J Po lue ran s ae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
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COMMUNICATION
Journal Name
a) 400
0 μM
b)400
demonstrated. To the best of our knowledge, this is the first
report on the real time visualization of in situ hydrazine release
as a metabolite of drug in animal using a fluorescent probe.
Keeping the requirements of a promising fluorescent probe
for the detection of hydrazine in biosystems in mind, a new type
of hemicyanine structure with terephthalaldehyde as the linker
of 3-(2, 3, 3-trimethyl-3H-indol-1-ium-1-yl) propane-1-sulfonate
DOI: 10.1039/C9TB00132H
0
μM
20 μM
40 μM
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8
0 μM
0 μM
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100μM
120μM
140μM
160μM
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200μM
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50
00
180μM
00μM
250
2
1
200
Y=-1.1113[N2H4]+394.18
50
2
R =0.9946
0
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0
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2 4
[N H ]/μM
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(1) and p-hydroxyacetophenone was designed. The resulting
Wavelength/nm
Fig. 2 (a) Fluorescence spectra of Hcy-Ac (10 μM) upon the addition of
increasing concentrations of hydrazine (0, 20, 40, 60, 80, 100, 120, 140, 160,
novel fluorophore (Hcy-OH) has a large D–π–A structure and the
phenolic hydroxyl could be masked by esterification easily. The
probe Hcy-Ac was synthesized via a four-step procedure as
outlined in Scheme S1 (see ESI†) and the experimental details
and structural characterizations are provided in the Electronic
Supporting Information.
1
80, 200 μM) when excited at 430 nm at 25℃. (b) Fluorescence intensity
changes of Hcy-Ac at 532 nm as a function of hydrazine concentration.
Phe, His), and biologically relevant amines (aniline, benzylamine,
ammonia,
thiourea,
ethanolamine,
cyclohexylamine,
To our delight, Hcy-Ac has a good water solubility and the
stock solution of Hcy-Ac in DMSO (20 mM, 100 μL) was
dissolved in complete aqueous buffer (10 mM PBS, pH=7.4, 200
mL), then, the diluted solution (10 μM) was used throughout
the experiments. The absorption spectral properties of Hcy-Ac
in the absence or presence of hydrazine in the buffer were
shown in Fig. 1a. Once 20 equiv of hydrazine was added, the
maximum absorption of Hcy-Ac at 420 nm decreased
remarkably, whereas a new absorption band centered at 320
nm appeared and increased gradually, with a well-defined
isosbestic point at 348 nm. At the same time, the color of the
solution changed from yellow to colorless, which could be seen
with the naked eye (inset of Fig. 1a). On the other hand, addition
of hydrazine also resulted in a remarkable fluorescent decrease
at 532 nm upon excitation at 430 nm (Fig. 1b).
diethylamine, ethylenediamine, hydroxylamine) to the
solutions of Hcy-Ac caused no or slight change in the
fluorescence spectra. Notably, INH and acethydrazine which is
one of the major metabolites of INH in vivo also brought about
no obvious change of fluorescence intensity. By contrast, the
addition of 20 equiv of hydrazine to Hcy-Ac led to a marked
fluorescence decrease at 532 nm upon excitation at 430 nm.
These results illustrated the good specificity of Hcy-Ac for
practical hydrazine detection and the potential to be used in
biosystems.
Based on the structural and spectral properties of Hcy-Ac, we
envisioned that the response of Hcy-Ac toward hydrazine was
attributed to its hydrazinolysis to release Hcy-OH, thus triggering the
chromogenic and fluorescence changes observed. The proposed
1
sensing mechanism was depicted in Scheme 1 and supported by H
Then, the fluorescence titration experiments of Hcy-Ac (10
μM) with different concentrations of hydrazine (0-200 μM) in
buffer solution were performed. As shown in Fig. 2a, the
fluorescence intensity at 532 nm decreased steadily and a good
linear relationship between fluorescence intensity and
1
NMR analysis (Fig. S14, ESI†). Compared with the H NMR spectrum
of pure Hcy-Ac, new proton signals at 8.97 ppm and 4.51 ppm
corresponding to the INH in acethydrazine (H
in Hcy-OH (H ) respectively were observed upon the addition of
hydrazine (20 equiv). Meanwhile, the methyl group at 2.39 ppm in
Hcy-Ac (H ) was completely disappeared and the methyl proton at
.58 ppm in acetohydrazide (H
b
) and phenolic hydroxyl
c
2
concentration of hydrazine with R = 0.9946 was observed (Fig.
2
b).
The detection limit (LOD, 3σ/k) of Hcy-Ac was thus calculated
to be 1.72 ppb, which is lower than the threshold limit value
TLV) of 10 ppb proposed by the U.S. Environmental Protection
a
1
a
’) was clearly found. Taken together,
1
the differences between the H NMR spectra of Hcy-Ac in the
absence and presence of hydrazine agree with the sensing
mechanism depicted in Scheme 1.
(
Agency. And the absolute fluorescent quantum yield of Hcy-Ac
was measured to be 0.20 in PBS buffer (10 mM, pH=7.4).
To examine the selectivity of probe Hcy-Ac, the fluorescence
responses of Hcy-Ac (10 μM) in the presence of different
analytes (40 equiv) in buffer solution were recorded. As shown
Encouraged by the promising results obtained, we carried out a
standard MTT assay to investigate the potential toxicity of Hcy-Ac
against HeLa cells. The results (Fig. S1, ESI†) indicated that HeLa cell
500
300
a)
b)
2
2
1
50
00
50
4
00
Probe and Others(2-28)
-
2-
-
2-
in Fig. 3, the addition of anions (Cl , SO
4
, NO
2 3
, CO ), cations
+
2+
3+
2+
2+
+
(
K , Ca , Fe , Cu , Mg , NH
4
), amino acids (Lys, Ile, Pro, Arg,
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b)
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40min
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min
min
min
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0.4
0.3
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0.1
0.0
0min
2 4
N H
0min
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25min
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35min
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40min
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
0min
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Wavelength/nm
Fig. 3 (a) Fluorescence responses and (b) fluorescence decrements at 532 nm
of Hcy-Ac (10 μM) upon addition of 20 equiv of hydrazine and 40 equiv of
50
+
2+
3+
2+
2+
+
0
4
interfering analytes (1, blank; 2, K ; 3, Ca ; 4, Fe ; 5, Cu ; 6, Mg ; 7, NH ;
300
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-
2-
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2-
8
1
2
2
4
4 2 3
, Cl ; 9, SO ; 10, NO ; 11, CO ; 12, Lys; 13, Ile; 14, Pro; 15, Arg; 16, Phe;
Wavelength/nm
Wavelength/nm
7, His; 18, Aniline; 19, Benzylamine; 20, Diethylamine; 21, Cyclohexylamine;
2, Ethylenediamine; 23, Ethanolamine; 24, Hydroxylamine; 25, Ammonia;
6, Thiourea; 27, INH; 28, Acetylhydrazine; 29, Hydrazine) when excited at
30 nm.
Fig. 1 Absorption spectral (a) and fluorescence spectral (b) changes of probe
Hcy-Ac (10 μM) in the absence and presence of hydrazine (20 equiv) in buffer
solution (10 mM PBS, pH=7.4) with λex at 430 nm at 25 ºC. Inset: color change of
the probe with the addition of hydrazine under natural light.
2
| J. Name., 2019, 00, 1-3
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