A Ratiometric Fluorescent Probe for Selective Detection of Hypochlorite Anion

Article abstract of DOI:10.1002/bkcs.11321

A new fluorescent probe based on rhodamine and naphthalimide was synthesized for the discrimination of hypochlorite anion. Upon addition of NaClO, the emission intensities ratio (I_{576 nm}/I_{528 nm}) of the probe 1 increased quickly accompanied with the obvious change of color. The probe 1 also exhibited highly selectivity and fast response to hypochlorite anion. Furthermore, the newly proposed probe has been applied for natural water samples, and a satisfied result was obtained.

Full text of DOI:10.1002/bkcs.11321

Article
DOI: 10.1002/bkcs.11321
BULLETIN OF THE
Z. Ou et al.
KOREAN CHEMICAL SOCIETY
A Ratiometric Fluorescent Probe for Selective Detection
of Hypochlorite Anion
†
†,‡,
†
†
Zhijian Ou,† Lei Shi,†,‡, Wenli Huang, Shengzhao Gong,
*
*
Haomei Liang, and Haojia Hong
†School of Chemical Engineering and Technology, Guangdong Industry Polytechnic, Guangzhou 510000,
China. *E-mail: 2016103059@gdip.edu.cn; 1996103022@gditc.edu.cn
‡Surfactant Detergent & Cosmetics Technology Research and Development Center, Guangdong Industry
Polytechnic, Guangzhou 510000, China
Received August 21, 2017, Accepted October 16, 2017
A new fluorescent probe based on rhodamine and naphthalimide was synthesized for the discrimination of
hypochlorite anion. Upon addition of NaClO, the emission intensities ratio (I576 nm/I528 nm) of the probe
1 increased quickly accompanied with the obvious change of color. The probe 1 also exhibited highly
selectivity and fast response to hypochlorite anion. Furthermore, the newly proposed probe has been
applied for natural water samples, and a satisfied result was obtained.
Keywords: Hypochlorite anion, Fluorescent probe, Ratiometric, Rhodamine B, Naphthalimide
Introduction
Experimental
The reactive oxygen species (ROS) are important signaling
molecules, which mediate a wide scope of physiological
processes.1,2 Unlike other ROS, hypochlorite anion (ClO−)
and hypochlorite acid (HClO) as its protonated form have
been extensively used in our daily life as an antimicrobial
agent and a bleaching agent.3 However, the concentrated
hypochlorite solution may result in damage to tissues and
lead diseases.4,5 As a consequence, many scientists have
paid great attention to explore the effective methods for
detecting HClO/ClO− in order to ascertain the physiological
and pathophysiological mechanisms of HClO/ClO− in these
diseases.
Reagents and Instrumentation. 1H NMR and 13C NMR
spectra were recorded using an INOVA-400 instrument
(Varian, California, USA) using tetramethylsilane as an
internal standard. Mass spectra were obtained by a
Trace2000 PolarisQ or LCQ-Advantage (ThermoFinnigan,
California, USA). Ultraviolet–visible (UV–vis) spectra were
obtained by a UV-2100 spectrophotometer (LabTech, East
Sussex, UK). Fluorescence spectra were recorded using an
F-6500 fluorescence spectrophotometer (Jasco, Tokyo,
Japan). 4-Bromo-1,8-naphthalic anhydride was obtained
from J&K Reagent Company (Beijing, China);
rhodamine B, KO2, POCl3, and NaClO were purchased
from Sinopharm Chemical Reagent Company (Shanghai,
China). All solvents were of analytical reagents.
In the past few years, many fluorescent probes for ClO−
were designed and reported due to their operational sim-
plicity, high selectivity, sensitivity, rapidity, nondestructive
methodology, high-sampling frequency, low cost of equip-
ment, and direct visual perception. However, most of these
probes were turn-on probes,6,7 and may be influenced by
the fluorescence intensity measurements. By contrast, the
ratiometric fluorescent probes can provide the observation
changes in the ratio of the intensities of the absorption or
the emission at two wavelengths, with the perceived color
changes for rapid visual sensing.8–11
Synthesis of Probe 2.
A mixture of 4-bromo-1,8-
naphthalic anhydride (3.0 g, 10.8 mmol) and aqueous
methylamine (50 mL) was stirred at 85ꢀC for 1 h. After
being cooled, the yellow precipitate was filtered to give the
2.66 g product as yellow solid with a yield of 85%.
m.p. 172–174ꢀC [176–177ꢀC]12; 1H NMR (400 MHz,
CDCl3): δ 3.56 (s, 3H), 7.86 (dd, J = 8.4 and 0.8 Hz, 1H),
8.05 (d, J = 7.6 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.58
(dd, J = 8.4 Hz & J = 1.2 Hz, 1H), 8.67 (dd, J = 8.4 and
1.2 Hz, 1H).
In this study, we report a new ratiometric fluorescent
probe, which was based on rhodamine and naphthalimide
structure and could be synthesized easily within a few
steps. This probe produced strong red fluorescence after the
addition of NaClO while the probe itself presented a green
fluorescence. The probe also showed highly selective detec-
tion toward ClO−, without interference from other ROS and
anion ions.
Synthesis of Probe 3. A mixture of compound 2 (2.4 g,
8.27 mmol) and 80% hydrazine hydrate (25 mL) was
heated at 130ꢀC for 1 h. Then this mixture was poured into
water to precipitate out a solid, which was collected by fil-
tration, washed with water, and dried. Then, 1.66 g of com-
pound 3 was obtained as a yellow solid with a yield of
1
83%. m.p. >260ꢀC [>300ꢀC]13; H NMR (400 MHz, d6-
DMSO): δ 3.35 (s, 3H), 4.67 (s, 2H), 7.24 (d, J = 8.4 Hz,
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1H), 7.63 (dd, J = 8.0 and 7.6 Hz, 1H), 8.29 (d,
J = 8.4 Hz, 1H), 8.42 (d, J = 7.6 Hz, 1H), 8.61 (d,
J = 8.0 Hz, 1H), 9.12 (s, 1H). MS (ESI):
Synthesis of Probe 1. To a solution of rhodamine B
(500 g, 1.13 mmol) in dry 1,2-dichloroethane (8.0 mL),
phosphorus oxychloride (650 mg, 4.23 mmol) was added
dropwise at room temperature within 10 min. After being
refluxed for 5 h, the reaction mixture was cooled and con-
centrated under vacuum to give rhodamine B acid chlo-
ride.14 This crude product was used directly in the next
reaction without further purification. The resulting rhoda-
mine B acid chloride was redissolved in dry 1,2-
dichloroethane (5 mL), and then was added dropwise to a
solution of compound 3 (409 mg, 1.70 mmol) and triethy-
lamine (0.1 mL) in dry DMF (10 mL). The solution was
stirred overnight at room temperature and concentrated in
vacuo. Then, the residue was purified by column chroma-
tography (CH2Cl2:CH3OH = 20:1) to give 286 mg com-
pound 1 as a yellow solid with a yield of 38%.
Scheme 1. The synthetic route of probe 1.
Results and Discussion
Synthesis of the Final Compound 1. Probe 1 was synthe-
sized as depicted in Scheme 1. 4-Bromo-1,8-naphthalic
anhydride was reacted with methylamine and hydrazine
hydrate to form compounds 2 and 3, respectively. Then,
rhodamine B acid chloride was added to the solution of
compound 3 to give probe 1. The probe 1 and intermediates
were characterized by 1H NMR, 13C NMR, and MS spectra
(Figures S5–S9, Supporting Information).
1
m.p. 181–184ꢀC; H NMR (400 MHz, CDCl3): δ 1.09 (t,
J = 7.2 Hz, 12H), 3.24–3.36 (m, 8H), 3.47 (s, 3H), 6.09 (s,
2H), 6.23 (d, J = 8.4 Hz, 2H), 6.45 (d, J = 8.8 Hz, 2H),
6.48 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.29 (d,
J = 7.6 Hz, 1H), 7.58–7.67 (m, 2H), 7.88 (d, J = 8.8 Hz,
1H), 7.97 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.2 Hz, 1H),
8.28 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ
12.45 (CH3), 26.88 (CH3), 44.36 (CH2), 67.35 (CH), 97.64
(CH), 104.74 (C), 107.83 (CH), 109.10 (CH), 113.72 (C),
120.07 (C), 122.03 (C), 123.55 (CH), 124.67 (CH), 125.12
(CH), 127.01 (CH), 128.43 (CH), 128.66 (CH), 130.37
(CH), 132.36 (CH), 133.57 (CH), 148.18 (C), 148.99 (C),
149.70 (C), 154.21 (C), 164.01 (C═O), 164.50 (C═O),
166.51 (C═O); EI-MS m/z 665.4 (M+); HRMS (EI) m/z
Calc. 665.2997 for C41H39O4N5, observed 665.3029.
Optical Response of Probe 1 to ClO−. As shown in
Figure 1(a) and (b), the initial probe 1 displayed almost no
considerable UV-absorption above 500 nm. And when
excitation at 410 nm, the featured emission of the naphtha-
limide moiety around 528 nm was observed instead of
characteristic emission of the rhodamine moiety at 576 nm,
which indicated the ring-closed structure of rhodamine
component. However, upon addition of NaClO, the emis-
sion of the naphthalimide at 473 nm gradually decreased,
and a strong emission band corresponding to the rhodamine
moiety at 576 nm appeared, implying that intramolecular
fluorescence resonance energy transfer (FRET) occurred
due to the ring-open mechanism of rhodamine. Remark-
ably, the fluorescence of the probe 1 showed a dramatic
change from green to red as well as the visible color change
(Figure S1).
Measurement Procedures. A 1.0 mM stock solution of
the probe was obtained by dissolving the appropriate
amount of compound 1 in DMF. The standard solutions of
ClO−, hydrogen peroxide, super-oxide, and hydroxyl radi-
cals were prepared according to the literature.15 Subse-
quently, to a 3 mL glass tube, various analytes including
ClO− and other species were added to the solution of probe
1 (10 μmol/L) in PBS/DMF (pH 7.4, v/v = 1:1), respec-
tively. And then, the UV absorption and fluorescence spec-
tra (excitation at 410 nm) of the resulting solution were
measured within 3.0 min.
To gain some insight into the reaction mechanism, the
reaction of the probe 1 with different quantities of NaClO
1
1
was studied by H NMR and MS spectra. As shown in H
NMR spectra (Figure S12), the signals of aromatic hydro-
gens at naphthalimide and the hydrogens of rhodamine’s
phenyl group (δH = 7.0–8.4) varied upon addition of
NaClO, indicting the structural change of hydrazine. Mean-
while, the mass spectra (Figures S8, S10 and S11) showed
clear peaks (m/z) of probe 1 as 665.4 (EI), and its oxidized
product 4 as 665.3 (M + H+, ESI), 733.3 (M + 79Br−, ESI),
and 735.3 (M + 81Br−, ESI). These results were identical
with the oxidized ring-open mechanism (Scheme 2) as pro-
posed by the literature.16
Determination of ClO− in Water Samples. The tap water
and commercial bottled water were collected at school on
May 15, and tested without further purification. The River
water was collected in the Zhujiang River (near Canton
Tower) on May 18, and was filtrated before analysis. These
water samples were diluted for five times by PBS/DMF
buffer solution (pH 7.4, v/v = 1:1), and then analyzed with
and without addition of NaClO solution.
As shown in Figure 2, the probe 1 exhibited a good line-
arity between the ratios (I576 nm/I528 nm) and the ClO− con-
centration (0–10 equiv), as well as the good sensitivity with
the detection limit of 96 nmmol/L (S/N = 3). The quantum
yields of 1 with and without NaClO (10 equiv) in
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BULLETIN OF THE
Article A Ratiometric Fluorescent Probe for Selective Detection of Hypochlorite Anion KOREAN CHEMICAL SOCIETY
18
16
14
12
10
8
y = a + b*x
No Weighting
2.12718
Equation
Weight
Residual Sum
of Squares
0.99622
0.99161
Pearson's r
Adj. R-Square
Value
Standard Erro
0.24398
Intercept
Slope
-0.18427
0.16011
B
0.00465
6
4
2
0
0
10 20 30 40 50 60 70 80 90 100
[NaClO] µmol/L
Figure 2. The emission ratio of I576/I528 of probe 1 (10 μM) in
PBS/DMF (pH 7.4, v/v = 1:1) to various concentrations of NaCIO
solution (0–90 μmol/L). Excitation was provided at 410 nm, and
the ratios of emission intensities at 528 and 576 nm were mea-
sured for three times.
Figure 1. The absorption (a) and fluorescence (λex = 410 nm,
10 μmol/L) (b) titration spectra of 1 in PBS/DMF (pH 7.4,
v/v = 1:1) upon addition of NaClO (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,
10 equiv).
PBS/DMF buffer solution were evaluated to be 0.33 and
0.62, respectively (rhodamine B was used as quantum
yields standard and its ΦF in PBS/DMF was defined as
1.0). The result demonstrated that the probe 1 can poten-
tially be used for quantitative detection of ClO−.
Meanwhile, the probe 1 showed a highly selectively in
respond to ClO− compared to other ROS (H2O2, OH•, O2 ,
•
and O2 ) and anion ions (Cl−, ClO4 , NO3 , and SO42−) in
the simulated physiological condition, and there was almost
no change in the fluorescent spectra with addition of these
species (Figure 3). The time-course studies also demon-
−
−
−
Figure 3. Emission intensity ratio (I576/I528) of 1 (10 μM) in
PBS/DMF (pH 7.4, 1:1) in the presence of ClO− (5.0 equiv) and
other analytes (10 equiv).
Scheme 2. The ring-opening mechanism for the reaction of probe
1 with CIO−.
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BULLETIN OF THE
Article A Ratiometric Fluorescent Probe for Selective Detection of Hypochlorite Anion KOREAN CHEMICAL SOCIETY
strated that the probe 1 had a rapid response to ClO−, and
the reaction between probe 1 and ClO− could be completed
within 2 min (Figure S2). Furthermore, the probe 1 was sta-
ble over a pH range of 7–10, even though medium changes
were still observed from pH 5.8 to 7. (Figure S3).
Application in Water Samples. For practical applicabil-
ity, tap water, Zhujiang River water and bottled water were
analyzed by the proposed fluorescent method. As shown in
Figure S4, only the tap water could be detected of ClO− at
7.23 μmol/L. Subsequently, a strong red fluorescence
enhancement was observed in these samples after addition
of NaClO solution (2 equiv.), and the recoveries of these
samples were from 95 to 106%, indicating this fluorescent
analytical method was reliable and practical for the com-
plex samples.
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A selective fluorescent assay for the hypochlorite anion
was developed based on the oxidized ring-open and FRET
strategy. The probe 1 showed a great selectivity to ClO−
and an obvious color change, as well as a good linearity
between the ratios (I576 nm/I528 nm) and the ClO− concentra-
tion (0–10 equiv). In addition, this fluorometric method
was also applied to complex water samples and a good per-
formance was achieved.
Acknowledgments. This work was supported by Research
Project (2A071508) provided by Guangdong Industry
Polytechnic.
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Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
Bull. Korean Chem. Soc. 2017
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