Sensing of bacterial endotoxin in aqueous solution by supramolecular assembly of pyrene derivative

Article abstract of DOI:10.1021/ol1016228

N,N-Dimethyl-N-(pyrenyl-1-methyl) dodecan-1-ammonium has been designed and applied as a fluorescent probe for sensing bacterial endotoxin. Upon assembly with bacterial endotoxin (lipopolysaccharide) in aqueous solution, the probe exhibits a ratiometric and sensitive fluorescence response with a low detection limit (100 nM). Moreover, the probe shows high selectivity for bacterial endotoxin over other related biological species.

Full text of DOI:10.1021/ol1016228

ORGANIC
LETTERS
2010
Vol. 12, No. 18
4014-4017
Sensing of Bacterial Endotoxin in
Aqueous Solution by Supramolecular
Assembly of Pyrene Derivative
Lintao Zeng,† Jiasheng Wu,† Qing Dai,† Weimin Liu,† Pengfei Wang,*,† and
Chun-Sing Lee*,‡
Key Laboratory of Photochemical ConVersion and Optoelectronic Materials,
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences,
Beijing 100190, People’s Republic of China, and Center of Super-Diamond and
AdVanced Film (COSDAF) & Department of Physics and Materials Science,
City UniVersity of Hong Kong, Hong Kong SAR, People’s Republic of China
wangpf@mail.ipc.ac.cn; apcslee@cityu.edu.hk
Received July 14, 2010
ABSTRACT
N,N-Dimethyl-N-(pyrenyl-1-methyl) dodecan-1-ammonium has been designed and applied as a fluorescent probe for sensing bacterial endotoxin.
Upon assembly with bacterial endotoxin (lipopolysaccharide) in aqueous solution, the probe exhibits a ratiometric and sensitive fluorescence
response with a low detection limit (100 nM). Moreover, the probe shows high selectivity for bacterial endotoxin over other related biological
species.
Sepsis and septic shocks cause about 150 000 casualties
annually in the United States.1 These are the results of
massive release of a bacterial endotoxin, lipopolysaccharides
(LPS), from the cell walls of Gram-negative bacteria.2,3 Due
to the high toxicity of LPS, considerable research has been
focused on developing specific LPS detection systems.
Currently, enzymatic limulus amebocyte lysate (LAL) assay
is the most commonly used method for detection and
quantification of LPS in clinical samples. While it is a
sensitive method, it is highly susceptible to changes in
temperature and pH environments.4 In addition, other
carbohydrate derivatives, such as ꢀ-glucans, also react
positively in this assay.4,5 It is thus highly desirable to
develop other simple and more selective assays for detection
of LPS in complex clinical samples.
† Chinese Academy of Sciences.
‡ City University of Hong Kong.
(1) Van Amersfoort, E. S.; Van Berkel, T. J.; Kuiper, J. Clin. Microbiol.
ReV. 2003, 16, 379.
(2) (a) Ulevitch, R. J. AdV. Immunol. 1993, 53, 267. (b) Seltmann, G.;
(4) (a) Voss, S.; Fischer, R.; Jung, G.; Wiesmu¨ller, K. H.; Brock, R.
J. Am. Chem. Soc. 2007, 129, 554. (b) Wei, A. P.; Blumenthal, D. K.;
Herron, J. N. Anal. Chem. 1994, 66, 1500.
Holst, O. The Bacterial Cell Wall; Springer: New York, 2002
(3) (a) Ulevitch, R. J.; Tobias, P. S. Curr. Opin. Immunol. 1994, 6, 125.
(b) Beutler, B.; Nietschel, E. T. Nat. ReV. Immunol. 2003, 3, 169
.
.
(5) Roslansky, P. F.; Novitsky, T. J. J. Clin. Microbiol. 1991, 29, 2477.
10.1021/ol1016228  2010 American Chemical Society
Published on Web 08/20/2010

Fluorescent sensors for biological species are receiving
considerable attention because of their high selectivity,
sensitivity, and simplicity.6 Various synthetic receptors have
been used for detecting biomedical analytes including
adenosine-triphosphate (ATP),6 heparin,7 protein,8 and IP3.9
However, for LPS, there has only been very limited
progress.10 The first synthetic sensor for LPS is a lipid-
functionalized polydiacetylene liposome, which was applied
to discriminate LPS from different species based on a
colorimetric change.10a However, the concentration of LPS
used in that experiment (around 100 µM) is much higher
than the IC50 value (10 µM). Another sensor for LPS is based
on carboxyltetramethylrhodamine/fluorescein-labeled CD 14
peptide,4 in which the sensor is expensive and requires a
complicated synthetic process. Therefore, we tried to develop
a simple, efficient and low-cost sensor that can detect LPS
at trace levels.
Scheme 1
.
Synthetic Pathway of Sensor (DMQA) and Model
Compounds (TEQA)
interaction in aqueous solution.12 We envision that the
negatively charged carboxylate and phosphate groups located
nearby in LPS molecule could catch two DMQA molecules
via electrostatic interaction. Meanwhile, the long alkyl chain
in DMQA could assemble with lipid A by the hydrophobic
interaction. In this process, pyrenyl groups from the two
DMQA molecules would stack onto each other via π-π
interaction. Upon photoexcitation, this molecular ensemble
should thus give an enhanced pyrene excimer photolumi-
nescence and applied as fluorescent probe for LPS detection.
UV-Vis spectrum of DMQA in HEPES solution displays
a characteristic absorption band of pyrene (Figure 1). In the
The primary toxic component of LPS is the lipid A core
(see Abstract Graphic). In most LPS molecules, two 2-keto-
3-deoxyoctonate units (each carrying carboxylic group) are
linked to lipid A.11 The sugar framework combined with 1
and 4′ phosphate moieties on the lipid A make LPS highly
negatively charged. Indeed, LPS has amphiphilic properties
as the glucosamine disaccharide core is modified with various
long-chain fatty acid ester and amides. As a result, these
long fatty chains of LPS would automatically arrange in order
in aqueous solution. When the concentration of LPS reaches
a certain extent, phospholipids bilayers would be formed in
aqueous solution. With the detailed feature of LPS in mind,
we designed a fluorescent molecule based on pyrenyl
quaternary ammonium for LPS sensing. The synthetic
pathway is described in Scheme 1, and experimental details
are given in the Supporting Information. Pyrenyl bromide
reacts with N,N-dimethyldodecylamine to yield a quaternary
ammonium (DMQA) with a long alkyl chain. Such a
modified DMQA can interact with the highly negatively
charged LPS by the electrostatic interaction and hydrophobic
(6) (a) Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Acc. Chem. Res.
2009, 42, 23. (b) Lee, D. H.; Kim, S. Y.; Hong, J.-I. Angew. Chem., Int.
Ed. 2004, 43, 4777. (c) Lee, D. H.; Im, J. H.; Son, S. U.; Chung, Y. K.;
Hong, J.-I. J. Am. Chem. Soc. 2003, 125, 7752. (d) Ojida, A.; Miyahara,
Y.; Wongkongkatep, J.; Tamaru, S.; Sada, K.; Hamachi, I. Chem. Asian J.
2006, 1, 555. (e) Mizukami, S.; Nagano, T.; Urano, Y.; Odani, A.; Kikuchi,
K. J. Am. Chem. Soc. 2002, 124, 3920.
Figure 1. Changes in UV-vis spectra of DMQA (8.0 µM) upon
addition of LPS in 10.0 mM CH3OH/HEPES (v/v ) 1/6, pH )
7.4).
(7) (a) Wright, A. T.; Zhong, Z.; Anslyn, E. V. Angew. Chem., Int. Ed.
2005, 44, 5679. (b) Zhong, Z.; Anslyn, E. V. J. Am. Chem. Soc. 2002, 124,
9014. (c) Wang, S.; Chang, Y.-T. Chem. Comm. 2008, 1173.
(8) (a) Lavigne, J. J.; Anslyn, E. V. Angew. Chem., Int. Ed. 2001, 40,
3119. (b) Chen, N.; Huang, Y.; Yang, L.; Liu, R.; Yang, J. J. Chem.sEur.
J. 2009, 15, 9311. (c) Honda, K.; Fujishima, S.; Ojida, A.; Hamachi, I.
ChemBioChem 2007, 8, 1370. (d) Suzuki, Y.; Yokoyama, K. Angew. Chem.,
Int. Ed. 2007, 46, 4097.
presence of LPS, band broadening and a small red-shift are
observed, which can be attributed to the intermolecular π-π
stacking of two pyrenyl groups in their ground state.13 Figure
2a shows the corresponding fluorescence spectra of DMQA
in the presence of LPS. Free DMQA exhibited a typical
pyrene monomer emission around 370-430 nm. Upon
(9) (a) Aoki, S.; Zulkefeli, M.; Shiro, M.; Kohsako, M.; Takeda, K.;
Kimura, E. J. Am. Chem. Soc. 2005, 127, 9129. (b) Oh, D. J.; Ahn, K. H.
Org. Lett. 2008, 10, 3539. (c) Hirose, K.; Kadowaki, S.; Tanabe, M.;
Takeshima, H.; Iino, M. Science 1999, 284, 1527. (d) Cheley, S.; Gu, L.-
Q.; Bayley, H. Chem. Biol. 2002, 9, 829. (e) Anslyn, E. V.; Shear, J. B. H.
Chem. Biol. 2002, 9, 779.
(12) (a) Peng, L.; Zhang, G.; Zhang, D.; Xiang, J.; Zhao, R.; Wang, Y.;
Zhu, D. Org. Lett. 2009, 11, 4014. (b) Peng, L.; Wang, M.; Zhang, G.;
Zhang, D.; Zhu, D. Org. Lett. 2009, 11, 1973. (c) Zeng, L.; Wang, P.; Zhang,
H.; Zhuang, X.; Dai, Q.; Liu, W. Org. Lett. 2009, 11, 4294.
(13) (a) Kim, H. J.; Kim, S. K.; Lee, J. Y.; Kim, J. S. J. Org. Chem.
2006, 71, 6611. (b) Kim, J. S.; Quang, D. T. Chem. ReV. 2007, 107, 3780.
(c) Jung, H. S.; Park, M.; Han, D. Y.; Kim, E.; Lee, C.; Ham, S.; Kim,
J. S. Org. Lett. 2009, 11, 3378.
(10) (a) Rangin, M.; Basu, A. J. Am. Chem. Soc. 2004, 126, 5038. (b)
Henderson, L. C.; Li, J.; Nation, R. L.; Velkov, T.; Pfeffer, F. M. Chem.
Commun 2010, 46, 3197. (c) Ganesh, V.; Bodewits, K.; Bartholdson, S. J.;
Natale, D.; Campopiano, D. J.; Mareque-Rivas, J. C. Angew. Chem., Int.
Ed. 2009, 48, 356.
(11) Ganesh, V.; Bodewits, K.; Bartholdson, S. J.; Natale, D.; Cam-
popiano, D. J.; Mareque-Rivas, J. C. Angew. Chem., Int. Ed. 2009, 48, 356.
Org. Lett., Vol. 12, No. 18, 2010
4015

Figure 3. Proposed assembly mechanism between DMQA and LPS
in aqueous solution.
carboxylate groups locate on the outer surfaces of the
phospholipids bilayer.14 In this circumstance, DMQA can
easily incorporate into the phospholipids bilayer through
hydrophobic effect and approach the negatively charged
groups via electrostatic interaction. As a result, the neighbor-
ing phosphate and carboxylate groups in a LPS molecule
can catch two DMQA molecules, resulting in excimer
emission. As show in Figure 3, there also exist isolated
carboxylate or phosphate groups along edges of the phos-
pholipids bilayer. When a single DMQA molecule interacts
with such isolated carboxylate or phosphate groups, it cannot
form the stacked pyrene structure, which gives the excimer
emission.
To further demonstrate the supramolecular assembly
mechanism, we measured the fluorescence responses of
DMQA to LPS in different solvents, and the results are
shown in Figure 4. Only in aqueous solution, DMQA
exhibits a distinct fluorescence response to LPS, while in
absolute methanol, it cannot give rise to any excimer
emission even at a higher LPS concentrations (5.0 µM). In
aqueous solution, when the water fraction reaches 82.5%,
addition of LPS can lead to the strongest DMQA excimer
emission, indicating that the supramolecular assembly is the
most efficient at this condition (Figure S8, Supporting
Information).
Figure 2. Changes in fluorescence emission spectra of (a) DMQA
(8.0 µM) and (b) TEQA (8.0 µM) upon addition of LPS in CH3OH/
HEPES (v/v ) 1/6) buffer solution (10 mM, pH 7.4). Excitation
wavelength: 346 nm. (Inset) Variation in fluorescence intensity of
DMQA (I486 upon addition of LPS (0-1.50 µM) in CH3OH/HEPES
solution (v/v ) 1/6, 10 mM, pH ) 7.4).
addition of LPS, a broad and structureless emission band
peaked at about 486 nm builds up at the expense of the
monomer emission peak. This spectral change was ac-
companied with an isoemissive point at 435 nm, indicating
coexistence of the two fluorescent species. This attribution
is further supported by time-resolved fluorescence decay
measurements. The fluorescence lifetime of free DMQA is
22.8 ns monitored at 376 nm. However, in the presence of
LPS, DMQA shows two fluorescence lifetimes (22.8 and
34.9 ns), which demonstrates that the excited molecules have
two decay pathways. Moreover, the fluorescence lifetime
monitored at 486 nm is 39.5 ns, which is also in favor of
the excimer formation in the excited state.
By plotting the fluorescence intensity at 486 nm (I486
)
versus the LPS concentration, a good linear relationship (I486
) 45.60 + 851.91 × [LPS], R ) 0.998) was observed with
the LPS concentration ranging from 0.10 to 1.50 µM (Figure
2a, inset). The detection limit of the present approach was
found to be at least as low as 100 nM. This result is
comparable to those obtained from previous sensor based
on CD 14 peptide.4
To investigate the binding model between DMQA and
LPS, we also synthesized a model compound (TEQA) whose
only difference to DMQA is the lacking of a long alkyl chain
(Scheme 1). As shown in Figure 2b, TEQA shows little
response to the addition of LPS. It is considered that without
the hydrophobic chain, TEQA cannot efficiently form an
assembly with LPS. LPS is an amphiphilic molecule which
would automatically arrange in order in aqueous solution,
as shown in Figure 3. LPS will form monolayer or phos-
pholipids in aqueous solution which its phosphate and
Fluorescence responses of DMQA (8.0 µM) to various
biological species are shown in Figure 5. The fluorescence
intensity of DMQA at 486 nm is unchanged in the presence
of phosphate, pyrophosphate, or triphosphate adenosine
(ATP) even at a higher concentration (5.0 µM), indicating
that the DMQA probe shows a good selectivity for LPS over
other simple anionic phosphates. Moreover, the sensor shows
no response to some coexisting proteins in practical samples,
such as immunoglobulin and glucose. Phosphatidylcholine,
which carries an ammonium end group, an anionic phosphate
group, and a long alkyl chain, cannot induce any significant
fluorescence enhancement at 486 nm. Furthermore, we also
(14) (a) Thomas, C. J.; Gangadhar, B. P.; Surolia, N.; Surolia, A. J. Am.
Chem. Soc. 1998, 120, 12428. (b) Clausell, A.; Garcia-Subirats, M.; Pujol,
M.; Busquets, M. A.; Rabanal, F.; Cajal, Y. J. Phys. Chem. B 2007, 111,
551. (c) Kashida, H.; Sekiguchi, K.; Liang, X.; Asanuma, H. J. Am. Chem.
Soc. 2010, 132, 6223.
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Org. Lett., Vol. 12, No. 18, 2010

Figure 4. Fluorescence responses of DMQA (8.0 µM) to LPS in
aqueous solution and absolute methanol. (Inset) Photographs of
DMQA in the presence of 1.50 µM LPS in CH3OH/HEPES (v/v
) 1/6) solution (10.0 mM, pH ) 7.4).
Figure 5. Selectivity of DMQA (8.0 µM) to some biologically
important species in 10.0 mM CH3OH/HEPES (v/v ) 1/6, pH )
7.4). Pi, PPi, ATP, phosphatidylcholine, BSA, immunoglobulin,
glucose, aspartic acid, glutamic acid, DNA, RNA, malic acid, oxalic
acid, dodecanoic acid, oleic acid, and LPS were added at 1.50 µM
into the sensor solution, respectively. Blank refers to free DMQA
(8.0 µM) solution in 10.0 mM CH3OH/HEPES solution (v/v ) 1/6,
pH ) 7.4).
checked responses of the DMQA probe to some ordinary
fatty acids including dodecanoic acid and oleic acid. Al-
though these fatty acids have long alkyl chain and negatively
charged end group, they cannot induce any DMQA excimer
emission. This can be simply explained by the fact that a
single fatty acid cannot effectively bind with two DMQA
molecules to form the stacked pyrene structure. In addition,
fluorescence titration curves of DMQA to LPS exhibit an
obvious excimer formation even in the presence of other
coexisting species, indicating the formation of the supramo-
lecular assembly and a good selectivity to LPS (Figure S9,
Supporting Information).
In summary, we have successfully developed a new
pyrenyl-derived long-chain quaternary ammonium (DMQA),
which exhibits a fluorescence response to LPS in aqueous
solution. The sensing mechanism is due to the formation of
pyrene excimer by supramolecular assembly of DMQA with
LPS. DMQA can be applied for a high sensitive detection
of LPS in aqueous solution with a low detection limit (100
nM). This work not only provides a simple method to detect
LPS but also opens a new perspective to rationally design
sensors via supramolecular assembly.
Acknowledgment. This work was supported by the
NNSF of China (No.20953002, 60778033), “863” Pro-
gram (No.2009AA03Z318), and the National Basic
Research Program of China (No. 2006CB933000, No.
2007CB936001).
Supporting Information Available: Synthetic procedures,
charicterization of new compounds, and additional spectro-
scopic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL1016228
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