Aptamer-based enantioselective competitive binding assay for the trace enantiomer detection

Article abstract of DOI:10.1021/ac070469d

The development of highly enantioselective assays and sensors has received much attention for the determination of enantiomeric impurities at a low level. For chiral compounds, the efficient monitoring of the in selection procedure has allowed the isolation of nucleic acid aptamers which are able to strongly discriminate the target enantiomers. In this paper, we demonstrated for the first time that an aptamer can be successfully used to design a highly enantioselective tool for the trace enantiomer detection. The aptamer-based stereoselective assay was developed using an affinity capillary electrophoresis-based competitive, homogeneous format and an on-capillary mixing approach. Detection of as low as 0.01% of the minor enantiomer in a nonracemic mixture can be achieved, in a short analysis time (<5 min).

Full text of DOI:10.1021/ac070469d

Anal. Chem. 2007, 79, 4716-4719
Aptamer-Based Enantioselective Competitive
Binding Assay for the Trace Enantiomer Detection
Josephine Ruta, Corinne Ravelet, Isabelle Baussanne, Jean-Luc De´ cout, and Eric Peyrin*
De´partement de Pharmacochimie Mole´culaire UMR 5063 CNRS, ICMG FR 2607, Universite´ Joseph Fourier,
UFR de Pharmacie de Grenoble, 5, Avenue de Verdun, 38240 Meylan, France
on highly enantioselective synthetic molecular receptors or
antibodies, has received much attention for the determination of
low enantiomeric impurities.6-8
The development of highly enantioselective assays and
sensors has received much attention for the determination
of enantiomeric impurities at a low level. For chiral
compounds, the efficient monitoring of the in selection
procedure has allowed the isolation of nucleic acid aptam-
ers which are able to strongly discriminate the target
enantiomers. In this paper, we demonstrated for the first
time that an aptamer can be successfully used to design
a highly enantioselective tool for the trace enantiomer
detection. The aptamer-based stereoselective assay was
developed using an affinity capillary electrophoresis-based
competitive, homogeneous format and an on-capillary
mixing approach. Detection of as low as 0.01% of the
minor enantiomer in a nonracemic mixture can be
achieved, in a short analysis time (<5 min).
Nucleic acid aptamers are single-stranded oligonucleotides with
binding properties originating from in vitro selection experiments
(SELEX methodology). They are able to bind the target molecules
with a very high affinity, equal or sometimes even superior to
those of antibodies, and their use as affinity reagents in bioana-
lytical techniques constitutes an interesting alternative to immu-
noassays and immunosensors.9-12 For chiral compounds, the
efficient monitoring of the SELEX methodology has allowed the
isolation of aptamers which are able to strongly discriminate the
target enantiomers. Extreme enantioselectivities have been re-
ported for various compounds including oligopeptides, amino acids
and derivatives, nucleosides, or drugs.13-17
Due to these binding features, aptamers appear to be excellent
potential elements for the development of new highly stereose-
lective assays and sensors, dedicated to the trace enantiomer
detection. In this paper, we describe for the first time an aptamer-
based enantioselective assay which allows the detection of the
minor enantiomer in a nonracemic mixture at a very low level.
The analytical methodology was established using affinity capillary
electrophoresis (ACE) through a competitive binding format and
an on-capillary mixing approach.
As a consequence of the chiral nature of living systems,
metabolic and regulatory processes mediated by biological sys-
tems are sensitive to stereochemistry, and different responses can
be observed when comparing the activities of enantiomers. Drug
enantiomers often differ in their pharmacological and toxicological
activity, resulting in stereoselective clinical responses. In some
cases, even minor enantiomeric impurities can cause severe toxic
side effects.1 In addition, it is now well-established that
D-amino
acids, found at a low level in higher order organisms in the form
of free amino acids, peptides, and proteins, can play a preponder-
EXPERIMENTAL SECTION
Synthesis of the Dansyl-Labeled
D
-Arginine. Briefly, the
ant role in biochemistry and physiology. Notably,
tions as an important neuromodulator, -aspartate is implied in
developmental and endocrine functions, and -arginine plays a
role in the urea cycle.2 The detection of
-amino acids is also of
D-serine func-
synthesis of the labeled
D-amino acid was accomplished in six
D
steps: (i) reaction of dansyl chloride with 2-aminoethanol, (ii)
alkylation of the hydroxyl function introduced with 2,2′-dichloro-
D
D
importance to estimate the chronological age in forensic sciences,
assess food quality, or analyze materials of extraterrestrial
origin.3-5 Therefore, it appears of great interest to design efficient
analytical methodologies which are able to detect traces of one
enantiomer in the presence of a high excess of the other
enantiomer, and the development of assays and sensors, based
(6) Tsourkas, A.; Hofstetter, O.; Hofstetter, H.; Weissleder, R.; Josephson, L.
Angew. Chem., Int. Ed. 2004, 43, 2395.
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(8) Zhao, J.; Davidson, M. G.; Mahon, M. F.; Kociok-Kohn, G.; James, T. D. J.
Am. Chem. Soc. 2004, 126, 16179.
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(10) Savran, C. A.; Knudsen, S. M.; Ellington, A. D.; Manalis, S. R. Anal. Chem.
2004, 76, 3194.
(11) Yoshida, W.; Sode, K.; Ikebukuro, K. Anal. Chem. 2006, 78, 3296.
(12) Tombelli, S.; Minunni, M.; Mascini, M. Biosens. Bioelectron. 2005, 20, 2424.
(13) Klussmann, S.; Nolte, A.; Bald, R.; Erdmann, V. A.; Furste, J. P. Nat.
Biotechnol. 1996, 14, 1112.
* To whom correspondence should be addressed. Phone: 33 476 041033.
Fax: 33 476 041007. E-mail: eric.peyrin@ujf-grenoble.fr.
(1) Kean, W. F.; Lock, C. J. L.; Howard-Lock, H. E. Lancet 1991, 338, 1565.
(2) Fuchs, S. A.; Berger, R.; Klomp, L. W. J.; de Koning, T. J. Mol. Genet. Metab.
2005, 85, 168.
(14) Michaud, M.; Jourdan, E.; Villet, A.; Ravel, A.; Grosset, C.; Peyrin, E. J.
Am. Chem. Soc. 2003, 125, 8672.
(3) Yekkala, R.; Meers, C.; Schepdael, A. V.; Hoogmartens, J.; Lambrichts, I.;
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(16) Shoji, A.; Kuwahara, M.; Ozaki, H.; Sawai, H. J. Am. Chem. Soc. 2007,
129, 1456.
(17) Geiger, A.; Burgstaller, P.; von der Eltz, H.; Roeder, A.; Famulok, M. Nucleic
Acids Res. 1996, 24, 1029.
Willems, G. Forensic Sci. Int. 2006, 159, S89.
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113.
(5) Cronin, J. R.; Pizzarello, S. Science 1997, 275, 951.
4716 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
10.1021/ac070469d CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/19/2007

diethylether for obtaining the chloro derivative in which (iii) the
chlorine atom was substituted for an iodine atom, (iv) reaction of
L-arginine at 2 mM and 20 mM concentrations (less than 5% of
difference).
the iodo derivative obtained with the amino group of a D-arginine
derivative carrying protected guanidine and carboxylic acid
functions (2,2,4,6,7-pentamethylbenzofuran-5-sulfonamide, Pbf, and
allyloxycarbonyl groups, respectively), (v) deprotection of the
carboxylic acid function under basic conditions and (vi) removal
of the Pbf group in TFA (14% yield for the six steps). Details on
the synthesis and characterization of the label can be found in
the Supporting Information.
RESULTS AND DISCUSSION
Design of the CE-Based Competitive Binding Assay. A
truncated 53-mer RNA aptamer, previously identified by a SELEX
procedure directed against L
-arginine,17 was used as a model
enantioselective aptamer. The aptamer-target association displays
high affinity and stereospecificity, characterized by a dissociation
constant Kd of 330 nM, a slow dissociation rate due to the selection
procedure based on a heat denaturation/renaturation step, and
an enantioselectivity of ∼12 000.17 As previously reported,13,18,19
Capillary Electrophoresis Experiments. The
D- AND L-
arginine enantiomers, Tris-HCl, KCl, and MgCl2 were supplied
by Sigma Aldrich (Saint-Quentin, France). Water was obtained
from an Elgastat option water purification system (Odil, Talant,
France) fitted with a reverse osmosis cartridge. Electrophoretic
experiments were carried out on a CE Agilent capillary electro-
phoresis system (Waldbronn, Germany) equipped with a diode
array detector. The running buffer (25 mM Tris-HCl, 25 mM
KCl, 10 mM MgCl2, pH 7.0) was prepared daily and degassed
using an ultrasonic bath. The solutions of single enantiomers or
the L-RNA aptamer, i.e., the mirror image of the “natural” D-RNA
aptamer, was used as a biostable receptor. In accordance with
the principle of chiral inversion, the mirror image of the “natural”
aptamer is expected to recognize with the same affinity and
specificity the mirror image of the target, i.e.,
Moreover, previous partial-filling CE experiments have shown that,
over a low-moderate temperature range, -arginine formed with
this -RNA aptamer a very stable complex, while the -arginine-
aptamer association was negligible.18
D
-arginine.13
D
L
L
nonracemic mixtures (sample) and labeled
prepared daily in the buffer. The truncated 53-mer
D
-arginine were
-RNA
As an alternative to the classical solid-phase techniques, ACE-
based assays represent a powerful technology for the detection
and quantification of analytes. For example, ACE immunoassays
offer several clear advantages over the commonly used immu-
noassays: they consume less sample and reagents, eliminate
washing steps, are compatible with automation and online analysis,
do not require antibody or analyte immobilization on a solid
support, avoid nonspecific binding of antibody or analyte to the
surface, and present a wide analyte applicability.20
L
oligonucleotide (5′-CAUGAUAAACCGAUGCUGGGCGAUUUC-
CUGAAGUAGGGGAAGAGUUGUCAUG-3′) was synthesized and
HPLC-purified by CureVac (Tubingen, Germany). A 380 µM
L
-RNA aptamer stock solution was prepared in the buffer and
stored at -20 °C. All solutions were filtered prior use through
0.20 µm pore size membranes. The working aptamer solutions
were obtained by dilution of the filtered stock solution with the
buffer to the final concentration (100 µM). Prior to the first
utilization, the aptamer was renaturated by heating at 90 °C for
10 min and left to stand at room temperature for 30 min. When
not in use, the working aptamer solutions were stored at 4 °C.
Under these storage conditions, the working solutions were stable
during more than 1 month due to the mirror image strategy.
The coated PVA capillary (Agilent, 50 µm i.d. with extended
light path (total length, 64.5 cm) was conditioned at the beginning
of the day using the following sequence: (i) 10 min of water and
(ii) 15 min of running buffer at 1000 mbar. A “short-end” injection
method was developed (effective length, 8.5 cm; negative polarity,
cathode at the inlet and anode at the outlet, applied voltage, -25
kV; temperature, 12 °C; UV detection at 250 nm). The species
solutions were injected hydrodynamically (-50 mbar) as follows:
20 s aptamer (100 µM) plug (∼24 nL), 32 s sample plug (∼41
nL), 4 s label (400 µM) plug (∼5 nL). Between runs, the capillary
was conditioned with the running buffer for 5 min at 1000 mbar.
The working aptamer solutions were put on ice between each
Taking into account all these attractive background features,
an ACE-based assay was designed using the anti-D-arginine L-RNA
aptamer as a target-specific receptor. A number of recent works
have focused on the use of aptamer-based ACE for the quantifica-
tion of various proteins such as thrombin, IgE, or HIV type 1
reverse transcriptase.21-23 These electrophoretic experiments
involved, in most cases, a direct (noncompetitive) format where
the aptamer was labeled and mixed with the target sample during
a sufficient incubation time to allow equilibrium between the
interacting species. The electrophoretic separation of the mixture
produced typically two distinct peaks corresponding to the free
labeled aptamer and the labeled complex, allowing the quantifica-
tion of the protein. In the present work, as arginine is a low
molecular mass species bearing only one net positive charge while
RNA is much heavier and highly negatively charged, the charge-
to-mass ratio of the analyte-aptamer complex is close to that of
the free aptamer.18 So, a direct ACE assay based on the separation
of the free aptamer from the complex cannot be easily achieved
so that a competitive binding format was developed. Furthermore,
an on-capillary mixing approach was carried out, where the
reaction and the separation occurred simultaneously in the same
capillary by introducing solutions of sample and reagents in the
electrophoretic run in order to maintain the
in its fully folded tertiary structure.18
L-RNA oligonucleotide
The injected solution volume (see above) was determined from
the time required for a given species, introduced in the capillary
under the constant pressure of -50 mbar, to reach the detector
window. The presence of D- or L-arginine enantiomers at different
(19) Brumbt, A.; Ravelet, C.; Grosset, C.; Ravel, A.; Villet, A.; Peyrin, E. Anal.
Chem. 2005, 77, 1993.
(20) Heegaard, N. H. H.; Kennedy, R. T. J. Chromatogr., B 2002, 768, 93.
(21) Pavski, V.; Le, X. C. Anal. Chem. 2001, 73, 6070.
concentrations in the running buffer did not affect significantly
the injected sample volume, even for the samples containing
(18) Ruta, J.; Ravelet, C.; Grosset, C.; Fize, J.; Ravel, A.; Villet, A.; Peyrin, E.
(22) German, I.; Buchanan, D.; Kennedy, R. T. Anal. Chem. 1998, 70, 4540.
(23) Huang, C.; Cao, Z.; Chang, H.; Tan, W. Anal. Chem. 2004, 76, 6973.
Anal. Chem. 2006, 78, 3032.
Analytical Chemistry, Vol. 79, No. 12, June 15, 2007 4717

Figure 1. Principle of the CE-based competitive binding assay using
the on-capillary mixing of the different species. Arrows indicate the
migration direction of the interacting species when the electric field
is applied.
Figure 3. (a) Typical electropherograms showing the free label
peaks obtained for blank (no enantiomer in the sample plug) and 10
µM D-arginine in the sample plug. (b) Standard curves obtained for
D- (square) and L- (diamond) arginine enantiomers over the 0.5-200
µM concentration range. The target curve was fitted by Table curve
2D (SPSS Science Software GmbH, Erkrath, Germany) to the data
plot using a sigmoidal equation. Additional standard curves obtained
for the D-enantiomer in the presence of 2 (circle) and 20 mM (triangle)
L-enantiomer are presented. Inset: assay response at low concentra-
tions of D-arginine (0-10 µM) in the sample plug. Error bars represent
standard deviations (n ) 5).
Figure 2. Structures of (a) dansyl-labeled D-arginine used in the
CE-based competitive binding assay and (b) immobilized arginine,
obtained by derivatization of epoxy activated agarose, used as target
in the SELEX methodology.
was first in contact with the aptamer zone, causing the formation
form of individual zones (plugs).24 The general scheme used is
illustrated in Figure 1.
of the
tion kinetics17,18 allowed separation of free from complexed target.
The unbound -arginine migrated toward the cathode, while the
target-aptamer complex and the free aptamer moved at a close
mobility toward the anodic end. Then, labeled -arginine passed
D-arginine-aptamer complex. The slow complex dissocia-
D
The electrophoretic experiments were conducted using a
polyvinyl alcohol (PVA)-coated capillary (i.d., 50 µm). This coating
shields the silanol groups of the fused silica and eliminates the
electroosmotic flow (EOF). The capillary was first prefilled with
the background electrolyte, and three different plugs were
introduced hydrodynamically using the “short-end” injection
D
through the aptamer zone and bound to the free aptamer binding
sites. As evoked above for the target, the unbound fraction of label
was separated from the label-aptamer complex zone. The free
labeled
detected at the UV detector window (Figure 1).
-Arginine was injected as a sample plug over a 0.5-200 µM
D-arginine band, moving toward the cathodic end, was
method: (i) a plug containing a known amount of the
aptamer, (ii) a sample plug containing an unknown amount of
target ( -arginine), and (iii) a plug containing a known amount
of the labeled target (referred as label).
For use as label, -arginine was tagged with a dansyl group
L-RNA
D
D
concentration range. Figure 3a shows a representative example
of electropherograms obtained for a blank (sample plug without
D
D-arginine) and a sample plug containing 10 µM of
D-arginine.
(Figure 2a, see the Supporting Information). The linker, its
attachment position, and the chemistry used for the labeling were
chosen in order to mimic the structure of the immobilized arginine
(obtained by derivatization of epoxy activated agarose) that was
used as target in the SELEX methodology (see Figure 2b).17 This
allowed avoiding possible alteration of the binding properties of
the labeled target. More, the label remained positively charged
under the running buffer conditions used.
The effect of the -arginine concentration on the assay response
D
is presented in Figure 3b. The curves were constructed by plotting
the relative peak height (ratio of free label peak height for the
sample to the free label peak height for the blank) versus the
solute concentration, as reported previously.25 The label free
fraction was enhanced with the sample target concentration
increasing, as a consequence of the fewer aptamer binding sites
available. The calibration curve presents a sigmoidal shape typical
When the electric field was applied, both the target and the
label moved toward the cathodic end, while the negatively charged
aptamer migrated in the opposite direction (Figure 1). The target
for competitive assays. At low D-arginine concentrations, the assay
response increased linearly as the target concentration varied from
(24) Haes, A. J.; Giordano, B. C.; Collins, G. E. Anal. Chem. 2006, 78, 3758.
4718 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
(25) Yang, W. C.; Yeung, E. S.; Schmerr, M. J. Electrophoresis 2005, 26, 1751.

0 to 10 µM (see the inset in Figure 3b; linear interpolation: y )
0.083x + 1.038 with a regression coefficient R ) 0.98). The
detection limit of D-arginine was calculated to be 1.5 µM through
compares very well with the very sensitive methods based on
other target-specific bioaffinity systems (such as enantioselective
immunosensors and immunoassays).6,29,30
the statistical analysis of the regression function.
Stereoselectivity and Trace Enantiomer Detection. The
CONCLUSION
binding curve for
the same methodology, i.e., with a sample plug containing
-arginine. As can be seen in Figure 3b, no binding of the
nontarget enantiomer to the aptamer was obtained over the 0.5-
200 µM concentration range. Furthermore, higher concentrations
L-arginine was subsequently determined using
In conclusion, we have demonstrated that an aptamer can be
successfully used to design a highly enantioselective tool for the
trace enantiomer detection. To the best of our knowledge, this
work reports the first example of an ACE-based assay dedicated
to the enantioselective analysis. Such assay combines a number
of attractive features including simplicity, flexibility, minute
consumption of material (only some nanoliters, see the Experi-
mental Section), and a homogeneous format. More, the on-
capillary mixing approach does not require any incubation time
and then allows very little time to conduct the assay relative to
that needed by a preincubated method.19-22 An analysis, including
the plug introduction in the capillary and the detection of the free
label peak, is completed in less than 5 min.
L
of L-arginine in the sample plug (2 and 20 mM) were analyzed
and did not cause any significant response. This demonstrated
that the cross-reactivity of the aptameric receptor with the opposite
enantiomer was negligible.
The enantioselective properties of the anti-
aptamer were accounted to analyze enantiomer mixtures of
arginine. The -arginine assay was performed in the presence of
high -arginine concentrations (2 and 20 mM) in the sample plug.
D-arginine L-RNA
D
L
As can be seen in Figure 3b, no significant changes were observed
in the standard curves of the target enantiomer. Moreover, the
The present assay is potentially applicable to a laser-induced
fluorescence detection system for improving sensitivity. In addi-
tion, for a high-throughput chiral analysis, parallel runs on a chip
or capillary array electrophoresis are possible.31,32 As aptamers
are able to recognize virtually any target of interest with high
affinity and specificity,33,34 it can be anticipated that this type of
CE-based enantioselective competitive binding assay could be
adaptable, by modifying some experimental settings, to any chiral
analyte that presents a charge-to-mass ratio different to that of
aptamers.
statistical limits of detection for
to 1.5 and 1.2 µM in presence of 2 and 20 mM of
respectively. So, as little as 2 µM of -arginine in the presence of
20 mM of -arginine can be detected. The assay allows the
D-arginine were found to be equal
L-arginine,
D
L
detection of 0.01% of the minor enantiomer in a nonracemic
mixture, 1 order of magnitude lower than the 0.1-1% detection
limits which are typically attainable with currently available
stereoselective analysis methods using NMR or conventional
separation techniques.26-28 In addition, the present sensing system
SUPPORTING INFORMATION AVAILABLE
Details on the synthesis and characterization of the dansyl-
labeled D-arginine. This material is available free of charge via
(26) Navratilova, H. Magn. Reson. Chem. 2001, 39, 727.
(27) Altria, K. D. J. Chromatogr., A 1999, 856, 443.
(28) Gika, H.; L¨ammerhofer, M.; Papadoyannis, I.; Lindner, W. J. Chromatogr.,
B 2004, 800, 193.
the Internet at http://pubs.acs.org.
(29) Hofstetter, O.; Hofstetter, H.; Wilchek, M.; Schurig, V.; Green, B. S. Nat.
Biotechnol. 1999, 17, 371.
(30) Dutta, P.; Tipple, C. A.; Lavrik, N. V.; Datskos, P. G.; Hofstetter, H.;
Hofstetter, O.; Sepaniak, M. J. Anal. Chem. 2003, 75, 2342.
(31) Paegel, B. M.; Emrich, C. A.; Wedemayer, G. J.; Scherer, J. R.; Mathies, R.
A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 574.
ACKNOWLEDGMENT
We thank the CNRS and the Ministe`re de l’enseignement
supe´rieur et de la recherche for financial support.
(32) Reetz, M. T.; Ku¨hling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Angew.
Chem., Int. Ed. 2000, 39, 3891.
(33) Sazani, P. L.; Larralde, R.; Szostak, J. W. J. Am. Chem. Soc. 2004, 126,
8370.
(34) Mann, D.; Reinemann, C.; Stoltenburg, R.; Strehlitz, B. Biochem. Biophys.
Res. Commun. 2005, 338, 1928.
Received for review March 7, 2007. Accepted
April 17, 2007.
AC070469D
Analytical Chemistry, Vol. 79, No. 12, June 15, 2007 4719