An imidazole-appended p-phenylene-Cu(II) ensemble as a chemoprobe for histidine in biological samples

Article abstract of DOI:10.1039/c4cc07274j

A tetra-imidazole-appended tetrakis(p-phenylene)ethylene 1-Cu²⁺ ensemble was found to enhance fluorescence upon addition of histidine, but not with any other amino acids. The 1-Cu²⁺ ensemble also selectively detected proteins contain

Full text of DOI:10.1039/c4cc07274j

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An imidazole-appended p-phenylene-Cu(II)
ensemble as a chemoprobe for histidine in
biological samples†
Cite this: Chem. Commun., 2014,
50, 15243
Received 15th September 2014,
Accepted 17th October 2014
Ka Young Kim, Sung Ho Jung, Joon-Hwa Lee, Shim Sung Lee and
Jong Hwa Jung*
DOI: 10.1039/c4cc07274j
www.rsc.org/chemcomm
A tetra-imidazole-appended tetrakis(p-phenylene)ethylene 1-Cu2+ synthesis procedures, which affords more practical and convenient
ensemble was found to enhance fluorescence upon addition of applications. Third, sensing of various analytes can be accomplished
histidine, but not with any other amino acids. The 1-Cu2+ ensemble by simply changing the receptor or the fluorescent indicator. There
also selectively detected proteins containing histidine residues are a few examples of an ensemble as a ‘‘turn-on’’ chemoprobe for
in a mixture of water and methanol (90 : 10, v/v%). The 1-Cu2+ His and proteins containing His moieties.9 Herein, we report a
ensemble-coated thin-layered chromatography (TLC) plate could tetrakis( p-phenylene)ethylene-based-Cu2+ ensemble-based chemo-
also detect histidine quantitatively. Furthermore, the fluorescence probe for proteins containing His in aqueous solution and in
emission recovery upon addition of five concentrations of His was biological fluids.
B80% with good linearity.
The tetra-imidazole-appended tetrakis(p-phenylene)ethylene
(1) was prepared using a slightly modified version of a previously
Histidine (His) is an essential amino acid required for human reported10 procedure as shown in Scheme S1 (ESI†). The ligand
growth.1 It can also act as a neurotransmitter in the central structure, based on the phenylethylene central core, facilitated the
nervous system of mammals.2 A deficiency of histidine may conversion of the initial self-assembled 1 into a stabilized form
result in the impaired nutritional state of patients with chronic through four part p–p stacking. The core was connected to the
kidney disease.3 Moreover, an abnormal level of histidine-rich imidazole by a solid-state reaction using an Ullmann coupling
proteins in the urine could indicate a variety of diseases such as protocol in 50% yield; the imidazole moiety enabled the coordina-
asthma4 and advanced liver cirrhosis.5 Accordingly, determina- tion of specific metal ions and the concomitant formation of a
tion of His in biological samples is of great importance in higher-order self-assembled nanostructure.
biochemical analysis and a number of methods have been
developed for its detection.6 In particular, fluorimetric detec-
tion has recently attracted considerable attention because of its
high sensitivity and spatial resolution.7
In chemical ensembles such as fluorogenic chemoprobes, the
receptor is noncovalently attached to a fluorescence moiety which
serves as an indicator.8 The binding between the receptor and the
analyte at the binding site results in either formation of a new
complex or displacement of the fluorophore from the receptor, both
of which would cause changes in fluorescence. These ensemble
systems have several advantages for this method of signaling.
First, the water solubility of an ensemble can be greatly improved,
Compound 1 exhibited a strong emission when dissolved in
especially those containing a metal center. Second, an ensemble can pure H2O (excitation wavelength = 345 nm). On the other hand,
usually be easily prepared, without the requirement for complicated the addition of methanol into these solutions decreased the
emission. As shown in Fig. 1, the luminescence properties of 1 were
observed at different ratios of water and methanol in composite
solutions (100 : 0–0 : 100 v/v%). When the water and methanol
solution was 90 : 10 v/v%, 1 showed an intense fluorescence emis-
Department of Chemistry and Research Institute of Natural Sciences, Gyeongsang
National University, Jinju, South Korea. E-mail: jonghwa@gnu.ac.kr;
Fax: +82-055-758-6027; Tel: +82-055-772-1488
sion band at 470 nm when photoexcited at 345 nm; compared to
the fluorescence spectrum acquired in pure water, the emission
† Electronic supplementary information (ESI) available: Experimental details and
spectroscopic analysis. See DOI: 10.1039/c4cc07274j
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Fig. 1 (A) Photoluminescence spectra of 1 (1.0 mM) at different ratios of
water and methanol in composite solutions (100 : 0–0 : 100 v/v%). (B) Plot
of fluorescence intensity against different ratios of water and methanol in
composite solutions (100 : 0–0 : 100 v/v%).
band was red shifted by 20 nm. On the other hand, the intensity
of the strong emission band of 1 dramatically decreased when
the content of water and methanol was changed to 60 : 40 v/v%, due
to changes in monomeric species by the increase in the fraction of
methanol. The mixture was transparent and homogeneous. Thus,
the SEM images of 1 prepared in both pure water and a mixture of
water/methanol (80 : 20 v/v%) were observed by scanning electron
Fig. 2 (A) Photograph of 1 (1.0 mM) upon addition of various concentrations
of Cu2+ (0–1.0 equivalent) under UV lamp. (B) Photoluminescence spectra
of 1 (1.0 mM) with various concentrations of Cu2+ (0–1.0 equivalent) in a
mixed water and methanol (90 : 10 v/v%).
microscopy (SEM). The SEM images of 1 in pure water and in a metal ions such as Hg2+, Co2+, Ni2+, Zn2+ and Mn2+ (Fig. S4, ESI†).
mixture of water/methanol (80 : 20 v/v%) showed nanoparticles No significant fluorescence changes of 1 were observed upon
of B20 nm diameter, which is homogeneous (Fig. S1, ESI†). addition of metal ions, because 1 did not form strong complexes
In contrast, we did not observe the aggregated morphology of 1 with other metal ions.
in a mixture of water/methanol (40 : 60 v/v%), suggesting that
molecules of 1 did not aggregate in an aqueous mixture with a intensity change of 1-Cu2+ ensemble was observed upon addition of
higher methanol fraction.
various amino acids (1.5 equivalents). As shown in Fig. 3A, 1-Cu2+
To investigate the selectivity for amino acids, the fluorescence
To gain insights into the forces that drive the assembly of 1, exhibited a strong emission with addition of histidine (His) when
we observed the 1H NMR spectra of 1 at different compositions the mixture was viewed under a UV lamp. The addition of other
of solvents (Fig. S2, ESI†). In pure water, the aromatic protons amino acids, in contrast, had no effect on the fluorescence changes
of 1 were shifted to the high field region and were broadened of 1-Cu2+. Thus, 1-Cu2+ responded selectively to histidine, a response
whereas in mixtures of methanol and water, the aromatic which can be attributed to the displacement of Cu2+ from 1 by His.
protons of 1 were shifted to the low field and sharpened as The fluorescence of 1-Cu2+ increased significantly upon addition of
the concentration of methanol increased. These results suggest His, with a fluorescence enhancement of up to B100-fold with the
that molecules of 1 form an aggregate by p–p stacking in pure addition of 1.5 equivalents of His (Fig. 3B). The addition of
H2O, but they exist as monomeric species in higher composi- His resulted in a linear increase in the fluorescence intensities of
tions of methanol in mixtures of methanol and water. Blue shift the 1-Cu2+ ensemble (Fig. 3C and Fig. S5, ESI†), indicating that Cu2+
of 1 with strong emission in pure water can be explained as became dissociated from the 1-Cu2+ complex, and then the His
follows. The first is due to restriction of intermolecular rotations molecule became bound to Cu2+ (Fig. 3D). The 1-Cu2+ complex also
of monomeric 1 by solvent. The second is due to p–p stacking showed excellent sensitivity with 20 ppb of histidine fluids (Fig. S6,
between molecules 1. Thus, p–p stacking of 1 definitely hinders ESI†), which is sensitive enough for its practical applications in
intermolecular rotation, as observed by the NMR experiment, so measurements of His in biological samples.
the strong emission of 1 would be induced by restriction of
intermolecular rotations with p–p stacking between molecules 1 1-Cu2+ ensemble upon addition of various amino acids. As shown
in pure water. in Fig. S7 (ESI†), the addition of 100 equivalents of other amino
We also observed the fluorescence intensity changes of the
We then examined the fluorescence response of 1 (1.0 mM) acids such as valine (Val), serine (Ser), phenylalanine (Phe), alanine
upon addition of Cu2+ (ranging from 0–1.0 equivalent) in a (Ala), cysteine (Cys), lysine (Lys), leucine (Leu), asparagine (Asp),
mixed water and methanol (90 : 10 v/v%). The fluorescence of 1 glutamic acid (Glu), glycine (Gly), methionine (Met) and arginine
at 470 nm dramatically decreased when 0.2 equivalents of Cu2+ (Arg) did not cause any obvious fluorescence enhancement. These
were added and became quenched completely when ca. 0.66 findings suggest that the highly selective nature of the fluorescence
equivalents of Cu2+ were added, indicative of formation of a 2 : 3 change of the 1-Cu2+ ensemble is due to the high selectivity of the
complex between Cu2+ and 1 (Fig. 2). The p–p stacking between assay for His.
the phenylene groups of 1 with Cu2+ induced a quenching effect
We further investigated the interaction between His and 1-Cu2+
by H-aggregation mode. A Job plot for complexation also gave a by using ESI spectroscopy (Fig. S8, ESI†). The ESI spectrum of the
2 : 3 stoichiometry (Fig. S3, ESI†). On the other hand, we also 1-Cu2+ ensemble upon addition of His corresponded to [2His + Cu2+]
observed the fluorescence changes of 1 upon addition of other at m/z B 373.08 while the peak at m/z B 597.50 was related to 1,
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Fig. 4 (A) Photograph of the TLC plate coated with the 1-Cu2+ ensemble
(1.0 mM) after addition of various concentrations of SPL 12 (0–1.0 mM)
under a UV lamp. (B) Photoluminescence spectra of 1 (1.0 mM) with various
concentrations of SPL 12 (0–1.0 mM) in a mixed water and methanol
(90 : 10 v/v%). (C) Plot of fluorescence intensity at 470 nm against the
concentration of SPL 12 (0–1.0 mM).
coated TLC plates were sprayed with solutions of proteins containing
His, the fluorescence intensity of the immobilized 1-Cu2+ ensemble
on TLC plates gradually increased in a linear manner with respect to
the increasing concentration of the protein. This result was attributed
to the displacement of Cu2+ from the receptor 1. The results are
consistent with the view that the 1-Cu2+ ensemble coated-TLC plates
not only detected His in a quantitative manner in a solid state system,
but also acts as a portable chemoprobe.
To apply this new material to a biological sample, we also
tested the ability of the 1-Cu2+ ensemble to detect His in urine
samples.7a The urine samples were collected from a single
subject in our University. Taking into account the possible
interferences in the determination of His in urine, we under-
took the quantification of His and the fluorescence emission
recovery of His by spiking samples with His over the range of
10 mM–50 mM. As shown in Fig. 5, we recovered B80% of the
His as determined by fluorescence emission when we tested
five concentrations of His and the response was linear with
respect to concentration. The percentage of recovery illustrates
the validity of the developed method. Thus, this 1-Cu2+ ensemble
would be useful as a quantitative detection method for His in the
biological samples.
Fig. 3 (A) Photograph of 1 (1.0 mM) upon addition of various amino acids
(2.0 equivalent) under UV lamp. (B) Photoluminescence spectra of 1
(1.0 mM) with various concentration of His (0–2.7 equivalent) in a mixed
water and methanol (90 : 10 v/v%). (C) Plot of fluorescence intensity at
470 nm against the concentration of histidine (0–10 ppm). (D) Schematic
illustration of the detection process for histidine by the 1-Cu2+ ensemble.
indicating that the binding between His and Cu2+ led to the
dissociation of the 1-Cu2+ ensemble. Thus, the selective detection
of His by the 1-Cu2+ ensemble was based on a displacement
approach. Furthermore, the appearance of a peak at 373.08
evidenced the formation of a typical complex for [2His + Cu2+].
To determine if the specificity of detection for His was still
functional in the presence of an excess of other amino acids, we
performed competition experiments to investigate the further
utility of the 1-Cu2+ ensemble (Fig. S9, ESI†). The addition of
various amino acids (100 equivalents) in water and methanol
(90 : 10 v/v%) to the solution of the 1-Cu2+ ensemble containing
1.5 equivalents of His did not induce any significant changes.
Hence, the 1-Cu2+ ensemble may serve as a selective chemoprobe
for His even in the presence of other relevant amino acids.
We also observed the microscopic image of the 1-Cu2+ ensemble
in the absence and the presence of His (Fig. S10, ESI†). Interest-
ingly, the microscopic image of 1-Cu2+ in the absence of His
revealed a cubic shape structure. In contrast, the microscopic
image of 1-Cu2+ in the presence of His showed a linear fibrillar
structure of 100 nm diameter. The fluorescence microscope image
of the 1-Cu2+ ensemble with His also showed a strong lumines-
cence that emitted blue light (Fig. S11, ESI†).
We evaluated the sensing abilities of 1-Cu2+ ensemble in the solid
state when the ensemble was coated onto TLC plate for use as a
portable chemoprobe under neutral conditions (pH 7) to detect
proteins containing His (Fig. 4). For the protein sensing experiments,
the 1-Cu2+ ensemble coated-TLC plates were dropped into protein
solutions (SPL 12), and then dried in air. Fluorescence changes were
monitored by measurement of the solid fluorescence spectra. As
expected, the TLC plates coated with only the 1-Cu2+ ensemble were
non-fluorescent in the apo state, due to the ACQ effect. When the
Fig. 5 Plot of fluorescence intensity upon addition of various concentra-
tions of histidine (green) and urine (red).
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This work was supported by a grant from the NRF
(2012R1A2A2A01002547 and 2012R1A4A1027750) supported
from the Ministry of Education, Science and Technology, Korea.
In addition, this work was partially supported by a grant from
the Next-Generation BioGreen 21 Program (SSAC, grant#:
PJ009041022012), Rural Development Administration, Korea.
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