A fluorescent probe for hypochlorite with colorimetric and fluorometric characteristics and imaging in living cells

Article abstract of DOI:10.1016/j.ab.2018.11.011

An excellent fluorescent probe with “turn on” phenomenon for sensitive monitoring hypochlorite (ClO^?) was prepared using the mild condensation reaction between coumarin and hydroxylamine (NH₂OH). The probe possessed potent selectivity to ClO^? with obvious color changes from yellow to light yellow and green to blue fluorescence emission, which could be noticed by the naked eye. Moreover, the probe has been succeeded in imaging ClO^? in living A549 cells and thus has the potential prospect in the visual detection of intracellular ClO^?.

Full text of DOI:10.1016/j.ab.2018.11.011

Accepted Manuscript
A fluorescent probe for hypochlorite with colorimetric and fluorometric characteristics
and imaging in living cells
Cunjie Hu, Jianping Li, Liqiang Yan
PII:
S0003-2697(18)30682-1
https://doi.org/10.1016/j.ab.2018.11.011
YABIO 13184
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 4 July 2018
Revised Date: 7 November 2018
Accepted Date: 9 November 2018
Please cite this article as: C. Hu, J. Li, L. Yan, A fluorescent probe for hypochlorite with colorimetric
and fluorometric characteristics and imaging in living cells, Analytical Biochemistry (2018), doi: https://
doi.org/10.1016/j.ab.2018.11.011.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
A fluorescent probe for hypochlorite with colorimetric and fluorometric
characteristics and imaging in living cells
a
a
a
Cunjie Hu, Jianping Li and Liqiang Yan, *
a
Guangxi Colleges and Universities Key Laboratory of Food Safety and Detection, Guangxi Key
Laboratory of Electrochemical and Magnetochemical Functional Materials, College of
Chemistry and Bioengineering, Guilin University of Technology, Guilin, Guangxi 541004 P. R.
China
*
E-mail: liqiangyan@glut.edu.cn
1

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Abstract
An excellent fluorescent probe with “turn on” phenomenon for sensitive monitoring
−
hypochlorite (ClO ) was prepared using the mild condensation reaction between coumarin and
−
hydroxylamine (NH OH). The probe possessed potent selectivity to hypochlorite (ClO ) with
2
obvious color changes from yellow to light yellow and green to blue fluorescence emission,
which could be noticed by the naked eye. Moreover, the probe has been succeeded in imaging
−
ClO in living A549 cells and thus has the potential prospect in the visual detection of
−
intracellular ClO .
Keywords: coumarin derivative; fluorescence probe, hypochlorite, colorimetric change,
fluorescence imaging
2

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1
. Introduction
−
Hypochlorite (ClO ) is one of the very important reactive oxygen species (ROS) mainly including
−
1
−
−
hydroxyl ion (OH ), oxygen ( O ), hydrogen dioxide (H O ), nitrite (NO ), hypochlorite (ClO )
2
2
2
2
-
and son on in the human body, which is biologically generated from chloride ions (Cl ) and H O
2
2
−
with the catalysis of enzyme myeloperoxidase.[1-3] ClO has an essential influence on cellular
defense system and organism immunity such as the prevention of infection and the destruction
−
of bacteria.[4-6]
ꢀ
However, when the concentration level of ClO is higher than normal, it will
bring about oxidative stress and tissue damage along with a lot of related risks including
immunodeficiency, myocardial damage, nerve injury, nephropathy, angiocardiopathy and
tumor.[7-12] Therefore, the design and development of the convenient and fast analysis
−
methods for the recognition of ClO is of vital significance.
−
At present, all kinds of detection methods have successfully been used to test ClO , such as
potentiomeric analysis, electrochemical method, chemiluminescence method and fluorescence
analysis.[13-17] In these methods, fluorescence probe has incomparable superiorities such as
user-friendly control, time saving, simple processing, good specificity, high sensitivity and cell
−
imaging.[18-20] Also some fluorescence probes of ClO have been successfully
developed.[21-24] Nevertheless, some of them took too long time in the detection process,
which limited their applications in real time detection,[25, 26] some of them possessed poor
−
detection limits,[27-29] some of them did not achieve ClO detection in live cells,[26, 30] and
others showed typical “turn off” characteristics, which is inconvenient to visual detection of
−
−
ClO .[31] For instance, Yin et. al developed two fluorescence probe for ClO based on
−
coumarin.[32] These both probes could be used in detecting intracellular ClO , however, they
showed serious “turn off” appearance, which hindered application in the visual detection of
−
ClO . Therefore, there are still a lot of works to be done in quantity and performance of
−
fluorescence probe of ClO .
3

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Herein we synthesized a simple compound (probe 1) based on coumarin and hydroxylamine
(
NH OH) without tedious chemical reactions,[33] which can be used as a dependable testing
2
−
implement of ClO with both ratiometric and turn on characteristics. Coumarin was selected as
a fluorescent chromophore to build up various kinds of fluorescence probes on the strength of
its prominent characteristics including good luminous performance, low cost, low biotoxicity,
easy to synthesis and excitation wavelength and emission wavelength are both in a visible light
area.[34] Hydroxylamine of probe 1 could transform into a positive derivative formation with
−
arresting fluorescence after interacting with ClO .[35] This probe has many advantages like as
−
the low limit of detection (0.21 μM), higher selectivity for ClO over other species, quick
response, and noteworthy transformations of color and fluorescence. More importantly, probe
−
1
2
2
has been implemented in the detection of ClO in living A549 cells.
. Experimental
.1 Reagents and chemicals
4
-(Diethylamino)salicylaldehyde (99%), Hydroxylamine hydrochloride (98.5%), Diethyl
malonate (99.5%), Hydrochloric acid (36-38%), Phosphorus oxychloride (99.5%), ethyl alcohol
99.5%), Triethylamine(99.5%), N,N-Dimethylformamide (99.9%), Acetic acid, glacial (99%)
were purchased from commercial suppliers and used directly without further purification.
(
-
2-
Double-distilled water was used to prepare solution throughout the experiment. ClO , SiO ,
3
2
-
-
-
−
-
S O , H PO , MnO , F, ClO and NO were severally prepared using their metal salts (NaClO,
2
3
2
4
4
4
2
−
- 1
Na SiO , Na S O , NaH PO , KMnO KF, NaClO and NaNO ). O , O and ·OH were produced
2
3
2
2
3
2
4
4,
4
2
2
2,
on the basis of relevant literatures.[36-38] The pH value of PBS solution (10 mM) was set as 7.4
by adjusting the amount of NaOH solution. A549 cells and MCF-7 cells were purchased from
ATCC.
2
.2 Apparatus
Fluorescence spectra were gained by Cary Eclipse fluorescence spectrophotometer (Varian,
4

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1
13
USA). H and C NMR spectra were measured using an AVANCE III HD NMR spectrometer (600
MHz, Bruker, Germany) and TMS as the internal standard. Melting point was examined by a
SGW X-4 melting point apparatus (Shanghai precision instruments Co., China). UV-visible
spectra were acquired using a Lambda 950 spectrometer (PerkinElmer, USA). The fluorescence
images were acquired by a laser scanning confocal microscopy (Olympus IX 71, Japan). The
testing conditions of all experiment were performed at room temperature.
2
.3 General Procedure
.3.1. Solution preparation
2
Probe 1 was easily dissolved in EtOH (10 mL) and was diluted with PBS solution (90 mL) to
obtain a stock solution of probe 1 (1 mM). Then the stock solution of probe 1 was diluted with
PBS-EtOH solution (9:1, v:v, 10 mM, pH = 7.4) to the final concentration according to the
testing requirement. The testing samples of analytes were prepared in PBS-EtOH solution and
were diluted to the target concentrations for test. The absorption and fluorescence spectra of
probe 1 with various analytes were measured after equilibrated for 3 minutes. The excitation
wavelength (λ ) of the fluorescence spectra was set as 365 nm.
ex
2
.3.2. Cell Imaging
A549 cells were cultivated for 30 minutes with probe 1 (10 μM) in DMEM culture at 37 °C, and
−
washed three times with PBS, and then incubated with ClO (20 μM) for 5 minutes. After then,
the cell images were photographed using green channel (500-600 nm) and blue channel (430-
4
70 nm), respectively.
.3.4. Synthesis
-diethylamino coumarin (3) and 7-diethylamino coumarin-3-aldehyde (2) were synthesized
according to the known methods (as shown in scheme 1).[39]
2
7
5

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Scheme 1. Synthesis of compound 1.
Compound 2 (0.49 g, 0.2 mmol), hydroxylamine hydrochloride (0.21 g, 0.3 mmol) and
triethylamine 0.5 mL were dissolved in anhydrous EtOH (20.0 mL). The precipitate taken shape
after refluxed for 16 h, and then filtered off by suction filtration, washed with ice EtOH once
again and dried in a vacuum drying chamber. The target product 1 as yellow solid could be
1
given. Yield 0.23 g (44.2%). m.p. 205.1-206.8℃. H NMR (DMSO-d , 600 MHz, δ ppm): 11.31 (s,
6
1
1
H), 8.16 (s, 1H), 7.99 (s, 1H), 7.56, 7.54 (d, J = 12 Hz, 1H), 6.75, 6.73 (d, J = 12 Hz, 1H), 6.56 (s,
13
H), 3.47-3.43 (q, J = 6 Hz, 4H), 1.14-1.12 (t, J = 6 Hz, 6H). C NMR (DMSO-d , 150 MHz, δ
6
ppm): 160.66, 156.72, 151.46, 143.32, 138.85, 130.81, 111.95, 109.96, 108.27, 96.84, 44.63,
+
1
2.81. EI-MS: 260.12 [M+H] . Elemental Analysis (%): C, 64.41; H, 6.32; N, 10.26, Calculated (%)
for C H N O : C, 64.59; H, 6.19; N, 10.78.
14
16
2
3
3
. Results and Discussion
−
3
.1. Ultraviolet-visible spectrum of probe 1 with ClO
−
The ultraviolet-visible spectra of probe 1 with ClO were tested firstly through the titration
experiment in PBS-EtOH solution (9:1, v/v, 10 mM, pH 7.4). As shown in Fig. 1, probe 1 (10 μM)
showed an obvious absorption ban around 397 nm. After titration with different dopamine
−
concentrations of ClO (0, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11
μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM), the
6

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ultraviolet-visible absorption bands at 397 nm gradually decreased. These changes suggested
−
that probe 1 possesses prominent ultraviolet-visible spectral response towards ClO .
Fig. 1. The ultraviolet-visible spectra of probe 1 (10 μM) in PBS-EtOH solution with different
−
levels of ClO .
−
3
.2. The fluorescence selectivity of probe 1 for ClO
−
The fluorescence selectivity of probe 1 for ClO was carried out and the situation was shown in
Fig. 2. Probe 1 displayed green fluorescence in the absence of other analytes. When 2 equiv. of
−
ClO was added to the solution of probe 1, an outstanding enhancement of fluorescence
−
2-
intensity with blue shift of the emission peaks from 506 nm to 465 nm. Whereas ClO , SiO ,
3
2
-
-
-
−
-
- 1
S O , H PO , MnO , F, ClO , NO , O , O ·OH and H O did not has an evident influence to
2
3
2
4
4
4
2
2
2,
2
2
the emssion spectra of probe 1 at the same concentrations. The results of these tests
−
demonstrated probe 1 has high selectivity for ClO over other ananlytes.
7

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.
Fig. 2. Fluorescence spectra of probe 1 (10 μM) in PBS-EtOH solution with various
analytes (20 μM).
−
The antijamming capability of probe 1 (10 μM) to the special recognition of ClO against other
analytes was also identified by means of competition experiment. The peak values of
−
fluorescence spectra of probe 1 with ClO (20 μM) and other competition analytes (20 μM)
−
2-
2-
-
-
−
−
−
−
1
(
including a: ClO , b: SiO , c: S O , d: H PO , e: MnO , f: F , g: ClO , h: NO , i: O , j: O , k:
3 2 3 2 4 4 4 2 2 2
·
OH, and l: H O ) were revealed in Fig. 3. Obviously, it can be easily observed that the
2 2
−
fluorescence intensities of probe 1 with ClO showed no evident changes between with and
without competition species. In other words, the tested competitive analytes didn't adversely
−
disturb the detection capability of probe 1 to ClO . It can thus be concluded that this probe has
−
high selectivity in the determination of ClO even when other species coexisted in abundance.
8

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−
Fig. 3. The peak values of fluorescence spectra of probe 1 (10 μM) with ClO coexisted
−
2-
2-
-
4
-
−
with other competing analytes. a: ClO , b: SiO , c: S O , d: H PO , e: MnO , f: F , g:
3
2
3
2
4
−
−
−
1
ClO , h: NO , i: O , j: O , k: ·OH, and l: H O .
4
2
2
2
2 2
−
3
.3. The fluorescence sensitivity of probe 1 for ClO
−
After verified the high selectivity of probe 1 towards ClO , we are more than happy to take our
interests to the sensitivity. The fluorescence spectra of probe 1 towards different gradient
−
concentrations of ClO (0, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM
,
20 μM) were collected and displayed in Fig. 4. The fluorescence intensities of probe 1 appeared
−
a trend of enhancement accompanied by the concentrations of ClO . In addition, the
−
fluorescence intensity reached the steady value when 2 equiv. of ClO was transferred to the
solution of probe 1. More importantly, the changes of the maximum fluorescence intensities of
−
the probe 1 linearly heightened accompanied by the accumulation of ClO concentrations. A
straight-line formula could be established between the fluorescence ratiometric signals Fmax/F0
−
2
and the concentrations of ClO on a scale of 0 to 18 μM: y=0.17+0.18x (R =0.9832, Fig. 4 Inset).
−
The detection limit (DL) of probe 1 for ClO was defined as 0.21 μM when the signal-to-noise
−
ratio (S/N) was 3.[40] Interestingly enough, with the concentration increasing of ClO , the
maximum emission wavelength of probe 1 solution also have a considerable blue-shift trend
−
drastically (Fig. 5). These results indicated that probe 1 is very sensitive to ClO and can be used
for the quantitative determination in biological systems.
9

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−
Fig. 4. Fluorescence spectra of probe 1 (10 μM) with different concentrations of ClO .
Inset shows the linearity between the peak values of fuorescence and the
−
concentrations of ClO .
Fig. 5. Linear relationship between the maximum emission wavelength of probe 1 (10
−
μM) in PBS-EtOH solution and the concentration of ClO .
−
The excellent recognition ability of probe 1 to ClO was visible with the naked eye. As shown in
−
Fig. 6, only when ClO (20 μM) was added, the color of the probe 1 solution became colorless
2
-
2-
4-
-
−
from obvious yellow, however, the other species including SiO , S O , H PO , MnO , F, ClO ,
3
2
3
2
4
4
-
-
1
NO , O , O ·OH and H O did not brought the color change. Moreover, the specific
2
2
2,
2
2
−
fluorescence color change of probe 1 towards ClO could be clearly distinguished under a
−
hand-held UV Lamp. Upon the addition of ClO , there was a significant fluorescence change of
1
0

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probe 1 from green to blue. However, all of the competing species have no noticeable influence
on the fluorescence color of probe 1. Based on the above results, the probe 1 could be used as
−
a simple and convenient visible probe for ClO .
Fig.6. Photographs of the color and fluorescent emission of probe 1 in H O-EtOH
2
solution after addition different common anions under sunlight and 365 nm UV-lamp,
−
2-
2−
3
4−
−
−
−
respectively. 1: probe 1, 2: ClO , 3: SiO , 4: S O , 5: H PO , 6: MnO , 7: F , 8: ClO ,
3
2
2
4
4
−
−
1
9
: NO , 10: O , 11: O , 12: ·OH, 13: H O .
2
2
2
2
2
−
3
.4. The detection time of probe 1 for ClO
The detection time of probe is an important factor in practical application. Therefore, the
−
response time of probe 1 to ClO was investigated. The kinetic characteristic of fluorescence
−
intensity over action time between probe 1 and ClO was displayed in Fig. 7. When 20 μM of
−
ClO was added to 10 μM of probe 1 solution, the emission maximum increased dramatically
and was nearly maximized in about 2 min. This demonstrated that probe 1 could be served as a
−
rapid identification tool for monitoring ClO .
1
1

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−
Fig. 7. The dynamic characteristic of probe 1 (10 μM) in the present of ClO (2 equiv.).
3
.4. Cell imaging and cytotoxicity assay
In order to examine the application performance in biological sample, the fluorescence imaging
−
experiments of probe 1 with and without ClO were further performed in living cells. When
A549 cells were incubated with probe 1 for 30 minutes, the strong green fluorescence could be
−
observed (Fig. 8b and 8c). And then incubated with 2 equiv. ClO , the green fluorescence (Fig.
8
e) of cells faded away completely and arresting blue fluorescence appeared after 5 minutes
(
Fig. 8f). In addition, A549 cells have almost no fluorescence when they were incubated with
−
ClO for 5 min (Fig. 8h and 8i). The cytotoxicity of the probe 1 was also evaluated via MTT assay
of MCF-7 cells.[41] The results of MTT assays indicated that cell viability was over 85% when
probe 1 was used below 80 μM (Fig. 9). These results demonstrated that the probe had
-
significant fluorescence change and low cytotoxicity, which is suitable for the detection of ClO
in living cells.
Fig. 8. Confocal microscopy images of A549 cells. Cells incubated with 10 μM probe 1:
bright-field image (a) and fluorescence image (b, c). Cells incubated with 10 μM probe
1
2

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−
1
and 20 μM ClO : bright-field image (d) and fluorescence image (e, f). Cells incubated
−
with 20 μM ClO bright-field image (g) and fluorescence image (h, i). Scale bar
represents 20 μm in every image.
Fig. 9. The changes of MCF-7 cell viabilities after incubation with probe 1 in range of 10
μM to 100 μM. Error bars represent the standard deviations of 3 trials.
4
. Conclusions
In conclusion, a coumarin-based spectroscopic probe with both colorimetric and fluorometric
-
−
features for the detection of ClO has been successfully prepared. Along with addition of ClO ,
the color of the probe solution showed a marked turnaround from yellow to colorless, while
other competing species hardly displayed any noteworthy color change. Additionally, the
fluorescence of the probe can turn into blue color from green color with the enhancement of
−
fluorescence intensity. The probe also has a fast response process for detection of ClO about 2
minutes. Based on the excellent virtues of rapid detection, remarkable color and fluorescence
changes, low toxicity, low limit of detection and high specificity, the probe has a good
−
application prospect in the fluorescence testing of ClO in living cells.
Acknowledgements
This work was supported by Guangxi Key Laboratory of Electrochemical and Magnetochemical
Functional Materials (EMFM20181107), Foundation of Guilin University of Technology
1
3

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(
GUTQDJJ2017), the Natural Science Foundation of Guangxi Province of China (Project
No.2015GXNSFFA139005) and High Level Innovation Teams of Guangxi Colleges & Universities
and Outstanding Scholars Program (Guijiaoren [2014]49).
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Highlights
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Observable colorimetric and fluorometric changes.
Fast response time;
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Highly selective and sensitive response to ClO ;
Strong contrast imaging in living cells.