Coumarin-DPA-Cu(ii) as a chemosensing ensemble towards histidine determination in urine and serum

Article abstract of DOI:10.1039/c2ob26955d

An ensemble-based fluorescent sensor HS-1 + Cu²⁺ for detection of histidine is reported. Complex HS-1 + Cu²⁺ sensitively senses histidine at pH 7.4 in aqueous media. The quantitative determination of histidine in urine and fetal calf serum is also conducted. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2ob26955d

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Coumarin–DPA–Cu(II) as a chemosensing ensemble
towards histidine determination in urine and serum†
Cite this: Org. Biomol. Chem., 2013, 11,
717
Ji-Ting Hou, Kun Li,* Kang-Kang Yu, Ming-Yu Wu and Xiao-Qi Yu*
Received 8th October 2012,
Accepted 3rd December 2012
DOI: 10.1039/c2ob26955d
www.rsc.org/obc
An ensemble-based fluorescent sensor HS-1 + Cu2+ for detection
of histidine is reported. Complex HS-1 + Cu2+ sensitively senses
histidine at pH 7.4 in aqueous media. The quantitative determi-
nation of histidine in urine and fetal calf serum is also conducted.
As the key constituents of proteins, amino acids are biologi-
cally important species.1 Among them, histidine (His) is
reported to be very essential for human growth.2 It can play
important roles in biological systems such as controlling the
transmission of metal elements in biological bases owing to
the high reactivity of its imidazole group.3 Also, it is found
that the deficiency of histidine may result in the impaired
nutritional state of patients with chronic kidney disease.4
Moreover, an abnormal level of histidine-rich proteins could
indicate a variety of diseases such as asthma5 and advanced
liver cirrhosis.6 Accordingly, determination of histidine in bio-
logical samples is of great importance in biochemical analysis
and a number of methods have been developed for this
purpose.7,8 In these studies, fluorimetric detection has recently
attracted considerable attention for its high sensitivity and
temporal and spatial resolution.9 Most of the molecular fluor-
escent sensors for histidine were prepared as metal–indicator
complexes, namely ensembles.7a–f,8
In a chemosensing ensemble, the receptor is noncovalently
attached to the indicator and the binding between the receptor
and the analyte results in either formation of a new complex
or displacement of the indicator, both of which will cause flu-
orescence changes.10 There are several advantages to this
method of signaling: (1) the water solubility of an ensemble
can be largely improved, especially containing a metal center;
(2) different species sensing can be achieved just by simply
changing the receptor (metal ion) or the binding site of the
Scheme 1
indicator at will; (3) an ensemble usually does not require com-
plicated synthesis procedures, which of course provides more
practical and convenient applications. To this end, numerous
chemosensing ensembles have been applied in the detection
of biological species such as cations,11 anions12 and thiols.13
Although several ensembles were reported for His detection,
most of them could not get rid of the interference of Cys and
few of them could be applied in urine and serum. Herein, we
would like to report a coumarin–DPA–Cu(II) ensemble-based
chemosensor HS-1 + Cu2+ to selectively detect histidine in
aqueous solution and biological fluids (see Scheme 1).
7-Diethylamino coumarin is chosen as the fluorophore due
to its good photostability, large Stokes shift and high quantum
yield.14 DPA (di(2-picolyl)amine) acts as the binding unit for
Cu2+ for its strong binding ability with divalent heavy metal
ions,15 which may improve the selectivity of the ensemble.
HS-1 was prepared via a simple process and characterized by
1H, 13C NMR and HRMS (see ESI†).
First, the fluorescence response of HS-1 (5 μM) toward Cu2+
was tested in HEPES (20 mM, pH = 7.4, containing 0.5%
DMSO as cosolvent). Upon the addition of Cu2+, the fluor-
escence of HS-1 at 500 nm gradually decreased and was
quenched completely when 1 equiv. Cu2+ was added, indicat-
ing the formation of a 1 : 1 bonding mode between HS-1 and
Cu2+ (Fig. S1†). Concomitantly, the quantum yield of HS-1
decreased from 41% to 0.85% (fluorescein in 1 N NaOH as
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of
Chemistry, Sichuan University, Chengdu, 610064, China.
E-mail: kli@scu.edu.cn, xqyu@scu.edu.cn; Fax: +(86)-28-85415886;
Tel: +(86)-28-85415886
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, additional plots of UV, fluorescence, NMR and mass analysis data. See
DOI: 10.1039/c2ob26955d
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Fig. 2 The fluorescence spectra of HS-1 + Cu2+ (5 μM) toward 100 equiv.
amino acids (50 equiv. for His) in HEPES (20 mM, pH = 7.4, containing 0.5%
DMSO as cosolvent) (λex = 410 nm, slits: 2 nm/2 nm). Inset: Fluorescence color
changes of HS-1 + Cu2+ upon addition of various amino acids under a UV lamp
(Ex = 365 nm). From top to bottom and left to right: HS-1, HS-1 + Cu2+, His, Ala,
Asn, Ile, Leu, Gln, Met, Thr, Gly, Arg, Lys, Ser, Phe, Val, Pro, Tyr, Trp, Asp, Cys, Glu.
Fig. 1 The titration profile of HS-1 + Cu2+ (5 μM) toward His in HEPES (20 mM,
pH = 7.4, containing 0.5% DMSO as cosolvent). Inset: The linear relationship
between HS-1 + Cu2+ (5 μM) and His in a low concentration range from
2–60 μM (λex = 410 nm, slits: 2 nm/2 nm).
reference, Φ = 0.85).16 A Job plot for the complexation also
showed a 1 : 1 stoichiometry (Fig. S2†). Based on the 1 : 1
binding mode, the binding constant derived from the fluor-
escence titration data was calculated to be 1.3 × 106 M−1
(Fig. S3†).
was also conducted, and only an ∼11-fold enhancement was
observed even though more Cys was added (Fig. S5†). By sharp
contrast, the fluorescence of HS-1 + Cu2+ at 500 nm was dra-
matically amplified with up to an ∼60-fold enhancement while
50 equiv. of His was added into the solution, which is pro-
found enough to distinguish His from Cys. Usually, a Cu2+/
Ni2+-ensemble sensing His can distinctly respond to Cys.7a–c,8
The spectroscopic studies suggest that the ensemble exhibits a
high selectivity over other amino acids including Cys, indicat-
ing the strong binding capacity of the imidazole group of His
to the copper ion. Since the formation constants of His with
Cu2+ were reported to be 10.16 (log β1) and 18.11 (log β2),17
which were far larger than those of HS-1 and Cu2+, a displace-
ment approach might be reasonable to explain the optical
changes above.
To further investigate the interaction between His and HS-1
+ Cu2+, the ESI spectra were obtained (Fig. S6†). Before the
treatment of His, an intense peak at m/z 533.1511 was found
to correspond to the [HS-1 + Cu2+ − H].12c Upon addition of 4
equiv. and 40 equiv. His respectively, the peak at m/z ∼
533.15 gradually weakened and the peak at m/z ∼ 472.23
related to HS-1 was observed, indicating that the binding
between His and Cu2+ led to the release of HS-1. Thus, the
selective His detection of the HS-1 + Cu2+ ensemble is based
on a displacement approach. In addition, the appearance of a
peak at 373.0664 evidenced the formation of the complex
[2His + Cu2+].
The above result that the fluorescence of HS-1 can be com-
pletely quenched by Cu2+, and the fact that the Cu2+-complex
can selectively discriminate His from other amino acids,7a–c,8b
allowed us to speculate that the HS-1 + Cu2+ ensemble is promi-
sing as a turn-on fluorescent sensor for histidine. To confirm
our assumption, HS-1 was preincubated with Cu2+, and the
titration of His to the resulting ensemble was conducted. As
Fig. 1 and Fig. S4† display, the fluorescence of HS-1 + Cu2+
increased significantly upon the addition of histidine and up
to an ∼80-fold enhancement was observed when 120 equiv.
His was added finally. Furthermore, the fluorescence intensity
of the ensemble at 500 nm increased linearly with the treat-
ment of His in a low concentration range from 2–60 μM (R2 =
0.983), and the detection limit (S/N = 3) of His from the titra-
tion profile was detected to be 3.1 μM,12c which is sensitive
enough for its practical applications in determination of His
in biological fluids.
To examine the selectivity, changes in the fluorescence
intensity of HS-1 + Cu2+ promoted by addition of excess
amounts of various amino acids were measured in HEPES
(20 mM, pH = 7.4, containing 0.5% DMSO as cosolvent). As
shown in Fig. 2, the addition of 100 equiv. (0.5 mM) of other
naturally occurring amino acids including tryptophan (Trp),
asparagine (Asn), lysine (Lys), leucine (Leu), isoleucine (Ile),
methionine (Met), threonine (Thr), tyrosine (Tyr), valine (Val),
aspartic acid (Asp), alanine (Ala), serine (Ser), glutamine (Gln),
glutamic acid (Glu), glycine (Gly), phenylalanine (Phe), cysteine
(Cys), proline (Pro) and arginine (Arg) did not cause any
obvious fluorescence enhancement except that the addition of
100 equiv. Cys could lead to an approximate 10-fold reinforce-
ment. A fluorescence titration experiment of HS-1 toward Cys
The competition experiments were conducted to investigate
the further utility of HS-1 + Cu2+ (5 μM) in the presence of
other amino acids in HEPES (20 mM, pH = 7.4, containing
0.5% DMSO as cosolvent). As shown in Fig. 3 and Fig. S7,† the
addition of 100 equiv. other amino acids to the solution of
HS-1 + Cu2+ containing 50 equiv. His did not induce any con-
siderable changes. Hence, HS-1 + Cu2+ seems to be useful for
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Fig. 4 Determination of His concentration in spiked (A) human urine and (B)
fetal calf serum using His as an internal standard. His concentrations measured
were 0, X, X + 4, X + 8 μM. The data represent the average of three independent
experiments.
In conclusion, an ensemble HS-1 + Cu2+ is reported to
selectively detect His in aqueous solution. No considerable
interference is observed in the presence of the other amino
acids and common biological species. Notably, as a common
interferent for chemosensing ensembles of His, Cys causes
negligible impact on the emission intensity of the HS-1 + Cu2+
ensemble, which enables more practical applications of HS-1 +
Cu2+. Ultimately, this ensemble was successfully applied for
the parallel measurement of His concentration in human
urine and fetal calf serum.
This work was financially supported by the National
Program on Key Basic Research Project of China (973 Program,
2012CB720603, 2013CB328900) and the National Science
Foundation of China (Nos. 20732004 and 21001077). We also
thank the Analytical & Testing Center of Sichuan University for
NMR analysis.
Fig. 3 Fluorescence response of HS-1 + Cu2+ (5 μM) toward amino acids (100
equiv.) in the absence (black bars) or presence (red bars) of His (50 equiv.) in
HEPES (20 mM, pH = 7.4, containing 0.5% DMSO as cosolvent) (λex = 410 nm,
slits: 2 nm/2 nm).
selectively sensing His even in the presence of other relevant
species.
As an essential bioactive amino acid component of many
proteins, the detection of histidine in biological fluids such as
urine and serum is in urgent demand. With previous results in
hand, we would like to explore the quantitative determination
of histidine in urine and serum samples using HS-1 + Cu2+.
We first studied the fluorescence response of HS-1 + Cu2+ to
His in the presence of some biological species, including K+,
Na+, Mg2+, Ca2+, Fe3+, glucose, ascorbic acid and urea
(Fig. S8†). No obvious fluorescence changes of HS-1 + Cu2+ in
the presence of His were observed when these biological
species were added, indicating the potential utility of HS-1 +
Cu2+ for the determination of His in biological fluids. Then,
urine was diluted before use and His was spiked into the urine
as an internal standard without further treatment. To the
spiked urine samples was added HS-1 + Cu2+ (5 μM) and then
the fluorescence of the mixtures was measured. The average
His concentrations in two urine samples were determined to
be 510 and 990 μM (the urine was totally diluted 150 times
before measurement), respectively, which is in good agree-
ment with its normal levels in human urine (normal level:
130–2100 μM in urine).18
Next, the His determination in fetal calf serum was con-
ducted. Fetal calf serum was deproteinized by methanol before
use and various amounts of the supernatant were added to the
HEPES solution containing HS-1 + Cu2+ (5 μM). An excellent
linear relationship between the amount of deproteinized fetal
calf serum and the fluorescence intensity of HS-1 + Cu2+ at
500 nm was obtained (Fig. S9†). Inspired by this result, we sub-
sequently measured the concentration of His in fetal calf
serum by spiking His into serum as an internal standard.
A good linear correlation was observed as well and the His con-
centration in fetal calf serum was measured to be 495 μM
(Fig. 4).
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