee efficient cell permeability, and then the free BDM dye were rinsed with
PBS buffer. Fluorescence images of SiHa cells, treated with/without Cu2+, were captured by a
Leica DMI8 inverted fluorescence microscope. As expected, the SiHa cells cannot be
differentiated under a dark field by blue channel laser excitation, as shown in the bright field
(Fig. 8a, 8c). Once the cells were treated with 1 equivalent of Cu2+, bright green fluorescence
were ignited in the cells, and a clear green cell image was obtained under the laser excitation
of the blue channel of a Leica DMI8 (Fig. 8b). In addition, the cells were still alive,
demonstrating no obvious toxic side effects from BDM and BMC during the photographic
operations process (approximately 2 hours). These results demonstrate that BDM has the
feature of detecting intracellular Cu2+ ions, establishing a biological tool.
3.5 Fluorescence detection of Cu2+ in zebrafish
To further validate the BDM as a fluorescent probe in biological systems, it was examined in
the vertebrate model live higher organism, the zebrafish (Danio rerio), due to the rapid
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development of embryo and formation of major organs. What is more, the size and
transparency of zebrafish meet the requirement of various chemical assays and in vivo
bioimaging. The 5-day-old zebrafish larva was subjected to Cu2+ (5 μM) in egg water and
subsequently the remaining Cu2+ ions were washed off. Finally, the larva was incubated in
BDM containing solution and a series of images were obtained. As shown in Fig. 9, there is
weak or no emission when the zebrafish treated with BDM and Cu2+ ions alone. By contrast,
the zebrafish exhibited bright green emission when treated with both Cu2+ and BDM. These in
vivo studies indicated that BDM was suitable for tracking the distribution of Cu2+ in a
tissue-specific manner in living organism. The green fluorescence was observable not only in
the intestine, but also in the brain. BDM is able to slip past the protective blood-brain barrier
of zebrafish (Fig. 10a). When BDM transformed into BMC in the presence of Cu2+, the brain
tissues were ignited by bright green fluorescence. Fig. 10b shows the brain-specific image of
a zebrafish. In addition, with neuronal biomarkers of pan-neuronal marker (HuC) and
Parkinson’s and Alzheimer’s disease-related genes (Fig. 10c, sncb and aplp1), it confirms that
BDM could effectively stain the neuronal cells and detect the existence of Cu2+. This
indicates that BDM benefits to this penetration and staining ability more than that of the
documented fluorescent probes [53–55]. The sncb image was obtained from the zebrafish
database (http://zfin.org/).
4. Conclusion
In summary, BDM, the condensed product of BODIPY and diaminomaleonitrile, was
demonstrated as a fluorescent probe toward Cu2+. The recognition mechanism is ascribed to
the typical Cu(II)-induced intramolecular cyclization reaction. The single crystal structure
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analysis further verified the generation of imidazole, resulting of the oxidative cyclization of
γ-aminoimine. Based on this fast reaction, BDM exhibited a specific fluorescence “turn-on”
when Cu2+ exists within the detection limit, calculated to be 2.92 × 10-7 M. The in vivo
detection of Cu2+ was also testified successfully by using SiHa cells and living vertebrate
model, zebrafish.
Acknowledgments
This work was supported by the National Natural Science Foundation of China [grant
numbers 21772034]; the Program for Innovative Research Team in Science and Technology
in Universities of Henan Province [grant numbers 19IRTSTHN023, 20IRTSTHN005].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://
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Declaration of interests
√ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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Credit Author Statement
Xiaochuan Li: Writing–Review and Editing, Conceptualization, Formal analysis(X-ray single
crystal diffraction and data solving), Supervision Yujie Han: Formal analysis, Visualization,
Investigation (Data and image processing, Single crystal cultivation) Saisai Sun: Investigation
(Organic preparation, Single crystal cultivation, Spectroscopy measurement, Data collection
and processing) Dandan Shan: Investigation (Organic preparation/Characterization,
Spectroscopy measurement and data processing) Xiaoming Ma: Resources Guangjie He:
Methodology (Cell culture and bio-image processing) Naveen Mergu: Methodology
(Establishment of vertebrate model, Bio-image processing) Jong-Su Park: Methodology
(Establishment of vertebrate model, Bio-image processing) Cheol-Hee Kim: Supervision,
Draft editing, Young-A Son: Reviewing and Editing, Supervision
Scheme 1. Synthesis of BMC.
Figure 1. UV-vis spectral changes for BDM upon the addition of Cu2+ (0-4.2 equiv.). Insert:
Dependence of absorption intensity at 530 nm with respect to the concentration of Cu2+
(0-3.75 equiv.).
Figure 2. UV-vis spectra of BDM (1.0 × 10−5 M) in acetonitrile with various metal ions (1
equiv.).
Figure 3. Changes in the emission intensity of BDM (1.0 × 10−5 M) in acetonitrile with the
continued addition of Cu2+ (0-3.15 equiv; λex = 470 nm). Insert: Dependence of emission
intensity at 530 nm with respect to the concentration of Cu2+ in acetonitrile (top right) and
fluorescence decay recorded at 530 nm (bottom right).
Figure 4. Emission spectra of BDM (1.0 × 10−5 M) in acetonitrile in the presence of various
metal ions 10 μM (Ag+, Ba2+, Mg2+, Al3+, Mn2+, Pb2+, Co2+, Cd2+, Ni2+, Zn2+, Hg2+, Fe3+, and
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Cr3+; λex = 470 nm).
Figure 5. Emission intensity of BDM (1.0 × 10−5 M) upon the addition of various metal ions
(3.0 equiv.) in acetonitrile with and without the presence of Cu2+.
Figure 6. Absorption (top) and fluorescence (bottom) changes of BDM in acetonitrile (10 μM)
with various metal ions (Cu2+, Hg2+, Ag+, Co2+, Ba2+, Mg2+, Cd2+, Al3+, Ni2+, Zn2+, Fe3+, Mn2+,
Pb2+, and Cr3+).
Figure 7. X-ray crystal structure of BDM (a) and BMC (b) with displacement ellipsoids
shown at the 30% probability level and crystal packing diagrams of BDM (c) and BMC (d).
Figure 8. Fluorescence images of SiHa cells incubated with BDM (2 μM) (a) and further
incubated with 1 equiv. of Cu(ClO4)2 (b) and their corresponding bright field images (c, d).
Figure 9. Images of zebrafish incubated with BDM before and after treatment with Cu2+.
Figure 10. Head images of zebrafish staining by BDM before and after treatment with Cu2+.
Side view (a), Top view (b), Images of brain structures (c). HuC-GFP transgenic zebrafish,
showing GFP (green fluorescent protein) signals under control of the pan-neuronal HuC gene
promoter (top). mRNA expression of Parkinson’s disease-related gene (bottom left), or
Alzheimer’s disease-related gene (bottom right) in zebrafish brain.
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Highlights
 Intramolecular oxidative cyclization reaction based sensor was established.
 Single crystal structure analysis was also demonstrated.
 Sensing property was validated in Siha cells and living vertebrate model, zebrafish.
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Figure 1

Figure 2

Figure 3

Figure 4