A turn-on fluorescence probe for imaging iodide in living cells based on an elimination reaction

Article abstract of DOI:10.1039/c5cc01130b

Based on a unique elimination reaction prompted by the iodide ion, a novel turn-on fluorescence probe (HCy-OMe-Br) has been developed for the first time. The probe emits in the near infrared region with a large Stokes shift, and can respond rapidly to iodide with high selectivity and sensitivity. This journal is

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DOI: 10.1039/C5CC01130B
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A turn-on fluorescence probe for imaging iodide in living cells based on
an elimination reaction
Fanpeng Kong, Xiaoyue Meng, Ranran Chu, Kehua Xu*, and Bo Tang*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Based on a unique elimination reaction prompted by the 50 can rapidly respond to iodide with high sensitivity and selectivity
and successfully image iodide in living cells.
iodide, a novel turn-on fluorescence probe (HCy-OMe-Br) has
been developed for the first time. The probe emits in the near
infrared region with a large Stokes shift, and can respond
rapidly to iodide with high selectivity and sensitivity.
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Iodide is one of the essential micronutrients for normal human
growth and plays an irreplaceable role in neurological
1, 2
55 Scheme 1. Structure and proposed reaction of the probe
development and thyroid gland function. Either a deficiency or
overabundance of iodide can lead to thyroid diseases, such as
thyroid enlargement, hypothyroidism and hyperthyroidism.
Therefore, to investigate the biological function of iodide, it is
important to develop a simple, feasible and reliable method for
the detection of iodide in living cells or in vivo.
HCyꢀOMeꢀBr was synthesized conveniently via Knoevenaꢀgel
condensation and Wittig reaction in moderate yields (Scheme 2),
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and the structure of the probe was confirmed by H NMR,
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NMR and HRMS (see ESI†). The fluorescence properties of
HCyꢀOMeꢀBr and HCyꢀOMe were also investigated. The results
showed that HCyꢀOMeꢀBr has very weak fluorescence at 530 nm
(λex = 350 nm) (Fig. S1, ESI†) and that the debromination
product HCyꢀOMe displayed very strong fluorescence in the
Fluorescence probes with high sensitivity, good selectivity, short
response time, and the advantage of direct observation are
3ꢀ6
powerful tools for identifying substances in biological systems.
To date, most of the reported fluorescence probes for iodide have
7ꢀ12
65 infrared region (λex / λem = 480 / 680 nm), with a large Stokes
shift of 200 nm (Fig S2. ESI†). The redꢀshift of fluorescence
emissions was attributed to the conjugated system extension.
been designed based on a fluorescence quenching response
because iodide has an intrinsic fluorescenceꢀquenching nature
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due to the heavy atom effect.
Only a few turnꢀon fluorescence
probes containing mercury (II) ion have been developed for
iodide. However, the introduction of a bioꢀtoxic heavy metal ion
and the emission wavelength of less than 550 nm has greatly
limited their application in biological systems. Hence, it is
challenging to construct probes that can exhibit an enhanced
fluorescence signal in response to iodide in living cells.
As a potential sensing system, a specific chemical reaction
between the probe molecule and the target species will give a
unique spectroscopic change and provide us with a versatile
means to investigate a wide range of analytes with superior
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sensitivity and selectivity.
Alkene is known to react with
bromine to form a vicinalꢀdibromide adduct, and the vicinalꢀ
dibromide can revert to the corresponding alkene treated by
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1
iodide via the debromination reaction. This property inspired us
to construct a vicinalꢀdibromide probe featuring the advantage of
low background and significant fluorescence enhancement upon
reaction with iodide. Hence, we designed and synthesized a
fluorescence probe containing a vicinalꢀdibromide group (HCyꢀ
OMeꢀBr, scheme 1). The probe showed very weak fluorescence,
not only because the two bromines displayed the heavy atom
effect but also because the πꢀconjugated system was broken by
the bromine compared to its precursor HCyꢀOMe. Upon the
addition of the iodide, the probe converted to the fluorescent
70
Scheme 2. Synthesis of HCyꢀOMeꢀBr
The emission intensity at 680 nm increased approximately 60ꢀ
fold after the addition of the iodide (Fig. 1a), and the fluoꢀ
rescence quantum yield increased from 0.027 to 0.190. The rapid
compound HCyꢀOMe. Experiments showed that HCyꢀOMeꢀBr 75 addition of iodide to a solution of HCyꢀOMeꢀBr caused the
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DOI: 10.1039/C5CC01130B
solution to turn orange and simultaneously caused bright red
fluorescence (Fig. 1b). The high signalꢀtoꢀnoise ratio in response
to iodide is advantageous in comparison with other existing
iodide probes.
concentrations in the range 0ꢀ300 ꢁM (Figure 3b), and the
regression equation was ꢂF = 914.03 + 38.91 [I ] ꢁM with a
linear coefficient of 0.9934. The low detection limit and good
40 linear correlation indicated that the probe was able to
qualitatively and quantitatively determine the iodide
ꢀ
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Fig. 1 (a) Fluorescence spectra of HCyꢀOMeꢀBr (10 ꢁM) (black
line) and the product upon addition of iodide (30 equiv) (red line).
Fig. 3 (a) Fluorescence emission spectra of HCyꢀOMeꢀBr (10 ꢁM)
treated with increasing concentrations of iodide (0–300 ꢁM) in a
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(b) Color and fluorescence changes of HCyꢀOMeꢀBr (10 ꢁM)
mixed solution of CH CN: HEPES buffer (6 : 4, 10 mM, pH 7.4).
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upon addition of 30 equiv of iodide in a mixed solution of
(b) The titration curve of HCyꢀOMeꢀBr reacted with iodide. Each
CH CN: HEPES buffer (6 : 4, 10 mM, pH 7.4). Digital images
3
spectrum was recorded at 680 nm.
were obtained with ambient light (left), and a handꢀheld UV lamp
To demonstrate the selectivity of HCyꢀOMeꢀBr for iodide, we
studied its responses toward competing anions, thiols and metal
ions. As shown in Fig. 4, the results showed that HCyꢀOMeꢀBr
exhibited high selectivity for iodide and was unperturbed upon
the addition of other anions, thiols and metal ions.
(right).
50
To explore the sensing mechanism of HCyꢀOMeꢀBr for iodide,
the reaction mixture of HCyꢀOMeꢀBr with sodium iodide was
characterized by HRMS spectrometry. The HRMS spectrum of
HCyꢀOMeꢀBr in Fig. 2a revealed a main peak at 568.0673 before
the addition of iodide, corresponding to the probe (m/z calcd =
5
68.0670). After the addition of iodide, a new peak at 408.2371
appeared, coinciding exactly with the debromination product (m/z
calcd = 408.2322), which indicated that HCyꢀOMeꢀBr was
completely converted into HCyꢀOMe (Fig. 2b)
55
Fig. 4 Fluorescence intensity of HCyꢀOMeꢀBr (10 ꢁM) treated
with different anions, reducing agents and metal ions in a mixed
solution of CH CN : HEPES buffer (6 : 4, 10 mM, pH 7.4) (a)
3
ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
2ꢀ
5
00 ꢁM for F ，Cl ，Br , SO , H PO , HPO , 400 ꢁM for
4 2 4 4
ꢀ
ꢀ
+
2+
60
AcO , NO , GSH, Cys, NAC, Hcy. (b) 300 ꢁM for K , Ca ,
3
2
+
3+
2+
2+
2+
2+
Mg , Al , Ni , 400 ꢁM for Zn and 500 ꢁM for Co , Fe ,
2
+
Cr . Black bars represent the addition of one of these
interferences to a 10 ꢁM solution of HCyꢀOMeꢀBr. Red bars
represent the addition of I plus one of these interferences to the
ꢀ
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probe solution, λex = 480 nm, λ = 680 nm.
em
The rapid response of HCyꢀOMeꢀBr to iodide was confirmed
by a kinetics experiment (Fig. S4, ESI†), in which the
fluorescence intensity rapidly increased upon adding iodide into
the probe solution, and the intensity remained at a relatively
stable level for at least 30 min, as shown in Fig. 5a. Furthermore,
the cytotoxicity of HCyꢀOMeꢀBr in HepG2 cells was determined
by a conventional MTT assay (Fig. 5b). Upon exposure to a 100
25
30
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Fig. 2 HRMS of HCyꢀOMeꢀBr before (a) and after (b) addition of
the iodide.
The sensing behavior of HCyꢀOMeꢀBr toward iodide was
investigated carefully under optimized conditions. A gradual
increase in the fluorescence intensity at 680 nm was obꢀserved
upon adding different concentrations of iodide into 10 ꢁM of
probe solution. Fluorescence intensity reached a plateau on
further addition of iodide up to 30 equiv (Fig. S3, ESI†). The
detection limit of HCyꢀOMeꢀBr for iodide was estimated to be as
low as 8.8 nM (standard deviation 3.5 %, n = 11). There was a
good linearity between the fluorescence intensities and iodide
ꢁM concentration of HCyꢀOMeꢀBr for 24 h, approximately 80%
of the cells remained viable. Therefore, these experiments
indicated that HCyꢀOMeꢀBr could be used as a viable probe for
the detection of iodide in biological samples.
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DOI: 10.1039/C5CC01130B
In summary, based on the unique elimination reaction, we have
for the first time designed and synthesized a novel turnꢀon
fluorescent probe (HCyꢀOMeꢀBr) that can detect iodide rapidly
with high selectivity and sensitivity. The advantages of nearꢀ
infrared emission, large Stokes shift and low background
fluorescence make it a promising candidate probe for in vivo
imaging applications. We anticipate that this probe will be a
useful tool for clarifying iodide’s roles in various diseases and
cellular processes.
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Fig. 5 (a) The time courses of the fluorescence intensities of 10
M HCyꢀOMeꢀBr with 300 ꢁM iodide (red line) and without
This work was supported by 973 Program (2013CB933800),
National Natural Science Foundation of China (21227005,
ꢁ
iodide (black line). (b) Cell viability of HCyꢀOMeꢀBr at different
concentrations.
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1390411, 91313302, 21275092 and 21405098).
5
As confirming that HCyꢀOMeꢀBr could detect iodide under
physiological conditions, the application of the probe to track the
intracellular iodide level was investigated via scanning
microscopy. HepG2 cells were grown with DMEM (containing
Notes and references
a
College of Chemistry, Chemical Engineering and Materials Science,
Collaborative Innovation Center of Functionalized Probes for Chemical
1
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2
0
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10 % fetal bovine serum and antibiotics) for 24 h and used at 50 Imaging in Universities of Shandong, Key Laboratory of Molecular and
Nano Probes, Ministry of Education, Shandong Provincial Key
subconfluency. The cells were first washed with 10 mM HEPES
buffer, pH 7.4, then treated with 10 µM CyꢀOMeꢀBr for 30 min in
the same buffer at 37 °C and washed three times with buffer
Laboratory of Clean Production of Fine Chemicals, Shandong Normal
University, Jinan 250014, P. R. China
E-mail: tangb@sdnu.edu.cn
before imaging. As shown in Fig. 6a, apparent red fluorescence 55 † Electronic Supplementary Information (ESI) available: [Details of the
was observed in HepG2 cells treated with sodium iodide. In
contrast, in a control experiment, no significant fluorescence
appeared in HepG2 cells incubated with only the probe (Fig. 6d).
The brightꢀfield images confirmed that the cells were viable
throughout the imaging experiments (Fig. 6b, 6e). The
fluorescence imaging results were consistent with the
observations made in the titration experiments and demonstrated
that probe HCyꢀOMeꢀBr could readily detect the presence of
iodide in cells by the turnꢀon fluorescent method.
general experimental section; Synthesis of the intermediate products and
HCyꢀOMeꢀBr.]. See DOI: 10.1039/b000000x/
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Fig. 6 Fluorescence and brightꢀfield images of HepG2 cells. (a)
Fluorescence image of cells pretreated with (30 ꢁM) sodium
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(b) Brightꢀfield image of cells shown in panel a; (c) overlay
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image of (a) and (b); (d) Fluorescence image of cells incubated
only with HCyꢀOMeꢀBr (10 ꢁM) for 30 min; (e) Brightꢀfield
image of cells shown in panel d; (f) overlay image of (d) and (e).
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Conclusions
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