A sensitive colorimetric and fluorescent sensor based on imidazolium-functionalized squaraines for the detection of GTP and alkaline phosphatase in aqueous solution

Article abstract of DOI:10.1039/c4cc00752b

Imidazolium-functionalized squaraine ImSQ8 is synthesized as a sensitive colorimetric and fluorescent chemosensor for GTP in aqueous solution. The detection limit of GTP reaches 5.4 ppb. Its applications in the live-cell imaging and enzyme activity assay have also been demonstrated. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00752b

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A sensitive colorimetric and fluorescent sensor
based on imidazolium-functionalized squaraines
for the detection of GTP and alkaline phosphatase
in aqueous solution†
Cite this: Chem. Commun., 2014,
50, 4438
Received 28th January 2014,
Accepted 4th March 2014
Ningjie Wu, Jingbo Lan,* Lipeng Yan and Jingsong You*
DOI: 10.1039/c4cc00752b
www.rsc.org/chemcomm
Imidazolium-functionalized squaraine ImSQ8 is synthesized as a In this report, we wish to present the design, synthesis and
sensitive colorimetric and fluorescent chemosensor for GTP in spectroscopic study of imidazolium-functionalized squaraines that
aqueous solution. The detection limit of GTP reaches 5.4 ppb. Its serve as a colorimetric and fluorescent sensor for the detection of
applications in the live-cell imaging and enzyme activity assay have GTP in aqueous solution. Imidazolium has been proven to be a
also been demonstrated.
potential receptor for anions with excellent water-solubility.9 Thus,
we speculated that the imdazolium unit would not only provide
Guanosine-50-triphosphate (GTP) is a vital member of nucleotides, effective binding sites for nucleotides, but also improve the water-
which acts as a substrate for RNA synthesis and provides energy for solubility of squaraine derivatives.
some metabolic reactions.1 Intracellular GTP levels are closely related
Scheme 1 shows the synthetic strategy of imidazolium-
to definite pathological states. Thus, it is important to monitor the functionalized squaraines. A 3,5-dihydroxyaniline bisimidazolium
concentration of GTP.2 Sensors based on analyte-induced colour derivative 6 was synthesized via a six-step process by using phloro-
changes or fluorescent changes are particularly attractive owing to glucinol as the starting material, followed by a condensation with
their simplicity, high sensitivity and real-time detection.3 A number semi-squaraine 7 to afford the target molecules with different alkyl
of colorimetric and/or fluorescent sensors have been designed for chains. It is noteworthy that two hydroxyls on the benzene ring of 6
various nucleotides in the past decades.4 In particular, the recogni- may be indispensable for increasing its reactivity or stabilizing the
tion of adenosine-50-triphosphate (ATP) has attracted much more resulting squaraines via intramolecular hydrogen bonding inter-
attention.5 However, there are relatively few reports on chemosensors action. The bisimidazolium-containing aniline derivatives with one
that selectively communicate with GTP.6 Moreover, due to the poor or no meta-hydroxyl group on the benzene ring could not deliver the
water-solubility of many hosts, a cosolvent has to be added. In desired squaraines (ESI,† Scheme S1).
addition, many known sensors are less sensitive, and as a result, a
Initially, the UV/Vis spectra of ImSQ4, ImSQ8 and ImSQ12 were
high concentration of GTP would usually be required to enhance studied in neutral buffer (ESI,† Fig. S1). The squaraines ImSQ4 and
spectral response. Therefore, there is still a demand for developing ImSQ8 showed good solubility in HEPES buffer and exhibited a
more sensitive fluorescent sensors for the detection of GTP in characteristic sharp and intense absorption band of the monomeric
aqueous solution.
squaraine chromophore at around 658 nm with a broad shoulder at
Squaraines are a well-documented class of organic dyes with approximately 615 nm. It was found that increasing the length of the
interesting photophysical properties such as sharp and intense alkyl chain led to poor water-solubility, which may presumably be
absorption bands in the red to near-infrared region and high
fluorescence quantum yields.7 Moreover, because of the sensitivity
to slight external stimulation, squaraine dyes have been extensively
used for the detection of various biomolecules.8 However, most
squaraines display poor water-solubility, and there are no
squaraine-based chemosensors for nucleoside phosphates so far.
Key Laboratory of Green Chemistry, Technology of Ministry of Education, College of
Chemistry, State Key Laboratory of Biotherapy, West China Medical School,
Sichuan University, 29 Wangjiang Road, Chengdu 610064, PR China.
E-mail: jingbolan@scu.edu.cn, jsyou@scu.edu.cn; Fax: +86-28-85412203
† Electronic supplementary information (ESI) available: Detailed experimental
Scheme 1 Synthetic routes of ImSQ4, ImSQ8 and ImSQ12.
procedures and analytical data. See DOI: 10.1039/c4cc00752b
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Fig. 1 Color changes of ImSQ8 (10 mM) upon addition of sodium salt of
various anions (1.0 equiv.) in HEPES buffer (10 mM, pH = 7.2).
attributed to the enhancement of the dye’s aggregation tendency. As
a result, the dodecyl-anchored squaraine ImSQ12 exhibited a very
weak monomeric absorption band centered on 650 nm and an
additional hypsochromic absorption band at around 513 nm. The
fluorescence spectra of imidazolium-functionalized squaraines were
also investigated upon excitation at 613 nm (ESI,† Fig. S2). ImSQ4
and ImSQ8 both displayed a strong emission in HEPES buffer
(10 mM, pH = 7.2), but the emission of ImSQ12 was hardly detected
due to its severe aggregation behavior.
Subsequently, colorimetric responses of ImSQ4 and ImSQ8 to a
range of anions were investigated. In the colorimetric test, no colour
change was observed during the addition of various anions into
buffer solutions of ImSQ4. In contrast, an apparent colour change
from azure to navy blue was observed upon addition of GTP into the
buffer solution of ImSQ8 (Fig. 1). The other anions including ATP,
PPi, GDP, GMP, phosphate, halide, acetate, bicarbonate, sulfate and
nitrate ions did not lead to appreciable color changes. These
observations clearly indicate that ImSQ8 exhibited a high selectivity
for naked-eye detection of GTP over other nucleotides and various
anions in aqueous solution.
Fig. 3 (a) Fluorescence emission of ImSQ8 (10 mM) upon addition of
sodium salt of various anions (1.0 equiv.) in HEPES buffer (10 mM, pH = 7.2).
lex = 613 nm. Fluorescence microscopy images of Bel-7402 cells treated
with ImSQ8 (20 mM): (b) before and (c) after addition of GTP.
Fluorescence responses of ImSQ8 to a range of various nucleo-
tides and anions are depicted in Fig. 3a. Upon addition of different
anions (1.0 equiv.), GTP induced the most remarkable fluorescence
change. The addition of 2.4 equiv. of GTP resulted in approximately
5.5-fold fluorescence quenching of ImSQ8 along with an emission
shift from 680 to 670 nm (ESI,† Fig. S4). Subsequently, the live-cell
imaging experiment was performed via incubation of Bel-7402 cells
with ImSQ8 (20 mM) in a physiological saline solution containing 1%
DMSO for 30 min at 37 1C. As shown in Fig. 3b, a clear red
fluorescence image was observed using fluorescence microscopy.
When GTP was added and incubated for another 30 min, a
significant fluorescence quenching phenomenon was observed
(Fig. 3c; ESI,† Fig. S5). The experimental results revealed that ImSQ8
would be a potentially useful reagent for detection of GTP in living
biological samples.
The selectivity of ImSQ8 for GTP over GDP and GMP enables us
to explore the possibility of applications in other areas of biology.
Alkaline phosphatase (ALP) is widely distributed in biological
tissues.12 The level of serum alkaline phosphatase is used as an
important detection index for several diseases.13 Given that ALP can
catalyze the hydrolysis of GTP to produce GDP, GMP, guanosine and
phosphate, we envisioned that ImSQ8 may be utilized as a real-time
fluorescence sensor for the detection of ALP because the hydrolytic
products have almost no influence on the spectral changes of
ImSQ8. As shown in Fig. 4, after adding ALP (133 mU mLꢁ1) into
the HEPES buffer (10 mM, pH = 7.2) containing ImSQ8 and GTP at
25 1C, the fluorescence intensity increased gradually by prolonging
the incubation time. The fluorescence intensity remained
unchanged after 18 min, which demonstrated that the ALP-
catalyzed hydrolysis was complete. The inset of Fig. 4 displays the
variation of the fluorescence intensity of ImSQ8 at 675 nm after
incubation with different amounts of ALP (0–200 mU mLꢁ1) for
different times. The corresponding calibration plot between the
fluorescence intensity at 675 nm and the ALP concentration at
8 min is also shown in the inset, which can be utilized to build
up a real-time analytical method to detect the enzyme activity.
To validate the aggregation behavior of ImSQ8 caused by the
host–guest interaction, a simple Tyndall effect experiment was
carried out.14 As illustrated in Fig. 5a, under laser irradiation
(532 nm), no Tyndall phenomenon was observed for HEPES buffer
with only ImSQ8, whereas the addition of GTP resulted in distinct
Tyndall scattering. The Tyndall phenomenon disappeared after
incubation with ALP. These results demonstrated that the aggrega-
tion of host molecules was triggered by GTP. TEM analyses were
To further confirm the spectral changes of ImSQ8 toward various
anions, the absorption spectra of ImSQ8 in HEPES buffer (10 mM,
pH = 7.2) were studied. As shown in Fig. 2a, with increasing amounts
of GTP, the absorption of monomeric ImSQ8 at approximately
658 nm was weakened significantly and the absorption of the
aggregates at approximately 557 nm was enhanced gradually. The
relative ratio of absorbance of ImSQ8 at 557 and 658 nm (A557/A658
)
increased linearly with GTP concentration at less than 8 mM.10 The
typical detection limit of GTP using this protocol was estimated
to reach 5.4 ppb (1.04 ꢀ 10ꢁ8 M), which exhibited a high
sensitivity.6a,b,11 The changes in the absorption spectra of ImSQ8
in HEPES buffer upon addition of other anions were also studied
(ESI,† Fig. S3). Fig. 2b shows the dependence of A557/A658 on the
different concentrations of various anions. It is clear that the
most striking effect is observed for GTP, and its A557/A658 value is
about 2-fold that of ATP.
Fig. 2 (a) Absorption spectra of ImSQ8 (10 mM) upon addition of different
equivalents of GTP in HEPES buffer (10 mM, pH = 7.2). Inset: linear
relationship between A557/A658 of ImSQ8 and the GTP concentrations.
(b) The dependence of A557/A658 of ImSQ8 on different anions at increasing
concentrations in HEPES buffer (10 mM, pH = 7.2).
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Fig. 6 The proposed model of the host–guest interaction.
Fig. 4 Fluorescence real-time detection of the ALP-catalyzed hydrolysis of
GTP (20 mM) with ImSQ8 (10 mM). Time trace (0–18 min) of the fluorescence
change of ImSQ8 with GTP after addition of ALP (133 mU mLꢁ1). Inset: (1)
Time-trace plots of ImSQ8-GTP with 0 (b), 17 (’), 33 (K), 67 (m), 133 (.),
200 (c) mU mLꢁ1 of ALP detected by the fluorescence intensity at 675 nm;
(2) the calibration plot between the fluorescence intensity at 675 nm and the
ALP concentration at 8 min. lex = 613 nm.
In conclusion, we have developed a sensitive colorimetric
and fluorescent sensor for the detection of GTP in aqueous
solution. The detection limit of GTP is up to 5.4 ppb. By adjusting
the length of the alkyl chain, squaraine ImSQ8 not only exhibits
good water-solubility, but is also capable of entering the cells as
an imaging reagent. The selective recognition of ImSQ8 for
GTP is attributed to the aggregation-caused spectral change. The
hydrolysis of GTP to GDP, GMP, guanosine and phosphate
catalyzed by ALP induces fluorescence turn-on, which enables
ImSQ8 to be applied to an enzyme activity assay.
This work was supported by grants from the National NSF of
China (No. 21372164, 21172155, 21025205, J1310008 and J1103315/
J0104), and the Sichuan Provincial Foundation (2012JQ0002).
Fig. 5 (a) Photographs of ImSQ8 (10 mM) in HEPES buffer (10 mM, pH =
7.2) before and after addition of GTP (2.0 equiv.) and subsequently ALP
(133 mU mLꢁ1) under laser irradiation (532 nm). TEM images of ImSQ8
(40 mM) in HEPES buffer (10 mM, pH = 7.2) (stained with sodium phospho-
tungstate): (b) before and (c) after addition of GTP (2.0 equiv.).
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