Accepted Manuscript

A highly sensitive and photo-stable fluorescent probe for endogenous intracellular

H2O2 imaging in live cancer cells

Fangyuan Xu, Wei Tang, Saisai Kang, Jinsheng Song, Xinrui Duan

PII: S0143-7208(17)32540-8
DOI: 10.1016/j.dyepig.2018.02.015
Reference: DYPI 6546

To appear in: Dyes and Pigments

Received Date: 13 December 2017

Revised Date: 6 February 2018
Accepted Date: 7 February 2018

Please cite this article as: Xu F, Tang W, Kang S, Song J, Duan X, A highly sensitive and photo-stable
fluorescent probe for endogenous intracellular H2O2 imaging in live cancer cells, Dyes and Pigments
(2018), doi: 10.1016/j.dyepig.2018.02.015.

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 ACCEPTED MANUSCRIPT

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A Highly Sensitive and Photo-Stable Fluorescent

Probe for Endogenous Intracellular H2O2 Imaging in
Live Cancer Cells

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Fangyuan Xu,‡,a Wei Tang,‡,a Saisai Kang,a Jinsheng Song,*,b and Xinrui Duan*,a

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a
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province and

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School of Chemistry and Chemical Engineering, Shaanxi Normal University, 620 Xi

Chang’an Street, Xi’an, Shaanxi 710119, P. R. of China.

b
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Engineering Research Center for Nanomaterials, Henan University, Kaifeng, Henan,

475004, P. R. China.
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*
Corresponding Author, E-mail: [email protected]; [email protected]
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Abstract
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Reactive oxygen species (ROS) are connected to aging and human diseases. The
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controlled generation of ROS, particularly H2O2, is necessary to maintain cellular

fitness. Fluorescent probe based imaging is the most popular approach for H2O2
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detection. The photobleaching of the fluorophores is the significant limitation towards

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better signal-to-noise ratio and imaging resolution. We design and synthesize a highly

sensitive and photo-stable fluorescent probe for detecting H2O2 by using the

2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as an electron

acceptor. After reacting with H2O2, the borate ester group in the probe was

transformed into phenol group to form red emitted fluorophore that is highly

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photo-stable under the irradiation of UV light and excitation laser. A good linear

relationship (R2 = 0.9930) in the range of 1 to 20 µM of H2O2 was obtained. The limit

of detection (3σ/slope) of our probe was 61 nM, which make our probe one of the

most sensitive fluorescent probes for H2O2 detection. And the probe doesn’t response

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to common other ROS species. The probe was successfully applied to endogenous

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intracellular H2O2 imaging in live A549 lung cancer cells. More importantly, the

imaging signal just had less than 10% difference in fluorescence intensity after 25 min

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continuous irradiation of excitation laser. Thus, we believe the proposed TCF-PB is

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very practical for sensing and imaging of trace amount of H2O2 that endogenously
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generated in the live cancer cells, which is essential for the study of H2O2 related cell

signaling and human diseases.

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Keywords: TCF, H2O2 sensing, Fluorescent probe, Cell imaging, Photo-Stability

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1. Introduction
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Reactive oxygen species (ROS) are generated in biological processes, which is a

product of oxidative metabolism and act as essential signaling molecules. It includes

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hydrogen peroxide (H2O2), singlet oxygen (1O2), hydroxyl (HO·), superoxide (O2-·),
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hypochlorous acid (HOCl) and peroxyl radical (ROO·).1-3 H2O2, a major member of
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ROS, is connected to aging and human diseases such as cardiovascular disorders,

Alzheimer’s, related neurodegenerative diseases, and cancer.4-6 Thus the controlled

generation of ROS, particularly H2O2, is necessary to maintain cellular fitness. Many

methods have been reported for detecting H2O2, for instance, electron spin resonance,7

electrochemical analysis,8,9 chemiluminescent,10,11 and UV-vis spectrophotometry.12

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To compare with these methods, fluorescent spectroscopy is of broad interest in

bio-analysis especially in cell imaging, because of its obvious advantages, such as

sensitivity, rapidity, and the ability of real-time monitoring.

Fluorescence probes for H2O2 detection have been developed during recent

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years.13,14 2,7-dichlorodihydrofluorescein (DCFH) is an indicator for ROSs.15-17 In the

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presence of H2O2, DCFH can be oxidized to DCF that emits strong fluorescence.

Based on the oxidation mechanism, a series of fluorescent probes have been

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synthesized, such as Amplex Red,18,19 DHR 123,20 and PF-H2TMRos (e.g. Redox

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Sensor Red).21 Since these probes can be oxidized by other ROS too, the selectivity is
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not satisfied. Chang and co-workers reported a highly selective fluorescent probe

based on borate ester deprotection for the imaging of H2O2, which shows selectivity
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toward H2O2 over other ROS.22 Subsequently, a series of borate ester protected
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fluorescent probes from various fluorophores23-28 have been developed. Although

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these probes show good selectivity, the photobleaching of the fluorophores is

significant limitation towards better signal-to-noise ratio and imaging resolution.29,30

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Endogenous H2O2 concentration in live mammalian cells is relatively low.27 To the

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best of our knowledge, almost of endogenous H2O2 fluorescence imaging in live cells
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are using stimulated/oxidative stress cells22,24-26. Sophisticated fluorescence

techniques, such as two-photon fluorescence microscopy, needed for endogenous

H2O2 imaging in live cells under normal culture condition2. Thus, highly sensitive and

selective fluorescent probe is highly demanded to monitor the endogenous H2O2 level

in live cells under normal culture condition by using traditional laser scanning

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confocal microscopy. We presume that developing such fluorescent probe based on

photo-stable fluorophore would be a practical strategy.

2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) based

fluorophores has attracted significant attraction due to its photo-stability and red/NIR

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fluorescence.31-45 TCF usually served as an electron acceptor and connected with an

electron donator group via conjugated structure in these fluorophores.32-34. Due to its

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excellent photo-stability under irradiation of excitation light, the photo-activatable

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version of the fluorophore has been used in single molecule and super-resolution

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imaging.35-38 Recently, TCF based fluorescent probes were successfully applied for
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imaging or sensing of pH39, suflite40, thiols41, hypochlorous acid33, metal ions42-44, and

human carboxylesterase 2.45

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In this work, we synthesized red fluorescent probe TCF-PB by combining

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4-(4,4,5,5-Tetramethyl-[1,3,2] dioxaborolan-2-yl)-benzaldehyde and TCF via

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Knoevenagel reaction (Scheme 1a).34 TCF-PB is almost nonfluorescent, but it can

produce fluorescence after reacting with H2O2 (Scheme 1b). In the presence of H2O2,
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we presume that the borate ester group in TCF-PB was transformed into hydroxyl
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group to facilitate the intramolecular charge transfer (ICT) process in the molecule,
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which results in strong fluorescence emission.

Scheme 1. (a) Synthesis of TCF-PB (b) The fluorescence response of TCF-PB

toward H2O2.

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2. Experimental section

2.1. Materials and Characterization

Lung cancer cell line A549 was purchased from the Type Culture Collection of the

Chinese Academy of Sciences, Shanghai, China. Fluorescein sodium salt, sodium

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nitroprusside dihydrate, and Dulbecco’s modified Eagle’s medium (DMEM) were

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purchased from Sigma-Aldrich. Other medium components were obtained from

Sigma-Aldrich too. Fetal bovine serum (FBS) was obtained from Hyclone

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Laboratories Inc. Ammonium iron (II) sulfate hexahydrate, pinacol, sodium

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hypochlorite solution, rhodamine 6G, and 4-hydroxy benzaldehyde were purchased
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from Aladdin Industrial Inc. 3-hydroxy-3-methyl-2-butanone, 4-formylphenylboronic

acid, sodium ethoxide, malononitrile, cresyl violet and TBHP were purchased from
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J&K Chemical (Beijing, China). Potassium phosphate (dibasic, anhydrous) was

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purchased from BBI Life Sciences. Resorufin was purchased from Tokyo Chemical
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Industry Co., Ltd (Shanghai, China). Potassium phosphate (monobasic, anhydrous)

was purchased from Sangon Biotech Co., Ltd (Shanghai, China). Purified water from
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a Millipore Simplicity 185 purification unit was used for rinsing and preparing all
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aqueous solutions. Hydrogen peroxide 30% was bought from Sinopharm Chemical
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Reagent Co., Ltd (Shanghai, China). All reagents were obtained commercially and

used without further purification.

2-{3-Cyano-4-[2-(4-hydroxy-phenyl)-vinyl]-5,5-dimethyl-5H-furan-2-ylidene}-malon

onitrile (TCFP), 4-(4,4,5,5-Tetramethyl-[1,3,2] dioxaborolan-2-yl)-benzaldehyde, and

48,49,34 1
TCF were synthesized according to literature , respectively. H-NMR

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spectroscopy was performed on Bruker Ascend 600 NMR spectrometer. Electrospray

ionization mass spectra (ESI-MS) were collected on a Bruker Maxis ESI-Q-TOF

instrument. Absorbance spectra were recorded on Purkinje General TU-1950 UV-vis

spectrophotometer. Fluorescence emission and excitation spectra were measured

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using Edinburgh FLS980 fluorometer. The excitation slit was 3.0 nm and the emission

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slit was 2.5 nm.

2.2. Synthetic Procedures

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Synthesis of TCF-PB. 2-(3-Cyano-5,5-dimethyl-4-{2-[4-(4,4,5,5-tetramethyl-[1,3,2]

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dioxaborolan-2-yl)-phenyl]-vinyl}-5H-furan-2-ylidene)-malononitrile (TCF-PB) was
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synthesized according to literature.41 4-Formylphenylboronic acid pinacol cyclic ester

(250 mg, 1.08 mmol) and TCF (257 mg, 1.29 mmol) were added to anhydrous EtOH
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(5 mL). Then, 1 mL pyridine was added to the mixture and stirred for 24h at RT in
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dark. The reaction mixture was cooled at 4 °C. The resulting solid was filtered and
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washed with a small amount of cold EtOH. The crude product was purified by

Cheetah MP 100 Flash chromatography using EtOAc/Petroleum ether=1:3 as the

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eluent, affording probe as a solid 130 mg (29% yield). The 1H NMR, 13C NMR, and
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HR-MS spectra of TCF-PB are shown in Figure S1-3. (see the Supporting
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Information). 1H NMR (600 MHz, DMSO-d6), δ 7.94 (d, 1H, J = 16.2Hz), 7.91 (d,

2H, J = 8.4Hz), 7.78 (d, 2H, J = 8.4Hz), 7.29 (d, 1H, J = 16.2Hz), 1.80 (s, 6H), 1.31 (s,

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12H). C NMR (600 MHz, DMSO-d6), δ 177.0, 174.8, 146.6, 136.8, 135.0, 128.5,

116.4, 112.5, 111.7, 110.8, 100.2, 99.5, 84.0, 54.8, 40.4, 25.0, 24.7. ESI-HRMS: calcd

[M+Na]+ m/z for [C24H24BN3O3+Na]+, 436.1803; found, 436.1800.

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2.3 UV-Vis Absorption and Fluorescence spectra for H2O2 detection.

H2O2 was added to a solution of TCF-PB in phosphate buffer (10 mM, pH 7.40).

Then, the mixture was incubated for 1 hour at 37 °C. UV-Vis absorbance spectra were

recorded, and the fluorescence spectra were measured at an excitation wavelength of

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560 nm.

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2.4 Photobleaching study under UV irradiation

TCFP, rhodamine 6G, fluorescein sodium salt, Resorufin, and cresyl violet were

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separately dissolved in phosphate buffer (10 mM, pH 7.40, the concentrations of each

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fluorophore were 2 µM). 1 mL of fluorophores solution were added into the
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fluorescent cuvette and exposed to UV irradiation (energy: 1 mJ/cm2) in the CL-1000

Ultraviolet Crosslinker. The fluorescence spectra were recorded using a fluorometer at

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each time point. Fluorescence intensities at each time point were then plotted against
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exposure time and normalized to the fluorescence intensity before irradiation.

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2.5 Photobleaching study under excitation laser on Confocal Laser Scanning

Microscopy.
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TCFP, rhodamine 6G, fluorescein, Resorufin and Cresyl violet were separately solved
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in phosphate buffer (10 mM, pH 7.40, the concentration of the fluorophores was 2
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µM). The solution of fluorophores was added on a glass slide and left to dry in the

dark to eliminate the influence of Brownian movement before imaging. Fluorescence

intensities were recorded by using Olympus FV-1200 confocal laser scanning

microscopy with the 40× objective lens. The signals were collected at 10.0 us/pixel

sampling speed. The excitation wavelength is 488 nm and emission wavelength are

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from 500 nm to 600 nm for fluorescein (HV, 520 V) and rhodamine 6G (HV, 265 V).

The excitation wavelength is 559 nm and emission wavelength are from 600 nm to

700 nm for TCFP (HV, 890 V), Resorufin (HV, 830 V), and Cresyl violet (HV, 880 V).

All photobleaching experiments were conducted in triplicate. Fluorescence intensities

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at each time point were then plotted against exposure time and normalized to the

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fluorescence intensity before irradiation.

2.6 Selectivity Study. Various ROS were added to a solution of TCF-PB in

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phosphate buffer (10 mM pH 7.40, [ROS] = 20 µM), which were incubated for 60

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min at 37 °C. The fluorescence spectra were recorded at an excitation wavelength of
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560 nm. Hydroxyl radical (·OH) and tert-butoxy radical (·OtBu) were generated by

reaction of 200 µM (NH4)2Fe(SO4)2·6H2O with 20 µM H2O2 or TBHP, respectively.

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and superoxide (O2-· ) was generated by the reaction of 10 mM Hypoxanthine (HPX)

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with 1.0 U·mL-1 Xanthine oxidase (XO), XO can catalyze the oxidation of HPX to
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uric acid and generate superoxide50,51.

2.7 Cell Culture.

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A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum at
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37 °C in a humidified incubator containing 5% CO2.

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2.8 Fluorescence Microscopic Cell Imaging.

A549 Cells were seeded on the cover glass and incubated overnight. For NAC52,53

(N-acetyl-L-cysteine, a common inhibitor of ROS, 10 mM) group: cells were treated

with NAC for 1 hour then incubated with TCF-PB (50 µM) for 1 hour at 37 °C. For

endogenous H2O2 imaging: cells were incubated with TCF-PB (50 µM) for 1 hour.

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Before imaging, NAC (10mM) was added and incubated for 1 hour at 37 °C to

quench the reaction. All samples were excited with 559 nm laser continuously for 25

min on Olympus FV-1200 confocal laser scanning microscopy with the 40× objective

lens. The signals were collected at 10.0 us/pixel sampling speed. The integral

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fluorescence intensities of cell imagine at each time point (a picture per minute) were

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plotted against scanning time and normalized to the integral fluorescence intensity of

initial imagine at time zero.

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2.9 Cell cytotoxicity assay. A549 cells were seeded into a 96-well plate and

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incubated for 24 h. TCF-PB (0-80 µM) were added to the wells. After 48 h, 20 µL of
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MTT (5 mg/mL in PBS) was added and incubated for 4 h. and 150 µL DMSO was

added after the supernatant was removed. Absorbance at 490 nm was measured by
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Biotek Epoch microplate reader.

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3. Results and discussion

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Since photobleaching of the fluorophores results in loss of fluorescence that

decreases the signal-to-noise ratio and imaging resolution.29,30 Two methods were
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used to validate the photo-stability of TCFP48: 1) measuring the fluorescence emission

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intensity by fluorometer after UV irradiation; 2) monitoring the fluorescence emission

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intensity under laser excitation by using laser confocal microscopy. And several

popular commercial available fluorophores (fluorescein, cresyl violet, resorufin, and

rhodamine 6G) that were widely used for constructing fluorescent probes were tested

for comparison as well. All the fluorophores were separately dissolved in phosphate

buffer (10 mM, pH 7.40, the concentration of the fluorophores was 2 µM).

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Fluorescence intensities that were normalized to the initial intensity of each

fluorophore were plotted against exposure time to generate each curve in Figure 1. As

we have predicted, the TCFP was very stable under the irradiation of excitation laser

and UV light. The TCFP has less than 5% loss in fluorescence after 25 min excitation.

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The time windows should be wide enough for most cell imaging applications. The

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difference became even dramatic under UV irradiation at the same wavelength as

shown in Figure 1b. The photo-stability of TCFP is significantly better than other

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common fluorophores that is consistent with previous reports, which claim TCF based

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fluorophores usually have very good photo-stability.35-38
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Figure 1 (a) Photobleaching curves for TCFP, resorufin, cresyl violet, fluorescein
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sodium salt and rhodamine 6G. Fluorescence intensities were recorded by using
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Olympus FV-1200 confocal laser scanning microscopy. (b) Photobleaching curves for
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TCFP, resorufin, cresyl violet, fluorescein sodium salt and rhodamine 6G.

Fluorophores were exposed to the CL-1000 Ultraviolet Crosslinkers (energy:

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1mJ/cm2). Errors bars represent standard deviation (n = 3).

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With the above results in mind, we are confident to develop a selective and

photo-stable fluorescence probe based on TCFP, namely TCF-PB. First, we studied

the response of TCF-PB to the H2O2 in a test tube. We measured the absorption and

fluorescence spectra of TCF-PB toward H2O2 in phosphate buffer (10mM pH 7.40).

As shown in Figure 2a, the TCF-PB solution showed one absorption peak at 404 nm

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in the UV-Vis absorption spectrum. The molar extinction coefficient of the TCF-PB

was 2.0 x 104 L mol-1·cm-1 at 404 nm. After reacting with H2O2, the maximum

absorption peak appeared at 570 nm and the color of TCF-PB changed from yellow

to red-violet. The clear color change can also make TCF-PB a “naked-eye” indicator

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for H2O2. In the fluorescence emission spectra (Figure 1b), the emission of TCF-PB

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was almost neglectable, which was due to the ICT progress was blocked by

introducing boric acid ester group. When added H2O2 to the solution of TCF-PB, the

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fluorescence emission was dramatically enhanced at 605 nm. The results indicated

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that TCF-PB could serve as a colorimetric and fluorescent indicator for H2O2.
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Figure 2 (a) UV-vis absorption spectra of TCF-PB (20 µM) upon addition of
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H2O2 (500 µM). (b) Fluorescence spectra of TCF-PB (20 µM) upon addition of H2O2
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(500 µM). The spectra were measured in phosphate buffer (10 mM, pH=7.40) at 37°
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for 60 min, and the excitation wavelength is 560 nm.

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Incubation time is an important factor for the reaction-based probe. As shown in

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Figure 3, the initial response of the probe towards H2O2 is almost instant, and the
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fluorescence intensity gradually increased and reaches a plateau at 60 min. Thus, 1

hour was chosen for further experiments.

Figure 3 (a) Emission response of TCF-PB (20 µM) to H2O2 (500 µM) under

different incubation time. (b) Fluorescence intensity of TCF-PB to H2O2 under

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different incubation time. The experiments were measured in phosphate buffer (10

mM, pH 7.40), and the excitation wavelength is 560 nm. Errors bars represent

standard deviation (n = 3).

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Sensitivity and dynamic range are the most important analytical parameters for a

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method, thus we studied sensitivity and dynamic range of our probe toward H2O2.

Results are shown in Figure 4, with the increasing concentration of H2O2, the

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fluorescence intensity gradually enhanced and reaches a plateau at 200 µM. A good

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linear relationship (R2=0.9930) between the fluorescence intensity at 605 nm and
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H2O2 concentrations in the range of 1 to 20 µM was obtained. The limit of detection

(3σ/slope) of TCF-PB was 61 nM, which make our probe one of the most sensitive
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fluorescent probe for H2O2 detection by comparing with previously reported

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probes.22-28
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Figure 4 (a) Emission spectra of TCF-PB treated with different concentrations of

H2O2 from 0 to 500 µM. (b) Fluorescence intensity of TCF-PB as a function of the
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H2O2. [TCF-PB] = 20 µM. The experiments were measured in phosphate buffer (10
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mM, pH 7.40). The incubation time is 60 min, and the excitation wavelength is 560
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nm. Errors bars represent standard deviation (n = 3).

High selectivity is a matter of necessity for any application in H2O2 detection of our

probe. Thus, we studied the response of our probe towards common other ROS

species. The results are shown in Figure 5, the fluorescence intensity at 605 nm was

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observed in the presence of H2O2 in comparison with other ROS species. H2O2

elicited a dramatic increase in fluorescence intensity, while other ROS species led to

negligible changes in fluorescence intensity. The results implied that TCF-PB can

serve as a high selectivity probe for H2O2 detection, which is consistent with

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previously reported boronate ester deprotection based fluorescent probes.22

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Figure 5 Fluorescence responses of TCF-PB towards common ROS species. The

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concentration of all species is 20 µM. The experiments were measured in phosphate

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buffer (10 mM, pH 7.40). The incubation time is 60 min, and the excitation
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wavelength is 560 nm. Errors bars represent standard deviation (n = 3).
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With a selective and sensitive red fluorescence probe in hand, we confidently

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conducted endogenous H2O2 imaging in live cancer cells. We chose lung cancer cell
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line A549 as model cancer cells. The cytotoxicity of our probe was investigated first,

and the result showed the probe has a negligible effect on cellular viability at less than
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50 µM (Figure S1). NAC treated A549 cells were used as a blank. NAC was a
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common inhibitor of ROS. NAC treatment will remove the endogenous intracellular
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reactive oxygen species.52,53 As shown in Figure 6b, NAC treated A549 cells was

almost nonfluorescent. A549 cells without NAC clearly gave fluorescence (Figure 6e).

The results are consistent with the previous report on endogenous intracellular H2O2

detection under normal culture condition, which has LOD of 90 nM and can

successfully imagine endogenous intracellular H2O2.2 Since the LOD of our probe (61

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nM) is at same level with previous work, it is reasonable to have clear imaging signal

on endogenous intracellular H2O2. We can observe a significant difference in the

histogram of the average fluorescence intensities of multiple images of each sample at

time zero as well as after 25 min excitation. More importantly, the fluorescence signal

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from endogenous H2O2 in A549 cells was only slightly changed (less than 10%),

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which certified that our probe was photo-stable under irradiation of the excitation

laser (Figure 6h). Also, as we can observe from Figure 6, the fluorescence intensity of

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A549 cells increases from time zero to time 25 min. We speculate that the NAC does

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not completely inhibit the production of reactive oxygen species since both groups
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with or without NAC shown the increase of fluorescence intensity after 25 min. Thus,

the probe will react with newly generated hydrogen peroxide to cause the slight
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increase in the fluorescence intensity. For most cell imaging applications, 25 min
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should be plenty. The results indicated that TCF-PB could easily penetrate cellular
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membrane and detect endogenous intracellular H2O2.

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Figure 6 (a-c) Images of NAC treated A549 cells; (d-f) Images of A549 cells; (g)
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Average fluorescence intensities of NAC treated and un-treated A549 cells. (h)
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Relative fluorescence intensity of A549 cells vs time. (a), (d) bright-field images; (b),

(c), (e), (f) fluorescence images (λex=559 nm, λem = 580-680 nm). NAC = 10 mM.

Scale bars denote 20 µm.

4. Conclusions

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In summary, we developed a sensitive and selective red emitted fluorescent probe

TCF-PB for detection of endogenous intracellular H2O2. Upon reaction occurring, the

fluorescence emission peak of TCF-PB was enhancement dramatically at 605 nm and

the color changes from yellow to red-violet, which could serve as a “naked-eye”

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probe for H2O2. A good linear relationship (R2=0.9930) between the fluorescence

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intensity at 605 nm and H2O2 concentrations in the range of 1 to 20 µM was obtained.

The limit of detection (3σ/slope) of TCF-PB was 61 nM, which make our probe one

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of the most sensitive fluorescent probe for H2O2 detection. And the probe does not

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response to common other ROS species. Since our probe has good sensitivity and
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selectivity for H2O2 detection, it was successfully applied to endogenous H2O2

imaging in live A549 lung cancer cells. The imaging signal just had less than 10%
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change in fluorescence intensity after 25 min continuous irradiation of excitation laser.

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Thus, we believe the proposed TCF-PB is very useful for sensing and imaging of
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trace amount of H2O2 that endogenously generated in the live cancer cells, which is

essential for the study of H2O2 related cell signaling and human diseases.
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Acknowledgements
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We thank the National Natural Science Foundation of China (Grant No. 21675108,
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21305083, U1704137, 21404031, U1204212), and Fundamental Research Funds for

the Central Universities (Grant No. GK201603028) for financial support.

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Captions

Scheme 1. (a) Synthesis of TCF-PB (b) The fluorescence response of TCF-PB

toward H2O2.

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Figure 1 (a) Photobleaching curves for TCFP, resorufin, cresyl violet, fluorescein
sodium salt and rhodamine 6G. Fluorescence intensities were recorded by using

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Olympus FV-1200 confocal laser scanning microscopy. (b) Photobleaching curves for
TCFP, resorufin, cresyl violet, fluorescein sodium salt and rhodamine 6G.

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Fluorophores were exposed to the CL-1000 Ultraviolet Crosslinkers (energy:
1mJ/cm2). Errors bars represent standard deviation (n = 3).

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Figure 2 (a) UV-Vis absorption spectra of TCF-PB (20 µM) upon addition of H2O2
(500 µM). (b) Fluorescence spectra of TCF-PB (20 µM) upon addition of H2O2 (500
M

µM). The spectra were measured in phosphate buffer (10 mM, pH=7.40) at 37°for
60 min, and the excitation wavelength is 560 nm.
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Figure 3 (a) Emission response of TCF-PB (20 µM) to H2O2 (500 µM) under
different incubation time. (b) Fluorescence intensity of TCF-PB to H2O2 under
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different incubation time. The experiments were measured in phosphate buffer (10
mM, pH 7.40), and the excitation wavelength is 560 nm. Errors bars represent
standard deviation (n = 3).
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Figure 4 (a) Emission spectra of TCF-PB treated with different concentrations of

H2O2 from 0 to 500 µM. (b) Fluorescence intensity of TCF-PB as a function of the
H2O2. [TCF-PB] = 20 µM. The experiments were measured in phosphate buffer (10
mM, pH 7.40). The incubation time is 60 min, and the excitation wavelength is 560
nm. Errors bars represent standard deviation (n = 3).

Figure 5 Fluorescence responses of TCF-PB towards common ROS species. The

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concentration of all species is 20 µM. The experiments were measured in phosphate

buffer (10 mM, pH 7.40). The incubation time is 60 min, and the excitation
wavelength is 560 nm. Errors bars represent standard deviation (n = 3).

Figure 6 (a-c) Images of NAC treated A549 cells; (d-f) Images of A549 cells; (g)

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Average fluorescence intensities of NAC treated and un-treated A549 cells. (h)
Normalized fluorescence intensity of A549 cells vs time. (a), (d) bright-field images;

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(b), (c), (e), (f) fluorescence images (λex=559 nm, λem = 580-680 nm). NAC = 10 mM.
Scale bars denote 20 µm.

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Scheme 1. (a) Synthesis of TCF-PB (b) The fluorescence response of TCF-PB

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toward H2O2.
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Figure 1 (a) Photobleaching curves for TCFP, resorufin, cresyl violet, fluorescein
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sodium salt and rhodamine 6G. Fluorescence intensities were recorded by using
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Olympus FV-1200 confocal laser scanning microscopy. (b) Photobleaching curves for
TCFP, resorufin, cresyl violet, fluorescein sodium salt and rhodamine 6G.
Fluorophores were exposed to the CL-1000 Ultraviolet Crosslinkers (energy:
1mJ/cm2). Errors bars represent standard deviation (n = 3).

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Figure 2 (a) UV-vis absorption spectra of TCF-PB (20 µM) upon addition of H2O2
(500 µM). (b) Fluorescence spectra of TCF-PB (20 µM) upon addition of H2O2 (500

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µM). The spectra were measured in phosphate buffer (10 mM, pH=7.40) at 37°for
60 min, and the excitation wavelength is 560 nm.

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Figure 3 (a) Emission response of TCF-PB (20 µM) to H2O2 (500 µM) under
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different incubation time. (b) Fluorescence intensity of TCF-PB to H2O2 under

different incubation time. The experiments were measured in phosphate buffer (10
mM, pH 7.40), and the excitation wavelength is 560 nm. Errors bars represent
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standard deviation (n = 3).

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Figure 4 (a) Emission spectra of TCF-PB treated with different concentrations of

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H2O2 from 0 to 500 µM. (b) Fluorescence intensity of TCF-PB as a function of the

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H2O2. [TCF-PB] = 20 µM. The experiments were measured in phosphate buffer (10
mM, pH 7.40). The incubation time is 60 min, and the excitation wavelength is 560

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nm. Errors bars represent standard deviation (n = 3).
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Figure 5 Fluorescence responses of TCF-PB towards common ROS species. The

concentration of all species is 20 µM. The experiments were measured in phosphate
buffer (10 mM, pH 7.40). The incubation time is 60 min, and the excitation
wavelength is 560 nm. Errors bars represent standard deviation (n = 3).

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Figure 6 (a-c) Images of NAC treated A549 cells; (d-f) Images of A549 cells; (g)
Average fluorescence intensities of NAC treated and un-treated A549 cells. (h)

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Normalized fluorescence intensity of A549 cells vs time. (a), (d) bright-field images;
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(b), (c), (e), (f) fluorescence images (λex=559 nm, λem = 580-680 nm). NAC = 10 mM.
Scale bars denote 20 µm.
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• Fluorescent probe for detecting endogenous intracellular H2O2 in live cancer cells
• Good photo-stability under continuous irradiation of excitation laser for 25 min
• High Sensitivity for H2O2 detection with limit of detection (3σ/slope) of 61 nM

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