Sensitive detection of acrolein in serum using time-resolved luminescence

Article abstract of DOI:10.1021/ol1002219

A novel lanthanide probe was designed, synthesized, and employed for a sensitive and reliable assay of acrolein based on time-resolved luminescence measurement, which suppresses the background signal of serum.

Full text of DOI:10.1021/ol1002219

ORGANIC
LETTERS
2010
Vol. 12, No. 8
1704-1707
Sensitive Detection of Acrolein in Serum
Using Time-Resolved Luminescence
Masataka Togashi,†,‡ Yasuteru Urano,§ Hirotatsu Kojima,| Takuya Terai,†,‡
Kenjiro Hanaoka,†,‡ Kazuei Igarashi,⊥ Yasunobu Hirata,‡,# and
Tetsuo Nagano*,†,‡,|
Graduate School of Pharmaceutical Sciences, Graduate School of Medicine, and
Chemical Biology Research InitiatiVe, The UniVersity of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan, CREST, Japan Science and Technology Agency,
4-8-1 Honcho, Kawaguchi, Saitama 332-0012, Japan, Department of Clinical
Biochemistry, Graduate School of Pharmaceutical Sciences, Chiba UniVersity,
1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan, and Department of AdVanced
Clinical Science and Therapeutics, Graduate School of Medicine, The UniVersity of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
tlong@mol.f.u-tokyo.ac.jp
Received January 29, 2010
ABSTRACT
A novel lanthanide probe was designed, synthesized, and employed for a sensitive and reliable assay of acrolein based on time-resolved
luminescence measurement, which suppresses the background signal of serum.
Acrolein (CH2CHCHO) is a lipid peroxidation product that
can be generated ubiquitously in biological systems.1 It is a
representative carcinogenic aldehyde formed endogenously
through oxidative stress, caused by reactive oxygen species
and lipid peroxide. Among these aldehydes, acrolein is one
of the strongest electrophiles, and it disrupts important
cellular functions by covalently modifying biomolecules,
including proteins, DNA, and lipids.2
Acrolein is becoming of great interest to medical scientists
as a potential marker of various diseases, including chronic
renal failure,3 stroke,4 and cancer.5 For example, acrolein
was reported to be increased in the plasma of stroke patients,
so that diagnosis may be possible by quantifying acrolein in
plasma or serum. In addition, acrolein is spontaneously
formed by polyamines and strongly inhibits cell growth
owing to its cytotoxicity.6 Therefore, a simple and convenient
† Graduate School of Pharmaceutical Sciences, The University of Tokyo.
‡ CREST, Japan Science and Technology Agency.
§ Graduate School of Medicine, The University of Tokyo.
| Chemical Biology Research Initiative, The University of Tokyo.
⊥ Graduate School of Pharmaceutical Sciences, Chiba University.
# Department of Advanced Clinical Science and Therapeutics, Graduate
School of Medicine, The University of Tokyo.
(3) Sakata, K.; Kashiwagi, K.; Sharmin, S.; Ueda, S.; Irie, Y.; Murotani,
N.; Igarashi, K. Biochem. Bioph. Res. Co. 2003, 305, 143.
(4) Tomitori, H.; Usui, T.; Saeki, N.; Ueda, S.; Kase, H.; Nishimura,
K.; Kashiwagi, K.; Igarashi, K. Stroke 2005, 36, 2609.
(5) Uchida, K. Trends CardioVas. Med. 1999, 9, 109.
(6) Sharmin, S.; Sakata, K.; Kashiwagi, K.; Ueda, S.; Iwasaki, S.;
Shirahata, A.; Igarashi, K. Biochem. Biophys. Res. Commun. 2001, 282,
228.
(1) (a) Esterbauer, H.; Schaur, R. J.; Zollner, H. Free Radical Bio. Med.
1991, 11, 81. (b) Girotti, A. W. J. Lipid Res. 1998, 39, 1529.
(2) (a) Kumagai, T.; Matsukawa, N.; Kaneko, Y.; Kusumi, Y.; Mit-
sumata, M.; Uchida, K. J. Biol. Chem. 2004, 279, 48389. (b) Matsumoto,
K.; Kumamoto, T.; Ishikawa, T.; Ishii, E.; Tomitori, H.; Igarashi, K.
Heterocycles 2006, 67, 361. (c) Kawai, Y.; Furuhata, A.; Toyokuni, S.;
Aratani, S.; Uchida, K. J. Biol. Chem. 2003, 278, 50346.
10.1021/ol1002219  2010 American Chemical Society
Published on Web 03/16/2010

detection method for low concentrations of acrolein would
find applications in medical, biological and other fields.
cent upon reaction with acrolein. Our design strategy is
summarized in Scheme 1. A lanthanide complex for TRF
A currently used conventional method for acrolein detec-
tion is fluorescence analysis based on the Skraup reaction
with m-aminophenol.7 In this method, the plasma of patients
is reacted with m-aminophenol, the product, 7-hydrox-
yquinoline, is separated by HPLC, and its fluorescence is
quantified. HPLC separation cannot be avoided, because (i)
the background fluorescence of plasma excited at 350-400
nm, which is the excitation wavelength of 7-hydroxyquino-
line, is quite high and (ii) the quantum yield of 7-hydrox-
yquinoline is quite low. However, HPLC separation is not
suitable for high-throughput measurement of multiple samples
using microplates. Acrolein-protein adducts can also be
detected using monoclonal antibody,5,8 but the method is
costly and needs complicated procedures. Additionally, there
is a lag of several hours between production of acrolein and
that of acrolein-protein adducts. Here we report a novel
luminescence-based method of acrolein detection that is
suitable for high-throughput microplate assays without HPLC
separation.
Scheme 1. (A) Schematic Representation of the Mechanism of
Our Probe. Skraup Reaction Forms an Extended-Conjugate
System. The Product Is Strongly Luminescent when Excited at
the Appropriate Wavelength. (B) Reaction of the Designed
Probe with Acrolein
The method of time-resolved fluorescence (TRF) measure-
ment was selected as a basis for the development of the assay.
It makes use of the characteristic long-lived luminescence
of certain compounds, especially lanthanide complexes.9 In
TRF measurement, the luminescence signal is collected for
a certain gate time after an appropriate delay time, following
a pulsed excitation. Lanthanide complexes have long lumi-
nescence lifetimes of the order of milliseconds, in contrast
to usual organic compounds, which have luminescence
lifetimes of the order of nanoseconds. By taking advantage
of this unique character, the influence of short-lived back-
ground fluorescence and scattered light can be reduced to a
negligible level, and the long-lived lanthanide luminescence
can be distinguished from background signals.10 Lanthanide
complexes also have large stokes shifts, which can further
decrease the background. Because of these advantages, TRF
measurements are employed in various fields where both
small assay scale and high sensitivity are required, especially
immunoassays11 and high-throughput screening.12
measurement usually consists of two parts, an antenna moiety
and a chelator moiety. We thought that the luminescence
properties could be precisely controlled if the absorbance
spectrum of the antenna moiety changed as a result of
reaction with acrolein. We chose an aniline structure as the
antenna and the Skraup reaction as the controlling reaction
(Scheme 1). The Skraup reaction of aniline is well-known
as being highly specific for acrolein, and the λmax of the
aniline moiety is greatly increased by quinoline formation.
In addition, the quinoline ring is a good energy transfer donor
to lanthanide complex.13 As the chelator, we selected
diethylenetriaminepentaacetic acid (DTPA) because (i) DTPA
has the ability to form a strong complex with europium ion
and to emit bright luminescence14 and (ii) it has high
solubility in aqueous solution. The probe composed of aniline
and DTPA moieties, p-NH2PhDTPA, was designed according
to Scheme 1. Before the Skraup reaction, the probe is
expected to emit no luminescence because it is not able to
absorb at the excitation wavelength for quinoline. In contrast,
the product of the reaction with acrolein can absorb the light
and emit luminescence via energy transfer to the europium
To achieve sensitive detection, we must control the
luminescence properties of our probe, which should have
no luminescence itself, but should become highly lumines-
(7) (a) Alarcon, R. A. Anal. Chem. 1968, 40, 1704. (b) Bohnenstengel,
F.; Eichelbaum, M.; Golbs, E.; Kroemer, H. K. J. Chromatogr. B 1997,
692, 163.
(8) Sakata, K.; Kashiwagi, K.; Sharmin, S.; Ueda, S.; Igarashi, K.
Biochem. Soc. Trans. 2003, 31, 371
.
(9) Mizukami, S.; Kikuchi, K.; Higuchi, T.; Urano, Y.; Mashima, T.;
Tsuruo, T.; Nagano, T. FEBS Lett. 1999, 453, 356.
(10) (a) Parker, D.; Dickins, R. S.; Puschmann, H.; Crossland, C.;
Howard, J. A. K. Chem. ReV. 2002, 102, 1977. (b) Døssing, A. Eur. J. Inorg.
Chem. 2005, 1425.
Table 1. Photochemical Properties of the Probes
a
(11) Johansson, M. K.; Cook, R. M.; Xu, J.; Raymond, K. N. J. Am.
Chem. Soc. 2004, 126, 16451.
Absmax (nm)
Em(max) (nm)
Φfl (%)
p- NH2PhDTPA-Eu
Q-DTPA-Eu
243
320
n.d.b
618
n.d.b
4.6
(12) Yuan, J.; Wang, G.; Majima, K.; Matsumoto, K. Anal. Chem. 2001,
73, 1869.
(13) Hanaoka, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am.
Chem. Soc. 2004, 126, 12470.
a Quantum yields were determined using [Ru(bpy)3]Cl2 (Φ ) 0.028 in
water) as a standard. Measurements were performed in 50 mM sodium
phosphate buffer (pH 3.5). b Not determined.
(14) (a) Moeller, T.; Thompson, L. C. J. Inorg. Nucl. Chem. 1962, 24,
499–510. (b) Terai, T.; Kikuchi, K.; Iwasawa, S. Y.; Kawabe, T.; Hirata,
Y.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2006, 128, 6938.
Org. Lett., Vol. 12, No. 8, 2010
1705

ion. Simultaneously, we also synthesized quinoline-DTPA
adduct (Q-DTPA), which is the expected product of the
Skraup reaction of acrolein and p-NH2PhDTPA.
The photochemical properties of the probe and product are
summarized in Table 1. As expected, λmax of Q-DTPA-Eu was
at 77 nm longer wavelength than that of p-NH2PhDTPA-Eu.
In addition, Q-DTPA-Eu emitted strong luminescence, indicat-
ing that energy transfer occurred from the quinoline ring to the
lanthanide complex. Luminescence of p-NH2PhDTPA-Eu was
too low to allow determination of the quantum yield. These
results showed that our rational design strategy worked very
well. Absorption and emission spectra of p-NH2PhDTPA-Eu
(green line) and Q-DTPA-Eu (red line) are shown in Figure 1.
Figure 2. Reaction of p-NH2PhDTPA (final concentration of 10
mM) with acrolein (0, 0.5, 1.0, 1.5, 2.0 mM). Emission spectra of
the reaction mixture diluted with 200 mM sodium phosphate buffer,
pH 3.5. Time-resolved luminescence intensity (ex ) 320 nm) was
recorded 30 min after the initiation of the reaction.
increase of luminescence intensity was observed. We also
carried out HPLC analysis to confirm that the reaction of
p-NH2PhDTPA and acrolein produced Q-DTPA. The main
product of the reaction was indeed Q-DTPA (Supporting
Information). These results suggest that the reaction pro-
ceeded as expected, and the luminescence was greatly
increased following the reaction of acrolein and
p-NH2PhDTPA. We thus considered that p-NH2PhDTPA is
potentially useful for the sensitive detection of acrolein and
next addressed acrolein assay with microplates. Microplates
(96-well, 384-well, etc.) are an indispensable tool for
biological assays. They can handle a large number of samples
simultaneously with a small sample volume (microliter order)
and are suitable for automated assay systems. They are,
however, more likely to suffer from autofluorescence of the
device and scattered light. Therefore, TRF measurements
Figure1.Absorption(A)andemission(B)spectraofp-NH2PhDTPA-
Eu (green) and Q-DTPA-Eu (red) 16.4 µM with europium ions in
aqueous solution (100 mM sodium phosphate buffer, pH 3.5).
The emission spectrum of Q-DTPA is characteristic of a
europium complex.
We investigated suitable reaction conditions of acrolein and
the probe. First, p-NH2PhDTPA and HCl were added to acrolein
in sodium phosphate buffer, and the mixture was heated for 30
min at 100 °C and then cooled in tap water. Europium was
added to the diluted mixture, and the luminescence intensity
was measured. Optimum conditions were evaluated by measur-
ing the luminescence of reaction solutions. The results indicated
that the optimum concentraion of the probe was 10 mM, and a
suitable reaction time was 30 min.
Figure 3. Microplate assays of acrolein (0, 1, 2, 3 µM) in aqueous
solution with p-NH2PhDTPA (final concentration of 10 mM). Time-
resolved luminescence intensity (ex/em ) 340/615 nm, measured
after dilution with 200 mM sodium phosphate buffer, pH 3.5) was
recorded 30 min after the initiation of the reaction. Data are shown
as mean ( SD (n ) 3).
Figure 2 shows the results of assays of various concentra-
tions of acrolein under optimal conditions. A dose-dependent
(15) (a) Yuan, J.; Wang, G.; Majima, K.; Matsumoto, K. Anal. Chem.
2001, 73, 1869. (b) Gudgin Dickson, E. F.; Pollak, A.; Diamandis, E. P. J.
Photochem.Photobiol. B 1995, 27, 3.
1706
Org. Lett., Vol. 12, No. 8, 2010

Serum itself has absorbance in UV-vis region and emits
fluorescence in the range of 300-500 nm. Sensitive and
reliable detection using the conventional method is prevented
by the background fluorescence of serum. However, in the
case of our probe, the background signal can be suppressed
by the use of TRF measurements. Thus, our probe should
be especially suitable for assays in serum. The following
experiments were performed with human serum spiked with
acrolein. As can be seen in Figure 4A, as little as 5 µM
acrolein could be detected with this probe, and the lumines-
cence increased linearly with respect to acrolein concentra-
tion. A concentration of 5 µM acrolein could not be detected
with m-aminophenol, which is the probe for the conventional
method, without HPLC separation (Figure 4B). In the
conventional method, reliable assays are impossible in the
range of low concentration of acrolein, because of the strong
background fluorescence of serum. With our method, the
detection limit of 0.96 µM was achieved. This is 1 order of
magnitude better than that of the conventional method (15
µM) without HPLC separation, and the detection limit
appears to be appropriate for measuring acrolein concentra-
tions found in disease states (normal, 0.5 µM; patient, 1.4
µM).8 In conclusion, we have developed a novel probe for
acrolein by using TRF measurement, which can eliminate
background fluorescence due to serum. Our probe permits
sensitive and reliable detection of acrolein in serum, and its
performance is superior to that of the conventional method.
Figure 4. (A) Microplate assays of acrolein (0, 5, 10, 15 µM) in
serum with p-NH2PhDTPA (final concentration of 10 mM).
Luminescence intensity (ex/em ) 340/615 nm, measured after
dilution with 200 mM sodium phosphate buffer, pH 3.5) was
recorded 30 min after the initiation of the reaction. (B) fluorescence
assay of acrolein in serum with the conventional method. Data are
shown as mean ( SD (n ) 3). ** indicates P < 0.01; * indicates
P < 0.05.
Acknowledgment. This research was supported in part
by a Grant-in Aid for JSPS Fellows (to M.T.), by the
Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan (Grant Nos. 19205021 and 20117003 to Y.U.,
20689001 and 21659024 to K.H., and 21750135 to T.T.),
and by the Industrial Technology Research Grant Program
in 2009 from the New Energy and Industrial Technology
Development Organization (NEDO) of Japan (to T.T.). K.H.
was also supported by the Mochida Memorial Foundation
for Medical and Pharmaceutical Research and Sankyo
Foundation of Life Science.
with low background signals are expected to be highly
advantageous compared with the use of organic fluorescent
compounds.15 Figure 3 clearly shows that p-NH2PhDTPA
worked as a reliable probe for acrolein. As expected, a dose-
dependent increase of luminescence intensity was observed.
The assay also showed good linearity over the concentration
range examined. The results showed that our probe worked
well in aqueous buffer. Therefore, we applied the method
to assays with human serum.
Supporting Information Available: Synthesis and ex-
perimental details. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL1002219
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