A highly sensitive and reductant-resistant fluorescent probe for nitroxyl in aqueous solution and serum

Article abstract of DOI:10.1039/c4cc01440e

A novel coumarin-based fluorescent probe, P-CM, for quantitative detection of nitroxyl (HNO) was developed. P-CM exhibits a selective response to HNO over other biological reductants and was also applied for quantitative detection of HNO in bovine serum with satisfactory results. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01440e

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A highly sensitive and reductant-resistant
fluorescent probe for nitroxyl in aqueous solution
and serum†
Cite this: Chem. Commun., 2014,
50, 5790
Received 25th February 2014,
Accepted 1st April 2014
Guo-Jiang Mao, Xiao-Bing Zhang,* Xue-Lin Shi, Hong-Wen Liu, Yong-Xiang Wu,
Li-Yi Zhou, Weihong Tan* and Ru-Qin Yu
DOI: 10.1039/c4cc01440e
www.rsc.org/chemcomm
A novel coumarin-based fluorescent probe, P-CM, for quantitative fluorescent probes have attracted particular interest due to their
detection of nitroxyl (HNO) was developed. P-CM exhibits a selec- advantages such as high sensitivity, fast analysis with high spatio-
tive response to HNO over other biological reductants and was also temporal resolution and non-destructive sample preparation.21–23
applied for quantitative detection of HNO in bovine serum with However, most of the previously reported fluorescent probes for
satisfactory results.
HNO were based on the reduction of Cu(II) to Cu(I),17 or nitroxide
to hydroxylamine19 by HNO. Therefore, these probes suffered from
Nitric oxide (NO) has been discovered to play a critical role in several interference from other biological reductants, such as glutathione
physiological and pathological processes, including anticancer and ascorbate, which are abundant in biological systems and limited
activity,1 vasodilation,2 neurotransmission,3 and regulation of the their applications in detection of HNO in practical biological samples.
immune response by macrophages.4 Nitroxyl (HNO), the one- The development of fluorescent probes that could discriminate HNO
electron reduced form of NO with a pKa of 11.4, which exists primarily from other biological reductants is desired.
in the protonated form under physiological conditions11a and exhibits
HNO was reported to be able to react with phosphines with a
biological effects distinct from NO,5 has gained increasing interest in high reaction rate constant (estimated to be 9 Â 105 MÀ1 sÀ1) to give
the past decade. For example, HNO reacts with protein thiols to the corresponding aza-ylide, intramolecular nucleophilic attack of
cause inhibition of aldehyde dehydrogenase,6 can activate voltage- which on the carbonyl carbon of the ester is expected to occur in the
dependent K+ channels in mammalian vascular systems,7 and is presence of a electrophilic ester, leading to the formation of alcohol
resistant to scavenging by superoxide.8 Moreover, biochemical studies and amide.14e,16a,20 On the other hand, 7-hydroxycoumarin was
suggest that HNO can be formed directly from nitric oxide synthase often chosen as a fluorophore of a probe since it possesses excellent
under appropriate conditions,9 and that NO and HNO may be able to photophysical properties, including good solubility, high fluores-
interconvert in the presence of superoxide dismutase (SOD).10 HNO cence quantum yield and large Stokes shift.24 Moreover, the fluores-
is a reactive molecule that could spontaneously dimerize and sub- cence properties of 7-hydroxycoumarin derivatives depend on their
sequently dehydrates to form nitrous oxide (N2O),11 making its direct molecular substitution, being influenced by the degree of intra-
detection in solution or biologically relevant samples difficult. As a molecular charge transfer from 7-substituents to the coumarin ring.
consequence, the studies on HNO chemistry and biology have been Based on these facts, we designed a new probe P-CM for HNO with
hampered by the lack of efficient detection methods. Thus, the 7-hydroxycoumarin as the fluorophore and a triphenylphosphine
development of highly sensitive and selective methods for the detec- group as the HNO recognition unit (Scheme 1).
tion of biological nitroxyl is of great importance.
Probe P-CM was synthesized following the synthetic route
Several methods, including mass spectrometry,12 high- shown in Scheme S1 (see ESI†). The structure of P-CM was
performance liquid chromatography,13 colorimetry,14 electrochemical confirmed by HNMR and MS (see Fig. S6 and S7, ESI†).
1
analysis,15 NMR,16 and fluorescent probes,17–20 have been developed
The fluorescent properties of the probe P-CM and its HNO-
for the detection of HNO in various samples. Among them, induced product 7-hydroxycoumarin were first assessed (see Fig. S1,
ESI†). A 76-fold fluorescence enhancement at 450 nm was observed
Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/
Biosensing and Chemometrics, College of Chemistry and Chemical Engineering,
College of Biology, Collaborative Innovation Center for Molecular Engineering and
Theranostics, Hunan University, Changsha 410082, China.
between P-CM and 7-hydroxycoumarin, indicating that P-CM is
suitable for constructing a ‘‘turn-on’’ fluorescence probe. Next, we
evaluated the capability of P-CM to detect HNO in aqueous buffer.
The time-dependent fluorescence response of P-CM to different
concentrations of AS was recorded (see Fig. S1, ESI†). The results
revealed that the response time of the probe toward AS increased
E-mail: xbzhang@hnu.edu.cn, tan@chem.ufl.edu
† Electronic supplementary information (ESI) available: Experimental details and
supplementary figures. See DOI: 10.1039/c4cc01440e
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ROS, including NO, GSNO, NO2À, NO3À, ONOOÀ, H2O2 and ClOÀ,
failed to induce significant fluorescence enhancement of P-CM.
Additionally, no obvious turn-on signal was induced when 100 equiv.
of K+, Na+, Ca2+, Mg2+, Zn2+, or Fe3+ was added. It is particularly
noteworthy that the fluorescence intensity of P-CM was hardly affected
by biological reductants, including GSH, AA, and Na2S (a hydrogen
sulfide source). The proposed probe P-CM seems to be greatly super-
ior to the previously reported Cu(II)-based probes which show high
fluorescence in the presence of large amounts of various biological
reductants.17 Moreover, the competitive experiments were conducted
with other testing species in the presence of 100 mM of AS (see Fig. S2,
ESI†). The fluorescence intensity of the probe did not vary much
except for Fe3+, GSH, AA and Na2S, which caused a decrease in the
fluorescence intensity. It may be because HNO was consumed by Fe3+,
GSH, AA or H2S since HNO is both an electrophile that can oxidize
thiols and a nucleophile that can reduce metal ions.25 All these results
demonstrated that P-CM probe is highly selective for HNO over other
biologically related species, and could meet the selective requirements
for detection of HNO in complex biological samples.
The effect of pH on the fluorescence intensity of P-CM in the
absence and presence of HNO was also investigated (see Fig. S3, ESI†).
The fluorescence intensity of P-CM was independent of pH over the
range of pH 5.0–10.0, indicating that the ester group is stable in
pH 5.0–10.0. After the probe was incubated with AS for 30 min, the
fluorescence intensity of P-CM notably increased with the increase in
pH between pH 5.0–9.0. It would be due to this that the reaction
product 7-hydroxycoumarin exhibits higher fluorescence intensity
under alkaline conditions because of deprotonation of the hydroxyl
group.26 At a pH value larger than 9.0, the fluorescence intensity of P-
CM showed a decreasing tendency, which might be ascribed to the
decreased decomposition rate of AS into nitroxyl under strong basic
conditions.27
Scheme 1 Structure and response mechanism of the probe P-CM for HNO.
with the increase in AS concentration. The P-CM itself shows a low
background fluorescence emission. However, the treatment of P-CM
(10 mM) with 10 equiv. of Angeli’s salt (AS, a HNO source) resulted in
a remarkable fluorescence enhancement, with a 57-fold fluores-
cence enhancement observed after 30 minutes. The changes in the
fluorescence spectra of P-CM upon the gradual addition of HNO
were further investigated (Fig. 1). The intensity increased linearly
with the concentration of AS ranging from 0.05 mM to 5 mM. A
detection limit of 20 nM was estimated for HNO (based on
3s/slope), which is much lower than the limits of previously
reported fluorescence probes for nitroxyl.17–19 The improved sensi-
tivity of the probe might be ascribed to the smaller size of probe
P-CM, which affords a weaker steric hindrance for HNO attaching.
For a probe with potential application in complex biological
samples, a highly selective response to the target species over other
potentially competing species is a necessity. Therefore, we assessed
the selectivity of P-CM for HNO over other biologically-related species
(Fig. 2). The responses of P-CM were found to be highly selective for
HNO over the reactive oxygen species (ROS), the reactive nitrogen
species (RNS), biological-related metal ions, and reductants. RNS and
To test the feasibility of the practical application of the probe
P-CM, we further conducted the HNO detection in 20% bovine serum
(Fig. 3). Upon addition of AS, the solution of P-CM in bovine serum
showed moderate fluorescence enhancements. The fluorescence
enhancement in bovine serum was not as large as that in aqueous
solution, which is because HNO can react with thiols and thiol
Fig. 1 (a) The fluorescence emission spectra of P-CM (10 mM) in the
presence of different concentrations of AS (0, 0.05, 0.2, 0.6, 1, 1.5, 3, 5, 10,
20, 40, 60, 100, 120, 150 mM) in buffered (pH 7.4) aqueous DMF solution.
Inset show the visual fluorescence of P-CM before (left) and after (right)
incubation with AS (100 mM) for 30 min (UV lamp, 365 nm). (b) Calibration
curve of P-CM to AS. The curve was plotted with the fluorescence intensity
vs. AS concentration after their incubation for 30 min. Inset shows the
linear responses at low AS concentrations.
Fig. 2 Fluorescence responses of the P-CM (10 mM) to testing species in
buffered (pH 7.4) aqueous DMF solution: (1) 100 mM AS; (2) 1 mM K+; (3)
1 mM Na+; (4) 1 mM Ca2+; (5) 1 mM Mg2+; (6) 1 mM Zn2+; (7) 1 mM Fe3+; (8)
3 mM GSH; (9) 4 mM AA; (10) 10 mM Na2S; (11) 500 mM H2O2; (12) 4 mM
ClOÀ; (13) 10 mM NO2À; (14) 10 mM NO3À; (15) 4 mM ONOOÀ; (16) 200 mM
NO; (17) 200 mM GSNO.
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reported fluorescence probes. Moreover, the probe was applied for
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tive detection of HNO in complex biological samples, demonstrating
its value in practical application.
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This work was supported by the National Key Scientific
Program of China (2011CB911000), NSFC (Grants 21325520,
21327009, 21221003, J1210040, 21177036, 21135001), National
Instrumentation Program (2011YQ030124), the Ministry of
Education of China (20100161110011), and Hunan Provincial
Natural Science Foundation (Grant 11JJ1002).
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5792 | Chem. Commun., 2014, 50, 5790--5792
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