A “reactive” turn-on fluorescence probe for hypochlorous acid and its bioimaging application

Article abstract of DOI:10.1016/j.saa.2018.07.090

An aza-BODIPY-CNOH probe attached aldoxime group demonstrated the specific detection for hypochlorous acid by the turn-on red emission signal. NMR and HRMS experiments confirmed that the fluorescence originated from the oxidation degradation of the non-fluorescence, aldoxime-based aza-BODIPY-CNOH probe into the red-fluorescence, nitrile oxide-based aza-BODIPY compound aza-BODIPY-CNO. The aza-BODIPY-CNOH probe showed good biocompatibility and was low toxic to living cells as shown from MTT experiments. Living RAW264.7 cells imaging indicated the aza-BODIPY-CNOH probe had good permeability and either exogenous or endogenous HClO caused the intracellular bright-red fluorescence, showing its potential hypochlorous acid-specific sensing ability in biological systems.

Full text of DOI:10.1016/j.saa.2018.07.090

Accepted Manuscript
A “reactive” turn-on fluorescence probe for hypochlorous acid
and its bioimaging application
Yunling Gao, Yong Pan, Yu Chi, Yuanyuan He, Haiyan Chen,
Victor N. Nemykin
PII:
DOI:
Reference:
S1386-1425(18)30754-6
doi:10.1016/j.saa.2018.07.090
SAA 16359
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Received date: 14 May 2018
Revised date:
Accepted
date:
23 July 2018
30 July 2018
Please cite this article as: Yunling Gao, Yong Pan, Yu Chi, Yuanyuan He, Haiyan Chen,
Victor N. Nemykin , A “reactive” turn-on fluorescence probe for hypochlorous acid and
its bioimaging application. Saa (2018), doi:10.1016/j.saa.2018.07.090
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ACCEPTED MANUSCRIPT
A “reactive” turn-on fluorescence probe for hypochlorous acid and its bioimaging
application
Yunling Gaoa,c*, Yong Pana, Yu Chia, Yuanyuan Heb, Haiyan Chenb**, Victor N. Nemykinc***
a State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical
Engineering, Zhejiang University of Technology, Hangzhou 310032, China.
b Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, Nanjing
210009, China.
c Department of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
*Corresponding author: Tel: +86-571-88320951; Email address: gaoyl@zjut.edu.cn
**Corresponding author: Tel: +86-25-83271080; Email address: chenhaiyan@cpu.edu.cn
**Corresponding author: Tel: +1-204-474-9265; Email address: Victor.Nemykin@umanitoba.ca
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Abstract
An aza-BODIPY-CNOH probe attached aldoxime group demonstrated the specific detection for
hypochlorous acid by the turn-on red emission signal. NMR, IR, and HRMS experiments confirmed that the
fluorescence originated from the oxidation degradation of the non-fluorescence, aldoxime-based
aza-BODIPY-CNOH probe into the red-fluorescence, nitrile oxide-based aza-BODIPY compound
aza-BODIPY-CNO. The aza-BODIPY-CNOH probe showed good biocompatibility and was low toxic to
living cells as shown from MTT experiments. Living RAW264.7 cells imaging indicated the
aza-BODIPY-CNOH probe had good permeability and either exogenous or endogenous HClO caused the
intracellular bright-red fluorescence, showing its potential hypochlorous acid-specific sensing ability in
biological systems.
Keywords: aza-BODIPY, aldoxime, turn-on probe, red-emission
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1. Introduction
Fluorescent dyes are widely applied in chemosensors [1-4], optical data storage[5, 6], photodynamic
therapy[7-9], and biological imaging[10-13]. Visible to near-infrared fluorescent dyes have attracted wide
interests in biological sensing filed, and well developed as fluorescence probes or sensors due to their high
sensitivity and specify, real-time vitalization, much deeper light penetration in the tissue and lower the
interference of auto-fluorescence than ultraviolet fluorescence dyes. Among these fluorescent dyes,
aza-BODIPYs [14-17] are excellent candidates, as they have good photo-stability, high molar absorption
coefficients, narrow absorption/emission band and good fluorescence quantum yield. In addition, their
electric structures and optical properties are easy to tailor, therefore various aza-BODIPYs were proposed as
optical sensors[18-20], photodynamic therapy agents[21-25], photoredox catalysts[26-28], solar energy
materials[29-31] and the super molecular building blocks[32].
Hypochlorous acid (HClO) is a significant reactive oxygen species (ROS) in living organisms mainly
produced from the myeloperoxidase-catalyzed (MPO) reaction of hydrogen peroxide and chloride ion[33,
34]. HClO plays a critical role to defense invading bacterial in living organisms. However high
concentration level of HClO would damage the tissue and cause various diseases, such as arthritis[35, 36],
kidney disease[37, 38], cardiovascular disease[39], and even cancer[40]. Therefore, the real-time monitoring
HClO level in living organisms is very important and highly demanded.
Fluorescence probes with the capability of the real-time visual detection of HClO, have been rapidly
developed in recent years [41, 42]. Various recognition groups sensitive to HClO, such as chalcogenide[43,
44], p-methoxyphenol[45], hydroxylamine[46, 47] and acylhydrazine[48, 49], were used in design and
synthesis of the HClO-specific probes. Among them, an aldoxime group[50, 51] frequently is employed to
successfully design turn-on HClO probes where the HClO oxidation of the aldoxime group removes its
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rotation and isomerization effects to light up the fluorescence. In this work, an aldoxime-based aza-BODIPY
core probe (aza-BODIPY-CNOH) was synthesized. The turn-on probe showed the excellent selectivity to
HClO over multiple oxidants and physiologically relevant ions. The sensing mechanism was well
established using NMR and HRMS. In addition, the probe was applied in living cells to explore exogenous
and endogenous HClO detection.
2. Results and discussion
2.1 Synthesis and characterization of the aza-BODIPY-CNOH probe
Scheme 1. Synthesis of the aza-BODIPY-CNOH probe.
The aza-BODIPY-CNOH probe was easily prepared from aza-BODIPY aldehyde reacted with
hydroxylamine sulfate in pyridine at 60 °C (Scheme 1), and characterized using 1H NMR, 13C NMR, IR, and
HRMS (Figs. S1-3). 1H NMR spectrum (Fig. S1) shows the aldehyde peak at 9.79 ppm has disappeared and
the HRMS confirmed the formation of the probe aza-BODIPY-CNOH with the m/z at 559.1927 ([M-H]-)
(Fig. S3).
2.2 Spectroscopic properties of the aza-BODIPY-CNOH probe
The aza-BODIPY-CNOH probe is almost non-fluorescence in the test solution (HEPES-acetonitrile, v/v =
1:1, pH = 7.2) with the quantum yield at 0.0012 (Fig. 1a). When 10 μM aza-BODIPY-CNOH was subjected
to the 300 μM of sodium hypochlorite solution (pH = 7.2), an increase in the red fluorescence was observed.
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The fluorescent intensity increase was 7.7 times compared to that of the probe alone, which suggested that
the aza-BODIPY-CNOH probe might be oxidized since the aldoxime H-C=N-OH group probably tends to
isomerize and rotate to quench fluorescence by a non-radiative deactivation[52, 53]. In addition, the
maximum emission wavelength was red-shifted to 667 nm from 660 nm (FL =0.0094). Uv-vis spectrum of
the aza-BODIPY-CNOH probe showed the decrease of absorbance at 636 nm and slight blue-shift to 631
nm with the addition of 300 μM of sodium hypochlorite solution (Fig. 1b).
Fig. 1. Fluorescence (a) and UV-vis spectra (b) of the aza-BODIPY-CNOH probe (10 μM) in the absence and presence of sodium
hypochlorite (300 μM) in HEPES/CH3CN (1:1, pH = 7.2) solution. Red stars: sodium hypochlorite plus the probe; black stars: the
probe alone.
The fluorescent titrations of the aza-BODIPY-CNOH probe with sodium hypochlorite solution were then
carried out shown in Fig. 2. The gradual increase in fluorescent intensity was observed with the addition of
sodium hypochlorite up to 150 μM (Fig. 2a). Meanwhile, the aza-BODIPY-CNOH probe has undergone a
distinct emission change from the non-fluorescence to red. The fluorescent intensity demonstrated the good
linearity with the concentration of sodium hypochlorite in the range of 0 to 120 μM based on the linear
equation y = 2.92x + 13.66 with a linear coefficient of 0.997. The limit of detection was calculated as 2.33
μM (Fig. 2b).
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Fig. 2. a) Fluorescent changes of the aza-BODIPY-CNOH probe with the addition of different concentrations of sodium
hypochlorite (0-150 μM) in HEPES/CH3CN (1:1, pH = 7.2) solution. b) The linear relationship of the fluorescence intensity and
the concentration of sodium hypochlorite at 667 nm.
2.3 Selectivity of the aza-BODIPY-CNOH probe
120
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Fig. 3. Fluorescence intensity changes of the aza-BODIPY-CNOH probe (10 μM) with the addition of individual ions (300 μM).
In order to determine the selectivity of the aza-BODIPY-CNOH probe, other ROS/RNS species including
-
-
- 1
H2O2, NO, NO2 , NO3 , O2 , O2, ·OH, ONOO- and t-BuOOH were added individually to the 10 μM probe
solution. As showed in Fig. 3, these ROS/RNS species caused no appreciable fluorescence changes.
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2-
2-
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2-
Similarly, common anions, like F-, Cl-, Br-, I-, HCO3 , CO3 , HSO4 , H2PO4 , ClO4 , S2-, SO32- and SO4 , did
not affect the fluorescence of the probe. Meanwhile, common metal cations were also explored (Al3+, Ca2+,
Zn2+, Pb2+, Cr3+, Fe3+, Cu2+, Hg2+, K+, Mn2+, Mg2+, Na+, Ni+ and Ag+) and showed negligible effect on the
probe. Hereby, the aza-BODIPY-CNOH probe showed the specific sensing ability for hypochlorous acid.
Fig. 4. Effects of various interfering ROS/RNS (300 μM) species on the fluorescence intensity of the aza-BODIPY-CNOH probe
at 667 nm. Grey bars: aza-BODIPY-CNOH plus sodium hypochlorite; Red bars: aza-BODIPY-CNOH plus sodium hypochlorite
followed by adding other ROS/RNS.
Next, the competition experiments were performed to test whether the aza-BODIPY-CNOH probe can
sense hypochlorous acid efficiently and the results were shown in Fig. 4 and Fig. S4. As expected, Figure 4
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shows that the ROS/RNS species have not obvious effect on the fluorescence of the aza-BODIPY-CNOH
probe in sensing hypochlorous acid except slight decrease in fluorescent intensity for ONOO-. Meanwhile,
the addition of hypochlorous acid caused distinct fluorescence increase after the aza-BODIPY-CNOH
probe was treated with different interfering ROS/RNS species (Fig. S4). Therefore, the
aza-BODIPY-CNOH probe demonstrated the good anti-interference ability to other ROS/RNS species and
could be an excellent turn-on fluorescence probe for detection of hypochlorous acid.
2.4 pH effect on the aza-BODIPY-CNOH probe
Aldoxime group in the structure of aza-BODIPY-CNOH might undergo protonation or deprotonation as a
function of pH values, therefore the relative fluorescence of the probe was studied in the presence and
absence of sodium hypochlorite solution. The pH titrations were performed with the 10 μM probe
aza-BODIPY-CNOH in the absence or presence of 50 μM sodium hypochlorite solution (Fig. S5). The
aza-BODIPY-CNOH probe kept the negligible changes of the weak fluorescence in the range of pH 2 to 13,
while in the presence of sodium hypochlorite, the fluorescence intensities were increased over the whole test
pH range, indicating the aza-BODIPY-CNOH probe has potential to sensing both HClO and ClO-.
Although the fluorescence intensities have got the extremely significant increase in the alkali region (pH 8 to
12) where the sensing species are probably dominated by hypochlorite (pKaHClO = 7.5[54]), the experiments
were still set up at pH = 7.2 because the physiological pH values are preferred at neutral in biological
system[55] where HClO can be detected by the probe using the fluorescence. Another reason to use pH 7.2
in all experiments was the probe may be less stable in more alkali pH, as was seen the decrease fluorescence
at pH 13 in Fig. S5.
2.5 Sensing mechanism
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NMR and HRMS experiments were carried out to explore the mechanism of the turn-on fluorescence of the
aza-BODIPY-CNOH probe by HClO (Fig. 5). The excess sodium hypochlorite was added to the probe
solution in chloroform monitored by TLC to monitor an appearance of a new compound with the red
fluorescence. The product was purified using flash column chromatography and characterized by NMR and
HRMS (Fig. 5).
Fig. 5. 1H NMR spectra of the aza-BODIPY-CNOH probe and isolated aza-BODIPY-CNO in CDCl3-d
Aldoximes as sensing groups for HClO/ClO- were reported to be transferred to aldehyde[56], nitrile
oxide[57], or the carboxylic acid[58]. Here, the aza-BODIPY nitrile oxide product was isolated
(aza-BODIPY-CNO) when aza-BODIPY-CNOH and sodium hypochlorite were mixed. Compared to the
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1H NMR spectrum of the aza-BODIPY-CNOH probe, the proton on pyrrole of aza-BODIPY-CNO was
shifted to 7.21 ppm vs 7.11 ppm of that on the probe and the proton peaks of the probe appearing at
7.78-7.66 disappeared accompanying an obvious new peak at 7.98 ppm. The MALI-TOF-MS demonstrated
the molecular ion peak of aza-BODIPY-CNO at m/z 538.1777 (calculated at 538.1788) (Fig. S6). Based on
the characterization of the isolated product including NMR and HRMS, the turn-on fluorescence of the
probe aza-BODIPY-CNOH in the presence of HClO might come from the oxidation degradation of the
aldoxime group of the aza-BODIPY core [57]. Here, the aldoxime group may be converted into the nitrile
oxide and hence the rotation and isomerization effects of the aldoxime group are removed. Additionally, the
rigid nitrile C≡N may slightly extend the π conjugation of aza-BODIPY core and therefore the
LUMO-HOMO energy gap agrees with the small red-shift in absorption and emission wavelengths showed
in Figs. 1-2.
2.6 Cell imaging
Fig. 6. Cell viability of the RAW264.7 cells in the presence of the aza-BODIPY-CNOH probe.
To demonstrate the potential application in biological fields, the MTT and cell imaging experiments were
explored and the results were shown in Figs. 6-7. As shown in Fig. 6, even high concentration (50 μM) of
the aza-BODIPY-CNOH probe was used, the cell variability was still above 80%, showing its low toxicity
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and good biocompatibility to living cells. Therefore, the probe has potential application in vivo experiments.
Next, the confocal fluorescence imaging experiments were conducted in living RAW264.7 cells (Fig. 7).
The probe demonstrated the good permeability and stained in cells with the very weak red-fluorescence as
shown in Fig. 7, a-c. Addition of 10 μM of sodium hypochlorite solution to the living cells pre-treated with
the probe for 10 min followed by wash thrice using PBS buffer caused the bright red fluorescence emission
from living RAW264.7 cells (Fig. 7, e and f). When the living cells were treated with 10 μM of the probe for
10 min and then 25 ng/mL of phorbol ester 12-myristate 13-acetate (PMA) was introduced and incubated for
2 h, PMA also induced the bright red fluorescence (Figs. 7, h and i). All the bright field and emerged images
confirmed the intracellular red emission. The results above indicated the aza-BODIPY-CNOH probe has
good sensing ability for exogenous and endogenous HClO in living cells.
Fig. 7. Fluorescence images of the RAW 264.7 cells incubated with 10 μM of aza-BODIPY-CNOH probe for 30 min (top) and
10 μM of aza-BODIPY-CNOH probe (10 min) followed by treatment of sodium hypochlorite (10 μM) for 1h (middle) or PMA
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(25 ng/mL) (bottom) for 2 h for exogenous and endogenous HClO imaging, respectively. a, d, g: bright field images; b, e, h:
fluorescence images ; c, f, i: emerged images of bright field and fluorescence images.
3. Conclusions
An aldoxime-based probe with an aza-BODIPY core has been synthesized and characterized using NMR, IR,
and HRMS spectra. The aza-BODIPY-CNOH probe can selectively detect HClO based on the turn-on red
-
-
-
fluorescence. Other ions including H2O2, NO, NO2 , NO3 , O2 , 1O2, ·OH, ONOO-, t-BuOOH, F-, Cl-, Br-, I-,
-
2-
2-
-
-
2-
2-
HCO3 , CO3 , HSO4 , H2PO4 , ClO4 , S2-, SO3 , SO4 , Al3+, Ca2+, Zn2+, Pb2+, Cr3+, Fe3+, Cu2+, Hg2+, K+,
Mn2+, Mg2+, Na+, Ni+ and Ag+ did not significantly affect the sensing of the probe to HClO. The oxidation
product isolated from the reaction of the probe and sodium hypochlorite were characterized, indicating the
aldoxime group of the probe aza-BODIPY-CNOH was oxidized to the nitrile oxide to form a new
aza-BODIPY-CNO compound which was responsible for the development of the turn-on red fluorescence.
The aza-BODIPY-CNOH probe can penetrate and stain in living cells. The probe inside living RAW264.7
cells showed good response to exogenous and endogenous HClO by adding sodium hypochlorite and PMA
induction. The survival rate experiments reflected low toxicity of the probe to living cells, suggesting the
potential in vivo experiments.
4. Experimental section
4.1 Materials and instruments
The cell line used was purchased from American Type Culture Collection (ATCC, USA). PMA and other
chemicals including organic solvents were obtained from Sigma-Aldrich and TCI. NMR, IR and
high-resolution mass spectra were recorded using the Bruker AVANCE III spectrometers, the Thermo Fisher
Nicolet 6700 and the DECAX-60000 mass spectrometer, respectively. Fluorescence and absorption spectra
were obtained using the Hitachi F-7000 fluorescence spectrometer and the GE Healthcare Ultraspec 3300
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spectrophotometer. An FV1000 confocal fluorescence microscope was used to collect the images of the
living cells.
4.2 Synthesis of the probe aza-BODIPY-CNOH
To a three-neck-round flask, aldehyde aza-BODIPY[59] (100 mg, 0.19 mmol) and 10 mL pyridine were
added and then the mixture was heated at 60 °C with agitation. Next, hydroxylamine sulfate
(65 mg, 0.76 mmol) was added at multiple times and the reaction was monitored using TLC. After 8 h, the
mixture was evaporated and the solid was dissolved in dichloromethane, followed by filtration. The filtrate
was concentrated and the product was purified using the silica gel column chromatography
1
(dichloromethane: n-hexane = 10:1) to obtain 24 mg of the aza-BODIPY-CNOH probe. Yield: 21.6%. H
NMR (500 MHz, CDCl3, ppm) δ 8.07-7.99 (m, 5H), 7.78-7.77 (m, 2H), 7.69-7.66 (m, 2H), 7.5-7.39 (m,
13
12H), 7.11 (s, 1H). C NMR (125 MHz, CDCl3, ppm) δ 144.34, 131.87, 131.70, 131.64, 131.60, 131.01,
130.67, 130.33, 130.22, 130.17, 130.01, 129.96, 129.91, 129.52, 129.26, 128.89, 128.84, 128.11, 127.98,
ESI-MS calculated for [M+H]+ at 541.2 and [M+Na]+ at 563.2; HRMS calculated for [M-H]- at 539.1866,
found: 539.1927.
4.3 General procedures for the fluorescence and UV-vis spectra measurement
The stock solution of the aza-BODIPY-CNOH probe (1.0 mM) was prepared in acetonitrile. The test
solutions were set up at 10 μM (HEPES-acetonitrile, v/v = 1:1, pH = 7.2). The competitive cations and aions
used in stock solutions (10 mM) are Al3+, Ca2+, Zn2+, Pb2+, Cr3+, Fe3+, Cu2+, Hg2+, K+, Mn2+, Mg2+, Na+, Ni+,
-
2-
2-
-
-
2-
2-
-
-
Ag+, F-, Cl-, Br-, I-, HCO3 , CO3 , HSO4 , H2PO4 , ClO4 , S2-, SO3 , SO4 , NO2 and NO3 . ROS and RNS
-
-
including HClO, H2O2, t-BuOOH, •OH, O2 , 1O2, ONOO- , NO and O2 were obtained from the commercial
reagents for HClO, H2O2, t-BuOOH and KO2[60], from Fenton reaction by mixing the same equivalent of
H2O2 and FeSO4·7H2O for hydroxyl radical (•OH)[61], by mixing NaOCl and H2O2 at a molar ratio of 1:2
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for singlet oxygen (1O2)[62], using sodium nitrite, acidic hydrogen peroxide and sodium hydroxide as
reagents for peroxynitrite (ONOO−)[41], and from sodium nitroferricyanide (III) nihydrate for nitric oxide
(NO)[63]. Fluorescence measurements were configured at room temperature, 2400 nm per min of the
scanning speed, 5 nm and 10 nm slit width for the excitation and emission, respectively.
4.4 Cell imaging
RAW264.7 cells were used in cell imaging, and incubated in the culture dishes until 70-80% space was
taken up. 10 μM of the aza-BODIPY-CNOH μM (HEPES-acetonitrile, v/v = 1:1, pH = 7.2) was added to
the culture dishes and incubated for 30 min at 37 °C and then cells were washed three times with PBS. In
order to get exogenous and endogenous HClO fluorescence imaging, 10 μM of sodium hypochlorite (pH =
7.2) and 25 ng/mL of PMA were added individually to the culture dishes where the cells has been pre-treated
with the probe for 10 min, and incubated for 1 h and 2 h at 37 oC, respectively. Each dish above was washed
three times with PBS and observed by confocal fluorescence microscope. The cell viability was also
performed using MTT colorimetric method. The cells were incubated with varied concentrations of the
probe aza-BODIPY-CNOH (100 μL) for 24 h at 37 °C and then washed PBS buffer thrice. Next, the cells
were incubated with MTT solutions (20 μL, 5 mg/mL) for 4 h at 37 °C. After removing the supernatant and
adding 100 μL DMSO, the cell viability was obtained by measuring the absorbance at 490 nm.
Acknowledgements
National Natural Science Foundation of China (21777143 and 81671803) and National Key Research and
Development Program (Grant No. 2017YFC0107700) are acknowledged for funding. VN would like to
acknowledge support from NSERC and CFI.
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Graphical abstract
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Highlights
 A turn-on aza-BODIPY-CNOH probe has been synthesized and characterized.
 The aza-BODIPY-CNOH probe showed specific fluorescence recognition for HClO.
 The turn-on fluorescence may originate from the aza-BODIPY nitrile oxide product oxidized by HClO.
 The aza-BODIPY-CNOH probe can detect exogenous and endogenous HClO in living cells.
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