Fluorescence imaging of lysosomal hydrogen selenide under oxygen-controlled conditions

Article abstract of DOI:10.1039/c8tb03169j

Hydrogen selenide (H₂Se), a central metabolite of Se supplements, displays critical biological functions in many physiological and pathological processes. To better understand its comprehensive function, especially those exerted in subcellular

Full text of DOI:10.1039/c8tb03169j

View Article Online
View Journal
Journal of
Materials Chemistry B
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. Tian, F. Xin, J.
Jing and X. Zhang, J. Mater. Chem. B, 2019, DOI: 10.1039/C8TB03169J.
This is an Accepted Manuscript, which has been through the
Volume
4 Number 1 7 January 2016 Pages 1–178
Royal Society of Chemistry peer review process and has been
accepted for publication.
Journal of
Materials Chemistry B
Accepted Manuscripts are published online shortly after
Materials for biology and medicine
www.rsc.org/MaterialsB
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

Page 1 of 14
Journal of Materials Chemistry B
View Article Online
Fluorescence imaging of lysosomal hydrogen selenide under
DOI: 10.1039/C8TB03169J
oxygen-controlled conditions
Yong Tian, Fangyun Xin, Jing Jing*, Xiaoling Zhang*
Beijing Key Laboratory of Photoelectronic/Electrophotonic Conversion Materials,
Key Laboratory of Cluster Science of Ministry of Education, Analytical and Testing
Center, School of Chemistry and Chemical Engineering, Beijing Institute of
Technology, Beijing 100081, PR China
* Corresponding authors: zhangxl@bit.edu.cn, hellojane@bit.edu.cn
Abstract
Hydrogen selenide (H2Se), a central metabolite of Se supplements, displays critical
biological functions in many physiological and pathological processes. To better
understand its comprehensive function, especially those exerted into subcellular
organelles, the development of specific assays thus are urgently needed. However,
methodology to detect H2Se is poorly developed. Here, we present the concise design
strategy to obtain activatable fluorescent probe (Se-1) for H2Se by utilizing
intramolecular photoinduced electron transfer (PET) process to switch the
fluorescence. The probe enables to selectively react with H2Se without interference
from intracellular reactive species, and has been successfully used to image the H2Se
content in lysosomes. Additionally, with the aid of Se-1, we demonstrated that
lysosomal H2Se can be generated and gradually accumulated in HepG2 cells under
hypoxic conditions. These applications make Se-1 a potential new candidate for
deciphering the biological effects of H2Se on lysosomes in the biology and pathology.

Journal of Materials Chemistry B
Page 2 of 14
View Article Online
DOI: 10.1039/C8TB03169J
Introduction
Selenium (Se) is an essential trace element for humans, which is important for many
biological processes.1-5 Up to now, evidence from numerous researches supports the
notion that Se has a wide range of pleiotropic effects, ranging from antioxidant and
anti-inflammatory effects to cancer prevention.6-12 Generally, it is accepted that Se
metabolites are responsible for the biological activities of Se supplements.13-17 As a
central metabolite of Se supplements, hydrogen selenide (H2Se) is intersected at
upstream and downstream of metabolic pathways, and has been implicated in various
biological functions in tissues and cells.18-23 In addition, it has been deduced that the
lysosomal functions are subtly regulated by H2Se, including the lysosome-involved
autophagy process and the lysosome-mediated apoptotic program.24-26 However, the
effects of H2Se on lysosome are far from being completely understood. Owing to the
dynamic transformation of the morphology and components of the highly
heterogeneous lysosomes, it is difficult to find solid evidence of direct interactions
between H2Se and components within lysosomes. So far, the method to determine
whether H2Se enters into lysosomes is still rare, highly targetable and specific
methods with good biocompatibility and high spatiotemporal resolution are thus
urgently needed.
Among various methods, fluorescence sensing has emerged as one of the most
powerful techniques to monitor targets and biological processes in the context of a
living system with high temporal and spatial resolution.27-31 As an indispensable
module in the fluorescence sensing system, fluorescent probe is able to provide a
reliable fluorescent response by interacting with the targeted analyte. Therefore, it is
essential to construct a desirable fluorescent probe for monitoring lysosomal H2Se.
There are two small-molecule fluorescent probes have been developed to trace
intracellular H2Se by now.32, 33 Nevertheless, none of these probes is well suited for
lysosomal applications, they all failed to specifically localize in lysosomes and
monitor lysosomal H2Se. In this regard, a lysosome-targetable small-molecule
fluorescent probe for lysosomal H2Se based on the principle of photoinduced electron
transfer (PET) has been proposed, and this strategy was rigorously validated in the
follow-up experiments.

Page 3 of 14
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03169J
The probe (Se-1) for lysosomal H2Se was integrated through H2Se-capturing
benzoselenadiazole, lysosome-targeting morpholine derivative, and fluorophore
naphthalimide (Scheme 1). Benzoselenadiazole was employed not only as a H2Se
trapper but also as a fluorescence quencher for fluorophore. Specific nucleophilic
attack of H2Se to the benzoselenadiazole moiety of Se-1 promotes the PET process
blocking, and enhancing the fluorescence emission. Various assays have been
employed to demonstrate that Se-1 displays high sensitivity and selectivity toward
H2Se. Furthermore, these novel properties of Se-1 enable its use in monitoring
endogenous H2Se in lysosomes.
Experimental section
Materials and instruments
Reagents were purchased at the highest commercial quality and used without
further purification, unless otherwise stated. Distilled water was used after passing
1
through a water ultrapurification system. H NMR spectra were recorded on Bruker
Advance 400, and tetramethylsilane (TMS) was used as a reference. Mass
spectrometry (MS) spectra were recorded on Bruker Apex IV FTMS using ESI. All
pH measurements were carried out with a pH acidometer (Mettle Toledo FE-30).
Fluorescence quantum yields were determined using Quinine sulfate (0.1 M H2SO4, Φ
= 0.577) as a reference. UV-visible spectra were obtained on a Beijing Purkinje TU-
1901. Fluorescence spectroscopic studies were performed on a Hitachi F-7000 at
room temperature. Cell imaging was performed in an inverted fluorescence
microscopy (OLYPUS) with a 60 objective lens.
Preparation of the test solution
Stock solution of Se-1 (1 mM) was prepared in ultrapure water and stored at
−4 °C until immediately prior to use. Stock solution of H2Se (1 mM) was freshly
prepared by the reaction of Al2Se3 with ultrapure water in an N2-filled glovebox at
room temperature before use. Stock solutions (10 mM) of other analytes including
•−
amino acids: Lys, Leu, Phe, Val; reactive oxygen species (ROS): O2 , NO, H2O2, t-
1
BuO•, O2, ClO−, OH• (all ROS were generated according to reported procedures);
sulfydryl-containing compounds: Na2S, dithiothreitol (DTT), cysteine (Cys),

Journal of Materials Chemistry B
Page 4 of 14
View Article Online
DOI: 10.1039/C8TB03169J
homocysteine (Hcy), glutathione (GSH); selenocompouds: Na2SeO3, selenocysteine
(Sec). For optical studies, a solution of Se-1 (10 μM) was prepared in acetic
acid/sodium acetate buffer solution (20 mM, pH 5). Then 3 mL of the Se-1 solution
was placed in a quartz cuvette (10 mm lightpath). The UV-vis or fluorescence spectra
were then recorded after addition of an analyte of interest at 25 °C (controlled with a
temperature controller). For measurements regarding to the spectra changes of Se-1
upon addition of various concentrations of H2Se (quantitative detection of H2Se), the
spectrum was collected 1 min after an indicated concentration of H2Se (0‒60 μM) was
added to the solution of Se-1 (10 μM) at 25 °C, and the data of these experiments
were reported as the mean ± standard deviation of triplicate experiments.
Colocalization fluorescence imaging
To evaluate the availability of Se-1 and Se-2 for subcellular location, the
colocalization imaging experiments in HepG2 cells were further performed. The
HepG2 cells were incubated with Se-1 and Se-2 for 30 min, respectively, washed
three times with PBS (pH 7.4), incubated with LysoTracker Blue (2 μM) for 30 min,
and washed three times with PBS. The fluorescence images were recorded in two
channels. The blue channel had excitation at 405 nm and a collection window of 420‒
480 nm, and the green channel captured images using a 445 nm laser and an emission
filter at 500‒600 nm.
Imaging of H2Se in living cells
Considering that the real solid tumor microenvironment is hypoxia because of
leaky and dysfunctional tumor vasculature, the detection of the changes in H2Se in the
lysosomes of HepG2 cells induced by Na2SeO3 was explored under normoxic (20%
O2) and hypoxic (1% O2) conditions, respectively. HepG2 cells were prepared in
glass-bottom culture dishes for 24 h. HepG2 cells were incubated with 0‒10 μM
Na2SeO3 for 12 h or 5 μM Na2SeO3 for 0‒12 h and then incubated with 10 μM Se-1 in
normoxic (20% O2) and hypoxic (1% O2) environments, respectively. Before each
step, the cells were washed three times with PBS. Fluorescence imaging was
performed by using a confocal microscope. The green channel captured images using
a 445 nm excitation. The procedures of controlling the O2 content for cells incubation
are (1) 20% O2, 75% N2 and 5% CO2 in volume were controlled by gas flowmeter for

Page 5 of 14
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03169J
the normoxic atmosphere; (2) 1% O2, 94% N2 and 5% CO2 in volume were controlled
by gas flowmeter for the hypoxic atmosphere.
Synthesis
The synthetic details of all compounds in this work are shown in Supporting
Information.
Results and discussion
Design and synthesis of Se-1
In order to realize the highly sensitive and selective detection of H2Se, a strategy
that the selenium‒nitrogen (Se‒N) bond contained molecular structure could be
specifically attacked by the H2Se with high nucleophilicity has been proposed. H2Se
could rapidly cleave the Se‒N bond and release Se species from the molecular
structure. In light of this, the benzoselenadiazole moiety (containing N‒Se‒N bond)
was chosen as the H2Se receptor to compose the fluorescent probe, and concomitantly
the introduction of benzoselenadiazole moiety could induce the PET process in
fluorescent probe resulting in the fluorescence quenching of fluorophore. Therefore,
we synthesized the probe Se-1 to investigate the feasibility of our design concept. The
principle of Se-1 for detection of H2Se is illustrated in Scheme 1. The effective
interaction between Se-1 and H2Se could block the PET process and switch on the
fluorescence. Additionally, the morpholine group tethered on Se-1 could directly
target lysosomes. Structural identification of Se-1 was confirmed by 1H NMR and MS
spectroscopy (Figure S1 and S2).

Journal of Materials Chemistry B
Page 6 of 14
View Article Online
DOI: 10.1039/C8TB03169J
Scheme 1 Proposed mechanism for H2Se activation of probe Se-1
UV-Vis and emission responses of Se-1 toward H2Se
Initially, we examined the absorption spectra of Se-1 in response to H2Se. Se-1
has two characteristic maximum absorbance at 335 (ε = 1.36×104 M−1 cm−1, in buffer)
and 435 nm (ε = 1.22×104 M−1 cm−1, in buffer), respectively. After the addition of
H2Se under the simulated lysosomal conditions, i.e., in 20 mM acetic acid/sodium
acetate buffer, pH 5, one absorbance at 435 nm was barely altered, whereas the other
at 335 nm was sharply decreased. Suggesting a clear reaction could be observed
between Se-1 and H2Se (Figure 1a). Then the reactivity of Se-1 with H2Se was
examined by fluorescence spectroscopy. It was unexpected that almost no emission
intensity was appeared in the absence or presence of H2Se when Se-1 was excited at
335 nm. We reasoned that the maximum absorbance at 335 nm could be ascribed to
the signal of benzoselenadiazole that is a non-fluorescence moiety. In this case, the
excitation wavelength at 435 nm was chosen for this experiment. From Figure 1b,
there was a relatively weak emission centered at 535 nm for Se-1 itself (ΦSe-1 = 0.02,
in buffer), whereas the emission increased remarkably within 1 min (Figure S3) upon
addition of H2Se when excited at 435 nm (ΦSe-2 = 0.3, in buffer). These results
confirm that the distinct and nearly instantaneous fluorescence response of Se-1
toward H2Se can be successfully achieved, owing to the inhibition of the PET process
from the benzoselenadiazole moiety to the naphthalimide fluorophore, which also
indicating the nucleophilic attack of H2Se to benzoselenadiazole resulting in the
decrease of the absorbance at 335 nm. Collectively, a ‘turn-on’ fluorescence mode of
Se-1 can be activated by the labile H2Se in this simulated media.

Page 7 of 14
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03169J
Se-1
(b)
(a)
Se-1
Se-1+H Se
Se-1+H Se
2
2
250
300
350
400
450
500
550
600
500
550
600
650
Wavelength (nm)
Wavelength (nm)
Figure 1 UV-vis spectra (a) and fluorescent spectra (b) of Se-1 (10 μM) and Se-
1+H2Se in acetic acid/sodium acetate buffer (20 mM, pH 5).
Next, we performed dose-dependent titration studies of Se-1 toward H2Se. As
can be seen in Figure 2a, the absorbance at 335 nm of Se-1 was finally reduced to a
marginal intensity over a H2Se concentration range of 0‒60 μM, and the absorbance at
435 nm showed only a slight drop. Concomitantly, the fluorescence intensity at 535
nm gradually enhanced with increasing concentration of H2Se (Figure 2b). In the
relationship between the relative fluorescence intensity and concentration of H2Se,
there was rapid emission increase in the presence of H2Se below 50 μM while the
reaction between Se-1 and H2Se was reduced due to saturation (Figure 2c). A linear
relationship was observed between the relative fluorescence and the H2Se
concentration (0‒15 μM, Figure 2d). Using this linear relationship, we obtained a
LOD (limit of detection) of 1.2 μM for the sensing of H2Se.

Journal of Materials Chemistry B
Page 8 of 14
View Article Online
DOI: 10.1039/C8TB03169J
0.16
0.12
0.08
0.04
0.00
3000
(b)
(a)
2500
2000
1500
1000
500
0
60 M
0 M
250
300
350
400
450
500
550
600
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
(c) 25
(d) 0.4
R2 = 0.98567
20
0.3
0.2
0.1
0.0
15
10
5
0
0
10
20
30
40
50
60
0
5
10
15
Concentration (M)
Concentration (M)
Figure 2 (a) UV-vis and (b) fluorescent spectra changes of Se-1 (10 μM) upon
addition of different concentrations of H2Se (0‒60 μM). (c) A plot of relative
fluorescence of Se-1 against H2Se (0‒60 μM). (d) The linear relationship between
relative fluorescence of Se-1 and H2Se concentration. All experiments were
performed in acetic acid/sodium acetate buffer (20 mM, pH 5), ex = 435 nm.
Sensing mechanism
As proposed, the spectral changes of Se-1 toward H2Se are initiated by the
nucleophilic attack of H2Se to benzoselenadiazole moiety. To confirm this reaction
1
13
mechanism, H NMR, C NMR and MS analyses of the product extracted from the
reaction mixture of Se-1 and H2Se were conducted. As shown in Figure S4 and S5,
the clear NMR signals could be in perfect accordance with all protons and carbons of
Se-2. Moreover, a peak of 432.2044 ([M+H]+) found in the HR-MS spectrum (Figure
S6) further proved the formation of Se-2 (Calc. 432.1957). Therefore, the mechanism
of Se-1 for sensing of H2Se is most likely the process as shown in Scheme 1, in which
the H2Se-triggered cleavage reaction causes the release of free Se-2, thus causing the
observed optical changes.

Page 9 of 14
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03169J
The pH effects
To establish the fidelity of the Se-1 under lysosomal conditions, we investigated
the pH-dependent fluorescence (Figure S7). By performing a pH titration ranging
from 3 to 7 and monitoring the fluorescence, we established that the Se-1 maintained
a constant emission in a wide pH range of 3‒7. Importantly, in the presence of H2Se,
the fluorescence enhancement of Se-1 displayed stable in the pH range of 4.5‒7,
which suggests that it can function under lysosomal conditions (pH 4.5‒5.5).
The selectivity
Since the Se-1 spectral signal is unambiguously switched on by H2Se in vitro, we
next determined if Se-1 could be selectively distinguished by H2Se. A panel of
common amino acids, ROS, sulfydryl-containing compounds and selenocompouds
were chosen to evaluate their possible interactions with Se-1. As shown in Figure 3a,
none of the tested agents caused significant decrease of absorbance signal at 335 nm.
GSH, Cys, Hcy and DTT, whose structures contain sulfydryl group with high
nucleophilicity, have little effect on the fluorescence signal of Se-1 in pH 5 buffer
(Figure 2a). Neither the common anions nor the ROS caused any significant changes
in fluorescence. The selenocompouds (Na2SeO3, Sec) also did not cause release of Se-
2 from the probe Se-1. A significant change in fluorescence was observed only upon
addition of 60 μM H2Se, providing further evidence for the selective nature of the
reaction between the probe and H2Se.

Journal of Materials Chemistry B
Page 10 of 14
View Article Online
DOI: 10.1039/C8TB03169J
0.20
0.15
0.10
0.05
0.00
(b) 10
(a)
Se-1 + others
Se-1 + H2Se
8
6
4
2
0
Na
Bu
1
O
H
O
OH
Na
D
VC
•
C
250
300
350
400
450
500
550
600
P
k
V
C
Bl
a
G
H
Ly Le
s
NO
H
S
2S
e
H
TT
h
2
l
2O
•
O
S
e
c
y
a
O
2Se
2S
e
s
c
l
u
2
n
•
−
y
2
−
O
3
Wavelength (nm)
Figure 3 (a) UV-vis and (b) fluorescence spectra changes of Se-1 (10 μM) toward
H2Se (60 μM) and other analytes (500 μM). All experiments were performed in acetic
acid/sodium acetate buffer (20 mM, pH 5), ex = 435 nm.
Bioimaging of H2Se in Living Cells
With the solid results in hand, we next wonder if Se-1 could image lysosomal
H2Se in living cells. Then the cellular localization experiments were performed with
confocal microscopy to confirm the intracellular location of probe Se-1 and product
Se-2. Lyso-Tracker Blue is a commercially available targeting dye for lysosomes and
was used in colocalization experiments. The Se-1 and Se-2 were coincubated for 30
min with HepG2 cells, respectively, and before imaging, lysosomes were labeled with
Lyso-Tracker Blue. In Figure 4, the blue channel detected the commercial dye at
420−480 nm, and the green channel detected the Se-1 and Se-2 at 500−600 nm. The
well-overlapped images were obtained for Se-1, Se-2 and Lyso-Tracker Blue in
HepG2 cells (Pearson’s correlation coefficient, PSe-1 = 0.91, PSe-2 = 0.96). These
results prove that the Se-1 and Se-2 are effective tools for targeting lysosomes.

Page 11 of 14
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03169J
Figure 4 (a‒d) Bright and fluorescence images of HepG2 cells co-stained with probe
Se-1 (10 μM) and Lyso-Tracker Blue (2 μM). (e‒i) Bright and fluorescence images of
HepG2 cells co-stained with Se-2 (10 μM) and Lyso-Tracker Blue (2 μM) for 30 min
at 37 °C.
Considering the hypoxic characteristic of solid tumors, we conclude that the
endogenous H2Se metabolism would be different in normoxic and hypoxic
environments due to the H2Se is closely associated with anticancer. To confirm this
hypothesis, the changes in the lysosomal H2Se contents in HepG2 cells induced by
Na2SeO3 (a precursor can be metabolized to H2Se) were detected with the aid of Se-1.
First, HepG2 cells were treated with 2‒10 μM Na2SeO3 for 12 h under hypoxia (1%
O2) and normoxia (20% O2), respectively, and H2Se were simultaneously monitored
using confocal imaging. The experimental results are shown in Figure 5a and 5c.
Higher H2Se contents were observed in lysosomes under hypoxic conditions, while
the H2Se contents were low and remained unchanged under normoxic conditions
during these parallel experiments. Next, HepG2 cells were exposed to 5 μM Na2SeO3
for different durations (0‒12 h) under hypoxia (1% O2) and normoxia (20% O2),
respectively, and then incubated with the Se-1 (10 μM), similar results were obtained
(Figure 5b and 5d). The above results indicate that, higher amount of H2Se
accumulated in lysosomes of cancer cells in a Na2SeO3 dose-dependent and
incubation time-dependent manner under hypoxic environments, and also validate that
Se-1 can be potentially used for monitoring the lysosomal H2Se in solid tumors.

Journal of Materials Chemistry B
Page 12 of 14
View Article Online
DOI: 10.1039/C8TB03169J
(c)
(d)
1% O2
20% O2
1% O2
20% O2
3
12
0
6
0
2
5
10
Na2SeO3 Concentration (M)
Incubation Time (h)
Figure 5 (a) Fluorescence images of endogenous H2Se in lysosome pretreated with
different concentrations of Na2SeO3 for 12 h, followed by incubation with 10 μM Se-
1 for 30 min under normoxic (20% O2) and hypoxic (1% O2) conditions. (b)
Fluorescence images of endogenous H2Se in lysosome pretreated with 10 μM
Na2SeO3 for different durations, followed by incubation with 10 μM Se-1 for 30 min
under normoxic (20% O2) and hypoxic (1% O2) conditions. (c, d) The quantitative
results of the corresponding imaging analyses. Scale bar: 20 μm.
Conclusions
To better understand the biological functions of lysosomal H2Se, a lysosome-
targetable small-molecule fluorescent probe, Se-1, has been developed to monitor

Page 13 of 14
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03169J
H2Se in living cells. The probe owns excellent sensitivity to H2Se and high selectivity
over other interferes. In vivo experiments, the probe displays decent biocompatibility
and targeting for lysosomes in HepG2 cells. Furthermore, the lysosomal H2Se content
accumulated gradually in a hypoxic environment, while the H2Se level almost
unchanged in normoxic condition. We anticipate that the current probe will provide
an ideal tool for further studies into the regulation effects of H2Se on lysosomes in the
biology and pharmacology.
Acknowledgment
This work was supported by the National Nature Science Foundation of China (No.
21575015 and 21505004).
References
1.
2.
3.
M. P. Rayman, Lancet, 2012, 379, 1256-1268.
L. H. Duntas and S. Benvenga, Endocrine, 2015, 48, 756-775.
S. J. Fairweather-Tait, Y. Bao, M. R. Broadley, R. Collings, D. Ford, J. E. Hesketh
and R. Hurst, Antioxid. Redox Signal., 2011, 14, 1337-1383.
M. Birringer, S. Pilawa and L. Flohe, Nat. Prod. Rep., 2002, 19, 693-718.
L. Vanda Papp, J. Lu, A. Holmgren and K. Kum Khanna, Antioxid. Redox Signal.,
2007, 9, 775-806.
4.
5.
6.
7.
M. A. Reeves and P. R. Hoffmann, Cell.Mol. Life Sci., 2009, 66, 2457-2478.
M. Kielczykowska, J. Kocot, M. Pazdzior and I. Musik, Adv. Clin. Exp. Med., 2018,
27, 245-255.
8.
9.
L. Letavayova, V. Vlckova and J. Brozmanova, Toxicology, 2006, 227, 1-14.
M. P. Rayman, Proc. Nutr. Soc., 2005, 64, 527-542.
10.
C. Sanmartin, D. Plano, A. K. Sharma and J. Antonio Palop, Int. J. Mol. Sci., 2012,
13, 9649-9672.
11.
12.
13.
H. Tapiero, D. M. Townsend and K. D. Tew, Biomed. Pharmacother., 2003, 57, 134-
144.
M. Wallenberg, S. Misra and M. Bjornstedt, Basic Clin. Pharmacol. Toxicol., 2014,
114, 377-386.
L. C. Clark, G. F. Combs, B. W. Turnbull, E. H. Slate, D. K. Chalker, J. Chow, L. S.
Davis, R. A. Glover, G. F. Graham, E. G. Gross, A. Krongrad, J. L. Lesher, H. K.
Park, B. B. Sanders, C. L. Smith and J. R. Taylor, JAMA-J. Am. Med. Assoc., 1996,
276, 1957-1963.
14.
15.
S. M. Lippman, E. A. Klein, P. J. Goodman, M. S. Lucia, I. M. Thompson, L. G. Ford,
H. L. Parnes, L. M. Minasian, J. M. Gaziano, J. A. Hartline, J. K. Parsons, J. D.
Bearden, III, E. D. Crawford, G. E. Goodman, J. Claudio, E. Winquist, E. D. Cook, D.
D. Karp, P. Walther, M. M. Lieber, A. R. Kristal, A. K. Darke, K. B. Arnold, P. A.
Ganz, R. M. Santella, D. Albanes, P. R. Taylor, J. L. Probstfield, T. J. Jagpal, J. J.
Crowley, F. L. Meyskens, Jr., L. H. Baker and C. A. Coltman, Jr., JAMA-J. Am. Med.
Assoc., 2009, 301, 39-51.
M. Mikalai, K. Sebastian, F. Lydia, V. Stefan, B. A. Carlson, S. Javier, J. Richard, D.
E. Handy, T. J. Park and L. Joseph, Antioxid. Redox Signal., 2015, 16, 185-192.

Journal of Materials Chemistry B
Page 14 of 14
View Article Online
DOI: 10.1039/C8TB03169J
16.
17.
18.
19.
20.
M. P. Rayman, Br. J. Nutr., 2008, 100, 254-268.
C. Ip, J. Nutr., 1998, 128, 1845-1854.
G. F. Combs and W. P. Gray, Pharmacol. Ther., 1998, 79, 179-192.
W. M, O. E, H. C, B. M and F. AP, Biochem. J., 2010, 429, 85-93.
Z. Veres, L. Tsai, T. D. Scholz, M. Politino, R. S. Balaban and T. C. Stadtman, Proc.
Natl. Acad. Sci. U. S. A., 1992, 89, 2975-2979.
21.
R. S. Glass, W. P. Singh, W. Jung, ., Z. Veres, ., T. D. Scholz and T. C. Stadtman,
Biochemistry, 1993, 32, 12555-12559.
22.
23.
C. M. Weekley and H. H. Harris, Chem. Soc. Rev., 2013, 42, 8870-8894.
K. Yayoi, O. Yasumitsu, I. Kazuya, T. Hiromitsu, A. Norio and K. T. Suzuki, Proc.
Natl. Acad. Sci. U. S. A., 2002, 99, 15932-15936.
24.
25.
H. M. Shen, C. F. Yang and C. N. Ong, Int. J. Cancer, 1999, 81, 820-828.
C. Yu, X. Huang, Y. Xu, H. Li, J. Su, J. Zhong, J. Kang, Y. Liu and L. Sun, Anat.
Rec., 2013, 296, 31-39.
26.
27.
K. A. Cupp-Sutton and M. T. Ashby, Antioxidants, 2016, 5, 42.
X. Chen, F. Wang, J. Y. Hyun, T. Wei, J. Qiang, X. Ren, I. Shin and J. Yoon, Chem.
Soc. Rev., 2016, 45, 2976-3016.
28.
29.
30.
Z. Guo, S. Park, J. Yoon and I. Shin, Chem. Soc. Rev., 2014, 43, 16-29.
W. Ralph and N. Vasilis, Nat. Med., 2003, 9, 123-128.
L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. Rev., 2013, 42, 622-
661.
31.
Y. Zhigang, C. Jianfang, H. Yanxia, Y. Jung Ho, K. Taeyoung, P. Xiaojun and K.
Jong Seung, Chem. Soc. Rev., 2014, 43, 4563-4601.
32.
33.
F. Kong, L. Ge, X. Pan, K. Xu, X. Liu and B. Tang, Chem. Sci., 2016, 7, 1051-1056.
F. Kong, Y. Zhao, Z. Liang, X. Liu, X. Pan, D. Luan, K. Xu and B. Tang, Anal.
Chem., 2017, 89, 688-693.