A dicopper complex chemiluminescence probe for the determination of thiols in the extracts of murine P388 lymphocytic leukemia cell

Article abstract of DOI:10.1039/b907844d

A chemiluminescence (CL) probe of peroxidase-like dicopper complex (Cu ₂L₂) for the determination of glutathione (GSH) and related cellular thiols was developed for the first time.

Full text of DOI:10.1039/b907844d

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A dicopper complex chemiluminescence probe for the determination
of thiols in the extracts of murine P388 lymphocytic leukemia cellw
Xiaoru Zhang, Huanran Zhou, Caifeng Ding and Shusheng Zhang*
Received (in Cambridge, UK) 20th April 2009, Accepted 30th July 2009
First published as an Advance Article on the web 18th August 2009
DOI: 10.1039/b907844d
A chemiluminescence (CL) probe of peroxidase-like dicopper
complex (Cu2L2) for the determination of glutathione (GSH)
and related cellular thiols was developed for the first time.
As shown in Scheme 1, the dicopper complex can catalyze
the CL of the luminol–H2O2 system and exhibits peroxidase-
like activity. When GSH or related thiols was added to the
solution of the dicopper complex and vortex mixed at room
temperature, the structure of the dicopper complex was
destructed due to the complexation of GSH with Cu(II)
derived from dicopper complex, which induced the losing of
the catalyzing function of the complex. Thus, the dicopper
complex could be used as a CL probe for the measurement of
thiols.
Analysis of thiols such as cysteine (Cys), homocysteine (Hcy),
cysteinylglycine (CysGly) and GSH, is important in the
diagnosis of inherited and acquired metabolic disturbances
because the amount of these thiols in body fluids changes
when the cellular processes do not work properly.1 GSH,
which has many essential functions, including protection of
cells against free radicals and oxidative stress, detoxification of
xenobiotics, synthesis of proteins and DNA, is a very
abundant free thiol compound in nearly all living cells.2
GSH levels in biological fluids or tissues have been associated
with the pathogenesis of a number of specific diseases such as
diabetes,3 leukemia4 and some kinds of cancer.5 Thus, the
quantitative detection of GSH and related thiols is very
important for investigating biological process.
The UV and visible absorption spectra were obtained to
investigate the competitive complexation between probe
Cu2L2 and GSH (Fig. 1). From the results we can see that
UV-vis spectra differed greatly before and after adding GSH
to probe Cu2L2 solution. The absorption spectra of a mixture
solution of probe Cu2L2 and GSH were almost the same with
that of the ligand L (curve a and curve c), whereas they were
both different with the absorption spectra of probe Cu2L2
(curve b). In the visible region (Fig. 1 insert), there is no
absorption of ligand L (curve a0), and the absorption peak of
probe Cu2L2 (curve b0) disappeared after the addition of GSH
(curve c0). All these results showed that GSH could complex
with Cu(II) derived from probe Cu2L2, which can destructed
the peroxidase-like function of probe Cu2L2.
Several techniques have been reported for the analysis of
GSH, including high performance liquid chromatography
(HPLC),6 fluorometry,7 electrochemistry,8 electrochemi-
luminescence (ECL),9 and so on.10 Although these methods
are currently available for GSH determination, most of which
are complicated and inconvenient to operate, chemiluminescence
(CL) methods for determination of GSH based on the
reduction property of GSH have been received more and more
attention recently.11 These methods are simple and sensitive,
combined with wide linear dynamic range, however, they were
interfered with by other reductants, for example, ascorbic
acid.11a,b Therefore, an approach for the detection of GSH
with good sensitivity, selectivity that could be applied for
analysis of real samples without the separation treatment is
highly deserved.
The conditions of the competitive complexation between
GSH and probe Cu2L2, and the CL reaction involving luminol
and H2O2 catalyzed by probe Cu2L2 were investigated
(Fig. S4–S9w). It should be noted that the catalytic activity
of Cu2L2 was proportional to the concentration of Cu2L2 at
no less than 2.0 ꢀ 10ꢁ6 M (Fig. S9w).
To evaluate the sensitivity of the assay, different concentrations
of GSH (from 0 to 2.0 ꢀ 10ꢁ7 M) were mixed with 2.0 ꢀ 10ꢁ6 M
Uzu and Sasaki found a new dicopper complex as a stable
artificial peroxidase model, which was facile to synthesise and
could produce efficient luminol CL with H2O2.12 However,
GSH has a great propensity for forming complexes with
copper(II) derived from the dicopper complex via a competitive
complexation.13 Based on these principles, we developed a
dicopper complex based CL probe for the determination of
thiols in cell extracts for the first time.
Key Laboratory of Eco-chemical Engineering, Ministry of Education,
College of Chemistry and Molecular Engineering, Qingdao University
of Science and Technology, Qingdao 266042, Shandong, P. R. China.
E-mail: shushzhang@126.com; Fax: +86 532-84022750;
Tel: +86 532-84022750
Scheme 1 CL reaction of luminol–H2O2–Cu2L2 system (A), and
w Electronic supplementary information (ESI) available: Experimental
details and additional figures. See DOI: 10.1039/b907844d
competitive complexation between Cu2L2 and GSH (B).
ꢂc
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Fig. 1 The UV absorption spectra of ligand L (a, 20 mM), probe
Cu2L2 (b, 20 mM), and the product of the reaction between probe
Cu2L2 and GSH (c, 20 mM Cu2L2 + 40 mM GSH). Inset are the visible
absorption spectra of compound L (a0, 300 mM), probe Cu2L2
(b0, 300 mM), and the product of reaction between probe Cu2L2 and
GSH (c0, 300 mM Cu2L2 + 600 mM GSH).
Fig. 3 CL responses of Cu2L2 (2.0 mM) to diverse bioanalytes. Na+
,
K+ (4.0 ꢀ 10ꢁ4 M); Ca2+, NH4 (8.0 ꢀ 10ꢁ5 M); Mg2+, Zn2+
(1.0 ꢀ 10ꢁ5 M); Cu2+, Fe3+ (1.0 ꢀ 10ꢁ6 M); Co2+, Ni2+ (4.0 ꢀ 10ꢁ7 M);
cholesterol (8.0 ꢀ 10ꢁ5 M); ascorbic acid (4.0 ꢀ 10ꢁ5 M); L-phenyl-
alanine (1.0 ꢀ 10ꢁ5 M); leucine, epinephrine (8.0 ꢀ 10ꢁ6 M);
dopamine, uric acid (6.0 ꢀ 10ꢁ6 M); glucose, alanine (2.0 ꢀ 10ꢁ4 M);
GSH (2.0 ꢀ 10ꢁ8 M.). The bars except GSH represent the subsequent
addition of 2.0 ꢀ 10ꢁ8 M GSH to the mixture. Other conditions were
the same as those described in Fig. 2.
+
of probe Cu2L2. After incubation at pH 6.5 for 10 min, the CL
was measured with 1.5 ꢀ 10ꢁ4 M luminol and 2.0 ꢀ 10ꢁ2
M
hydrogen peroxide at pH 10.0. The CL signal of luminol–
H2O2–Cu2L2 decreased with the increasing of the concentration
of GSH as shown in Fig. 2. With the GSH concentration
changed in the range of 8.0 ꢀ 10ꢁ9 B 2.0 ꢀ 10ꢁ7 M, a
good linearity was plotted with the regression equation as
DICL (106) = 0.2465 + 1.0821CGSH (10ꢁ8 M) (n = 12, R =
0.9993). The relative standard deviation for 11 repetitive
measurements of 2.0 ꢀ 10ꢁ8 M was 4.6%, and the detection
limit was 1.8 ꢀ 10ꢁ9 M (3s) under the optimum conditions.
The results indicated that probe Cu2L2 could be used to detect
GSH sensitively.
2.0 ꢀ 10ꢁ8 M GSH. The tolerance limit of interference was
taken as a relative error not greater than ꢃ5% variation in the
average DICL, calculated for the established level of GSH as
shown in Fig. 3 and Table S1w. It can be seen that most
bioanalytes yielded no or negligible interference with GSH
detection. 2000-fold excess of ascorbic acid relative to GSH
did not interfere with the detection of GSH. When concentrations
of Cu2+, Fe3+, Co2+ and Ni2+ were 50 or 20 times more than
that of GSH, interference was observed, but compared to the
trace levels of the metal ions in murine P388 lymphocytic
leukemia cell (Table S3w), this method can be used for
the determination of thiols in cell extracts free from the
interference of other constituents.
To testify the selectivity of present method, the influence of
biorelevant analytes was also studied for control experiments.
Ten kinds of cation (including Na+, K+, Ca2+, NH4
,
+
Mg2+, Zn2+, Cu2+, Fe3+, Co2+ and Ni2+) and nine kinds
of non-thiol compounds (including cholesterol, ascorbic acid,
L-phenylalanine, dopamine, leucine, uric acid, epinephrine,
glucose and alanine) were tested on the determination of
The competitive complexation can occur between not only
GSH but also the other thiols and probe Cu2L2. So, reactivity
of Cu2L2 with 5 ꢀ 10ꢁ7 M different free thiols was also
measured as quantifiably decreasing CL intensity. As shown
from Fig. 4, the CL intensities decreased more obviously after
probe Cu2L2 reacted with GSH than with any other studied
free thiols, such as dithiothreitol (DTT), thioglycolic acid (TA),
Fig. 2 CL responses of the luminol (1.5 ꢀ 10ꢁ4 M)–H2O2 (2.0 ꢀ 10ꢁ2 M)
reaction catalyzed by the probe Cu2L2 (2.0 mM), which was then
reacted with different concentrations of GSH (final concentration
from a to l): 0, 0.008, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16,
0.18, 0.2 mM) for 10 min in 0.1 M H3BO3–NaOH buffer (pH 6.5)
at room temperature. CL measurement was performed in 0.1 M
H3BO3–NaOH buffer solution (pH 10.0).
Fig. 4 Decreased CL intensity toward different kinds of non-protein
thiols (5 ꢀ 10ꢁ7 M). Other conditions were the same as those described
in Fig. 2.
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analyzed with the method proposed. From the results given in
Table S2w, we can see that the precision and recovery of our
method for determining GSH in cell extracts are satisfactory.
In conclusion, we provide a dicopper complex CL probe for
the determination of GSH and related cellular thiols for the
first time. GSH was able to form complexes with copper (II)
derived from probe Cu2L2. This competitive complexation
with copper (II) may lead to the destruction of the
peroxidase-like dicopper complex Cu2L2, then decreases in
CL intensity were observed accordingly. The relatively low
detection limit of 1.8 nM and a broad dynamic range of
8.0 ꢀ 10ꢁ9B2.0 ꢀ 10ꢁ7 M were achieved. The developed
CL assay based on CL probe Cu2L2 was considerably
attractive for rapid, sensitive and selective detection of cellular
thiols. The novel strategy provides an alternative method for
molecule recognition using CL probe.
Fig. 5 CL responses of luminol–H2O2 catalyzed by probe Cu2L2,
after which was reacted with the supernatant of murine P388 lympho-
cytic leukemia cells (A) and with the protein extracts of murine P388
lymphocytic leukemia cells (B). The other conditions were the same as
Fig. 2.
This work was supported by the National Natural Science
Foundation of China (No.20775038), Doctoral Fund of
Ministry of Education of China (No. 200804260001), and
the Excellent Young Scientists Foundation of Shandong
Province (JQ200805).
2-mercaptoethanol (b-ME) and Cys. The results are in good
agreement with the results reported previously.7a,8
Two protein thiols of metallothionein (MT) and glutathione
reductase (GR) were further testified with the CL method.
When 2.0 ꢀ 10ꢁ6 M probe Cu2L2 was used, the DICL increases
when the concentration of MT (Fig. S10Aw) ranges from
1.0 nM to 80 nM and GR (Fig. S10Bw) from 10.0 mU Lꢁ1
to 1500 mU Lꢁ1, respectively, and reached a plateau since
then. It was noteworthy that the probe Cu2L2 could still
achieve sensitive detection even at 1.0 nM MT, this indicated
that the probe Cu2L2 is more sensitive to protein thiols than
free thiols.
Notes and references
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In order to evaluate the applicability of the proposed
method in clinical diagnosis, CL determination of free thiols
and protein thiols in the extracts of murine P388 lymphocytic
leukemia cells was performed, respectively. The experimental
details were described in the ESI and results were shown in
Fig. 5.w From the results we can see that probe Cu2L2 showed
much higher reactive responses toward protein thiols in the
protein extracts of murine P388 lymphocytic leukemia cells
than those of free thiols in the supernatant. This may caused
by the large amount of sulfhydryl groups that exist with
protein thiols.
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The total concentration of thiols in cell extracts was counted
by GSH and the GSH content of the cell extracts was derived
from the standard curve and the regression equation.
Compared with electrochemical method developed by our
group,8 the results using our present approach were in good
agreement with those obtained using electrochemical method
(experimental details, see ESIw). The average recovery test was
made using the standard addition method. All the cell extracts
were spiked with GSH at different concentration levels and then
ꢂc
This journal is The Royal Society of Chemistry 2009
5626 | Chem. Commun., 2009, 5624–5626