A novel cholesterol conjugated fluorescence probe for Cu2+ detection and bioimaging in living cells

Article abstract of DOI:10.1016/j.saa.2019.117530

A cholesterol conjugated fluorescence probe T was designed and synthesized. The probe T can be used for recognition of Cu²⁺ by the absorption spectrum, fluorescence spectrum, and naked eyes respectively in aqueous solution. The cell imaging experiments showed that the probe has good membrane permeability and a huge potentiality for the detection of Cu²⁺ in living cells.

Full text of DOI:10.1016/j.saa.2019.117530

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A novel cholesterol conjugated fluorescence probe for Cu2+
detection and bioimaging in living cells
Jia Ting, Fu Mei, Zhang Mengyao, Qiu Jianwen, Zhu Hu, Gao
Yong
PII:
S1386-1425(19)30920-5
https://doi.org/10.1016/j.saa.2019.117530
SAA 117530
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 31 July 2019
Revised date:
9 September 2019
12 September 2019
Accepted
date:
Please cite this article as: J. Ting, F. Mei, Z. Mengyao, et al., A novel cholesterol
conjugated fluorescence probe for Cu2+ detection and bioimaging in living cells,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2019),
https://doi.org/10.1016/j.saa.2019.117530
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© 2019 Published by Elsevier.

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A novel cholesterol conjugated fluorescence probe for
Cu2+ detection and bioimaging in living cells
Jia Ting1, Fu Mei1, Zhang Mengyao1, Qiu Jianwen1, Zhu Hu1,2, Gao Yong1,2*
(1. College of Chemistry and Materials Science, Fujian Normal University, Fuzhou 350007, China;
2. Fujian Provincial University Engineering Research Center of Industrial Biocatalysis, Fujian
Normal University, Fuzhou, 350117, China)
Abstract
A cholesterol conjugated fluorescence probe T was designed and synthesized. The
probe T can be used for recognition of Cu2+ by the absorption spectrum, fluorescence
spectrum, and naked eyes respectively in aqueous solution. The cell imaging
experiments showed that the probe has good membrane permeability and a huge
potentiality for the detection of Cu2+ in living cells.
Keywords
Cholesterol; Fluorescence; Probe; Cu2+; Bioimaging
1. Introduction
For organic living organisms, many transition metal ions play a vital
role[1]. Copper as the third most abundant transition metal element in
human body after iron and zinc plays an important role in physiological
process[2]. As a catalyst cofactor, it plays a key role in the synthesis of
many metal enzymes. Copper is distributed in all tissues and body fluids
of the human body, and the highest copper content is found in the liver[3].
However, excessive Cu2+ can lead to neurasthenia, such as Alzheimer's
disease, Wilson's disease and elicit uncontrolled Fenton oxidation which
is the important reasons of cancerization[4-5]. Considering the multifaceted
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physiology functions, many techniques have been reported for the
detection of trace copper ions in biological systems, such as, atomic
absorption spectrometry (AAS)[6], atomic fluorescence spectrometry
(AFS)[7], ion-coupled plasma-mass spectroscopy (ICP-MS)[8] and high
performance liquid chromatography (HPLC)[9]. However, these methods
of copper determination require extraction and pre-concentration step
before analysis, which is time-consuming and expensive[10-11]. Therefore,
it is urgent to find a new analytical technique for the detection of copper
ions in living organisms. Recently, fluorescent probes have been
developed as highly sensitive, rapid, visual, lossless and inexpensive tools
for copper determination in biology research[12]. These fluorescent probes
have a significant fluorescence response to Cu2+ with specific selectivity
and high sensitivity, but few of them can be used in biological samples by
the lack of biocompatibility[13-14] and membrane permeability[15].
As a kind of biological amphiphile, cholesterol is an important
component of mammalian cell membranes. It plays an indispensable role
in regulating cell membrane properties in mammalian cells[16]. It is
reported that cholesterol moiety can greatly improve the biocompatibility
and membrane permeability of a drug delivery system.[17] But it is
surprising that there are seldom reports about the utility of cholesterol in
fluorescent probe[18].
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After the first rhodamine-based copper ion fluorescent probe[19] was
reported in 1997, rhodamine hydrazide has been considered as a typical
framework to develop many copper ion fluorescent probes by utilizing
the property of spirocyclic closed and ring-opened forms, which exhibits
a “turn-on” signal response[20-24]
.
In this work, we designed and synthesized a novel cholesterol conjugated
fluorescent Cu2+ probe T (Scheme1). Based on the homoousian of
cholesterol with membrane lipids, we envisage that probe T should have
better biocompatibility and membrane permeability.
On the other hand, recent studies have shown that cholesterol and its lipid
regions composed of other lipid components of membranes are not only
involved in the distribution of membrane proteins on the cell membrane
surface but also play an important role in many cellular events such as
cell signal transduction, immune response activation and substance
transport.[25] A deep understanding of cholesterol metabolism is key
toward developing efficient and targeted drugs for overcoming
cholesterol-related disorders. Therefore, research for visualization of
cholesterol in cells has caused widespread concern[26-27]. Interestingly, the
probe T we designed can also be considered as visualized cholesterol by
triggering with Cu2+.
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Scheme 1. The synthesis process of T
2. Experimental
2.1 Materials and physical methods
All reagents for synthesis were purchased from commercial suppliers and
stored in a vacuum desiccator. They were used without further
purification. The solutions of metal ions were prepared from their
perchlorate salts. Tris-HCl buffer solutions (50 mM, pH=7.4) were
prepared in deionized water. Fluorescence spectra were recorded on a
RF-5310 PC Fluorescence Spectrometer. UV–visible spectra were
1
13
recorded on a UV-1750UVspectrometer. H NMR and C NMR spectra
were recorded on a VarianUnity-400 spectrometer operating at 400 MHz
and 100 MHz, respectively. Chemical shifts are reported in ppm
downfield from tetramethyl silane (TMS, δ scale) with the solvent
resonances as internal standards. High-resolution mass spectra were
conducted on a Bruker microTOF-QII ESI-MS. Cell image was
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performed on Olympus FV1000-IX81 Fluorescence imager (100-fold oil
mirror).
2.2 Synthesis of intermediate 1
A solution of cholesterol (2.0718g, 36mmol) in DCM (50ml) was added
chloroacetyl chloride (2.5mL) at room temperature. The reaction solution
was heated to reflux for 10h. After the reaction was completed, the
mixture was cooled to room temperature and washed with H2O (30
mL×3). The organic phase was dried over anhydrous MgSO4 and
evaporated in vacuum. The crude product was further purified by column
chromatography (Petroleum ether: DCM = 50:1) to afford intermediate
1
1(1.2848g, 51.77%). HNMR (400 MHz, CDCl3, ppm) δ: 5.41(s, 1H,
=CH), 4.05 (s, 2H, -CH2CO-), 2.38 (d, 2H), 2.10-0.8(m, 19H), 0.69 (s, 3H,
-CH3). (Fig.S1)
2.3 Synthesis of intermediate 2
Intermediate 1 (0.2972g, 0.64mmol), potassium carbonate (0.1838g,
1.33mmol) and potassium iodide (0.1077g, 0.64mmol) were combined in
acetonitrile (10 mL) and stirred. Salicylaldehyde (0.2mL, 1.9mmol) was
added dropwise. The reaction mixture was heated to reflux for 7h. The
mixture was cooled and concentrated by evaporation under reduced
pressure. The residue was dissolved in DCM (20mL) and was washed
with H2O (30 mL×3). The organic phase was dried over anhydrous
MgSO4 and the solvent was evaporated under reduced pressure, afforded
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1
the intermediate 2 (0.3222g, 91.48%). HNMR (400 MHz, CDCl3, ppm)
δ: 10.59(s, 1H, CHO), 7.88(d, 1H, ArH), 7.55(t, 1H, ArH), 7.10(t, 1H,
ArH), 6.88(d, 1H, ArH), 5.37(s, 1H, =CH), 4.73 (s, 2H, -CH2CO-), 2.38
(d, 2H, ), 2.10-0,8(m, 19H), 0.65 (s, 3H, -CH3). (Fig.S2)
2.4 Synthesis of T
Rhodamine hydrazide was synthesized according to the literature
procedures [21]. Rhodamine hydrazide (0.2121g, 0.46mmol) was dissolved
in absolute ethanol (20mL) and stirred. The intermediate 2 (0.3016g,
0.55mmol) in DCM (10mL) was added dropwise. The reaction mixture
was heated to reflux for 4h. After cooling to room temperature, the
reactant was stored for several days, and a white precipitate was formed.
The precipitate was filtrated and dried in vacuum to afford T (0.2096g,
1
45.71%). HNMR (400 MHz, CDCl3, ppm) δ: 7.99(d, 2H, ArH), 7.88(d,
1H, ArH), 7.49(m, 2H, ArH), 7.20(m, 2H, ArH), 6.93(t, 1H, ArH), 6.73(d,
2H, ArH), 6.53(d, 2H, ArH), 6.43(d, 2H, ArH), 5.37(s, 1H, =CH), 4.73 (s,
2H, -CH2CO-), 3.34(d, 8H, -CH2-) 2.38 (d, 2H, ), 2.10-0,8(m, 31H), 0.65
13
(s, 3H, -CH3). (Fig.S3) CNMR (100 MHz, CDCl3, ppm) δ: 189.66,
168.39, 167.63, 164.78, 160.52, 157.78, 155.94, 153.94, 151.82, 148.83,
143.65, 140.83, 139.40, 135.72, 133.18, 132.45, 131.14, 130.83, 129.64,
128.85, 128.12, 126.59, 125.32, 124.14, 123.88, 122.90, 121.84, 113.57,
112.67, 107.96, 106.45, 97.89, 76.76, 75.61, 71.76, 66.96, 65.88, 56.79,
50.16, 50.03, 44.34, 42.33, 39.81, 37.96, 36.95, 36.21, 35.81, 31.93,
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31.86, 29.73, 28.26, 28.03, 27.65, 24.32, 23.86, 22.85, 22.59, 21.11,
21.05, 19.42, 19.32, 18.75, 12.68, 11.89. (Fig.S4) HRMS (ESI) calcd. for
C64H81N4O5Na[M+Na]: 1009.3417; found:1009.3478. (Fig.S5)
2.5 General procedure for spectra experiments
A stock solution of T (5×10-4mol·L-1) was prepared in DMF. The solution
of T in the test system was then diluted to 1×10-5mol·L-1 with
DMF/Tris-HCl buffer solution (1:3, V/V). All fluorescence spectra are
collected at an excitation wavelength of 510 nm (slit width: 2/2 nm).
2.6 Cell imaging
MCF-7 cells were incubated with 20μM T in DMSO/PBS (1:100, v/v) for
30 min at 37oC and 5% CO2 and then washed with PBS three times. After
incubating with 5μM of Cu2+ for 30 min at 37oC and 5% CO2 and then the
cells were washed with PBS three times again. Cell imaging of Cu2+ in
MCF-7 cells was conducted by using a confocal fluorescence microscopy.
The excitation light was excited at 559nm and the 575-620nm band was
collected.
3. Results and discussions
3.1 Selectivity of the probe T to Cu2+
As the introduction of amphipathic cholesterol, the first thing we want to
know is the solubility of this new compound in aqueous solution.
Absorption spectra were recorded from 200 to 800 nm for the solutions of
T (10 μM) in a serious of different volume ratio of DMF and Tris-HCl
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buffer. (Fig.S6) There are no absorption peak from 450 nm to 800 nm
when the volume ratio of DMF and Tris-HCl buffer is 1:1, 1:2 or 1:3. It
means the probe is soluble and stable in the solutions. But when the ratio
is larger than 1:3, the absorptions are significantly enhanced. Based on
that experiment, we selected the DMF/Tris-HCl buffer solution (1:3, V/V)
as the solvent for T to do spectroscopic properties research.
The spectroscopic properties of probe T were primarily determined by the
selectivity of the probe T to Cu2+ with UV-vis absorption and
fluorescence spectra, respectively. As shown in UV-vis absorption
spectrum (Fig.1), there was no absorption peak at 562nm for the probe T.
After adding 2 equiv. amount of Cu2+, the absorbance increased obviously
at 562nm and the color changed from colorless to orange-red. But under
the same conditions, the addition of 2 equiv. amount of K+、Na+、Ca2+、
Mg2+、Zn2+、Cd2+、Mn2+、Pb2+、Fe3+ did not cause any absorption peak
and color variation.
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0.20
0.15
0.10
0.05
0.00
2+
Cu
3+
2+
2+
+
2+
Cd
，
Fe , Ca , Mg , K
2+
+
+
2+
Mn , Zn , Na , Pb ， T
450
500
550
600
650
700
750
Wavelength/nm
Fig.1.The UV-vis absorption spectra of probe T(10μM) in the presence and absence of
different metal ions (20μM) in DMF/Tris-HCl (1:3, v/v)
As shown in the fluorescent spectrum (Fig.2), T itself had no significant
fluorescence intensity. Once the probe was exposed to Cu2+, a strong
emission band appeared at 586nm accompanied by fluorescence
enhancement. While other cationic solutions were added under the same
conditions, the fluorescence spectra did not change significantly. The
results show that probe T has good selectivity for Cu2+, and the probe can
be used as a naked eye chemical sensor for the detection of Cu2+.
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50
40
30
20
10
0
2+
Cu
3+
2+
2+
+
2+
Fe , Ca , Mg , K ,Cd
2+
2+
+
2+
Mn , Zn , Na , Pb ,T
550
600
650
700
750
Wavelength/nm
Fig.2. The fluorescent emission spectra of probe T(10μM) in the presence and
absence of different metal ions (20 μM) in DMF/Tris-HCl (1:3, v/v).
3.2 The anti-interference of the probe T
The anti-interference of the probe to competing ions is very important for
the practical application of a probe. So the anti-interference experiments
were conducted with the common metal ions mentioned above,
respectively. As shown in Fig.3, the probe T does not have obvious
fluorescence in the presence of 10 equiv. interfering ions. After adding 3
equiv. Cu2+, the fluorescence emission induced an obvious fluorescence
intensity enhancement, while the other metal ions did not cause any
discernible changes. This phenomenon indicates that these co-existent
ions had a negligible interfering effect on Cu2+ sensing by T.
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50
40
30
20
10
0
2+
T + other ion + Cu
T + other ion
2+Ca2+ Mn2+
2+ Mg
Cd
Zn2+ K+
Na+ Fe3+
Cu2+
Pb2+
Fig.3. The Fluorescent emission spectra of probe T(10μM) in DMF/Tris-HCl (1:3, V/V)
solution with the addition of 10 equiv. interfering ions and 3 equiv. copper ion.
3.3 UV-vis and fluorescent titration experiments
The spectroscopic properties of T at different concentrations of Cu2+ in
DMF/Tris-HCl (1:3, V/V) solution were carried out. As shown in Fig.4,
with the adding of Cu2+a new absorption band appeared at 562nm and the
absorbance increased with the increase of copper ion concentration.
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0.28
0.21
0.14
0.07
0.00
0.19
0.18
0.17
0.16
0.15
0.14
0.13
150 160 170 180 190 200
C/μM
250μM
0μM
450
500
550
600
650
700
750
Wavelength/nm
Fig.4. Absorption spectra of T (10 M) in DMF/Tris-HCl (1:3, V/V) upon addition of
different amounts of Cu2+ ions.
Similarly, as shown in Fig.5, with the increase of copper ion
concentration (0-250μM), a fluorescence emission band appeared at
586nm and the fluorescence intensity increased gradually with the adding
of copper ion concentration. The fluorescence intensity has a good linear
relationship with Cu2+ concentration ranging from 150 to 220μM
(R2=0.98462). At this condition, the detection limit of copper ion is
5.7μM. The above results indicate that the T could serve as a good
fluorescent probe for Cu2+ detection.
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400
300
200
100
0
300
270
240
210
180
150 165 180 195 210 225
C/μM
250μM
0μM
550
600
650
Wavelength/nm
700
750
Fig.5. The fluorescent emission spectra of T (10 μM) in DMF/Tris-HCl (1:3, V/V)
upon addition of different amounts of Cu2+ ions.
3.4 Reaction mechanism of T with Cu2+
To explore the reaction mechanism of T with Cu2+, the stoichiometry of
the copper-T complex and reversibility of the reaction were investigated.
Job’s method for absorbance measurement was applied to determine the
stoichiometry. The total concentration of the probe T and copper ions was
immobilized as 2×10-4M and the probe and copper ions were formulated
to a concentration ratio of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9.
As shown in Fig.6, when the mole fraction of probe T reaches 0.5, the
maximum absorbance value appears. This indicates a 1:1 stoichiometry of
the Cu2+ to T in the complex.
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0.16
0.12
0.08
0.04
0.00
0.0
0.2
0.4
0.6
0.8
1.0
2+
2+
[Cu ]/[T+Cu
]
Fig.6 Job-plot of probe T for Cu2+ ion in DMF/Tris-HCl (1:3, V/V)
Reversibility experiments were conducted to indicate the possible mode
of interaction between T and Cu2+. As shown in Fig. S7, after the addition
of 4 equiv. EDTA, the absorption peak at 562 nm decreased to the
original pre-complex state. While re-adding 2 equiv. Cu2+ to the solution,
the signals were almost recovered. The consecutive additions of EDTA
and Cu2+ reveal that this cycle can be repeated at least three times. This
indicates that the recognition of Cu2+ by probe T is based on reversible
coordination.[28-29] According to the experiments and previously reported
results[30-31], we assumed that the T most likely to chelate with Cu2+ ions
to form a ring-opened complex (Fig. 7).
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Fig. 7. Proposed mechanism for the detection of Cu2+ by T
3.5 Cell imaging
In order to verify the ability of the probe in bioimaging, probe T was used
to detect Cu2+ in MCF-7 cells. From the bright field diagram (Fig.8A), it
can be seen that the cell morphology is healthy in the confocal imaging
experiment, indicating that the probe has excellent biocompatibility and
low cytotoxicity. Observed under confocal fluorescence microscopy,
there was significant fluorescence in the cell area. The overlay image of
bright field and fluorescence shows that the fluorescent signal is in the
cytoplasmic region, indicating that the probe has good cell membrane
permeability. In contrast, the control sample didn’t have fluorescence
(Fig.8B). These results indicate that the probe has a huge potentiality for
the detection of Cu2+ in biological cells.
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Fig.8. Fluorescence image of MCF-7 cells preloaded with T (20 μM) and incubated
with (A) 5μM Cu2+; (B) 0μM Cu2+.
4. Conclusions
In summary, a novel cholesterol conjugated fluorescent probe T for Cu2+
was synthesized and characterized. The probe shows good selectivity and
high sensitivity towards Cu2+ via a reversible binding mode over a wide
range of tested metal ions. The cell image shows that the probe T has
good cell membrane permeability and a huge potentiality for the detection
of Cu2+ in biological cells. At the same time, the fluorescent image which
was triggered by Cu2+ is also present the position of cholesterol. In this
point, the probe T has potential use in tracking cholesterol in cells. This
work is in progress by collaborators of our group and will be reported in
due time.
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Acknowledgments
This work was supported by the Natural Science Foundation of Fujian
Province (No. 2018J01671, JK2017009).
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Graphical abstract
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Highlights
● A novel cholesterol conjugated fluorescence probe T was designed and synthesized.
● The probe T exhibited a selective and sensitive response to Cu2+ in aqueous
solution.
● The cell imaging experiments exhibited that the probe has good membrane
permeability and huge potentiality for the detection of Cu2+ in living cells.
21