A merocyanine-based dual-mode optical probe for detection of hydrazine and its bioimaging application in vitro and vivo

Article abstract of DOI:10.1016/j.saa.2019.117625

In this paper, a Merocyanine-based turn-on probe (McyA) has been developed for colorimetric and fluorescent dual-mode detection of N₂H₄ via an intra-molecular charge transfer (ICT) mechanism. In the presence of N₂H₄

Full text of DOI:10.1016/j.saa.2019.117625

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117625
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A merocyanine-based dual-mode optical probe for detection of
hydrazine and its bioimaging application in vitro and vivo
Xiumei Guo , Shanshan Li , Shuai Mu , Yintang Zhang , Xiaoyan Liu , Haixia Zhang a
a
a
a
b
a, *
a
State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and
College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China
b
Henan Key Laboratory of Biomolecular Recognition and Sensing, College of Chemistry and Chemical Engineering, Shangqiu Normal University, Shangqiu,
476000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 February 2019
Received in revised form
In this paper, a Merocyanine-based turn-on probe (McyA) has been developed for colorimetric and
fluorescent dual-mode detection of N
presence of N , the probe shows an obvious chromogenic response and fluorescent enhancement.
Based on this feature, the synthesized McyA can be applied to quantify N concentration from 0.12 to
.0 M and 0.5e10 M with a detection limit of 0.042 M (4.5 ppb, S/
M (1.3 ppb, S/N ¼ 3) and 0.140
N ¼ 3), respectively. Moreover, McyA has also be successfully employed for imaging analysis of N
distribution in different organs from the N ingested mice model, revealing the potential application of
McyA as a powerful fluorescent sensor for tracking detection and risk assessment of hydrazine in vivo.
2019 Elsevier B.V. All rights reserved.
2 4
H via an intra-molecular charge transfer (ICT) mechanism. In the
2 4
H
20 July 2019
2 4
H
Accepted 6 October 2019
Available online 7 October 2019
5
m
m
m
m
2 4
H
2 4
H
Keywords:
Dual-mode detection
Hydrazine
©
Fluorescence probe
Bioimaging
In vivo
1. Introduction
[12,13]. Therefore, designing a facile and visualized detector for on-
site and real-time recognition of N
very desirable.
2 4
H in a variety of matrices is
Hydrazine (N
2 4
H ) is an essential reactive reactant in the prep-
aration of polymers, pesticides, pharmaceuticals intermediated [1],
emulsifiers and textile dyestuff [2e4]. Its strong reducing property
enables its use as an oxygen scavenger and corrosion inhibitor in
various applications including electric utilities [5], new materials,
as well as an energetic fuel in missile and rocket propulsion system
To date, several different analytical methods for determination
of N have been developed, including chromatography [14e17],
2 4
H
electrochemistry [18], titrimetry [19], fluorometry [20e22] and so
on. Among these analytical approaches, fluorescent techniques
based on fluorescent probes [23e27] have received tremendous
attention due to their high sensitivity, specificity, economy,
simplicity for implementation, noninvasive and real-time detection
[
6,7]. Besides, it is also used as an indispensable precursor in syn-
thesis of hydrazine fuel cells in power generation sector [8]. How-
ever, N is extremely toxic and can be readily absorbed by
2
H
4
2 4
in live cells or tissues. So far, numerous fluorescent probes for N H
biological systems during manufacture, usage, transportation and
disposal [9,10], which cause carcinogenic and mutagenic effect [11].
Even worse, N H has a volatile and high water solubility, which
2 4
caused its ubiquitous distribution in the environment as well as in
turn enters a route into human body through respiratory, dermal
contact or food chain transfer. Thus, even for persons who are not
chemists or industrial workers, the potential risk of them exposure
detection have been explored based on the strong nucleophilicity of
hydrazine toward the recognition moiety such as acetyl [28e32],
formyl phenol [33], bromo butylate [34e36], levulinate [37e39],
benzothiazole acetonitrile [40] and 2,4-dinitrophenyl [41]. Such
probes are typically composed of an analyte-triggered site and a
fluorophore. Undoubtedly, some of them show good selectivity and
2 4
high sensitivity for detection of N H . However, from an economic,
to N
H
2 4
is still existed. For these reasons, the U.S Environmental
convenient and practical perspective, extra strategy such as con-
Protection Agency (EPA) has been implicated as a probable
carcinogen with an allowable threshold limit value of 10 ppb
struction of “naked-eye” and fluorescent dual-mode sensors for
2 4
detection and bioimaging of N H in vitro and vivo are still
imperative.
Merocyanines are donor-acceptor-substituted conjugated sys-
tems (D- -A) and shows a strong intramolecular charge transfer
(ICT) character [42], which endow merocyanine unit potential
p
*
Corresponding author.
E-mail address: liuxiaoy@lzu.edu.cn (X. Liu).
https://doi.org/10.1016/j.saa.2019.117625
386-1425/© 2019 Elsevier B.V. All rights reserved.
1

2
X. Guo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117625
applications in optoelectronics [43], and as fluorescent sensors for
biology imaging or medicine detection [44]. Driven by above
requirement, we designed and synthesized a simple ratiometric
colorimetric and fluorescent dual-mode probe (McyA) based on
Merocyanine as fluorophore and acetyl group as recognition site for
digital pH-meter (PHSJ-3F, Leici, Shanghai, China). Cell imaging was
performed by Confocal fluorescence microscopy (FV1000,
Olympus, Japan) and bioimaging was carried out with IVIS Spec-
trum (Canada) equipped with molecular imaging software 5.0.6.20.
Chromatographic analysis was performed by Thermo Fisher's Dio-
nex UltiMate 3000 HPLC (UHPLC, Chromatographic conditions:
A ¼ acetonitrile, B ¼ ammonium acetate buffer solution, A ¼ 90%,
B ¼ 10%; flow rate of 0.8 mL minꢀ , detection wavelength: 445 nm)
2 4
specific detection of N H . As depicted in Scheme 1, in the present
of N , N will attack the carbonyl group of the acetyl group and
2
H
4
2 4
H
1
then lead to cleavage of the ester group, which produce the optical
structure accompanying by changes in color and spectral properties
under the physiological conditions [36]. Furthermore, the sensing
2.2. Synthesis of probe(McyA)
mechanism of McyA for N
tional theory (DFT) and high performance liquid chromatography
HPLC). Finally, McyA was employed for bioimaging of intracellular
and investigation of its bioaccumulation in animals.
2 4
H was demonstrated by density func-
The synthetic routes of probe McyA are shown in Fig. 1 and the
details of procedure are described as follows.
(
2 4
N H
2.2.1. Synthesis of McyOH
2
2
2
. Experimental section
1,2,3,3-tetramethyl-3H-indoleiodide (903.5 mg, 3.0 mmol) and
p-hydroxybenzaldehyde (439.6 mg, 3.6 mmol, 1.2 equiv) were
mixed in 100 mL round-bottomed flask containing 40 mL of anhy-
drous ethanol. The mixture was heated to reflux for 12 h and
monitored by thin layer chromatography (TLC). After the reaction
was completed, the mixed solution was filtered and the filter cake
was washed with petroleum ether to remove residual p-hydrox-
.1. Chemical reagents and instruments
.1.1. Chemical reagents
1
,2,3,3-Tetramethyl-3H-indole
ybenzaldehyde (C ,99%), bromo acetyl (C
purchased from Anagji Chemicals (Shanghai, China). N
(C12
H
16IN,99%),
BrO, 98%) were
was
p-hydrox-
7
H
6
O
2
2 3
H
ꢁ
H
2 4
ybenzaldehyde. Then, the crude product was dried at 60 C. Sub-
purchased from Saan Chemical Technology Co., Ltd. (Shanghai,
China). Ultrapure water is purified by the ALH-6000-U instrument
sequently, the product was purified and obtained the compound
1
McyOH (yield 76%). H NMR (400 MHz, DMSO-d )
6
d
¼ 10.68 (s, 1H),
(
America). other drugs and reagents were of analytical grade
8.35 (d, J ¼ 16.1 Hz, 1H), 8.10 (d, J ¼ 8.7Hz,2H), 7.83 (d, J ¼ 7.1 Hz,
without further purification.
2H), 7.60 (dt, J ¼ 7.2, 5.8 Hz, 2H), 7.45 (d, J ¼ 16.2 Hz, 1H), 6.97 (d,
13
The stock 1.0 mM solution of McyA was prepared in DMSO. The
following solutions (10.0 mM) were prepared in ultrapure water:
J ¼ 8.6 Hz, 2H), 4.09 (s,3H), 1.78 (s, 6H). C NMR (101 MHz, DMSO-
d )
6
d
¼ 181.38, 163.19, 153.72, 143.17, 141.84, 133.56, 128.78, 125.98,
þ
2þ
ꢀ
þ
N
2
H
4
, Cys, GSH, Glu, NH
2
OH, urea, Aniline, thiourea, K , Ca , Na ,
122.76, 116.40, 114.64, 109.30, 51.71, 34.05, 25.65.MS (ESI, m/z)
2
þ
2þ
2þ
3þ
3þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
-
Mg , Cu , Zn , Al , Fe , Hg , F , Br , Cl , I , ClO
H
4
, ACO , CO
M in phosphate
3
,
Calcd.for [C19H20INO-I ] ¼ 278.3745, found: m/z278.3768.
ꢀ
2
PO
4
. The final concentration of McyA was 20
m
buffer saline (PBS) buffer (10 mM, pH ¼ 7.4, 20% DMSO).
2.2.2. Synthesis of probe McyA
Compound McyOH (80.8 mg, 0.2 mmol) was dissolved in 20 mL
2
.1.2. Instruments
NMR spectra were measured using a JEOL400 MHz instruments
CH
2
Cl
2
, then triethylamine (Et
3
N, 45
m
L, 0.32 mmol, 2.0 equiv) was
ꢁ
added. The mixture was stirred at 0 C, and acetyl bromide
(
Japan). Mass spectra were analyzed using Brukermicr OTOF II with
2 3
(C H BrO, 30 mL, 0.4 mmol, 2.0 equiv) was added. After the reaction
ESI mode (America). Absorption spectra were determined on a
UVevisible spectrophotometer (TU-1810, China). Fluorescence
spectra were recorded on a Fluorescence Spectrometer (RF-5301pc,
Japan) with a Xenon lamp. The pH values were measured using a
was finished (about 10 min), solvents were evaporated under
reduced pressure and the solute was purified on a silica gel column
1
(CH
2
Cl
2
/CH
3
OH ¼ 90:1) to give the desired product (yield 78%). H
NMR (400 MHz, DMSO-d
6
)
d
¼ 8.21 (dd, J ¼ 12.4, 8.4 Hz, 3H), 7.76
2 4
Scheme 1. Proposed Reaction of the Probe with N H .

X. Guo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117625
3
Fig. 1. Synthesis of fluorescent probe McyA.
(
7
d, J ¼ 16.2 Hz, 1H), 7.67 (dd, J ¼ 5.9, 3.0 Hz, 1H), 7.62 e 7.56 (m, 3H),
2.5. In vivo imaging
13
.29 (d, J ¼ 8.7 Hz, 2H), 4.46 (s, 3H), 2.33 (s, 3H), 1.87 (s, 6H).
C
NMR (101 MHz, DMSO-d
1
4
6
)
d
¼ 181.87, 168.84, 154.03, 151.76, 143.61,
2.5.1. Cell culture and imaging
41.79, 132.11, 131.86, 129.47, 128.95, 122.81, 115.31, 113.29, 52.26,
HepG2 cells, grown in DMEM supplemented with heat-
ꢀ
1
5.67, 34.72, 25.64, 25.18, 20.94. HRMS (ESI, m/z) Calcd. for
inactivated 10% (v/v) fetal bovine serum, 100 U mL
penicillin
-
ꢀ1
ꢁ
[C
21
H
22INO
2
-I ] ¼ 320.4115 m/z, found: 320.2164 m/z.
and 100
m
g mL streptomycin at 37 C in 95% humidity and 5% CO
2
environment, were incubated with different concentrations of
ꢁ
2
.3. General procedure for spectral measurement
N
2
H
4
(0.0, 3.0, 5.0, 50.0, 150.0
m
M) at 37 C for 20 min. Then, the
cells were washed with PBS buffer to remove excrescent N
Furthermore, the cells were treated by McyA (10 M) for another
0 min. Finally, the cells were rinsed with PBS to remove the
2 4
H .
A typical test solution was prepared by mixing 20
mL of McyA
m
2
(
1.0 mM) and 180 mL of DMSO, appropriate aliquot of each analyte
stock solution and then supplementing to 1.0 mL with PBS buffer
excessive McyA and the imaging of the cells was observed under
Confocal microscopy at different times. The McyA emission was
collected at 535e590 nm.
ꢁ
(
10 mM, pH ¼ 7.4). The resulting solution was shaken well at 37 C
for 20 min before the fluorescence and ultravioletevisible (UV/Vis)
absorption spectra were recorded. The fluorescence excitation and
emission wavelength were 520 and 553 nm, respectively. The
2.5.2. Mice skin-poptest
absorbance (
.4. Cytotoxicity assay
The cytotoxicity assay was manipulated with 3-(4,5-dimethyl-2-
l
530
/
l
385) was measured in an UV/Vis spectrograph.
All animal experiments were performed in accordance with the
guidelines issued by The Ethical Committee of Gansu College of
Traditional Chinese Medicine. Kunming mice (KM, 7e8 weeks old)
2
were given a skin-pop injection of McyA(100
of PBS buffer (pH ¼ 7.4, 10 mM, 20% DMSO), and given a subsequent
skin-pop injection of N2H4 (50 L, 500 M in a mixture of PBS
buffer (pH 7.4, 10 mM, 20% DMSO). The fur was removed by using
mL, 50 mM in a mixture
m
m
thiazolyl)-2,5-diphyl-2H-tetrazolium bromide (MTT) [45]. HepG2
4
cells (1 ꢂ 10 cells/pore plate) were seeded in a 96-well plate with
ꢁ
8% Na S aqueous solution and the mice were placed under general
180
m
L of cell suspension and incubated in 5% CO
2
at 37 C for 24 h.
2
anesthetic through intraperitoneal injection with 10% chloral hy-
drate before imaging.
The cells were treated with various concentration of McyA (0.0, 5.0,
0.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0 M) for 24 h, respectively.
After washing twice with PBS buffer, MTT solution (5.0 mg mL
1
m
ꢀ
1
,
ꢀ1
PBS) was added into each well (10
residual MTT solution was removed by PBS solution after 4 h.
Subsequently, 100 M of DMSO was added into each well to
m
L/well, 0.5 mgmL ) and the
2 4
2.5.3. Bioaccumulation tests of N H in mice
For tissues imaging, the mice were divided into four groups. The
first group of mice was the control group, which were feed with
normal drinking water for two days. The other three groups of the
mice were feed with drink water containing the different concen-
m
dissolve the formazan crystals. After shaking for 10 min, the
absorbance values of the wells were recorded using a microplate
reader. The cytotoxic effect (VR) of probe McyA was assessed using
tration (5, 10, 15 ppb) of N
euthanized and the organs were dissected and directly soaked in
50
M McyA in PBS buffer (10 mM, pH ¼ 7.4, 20% DMSO) solution
for 20 min. Subsequently, these treated organs (heart, liver, lung,
2 4
H for two days. Then, these mice were
the following equation: VR ¼ A/A ꢂ100%. The assays were per-
0
formed six replicates. The statistic mean values and standard
derivation were utilized to estimate the cell viability.
m
Fig. 2. (A) UV/Vis absorption (A) and fluorescence emission spectra (B) of the McyOH and McyA probe (20
2 4
mM) before (black line) and after reacted with N H (200 mM) (red line),
and McyOH (blue line). The detection system was DMSO/H
2
O solution (1:4, v/v, 10 mM PBS, pH ¼ 7.4, Ex ¼ 520 nm).

4
X. Guo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117625
2 4 2 4
Fig. 3. (A) Fluorescence spectra of probes (20 mM) in response to N H and other analytes (150 mM). 1. Blank probe, 2. N H , 3. Cys, 4. GSH, 5. Gln, 6. Hydroxylamine, 7. Urea, 8.
þ
2þ
þ
2þ
2þ
2þ
3þ
3þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
4
PO . Fluorescence
Aniline, 9. Thiourea, 10. K , 11. Ca , 12. Na , 13. Mg , 14. Cu , 15. Zn ,16. Al , 17. Fe , 18. Hg , 19. F , 20. Br , 21. Cl , 22. I , 13. ClO
4
, 24. Ac , 25. CO
3
, 26. H
2
ꢁ
changes were recorded after incubation at 37 C for 20 min. The error bars had three groups of standard deviations with parallel data.
kidney, spleen and stomach) were observed under Confocal mi-
croscopy and the emission was collected at 535e590 nm.
presence and absence of N
DMSO), respectively. As shown in Fig. 2A, the McyA itself exhibits a
2
H
4
in PBS buffer (10 mM, pH ¼ 7.4, 20%
strong absorption band at 385 nm. However, upon reaction with
2 4
N H for 20 min, the absorption band at 385 nm almost disappears
3
. Results and discussion
and subsequently a new band at 530 nm appears evidently, which
corresponds to the UV characteristic peak of McyOH. Simulta-
neously, the color of the probe changed from faint yellow to pink
under natural light (Inset of Fig. 2A), allowing ratiometric colori-
3
.1. Synthesis of probe McyA
Based on the concept of simple and fast, the probe (McyA) was
2 4
metric detection of N H by the naked eye. Accordingly, an obvious
synthesized by one-step reaction of McyOH with acetyl bromide as
described in Fig. 1. The structures of McyOH and McyA were
enhancement fluorescent signal can be observed at 553 nm upon
excitation at 520 nm (Fig. 2B). These results are in line with our
design concept.
1
13
demonstrated by analysis of H NMR, C NMR, and ESI-MS data
Figs. S1, S2, S3, S4 and S5 in the Electronic Supplementary
Information).
(
3.3. Selectivity of McyA to N
2 4
H
3.2. UVevis absorption and fluorescence spectra
To verify the selectivity, the fluorescence responses of McyA
In order to verify the feasibility of detecting the N
probe, we first evaluated spectral properties of McyA in the
2
H
4
by the
toward different analytes were performed in PBS buffer solution
(10 mM, pH 7.4, 20% DMSO). As shown in Fig. 3A and B, Addition of
Fig. 4. McyOH (A) and McyA (B) optimize structure and frontier orbital calculations through density functional theory (DFT) (B3LYP/6-311G (d, p)/level, Gaussian 09). In the stick
model, carbon, oxygen, and nitrogen atoms are labeled as gray, red, and blue, respectively.

X. Guo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117625
5
N
2
H
4
to the McyA solution leads to a strong fluorescence
composed of a phenol latent donor (push) in conjugation with one
acceptor (2,3-dihydro-1H-xanthene-indolium, pull). Upon forma-
tion of a phenolate ion, the latent donor is turn on, which enables
an ICT from the activated donor to the acceptor to form a new push-
pull conjugated system. Thus, the push-pull system of McyOH
molecule can emit strong fluorescence. However, since the
electron-withdrawing acetyl group in McyA hinders the progress of
ICT, it weakens the fluorescence of the original fluorophore. In or-
der to verify this speculation, we performed a computational study
using a suit of Gaussian 09 programs [density functional theory
enhancement at 553 nm. However, various analytes (150
GSH, Gln, hydroxylamine, urea, aniline, thiourea, K , Ca , Na ,
Mg , Cu , Zn , Al , Fe , Hg , F , Br , Cl , I , ClO
H
the competitive experiments demonstrate that the specificity of the
probe McyA for N is prominent over other interfering analytes
under physiological conditions (Figs. S6A and S6B).
m
M, Cys,
þ
2þ
þ
2
þ
2þ
2þ
3þ
3þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
4
, Ac , CO ,
3
ꢀ
2
PO
4
) exhibit almost no interaction with the probe. Additionally,
2 4
H
2 4
3.4. The proposed mechanism of probe in sensing N H
(DFT) at the B3LYP/6-311G (d, p)level] [46e48]. The structures of
McyA and McyOH were optimized and their frontier molecular
orbital energies were calculated (Fig. 4). The results show that the
In theory, the merocyanine chromophores have conjugated
electrons and a push-pull structural element. McyOH molecule is
p-
Fig. 5. (A) Fluorescence changes were recorded after addition of N
The linear relationship between fluorescence intensity and N concentration (excited at 520 nm). (C) UVeVis absorption spectra were recorded after addition of N
.5, 4.0, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 M) to the probe (20 M). (D) Linear relationship between UV/Vis absorbance ratio R ( 385) and N concentration; (E) The
color change of McyA upon addition of N (1e10: 0.0, 0.5, 1.5, 2.5, 4.0, 5.0, 10.0, 15.0, 25.0, 50.0 M) under nature light conditions. Each picture was recorded at 20 min after the
addition of N . Conditions: DMSO/H
O solution (1:4, v/v, 10 mM PBS, pH ¼ 7.4).
2
H
4
(0.0, 0.125, 0.25, 0.5, 1.50, 2.5, 4.0, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0
mM) to the probe (20
mM). (B)
H
2 4
2 4
H
(0, 0.5, 1.5,
2
m
m
l
530
/
l
2 4
H
H
2 4
m
H
2 4
2

6
X. Guo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117625
relatively low pH value, which hinders the nucleophilic attack
ability of N for the probe [41]. Delightedly, Fig. S8A also exhibits
a strong fluorescence signal of the probe treated with N and it is
2 4
H
2 4
H
primary stable in the pH range of 7.0e10.0, which indicates that the
probe McyA can provide a stable fluorescence response for detec-
tion of N H under physiological conditions.
2 4
3
.5.2. Response time study
The response time of the synthesized probe to N
examined. Fig. S8B shows that the addition of N (100
McyA (20 M) leads to a great fluorescence enhancement at
2
H
4
was
2
H
4
mM) to
m
5
2
53 nm with an increase of time until equilibrium is reached after
0 min. Therefore, 20 min was selected as the response time of the
2 4
probe to N H .
Fig. 6. Confocal fluorescence imaging of N
McyA; Control group: cells were treated with only McyA (10
2
H
4
in the HepG2 cells stained with 10
m
M
ꢁ
mM) at 37 C for 20 min (at
3.6. The fluorescence titration experiments
the top). Tested group: Cells were pretreated with N
2
H
4
(50
m
M) and then added McyA
ꢁ
(
10
mM) at 37 C for 20 min (at the bottom). Excited at 514 nm; emission was collected
at 535e590 nm.
Under the optimal detection conditions, the mixture solution of
the probe (20 M) and different concentration of
0,0.125,0.25, 0.5, 1.5, 2.5, 4.0, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0,
m
2 4
N H
(
energy gap (HOMO-LUMO) of McyOH (2.086 eV) is significantly
lower than that of McyA (2.305 eV), which allows us to predict the
recovery of the D-
500.0
m
M) in PBS buffer (10 mM, pH ¼ 7.4, 20% DMSO) were per-
formed to fully assess the viability of McyA as a fluorescent probe.
Fig. 5A displays that the fluorescence intensities of the probe are
nearly proportional to N H concentration range from 0.125 to
p-A feature of McyOH and the red-shift character
by N -caused cleavage in absorption [49]. Therefore, the theory
2 4
H
2
4
2
calculations are support the above anticipation, which rationalize
the ICT process. Furthermore, UHPLC analysis was also employed to
testify the reactive product of McyA and N
the retention time of the McyA is 1.58 min. When the different
equivalents (0, 2.5, 5.0 and 12.5 equiv) of N were added, the
5.0
deviation (
fluorescence titration experiment, the detection limit (LOD) for
N H is 0.042
mM with a correlation coefficient (R ¼ 0.9917) and a standard
s
¼ 0.23 M) (Fig. 5B). Based on the linearity of the
m
2 4
H . As shown in Fig. S7,
2
4
m
M (1.3 ppb, S/N ¼ 3, at pH 7.4 in DMSO/H O (1:4, v/v,
2
2
H
4
PBS, 10 mM), which is sufficiently lower than the threshold limit
intensity of the McyA chromatographic peak gradually reduced and
the chromatographic peak of the McyOH (at retention time of
value (TLV) of N H in drinking water (10 ppb) [13]. Meantime, a
2
4
gradually increase of the peak ratio (l530/l385) in UV/Vis absorption
2
.40 min) gradually increased. It agrees with the previous results
spectra can be observed with the increase of N H concentration,
2
4
2
(
in section 3.2) and illustrates the product of McyOH.
obtaining a good linear equation with R of 0.9928 and LOD of
.14 M (4.5 ppb) (Fig. 5C and D). As shown in Fig. 5E, with
increasing concentrations of N , the color of mixture solution
0
m
3
3
.5. Optimization of detection conditions
2 4
H
increases gradually under natural light. These results indicate that
McyA can be utilized as fluorescent and ratiometric colorimetric
.5.1. pH stability study
It is vital that the proposed probe can be operated in the wide
2 4
dual-mode sensor for quantitative detection of N H . Finally, we
pH range, especially in the physiological environment. Thus, the pH
effect on fluorescence response of the probe McyA to N was
examined at different pH values. Fig. S8A displays the low fluo-
rescence signal of the probe in the presence of N in the pH range
from 4.0 to 6.0. It should be attributed to the protonation of N at
compared the probe McyA with other probes based on an acetyl
group as a recognition group (Table S1). It is found that the probe
McyA has certain advantages in the detection limit compared with
the reported work (Table S1). However, the shorter stokes shift
needs to be further improved.
2 4
H
2 4
H
2 4
H
Fig. 7. (A) Representative fluorescence images (pseudocolor) of a Kunming mouse given a skin-pop injection of McyA (100
DMSO). (B) Mouse given a skin-pop injection of McyA (100 L, 50 M) and given a subsequent skin-pop injection of N
incubation for 0, 5, 10, 15, 20 min, respectively. (C) In vivo images of heart, liver, lung, spleen, stomach and kidney from mouse (a, the control group) feed with drinking water and
from mouse (bed) feed with drinking water containing 5 ppb (b), 10 ppb (c) and 15 ppb(d) N , respectively. Incubated with McyA (50 M) for 20 min, respectively. Excited at
14 nm; emission was collected at 535e590 nm.
m
L, 50
m
M in a mixture of PBS buffer (pH 7.4, 10 mM, 20%
m
m
H
2 4
(50 L, 500
m
mM). Images of (A) and (B) were taken after
2
H
4
m
5

X. Guo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117625
7
3
.7. Determination of N
2
H
4
in real samples
established approach can be utilized as an efficient means for
convenient detecting, tracking hydrazine in environmental and
biological sample.
In order to validate the practicality of the probe, the proposed
method was applied for determining N in Yellow river water,
tap water and diluted human serum samples. Furthermore, the
recovery experiments of N in the spiked samples were carried
2 4
H
Acknowledgement
2 4
H
out and recoveries were calculated, which shows satisfactory re-
coveries as well as relative standard deviation (RSD) values
This work was supported by the National Natural Science
Foundation of China (NSFC) Fund (No.21575055), the Fundamental
Research Funds for the Central Universities (lzujbky-2017-k09) and
Henan Key Laboratory of Biomolecular Recognition and Sensing
(Table S2), indicating that McyA can be used as the probe to qualify
2 4
N H
in environmental and biological samples.
(
HKLBRSK1802).
3
3
2 4
.8. Bioimaging of N H in living cells and tissues
Appendix A. Supplementary data
.8.1. Imaging of N
2 4
H in living cells
In order to further extend the application of McyA in biological
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2019.117625.
system, the cytotoxicity experiments in living cells were performed
by MTT assay using standard cell viability protocols (Fig. S9). The
result shows that more than 98% cells survive in the present of
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mM of McyA, the cell viability still remained
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