An unprecedented strategy for selective and sensitive fluorescence detection of nitric oxide based on its reaction with a selenide

Article abstract of DOI:10.1039/c1cc12174j

A new strategy that utilizes the interaction between NO and a selenide is reported for fluorescence detection of NO, in which rhodamine B selenolactone serves as a model selenide. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc12174j

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COMMUNICATION
An unprecedented strategy for selective and sensitive fluorescence
detection of nitric oxide based on its reaction with a selenidew
Chengdong Sun, Wen Shi, Yanchao Song, Wei Chen and Huimin Ma*
Received 15th April 2011, Accepted 20th June 2011
DOI: 10.1039/c1cc12174j
A new strategy that utilizes the interaction between NO and a
selenide is reported for fluorescence detection of NO, in which
rhodamine B selenolactone serves as a model selenide.
heavy-atom effect can quench the fluorescence of an appended
fluorophore. Upon reacting with NO, however, the quenching
effect could be prohibited by the metal reduction and/or release,
resulting in the recovery of fluorescence.18–22 A number of this
type of probes have been proposed, one of which has been
successfully used to detect NO produced by both constitutive
and inducible NO synthases in live neurons and macrophages.23
Despite the progress in the metal-based complex probes, there still
exist several disadvantages such as biological incompatibility,19,20
easy leakage from cells,3 or side effect of the metal ion.13 There-
fore, NO fluorescent probes with high selectivity, sensitivity and
practicability for biological applications need to be developed.
Towards this end, very recently Anslyn and co-workers have
developed a superior fluorescent probe with high specificity for
NO. The probe employs a single reactive amino group rather than
highly electron-rich ortho diamines to detect NO, generating a
six-membered diazo ring structure.8 To the best of our knowledge,
however, there is no other strategy reported which could be used
to design a fluorescent probe for NO.
Nitric oxide (NO) plays an important role in the field of neuro-
science, physiology, and immunology.1–6 Appropriate levels of
NO are necessary for protecting organs such as the liver from
ischemic damage. However, sustained levels of NO production
result in direct tissue toxicity.7 Therefore, methods of sensing
and visualizing NO would be critical to reveal its biological
function and might eventually be useful for diagnosis as well.
A number of approaches have been reported for detecting NO,
but fluorescence methods have apparent advantages over other
ones in sensitivity and easy use in biological research, such as
real time monitoring or imaging in living systems.8
We note that nearly all the reported small molecular fluorescent
probes for NO fall into the following two types: ortho aromatic
diamines and transition-metal complexes (Scheme S1, ESIw).3,9,10
In ortho aromatic diamines, the electron-rich amino groups
effectively quench the fluorescence of the fluorophores by a
process known as photoinduced electron transfer (PET). Upon
reaction with NO under aerobic conditions, the vicinal aromatic
diamines are transformed into an electron-deficient triazole, which
terminates the PET process and restores the fluorescence.9,11,12
Although the past several years have witnessed great progress in
ortho diamine fluorescent probes, they still have some short-
comings. First, vicinal aromatic diamine probes are susceptible
to the interference by both oxidants and antioxidants,13 for
example, dehydroascorbic acid and ascorbic acid can react with
this kind of probes and turn on the fluorescence.14,15 Second, some
common influencing factors such as light,13 Mg(II)16 and Ca(II)17
also interfere with the detection of NO. Third, ortho aromatic
diamine probes actually react with NO+ or N2O3, rather than NO
itself, implying indirect detection.8 In transition-metal complex
probes (Scheme S1w), either the paramagnetism of the metal or the
Herein we present a new strategy that utilizes the interaction of
NO with a selenide. As is known, the NO-mediated cellular
functions are concerned with the interaction between NO and the
SeH groups of selenoproteins,24–26 and the formation of the
Se–NO bonds has been also reported (Scheme S2, ESIw).27,28
This inspires us to develop a NO fluorescent probe containing a
selenium atom. On the other hand, spectroscopic responses of
rhodamine probes to analytes are usually based on the opening
of their spirocyclic structures,29,30 and this structural fragility
had motivated us to prepare rhodamine B selenolactone (RBSe;
Scheme 1) by incorporating a Se atom into rhodamine B.
Because of the high affinity of the Se atom for Hg(II) and
Ag(I), the as-prepared RBSe can be applied to the detection of
Hg(II), CH3HgCl and Ag(I).31 In this work, we envision that the
formation of the Se–NO bonds27,28 may switch on the fluores-
cence of RBSe. Specifically, we here use RBSe as a model selenide
to exemplify the feasibility of our strategy for NO detection.
As expected, the probe RBSe itself has nearly no absorption
in the visible region and emits very weak fluorescence due to
its spirocyclic structure. This low-background spectroscopic
signal is extremely desirable for sensitive detection. Upon addi-
tion of NO (its saturated concentration in deionized H2O is
2.057 mM)32 into the solution of RBSe, a pink color and a large
fluorescence enhancement are immediately produced, with a
maximum absorption at 560 nm (Fig. S1, ESIw) and an emission
Beijing National Laboratory for Molecular Sciences, Key Laboratory
of Analytical Chemistry for Living Biosystems, Institute of Chemistry,
Chinese Academy of Sciences, Beijing 100190, China.
E-mail: mahm@iccas.ac.cn; Fax: +86-10-62559373;
Tel: +86-10-62554673
w Electronic supplementary information (ESI) available: Apparatus
and materials, fluorescence imaging of NO, optimization of experi-
mental variables, high-resolution FTICR mass spectra for the reaction
mechanism. See DOI: 10.1039/c1cc12174j
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Fig. 2 The plot of the fluorescence intensity change of RBSe (20 mM)
against the concentration of NO from 0–70 mM. DF = F À F0, where
F0 and F are the fluorescence intensity at 580 nm before and after NO
is added to the RBSe solution, respectively.
As shown in Fig. 3, only NO produces a large fluorescence
enhancement, whereas the other analytes do not show such
behavior. Moreover, the effects of various species on the
detection of NO were investigated. The results (Table S1, ESIw)
show that the species such as common inorganic salts, RONS,
hemoglobin, superoxide dismutase, reduced glutathione and
pyruvic acid at considerable concentrations hardly interfere
with the NO assay. These results indicate that RBSe has a high
selectivity for NO. Although Ag(I) and Hg(II) can react with
RBSe,31 the two ions at their normal physiological concentra-
tions actually would not influence the measurement of NO.
Thus, it is anticipated that RBSe may be used for NO detection
in some real biological samples.
Scheme 1 Possible reaction mechanism of RBSe with NO.
at 580 nm (Fig. 1), both of which rather resemble the spectro-
scopic feature of rhodamine B. This suggests that the reaction
may lead to the ring-opening of RBSe. The effect of pH of the
media varying from 4.8 to 9.8 was examined, revealing that the
fluorescence intensity of both the probe and its reaction products
is hardly affected by the pH change in the range of about pH
6.8–7.8 (Fig. S2w), indicating that RBSe is suited for physio-
logical environment applications. Time course studies showed
that the reaction of the probe with NO is fast; the fluorescence
intensity rises to a plateau in 15 min, and then increases only
slowly (Fig. S3w). For the purpose of reproducibility, a reaction
time of 30 min was employed in our experiments.
In order to explore the reaction mechanism of the present
system, detailed high-resolution mass spectral analyses were con-
ducted for the reaction products of RBSe with NO (Table S2w).
In a short 5 min reaction time (Fig. S4w), two major peaks at m/z
507.155 and 537.155 were observed, which are identified to be the
remaining RBSe (calcd m/z 507.155 [M + H]+) and compound 1
(calcd m/z 537.153 [M + H]+), as depicted in Scheme 1. With the
increase of reaction time to 30 min (Fig. S5w), the two peaks for
RBSe and compound 1 completely disappeared, but two new
ones appeared at m/z 443.234 and 506.149 (the latter one was
proven to be a doubly charged ion peak), which were character-
ized as rhodamine B (calcd m/z 443.233 [M + H]+) and a
Then, the fluorescence response of RBSe to NO at varied
concentrations was studied. A good linearity between the change
in fluorescence intensity and the NO concentration in the range
of 2.5 to 30 mM is observed (Fig. 2), which gives a regression
equation of DF = 48.5 Â [NO] (mM) À 9.5 (g = 0.995), as
depicted in the inset of Fig. 2. The limit of detection (3S/m,
in which S is the standard deviation of blank measurements,
n = 11, and m is the slope of the regression equation) was
determined to be 38 nM NO, also showing a high sensitivity.
Common coexisting substances including other reactive oxygen/
nitrogen species (RONS) were examined in parallel under the same
conditions to assess the specificity of the fluorescence-on reaction.
Fig. 3 Fluorescence spectra (lex = 520 nm) of RBSe (20 mM) only
and its reaction solutions with various species: 20 mM of NO; 50 mM
of ClOÀ;À100 mM of NaNO3, NaNO2, Na2SO4, KCl, CaCl2, MgCl2,
H2O2, O2
,
1O2, ONOOÀ, ꢀOH, L-cysteine, pyruvic acid or reduced
Fig. 1 Fluorescence spectra (lex = 520 nm) of RBSe (20 mM) with
varied concentrations of NO from 0 to 70 mM in 20 mM HEPES
buffer (pH 7.2) containing 20% (v/v) of ethanol at 37 1C.
glutathione; 10 mM of CuCl2, ZnCl2, FeCl3, hemoglobin or super-
oxide dismutase. The reactions were carried out for 30 min at 37 1C in
20 mM HEPES buffer (pH 7.2) containing 20% (v/v) of ethanol.
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unique detection strategy may provide a perspective to design
new selenide probes for NO sensing.
This work is supported by grants from the NSF of China (Nos.
20935005, 90813032, and 20875092), National Basic Research
Program of China (Nos. 2010CB933502, and 2011CB935800),
and the Chinese Academy of Science (KJCX2-EW-N06-01).
We thank Prof. W. Thiemann for helpful discussions on the
manuscript.
Fig. 4 Fluorescence images (top) and corresponding differential inter-
ference contrast (DIC) images (bottom) of RBSe-loaded Hela cells.
The RBSe-loaded Hela cells were incubated in the absence (A, C) and
presence (B, D) of 20 mM NOC 5 for 30 min (A, B) and 60 min
(C, D), respectively. In image (E), the RBSe-loaded Hela cells were
pre-treated with a NO scavenger PTIO (100 mM) for 20 min, and then
incubated with 20 mM NOC 5 for 60 min (see ESIw). Scale bar, 20 mm.
Notes and references
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diselenide compound (calcd m/z 506.147 [M]2+; Scheme 1),
respectively. Moreover, rhodamine B was further verified as a
major final product by HPLC analysis (Fig. S6w). In addition, the
weak signals at m/z 545.109, 585.067 and 625.026 (Fig. S5w) were
also proven to be doubly charged ion peaks, which may be
ascribed to the formation of tri- (calcd m/z 545.106 [M]2+), tetra-
(calcd m/z 585.064 [M]2+), and penta-selenide (calcd m/z 625.022
[M]2+) compounds, respectively. Based on the above results, we
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To evaluate the applicability of RBSe to biosystems, its
response to NO in Hela cells was investigated (see ESIw). Fig. 4
shows fluorescence images and corresponding differential
interference contrast images of RBSe-loaded Hela cells in the
absence and presence of NOC 5 [3-(aminopropyl)-1-hydroxy-
3-isopropyl-2-oxo-1-triazene, a NO donor]. As can be seen, the
RBSe-loaded Hela cells, incubated with NOC 5 for 30 min,
show a noticeable fluorescence (Fig. 4B), and the cells with a
longer incubation time of 60 min emit a much stronger
fluorescence (Fig. 4D). This indicates that the increase of
incubation time leads to more NO entering the cells. To
confirm that the fluorescence changes of the cells were caused
by NO, the RBSe-loaded Hela cells were pre-treated with a
NO scavenger PTIO [2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-
imidazoline-1-oxyl-3-oxide],33 and then incubated with NOC 5
for 60 min. As depicted in image E of Fig. 4, the fluorescence
intensity is indeed markedly suppressed. These results indicate
that RBSe, a less polar lactone derivative, is cell membrane-
permeable, and could well image the concentration change
of NO in intracellular environments. In addition, the major
reaction product of the probe is rhodamine B, which is a more
polar zwitterion and thus may be well-retained in cells. This
superior property makes RBSe a candidate with great potential
for imaging NO in biosystems.
In conclusion, we have developed a new strategy for NO
detection, which utilizes the interaction of NO with a selenide.
As a model probe for NO, RBSe exhibits excellent selectivity
and sensitivity, and its applicability to biosystems has been
demonstrated in fluorescence imaging of NO in Hela cells. The
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8640 Chem. Commun., 2011, 47, 8638–8640
This journal is The Royal Society of Chemistry 2011