A near-infrared xanthene-based fluorescent probe for selective detection of hydrazine and its application in living cells

Article abstract of DOI:10.1016/j.saa.2019.117344

Developing fluorescent probes for selective determination of the toxic and carcinogenic hydrazine are pretty significant. Herein, a rhodamine dye coupled to naphthalene was selected as a near-infrared fluorophore and acetyl group as a trigger unit for hydrazine sensing with a Stokes shifts of 62 nm. The probe showed about 77-fold NIR fluorescence enhancement in the presence of hydrazine. In addition, the detection limit was as low as 3.4 ppb, and the fluorescence intensity at 654 nm showed a satisfactory linearity with the concentration range of hydrazine from 0 to 120 μM. More importantly, the practical utility of probe has been successfully proved through the fluorescence bioimaging of hydrazine in living cells with low cytotoxicity and quantitative N₂H₄ detection in environmental water samples.

Full text of DOI:10.1016/j.saa.2019.117344

Accepted Manuscript
A near-infrared xanthene-based fluorescent probe for selective
detection of hydrazine and its application in living cells
Shao-Hua Guo, Tao-Hua Leng, Kai Wang, Yong-Jia Shen, Cheng-
Yun Wang
PII:
S1386-1425(19)30734-6
DOI:
https://doi.org/10.1016/j.saa.2019.117344
Article
Number:
Reference:
1
17344
SAA 117344
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
29 October 2018
27 June 2019
7 July 2019
Please cite this article as: S.-H. Guo, T.-H. Leng, K. Wang, et al., A near-infrared xanthene-
based fluorescent probe for selective detection of hydrazine and its application in living
cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,
https://doi.org/10.1016/j.saa.2019.117344
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ACCEPTED MANUSCRIPT
A near-infrared xanthene-based fluorescent probe for selective
detection of hydrazine and its application in living cells
a
b,
a
a
a,
Shao-Hua Guo , Tao-Hua Leng *, Kai Wang , Yong-Jia Shen , Cheng-Yun Wang 
a
Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular
Engineering, East China University of Science & Technology, Shanghai 200237, P. R. China.
b
National Food Quality Supervision and Inspection Center (Shanghai) / Shanghai Institute of Quality Inspection
and Technical Research, Shanghai, China.
Abstract
Developing fluorescent probes for selective determination of the toxic and
carcinogenic hydrazine are pretty significant. Herein, a rhodamine dye coupled to
naphthalene was selected as a near-infrared fluorophore and acetyl group as a trigger
unit for hydrazine sensing with a Stokes shifts of 62 nm. The probe showed about
7
7-fold NIR fluorescence enhancement in the presence of hydrazine. In addition, the
detection limit was as low as 3.4 ppb, and the fluorescence intensity at 654 nm
showed a satisfactory linearity with the concentration range of hydrazine from 0 to
1
20 μM. More importantly, the practical utility of probe has been successfully proved
through the fluorescence bioimaging of hydrazine in living cells with low cytotoxicity
and quantitative N detection in environmental water samples.
2
H
4
Keywords: Near-infrared fluorescence, Hydrazine, Bioimaging, High selectivity

Corresponding author: e-mail: length@sqi.org.cn (T-H Leng), cywang@ecust.edu.cn (C-Y Wang)

ACCEPTED MANUSCRIPT
1
. Introduction
Hydrazine (N
2
4
H ), which plays a key role in the preparation of emulsifiers,
pharmaceuticals, paints and pesticides, has been extensively applied to missile and
rocket propulsion systems due to its high enthalpy [1-4]. More importantly, excess
exposure to hydrazine might lead to serious organ damage, including kidney, liver,
lung, and especially the central nervous system [5, 6]. Thus, the governmental
regulations have been issued to control the residual hydrazine content in the final
products for the prevention of health problems, for instance, its threshold in drinking
water is 10 ppb [7]. In view of this, it is of great significance to develop a highly
effective method for hydrazine sensing in the fields of chemistry, biology and
environment. Up to now, a variety of techniques, such as chemiluminescence [8],
surface enhanced Raman spectroscopy [9], chromatography [10], electrochemistry
[
11-14], have been developed to determine hydrazine. However, they often need
complicated instruments and are inappropriate to intracellular detection. The
fluorescence spectra analysis has attracted a lot of attention because of its
non-invasiveness, good compatibility to biological samples and excellent selectivity.
So, developing simple, selective and non-invasive methodologies for determination
and imaging of hydrazine is extremely urgent.
Based on three main mechanisms, including specific deprotection of acetyl
groups, specific reaction with arylidenemalononitrile and condensation with

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arylaldehydes, many kinds of fluorescent chemosensors for hydrazine detection have
been reported [15-30]. However, only a few near-infrared fluorescent probes for
hydrazine sensing have been developed. For the first time, Peng et al. used
cyanine-based probe for visualizing hydrazine in living mice [31]. This probe shows
lower detection limit and good selectivity. By selecting a hemicyanine skeleton as
fluorophore and acetyl group as a recognizing group, Zhang and co-workers
developed a near-infrared fluorescent probe for hydrazine sensing [32]. The probe
exhibits a low limit of detection (LOD) (6.1 ppb as UV sensor and 5.4 ppb as
fluorescence sensor). Li’s group selected hemi-cyanine as a chromophore and
2
-thiophenecarbonyl unit as a recognizing moiety for imaging the hydrazine in living
cells and tissues [33]. These sensors displayed good selectivity and sensitivity.
However, some of them had relatively long synthetic route, low fluorescence intensity
or long detection time, which limited their application. In view of this, developing
high performance near-infrared fluorescent probes for hydrazine sensing is still highly
demanded.
Emission wavelength of most rhodamine derivatives is less than 600 nm [34,
3
5], and they usually possess small Stokes shifts (less than 35 nm) [36, 37]. Notably,
the relatively small Stokes shifts is unfavourable to its application in biology because
of excitation backscattering effects. In addition, compared with fluorescent probes in
the visible region, NIR-emitting (650−900 nm) fluorescent probes possess some
advantages in biological applications, such as relatively low background
auto-fluorescence in living systems, deep photon penetration in tissue and minimum

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photodamage in biological samples [38-40]. A particular challenge is to develop
hydrazine probes that are biocompatible [41,42], and probes could be used in cellular
milieus since this might allow toxic events to be monitored in real time. Herein, a new
near-infrared fluorescent probe based on xanthene has been developed for hydrazine
sensing. In our design, in order to enlarge the π-electron conjugated system,
naphthalene was introduced into the xanthene ring to obtain fluorescent dye RN
(Scheme 1). Notably, RN not only has the excellent performance of the rhodamine
dyes, such as strong fluorescence and good water solubility (Fig. S1), but also has an
emission wavelength of 654 nm along with a large Stokes shifts (>60 nm). Therefore,
RN was selected as a near-infrared fluorophore and acetyl group as a trigger unit for
hydrazine sensing. RN-Probe not only showed excellent colorimetric (pink to blue)
and NIR “off-on” detection process toward hydrazine, but also had a low detection
limit (3.4 ppb) and dramatic NIR fluorescence enhancement (about 77-fold).
Moreover, the probe was able to detect hydrazine in living cells and determine
hydrazine in environmental water samples quantitatively.
2
. Experimental section
2
. 1 Materials and instruments
All chemical reagents and solvents were analytical grade and commercially
1
3
1
available. Bruker AM 400 spectrometer was used to obtain C NMR and H NMR
spectra. Agilent 1100 Series chromatographic instrument was used to collect HPLC
data. We used Waters LCT Premier XE spectrometer to collect HRMS data.
Fluorescence emission spectra and UV-vis absorption spectra were performed on

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F97pro fluorospectrophotometer and Varian Cary 500 spectrophotometer respectively.
2
. 2 Synthetic experiments
The synthesis process was shown in Scheme 1. The characteristic data of each
compound were shown in the supporting information.
Insert Scheme 1
2
. 2.1 Synthesis of Compound 1
The mixture of m-diethylaminophenol (990 mg, 6 mmol) and o-phthalic
o
anhydride (890 mg, 6 mmol) in toluene was stirred at 110 C for 8 h at N
2
atmosphere.
Subsequently, the reaction mixture cooled down to room temperature and filtered.
After being washed with absolute alcohol and dried, Compound 1 was produced as a
pale pink solid (1.17 g, 62.5% yield) which was used for the subsequent synthesis
1
without further purification. H NMR (400 MHz, DMSO-d
6
) δ (ppm) 13.08 (s, 1 H),
1
2.57 (s, 1 H), 7.95 (d, J = 6.8Hz, 1 H), 7.66 (t, J = 7.5Hz, 1 H), 7.59 (t, J = 7.6Hz, 1
H), 7.37 (d, J = 7.4 Hz, 1H), 6.78 (d, J = 9.1 Hz, 1 H), 6.17 (dd, J = 9.2 Hz, J = 2.4 Hz,
1
H), 6.07 (d, J = 2.4 Hz, 1 H), 3.36 (q, J = 7.0 Hz, 4 H), 1.08 (t, J = 7.0 Hz, 6 H).
. 2. 2 Synthesis of RN
2
In a 100mL round bottomed flask, Compound 1 (949 mg, 3 mmol) and
1
,6-dihydroxynaphthalene (460 g, 3 mmol) were dissolved in 40 mL methanesulfonic
o
acid. The resulting mixture was stirred at 90 C for 7 h. After cooling to the room
temperature, the reaction mixture was poured into NaCl solution (100 mL, 10%). The
resulting purplish red precipitate was collected by filtration and dried under vacuum.
The residue was further purified by silica gel chromatography using petroleum

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ether/dichloromethane/absolute alcohol (v/v/v, 3:3:1) as an eluent to give pure
1
compound RN with yield of 60%. H NMR (400 MHz, DMSO-d
6
) δ (ppm) 10.10 (s, 1
H), 8.45 (d, J = 9.0 Hz, 1 H), 8.04 (d, J =7.4 Hz, 1 H), 7.80-7.71 (m, 2 H), 7.37 (d, J =
8
6
.8 Hz, 1 H), 7.28-7.24 (m, 2 H), 7.14 (d, J = 2.3 Hz, 1 H), 6.73 (d, J = 1.7 Hz, 1 H),
.6 (d, J = 8.8 Hz, 1 H), 6.56-6.50 (m, 2 H),3.43 (q, J = 7.0 Hz, 4 H), 1.13 (t, J = 7.0
+
Hz, 6 H). HRMS (ESI): Calc. Mass for [M+H] 438.1705, found 438.1702.
2
. 2. 3 Synthesis of RN-Probe
Acetyl bromide (492 mg, 4 mmol) and compound RN (437 mg, 1 mmol) were
dissolved in 30 mL of CH
2
Cl . Subsequently, triethylamine was added and stirring for
2
2
h. Then, the combined residue was subjected to silica gel chromatography to
1
generate RN-Probe as pink powder with yield of 75%. H NMR (400 MHz, DMSO-d
δ (ppm) δ 8.65 (d, J = 9.1 Hz, 1H), 8.05 (d, J = 7.3 Hz, 1 H), 7.84-7.69 (m, 3 H),
.69-7.56 (m, 2 H), 7.29 (d, J = 7.5 Hz, 1 H), 6.78 (dd, J = 5.3, 3.3 Hz, 2 H), 6.64-6.49
6
)
7
1
3
(
m, 2 H), 3.42 (q, J = 7.0 Hz, 4 H), 2.35 (s, 3 H), 1.14 (t, J = 7.0 Hz, 6 H). C NMR
100 MHz, DMSO-d ) δ 169.23, 168.63, 152.86, 151.70, 149.90, 149.28, 147.49,
46.54, 135.63, 134.52, 130.13, 128.62, 126.05, 124.79, 124.65, 124.09, 123.70,
(
6
1
1
22.81, 122.38, 121.04, 118.05, 97.02, 83.39, 43.77, 20.90, 12.36. HRMS (ESI): Calc.
+
Mass for [M+H] 480.1811, found 480.1855.
2
. 3 Absorption and fluorescence spectra analysis
Fluorescence and absorption spectra analysis were conducted in PBS-DMSO
(pH 7.4, 20 mM, v/v = 1/1). Test solutions were adjusted to a suitable concentration
by PBS-DMSO (pH 7.4, 20 mM, v/v = 1/1). The final samples mixed with N were
2
H
4

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o
stirred at 30 C for 10 min, and then fluorescence and UV test were carried out. 592
nm was selected as the excitation wavelength in the fluorescence test.
2
. 4 Determination of hydrazine in real water samples
The real water samples were from the tap water, Qingchun River (ECUST) and
Huangpu River in Shanghai. We used a microfiltration membrane to filter the real
water samples before being used in the test. PBS solution was used to adjust the pH of
water samples. Four different concentrations of N
2
4
H (0, 10, 50 and 90 μM) were
spiked in each sample. Unless clearly stated, all of the fluorescence tests were
o
conducted after mixing the samples at 30 C for 10 min. Eventually, RN-Probe was
used for determining the concentration of N
2
4
H through the calibration curve.
2
. 5 Cell imaging
o
Initially, the Hela cells incubated with RN-Probe (10 μM) for 30 min at 37 C,
and then bioimaging after the Hela cells being washed three times with PBS (pH 7.4,
2
0 mM). Next, the cells co-cultured with N
2
H
4
(250 μM) for 30 min and were treated
o
by RN-Probe (10 μM) for another 30 min at 37 C. The Hela cells were washed by
PBS (pH 7.4, 20 mM) for three times before conducting bioimaging experiments. All
the measurements of fluorescence spectra were performed on the confocal laser
scanning microscopy (Nikon A1R). The emissions were recorded within the range of
6
40 − 700 nm (red channel).
3
. Results and discussion
3
. 1 Absorption and fluorescent detection of hydrazine

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Initially, we investigated the response of RN-Probe toward N
2
4
H in PBS-DMSO
o
(
pH 7.4, 20 mM, v/v = 1/1) at 30 C. As it can be seen in Fig. 1A, the probe itself
displayed a pink color. But upon treatment with N H4, we could find a new absorption
2
peak around 592 nm, and there was a clear color change from pink to blue under
natural light. This result indicated that the RN-Probe was able to serve as a
“
naked-eye” colorimetric indicator for N
2
4
H . As shown in Fig. 1B, the probe shows a
extremely low fluorescence emissions at 654 nm. However, within several minutes, a
red remarkable up-to-77-fold fluorescence enhancement was clearly observed at 654
nm after addition of N
2
4
H . These results implied that the probe not only can play the
part of a promising turn-on NIR fluorescent indicator, but also can detect N
2
4
H in
minutes.
Insert Fig. 1
3
. 2 The sensitivity
The probe solution were handled with different concentrations of N
2
4
H to
investigate the sensitivity of the probe. As shown in Fig. 2, the fluorescence intensity
of the probe solution increased upon gradual addition of N
2
4
H . The relative
2
fluorescence intensity at 654 nm showed a satisfactory linearity (R = 0.9906) with the
N
2
H
4
concentration ranging from 0 to 120 μM. Based on the definition by IUPAC
DL = 3σ / S) [43-46], the detection limit was calculated to be about 3.4 ppb in
PBS-DMSO (20 mM, pH 7.4, v/v = 1/1), which was lower than several reported
fluorescent probes for N (Table 2), these results indicated that the probe is highly
(C
2
H
4

ACCEPTED MANUSCRIPT
sensitive toward N
was investigated by carrying out time-dependent fluorescence experiments. As
it can be seen in Fig. S2, after addition of 25 equiv. of N , fluorescence intensity (at
54 nm) of probe (10 μM) was gradually increased and appeared a fluorescent
maximum around after 10 min. These results showed that the probe could achieve a
2
4
H . At the same time, the reaction time of RN-Probe probe with
N
2
H
4
2
H
4
6
rapid detection of N
2
H
4
and was able to be used in practical application.
Insert Fig. 2
Insert Table 1
3
. 3 Selectivity and effect of pH
Besides highly sensitive, a satisfactory N
2
H
4
probe should also have high
, a wide variety
selectivity. In order to investigate the selectivity of the probe for N
2
H
4
of analytes were added to the probe solution. As it can be seen in Fig. 3, the
fluorescence spectra displayed almost no changes after addition of various other
2
+
3+
3+
+
2+
2+
2+
+
-
analytes, including Cu , Fe , Al , Na , Fe , Ba , Zn , K , HS , Asparagine (Asn),
Aspartic acid (Asp), Alanine (Ala), Arginine (Arg), Glutamine (Gln), Glutathione
(GSH), Homocysteine (Hcy), Cystine (Cys), Glutamic acid (Glu), Glycine (Gly),
Phenylalanine (Phe), Proline (Pro), Tryptophan (Trp), Histidine (His), Leucine (Leu),
Threonine (Thr) and Valine (Val). However, a significant fluorescence enhancement
(
about 77-fold) was observed upon addition of N
selectivity toward N over these analytes. In addition, competing experiment
showed that other analytes could hardly interfere with the detection process (Fig. 4).
2
H
4
, indicating the probe had good
2
H
4

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A suitable pH working range is an important criterion for measuring the utility of
fluorescent probes in biology. Therefore, the probe for N
2
H
4
detection was
investigated in wide pH range 5.0-12.0. As shown in Fig. S3, the appropriate working
pH was from 6.0 to 10.0. The result implied that the probe could work over a
relatively wide pH range.
Insert Fig. 3
Insert Fig. 4
3
. 4 Determination of hydrazine in real water samples
As a suspected carcinogen, hydrazine has been extensively applied to various
industrial manufactures. Even worse, owing to the perfect water solubility of N
2
4
H ,
biological systems can absorb N easily through inhalation or skin contact. Thus, it
2
H
4
is of great significance to determine hydrazine in real water samples. We used
standard addition method to deal with the real water samples from the tap water,
Qingchun River (ECUST) and Huangpu River in Shanghai, then the samples were
used in the test [47, 48]. As shown in Table 2, the recovery rates were in the range of
9
6%-103%. The result indicated that RN-Probe was able to determine hydrazine in
environmental water samples quantitatively.
Insert Table 2
3
. 5 Proposed Sensing mechanism
In order to investigate the mechanism of RN-Probe for N
2
4
H detection, TLC,
mass and HPLC analysis were conducted. TLC analysis (Fig. S4) showed that when

ACCEPTED MANUSCRIPT
reacted with N
product formed, which had the same retention factor (R
indicated that the reaction product was RN. When RN-Probe reacted with N
min, the reaction mixture of RN-Probe and N was isolated by a silica gel column
product A). Then product A was subjected to mass analysis. The peak was found at
m/z 438.1702 correponding to RN (Fig. S5). HPLC analysis (Fig. 5) showed that
upon addition of N , a decrease in the RN-Probe signal was observed and a new
signal corresponding to RN appeared. All the results indicated that the mechanism of
RN-Probe for N detection might be as follows: RN-Probe reacted with hydrazine
2
H
4
for 10 min, RN-Probe disappeared. Meanwhile, a new fluorescent
) value as RN. This result
for 10
f
2
H
4
2
H
4
(
2
H
4
2
H
4
due to the specific deprotection of acetyl group of the probe, to deliver RN, and
eventually caused the appearance of fluorogenic signaling (Scheme 2).
Insert Fig. 5
Insert Scheme 2
3
.6 Cellular imaging
In order to extend the application of RN-Probe into biological systems, the
CCK8 assay was used to estimate the cytotoxicity of RN-Probe in HeLa cells [49-52].
As it can be seen in Fig. S6, the cell viability still remained more than 80 % even cells
cultured with RN-Probe (40 μM) for 12 h. This result indicated that RN-Probe has
low cytotoxicity. Then, RN-Probe (10 μM) was used to carry out the bioimaging
experiments because of its high fluorescence in the presence of N
2
H
4
and good
cytotoxicity. As shown in Fig. 6, we found that no intracellular fluorescence was

ACCEPTED MANUSCRIPT
detected under excitation at 592 nm after HeLa cells were incubated only with
RN-Probe (a1). However, in the control experiment, when cells co-cultured with
N
2
4
H for first 30 min, and then treated with RN-Probe for another 30 min, a
remarkable red fluorescence enhancement was able to be observed (b1). All of these
results demonstrated that RN-Probe could determine intracellular hydrazine in living
cells.
Insert Fig. 6
4
. Conclusion
In summary, a NIR xanthene-based fluorescent probe has been developed for
N
2
H
4
sensing. The probe showed a dramatic NIR fluorescence enhancement (about
7-fold) toward N . In addition, it had a Stokes shifts of 62 nm (λem – λabs = 62 nm),
and the fluorescence intensity at 654 nm exhibited a satisfactory linearity with the
7
2
H
4
concentration of N
selectively detect N
2
H
4
in the range of 0-120 μM. The probe was also able to
2
H
4
in minutes against other interfering analytes and the detection
limit was as low as 3.4 ppb. More importantly, we have successfully proved the
practical utility of RN-Probe through the fluorescence bioimaging of N in living
2
H
4
cells and quantitative N
2
4
H detection in real water samples.
Acknowledgements
The authors are grateful to financial support from Natural Science Foundation of
Shanghai (16ZR1408000) and the National Natural Science Foundation of China (No.

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2
1576087).
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o
Reagents and conditions: (a) o-phthalic anhydride, toluene, 110 C; (b) methanesulfonic acid,
o
o
1
,6-dihydroxynaphthalene, 90 C; (c) acetyl bromide, triethylamine, CH
2
Cl , 0 C.
2
Scheme 1 Synthesis of RN-Probe

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Fig. 1 (A) Absorption spectra properties of RN-Probe upon treatment with 25 equiv. of N
2
H ; (B)
4
Fluorescent spectra changes of different concentration of N
2
4
H (0–30equiv.) in a mixed
o
PBS-DMSO solution (pH 7.4, 20 mM, v/v = 1/1) at 30 C; All the spectrum were recorded at 10
min. Insert: Emission color and color change of RN-Probe before and after addition of N
92 nm. Slit width:10 nm/10 nm.
2
H . λex =
4
5

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Fig. 2 The linearity of fluorescence intensity (at 654 nm) versus N
2
4
H concentrations

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Table 1. Comparation of fluorescent probes for N
2
H
4
Detection
medium
λ
ex/λem
Detection
time
References
Probe
Detection limit
Not mention
(nm)
PBS–DMSO
(
v/v = 1/9, pH
3
2
2
0
4
1
510/559
60 min
7 min
7
.4,
1
0 mM)
PBS
( pH 7.4,
0 mM)
341/400
1007 ppb
5.09 ppb
1
DMSO/H
(1/19, v/v, pH
.4, 20 mM).
2
O
λ
ex = 685 nm
30 min
λ
em = 715 nm
7
DMSO-Hepes
(4/6, v/v, pH
λ
ex = 403 nm
2
6
λem = 462/524
60 min
4.5 ppb
7
.4, 10 mM)
nm
DMSO-Hepes
(1/1, v/v, pH
λ
ex = 450 nm
em = 510/660
2
3
8
1
λ
24 h
18.2 ppb
5.4 ppb
7
.4, 10 mM)
nm
DMSO-Hepes
(4/1, v/v, pH
λ
ex = 675 nm
em = 706 nm
10 min
λ
7
.4, 10 mM)
DMSO–PBS
This work
(v/v = 1/1, pH
592/654
10 min
3.4 ppb
7
.4, 20 mM)

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Fig. 3 (A) Fluorescent spectra changes of RN-Probe (10 μM) for various analytes (25 equiv.) in a
o
mixed solution PBS-DMSO (20 mM, pH 7.4, v/v = 1/1) at 30 C; (B) Flurescence intensity (at 654
2
+
3+
3+
nm) of RN-Probe for various analysts (25 equiv.). Analytes are: 1: black, 2: Cu , 3: Fe , 4: Al ,
+
2+
2+
2+
+
-
5
: Na , 6: Fe , 6: Ba , 8: Zn , 9: K , 10: HS , 11: Asn, 12: Asp, 13: Ala, 14: Arg, 15: Gln, 16:
GSH, 17: Hcy, 18: Cys, 19: Glu, 20: Gly, 21: Phe, 22: Pro, 23: Trp, 24: His, 25: Leu, 26: Thr, 27:
Val, 28: N . λex = 592 nm. Slit width:10 nm/10 nm.
2
H
4

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Fig. 4 The selectivity of RN-Probe (10 μM). The black bars represent the emission intensity of
RN-Probe in the presence of other analytes (250 μM). The red bars represent the fluorescent
2
+
intensity (at 654nm) that occurs upon addition of (250 μM) N
2
H
4
to the above solution. 1: Cu , 2:
3
+
3+
+
2+
2+
2+
+
Fe , 3: Al , 4: Na , 5: Fe , 6: Ba , 7: Zn , 8: K , 9: NaHS, 10: Asn, 11: Asp, 12: Ala, 13: Arg,
1
2
4: Gln, 15: GSH, 16: Hcy, 17: Cys, 18: Glu, 19: Gly, 20: Phe, 21: Pro, 22: Trp, 23: His, 24: Leu,
5: Thr, 26: Val, 27: ascorbic acid

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2
Table 2. Analysis of the N H
4 concentrations in real water samples a
N
2
H
4
2 4
N H
samples
recovery (%)
spiked (M)
0
recovered (M)
Not detected
—
1
×10-5
5×10-5
(0.97±0.22)×10-5
(5.06±0.07)×10-5
(9.27±0.20)×10-5
97
tap water
101.2
103
9
×10-5
0
Not detected
—
1
5
9
×10-5
(0.98±0.10)×10-5
98
Qingchun River
×10-5
(4.95±0.16)×10-5
99
×10-5
(9.30±0.14)×10-5
102.5
0
Not detected
—
96
1
5
9
×10-5
(0.96±0.15)×10-5
Huangpu River
×10-5
(5.12±0.19)×10-5
102.4
101.3
×10-5
(9.16±0.12)×10-5
a
Note: the results shown in the table are reported as the mean ± standard deviation of triplicate
experiments.

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Fig. 5 HPLC analysis. (A) and (B) are the HPLC traces of the reference samples for RN and
RN-Probe, respectively. (C) HPLC traces of the reaction of the RN-Probe (20μM) with
500 μM) in PBS-DMSO (20 mM, pH 7.4, v/v = 1/1) after 20 min. Conditions: eluent,
O/CH CN (v/v, 1/9), flow rate, 0.2 mL/min; detection wavelength, 270 nm; injection volume,
0.0 μL
N
2
H
4
(
H
2
3
2

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Scheme 2 The proposed sensing mechanism of RN-Probe for N
2
H
4