Aptamer-Modified Micellar Electrokinetic Chromatography for the

Anal. Chem. 2009, 81, 1169–1176

Aptamer-Modified Micellar Electrokinetic Chromatography for the Enantioseparation of Nucleotides Josephine Ruta,† Sandrine Perrier,† Corinne Ravelet,† Beátrice Roy,‡ Christian Perigaud,‡ and Eric Peyrin*,† De´partement de Pharmacochimie Molećulaire UMR 5063, Institut de Chimie Molećulaire de Grenoble FR 2607, CNRS-Universite´ Grenoble I (Joseph Fourier), 38041 Grenoble Cedex 9, France, and Institut des Biomolećules Max Mousseron UMR 5247, CNRS, Universite´s Montpellier 1 et 2, case courrier 1705, Universite´ Montpellier 2, Place Euge`ne Bataillon, 34095 Montpellier cedex 5, France In this paper, a new aptamer-based capillary electrophoresis (CE) method, which was able to separate the enantiomers of an anionic target (adenosine monophosphate, AMP) displaying the same electrophoretic mobility as that of the oligonucleotidic chiral selector, is reported. The design of the aptamer-modified micellar electrokinetic chromatography (MEKC) mode consisted of nonionic micelles which acted as a pseudostationary phase and a hydrophobic cholesteryl group-tagged aptamer (Chol-Apt) which partitioned into the uncharged micellar phase. Under partial-filling format and suppressed electroosmotic flow conditions, the strong mobility alteration of Chol-Apt permitted AMP enantiomers to pass through the micelle-anchored aptamer zone and promoted the target enantioseparation. The influence of several electrophoretic parameters (such as concentration and nature of the nonionic surfactant, preincubation of the Chol-Apt and surfactant, capillary temperature, and applied voltage) on the AMP enantiomer migration was investigated in order to define the utilization conditions of the aptamermodified MEKC mode. The chiral resolution, in a single run, of three adenine nucleotides, i.e., AMP, ADP (adenosine diphosphate), and ATP (adenosine triphosphate), was further accomplished using such methodology. This approach demonstrates the possibility to extend the CE applicability of aptamer chiral selectors to potentially any target, without restriction on its charge-to-mass ratio. Because of the numerous attractive analytical features of capillary electrophoresis (CE) and the remarkable molecular recognition properties of the nucleic acid aptamers, aptamer-based CE has become a powerful tool during the last years.1 Several examples of applications have been reported for the evaluation of the protein-nucleic acid interactions,2-4 the design of sensitive * To whom correspondence should be addressed. E-mail: eric.peyrin@ ujf-grenoble.fr. † Universite´ Grenoble. ‡ Universite´ Montpellier. (1) Ravelet, C.; Grosset, C.; Peyrin, E. J. Chromatogr., A 2006, 1117, 1. (2) Huang, C. C.; Cao, Z.; Chang, H. T.; Tan, W. Anal. Chem. 2004, 76, 6973. (3) Berezovski, M.; Krylov, S. N. Anal. Chem. 2005, 77, 1526. 10.1021/ac802443j CCC: $40.75  2009 American Chemical Society Published on Web 01/07/2009

protein5-7 and small molecule8 assays, and the development of enantiomeric separation systems.9-11 In CE chiral analysis, the difference between the electrophoretic mobility of the free enantiomers (µf) and their complexed forms (µc) constitutes the prerequisite for the analyte enantioseparation.12 Assuming equal mobilities of the diastereomeric complexes, the observed mobility difference between the enantiomers (∆µ) is proportional to the (µf - µc) difference. In the particular case of a CE assay using an aptamer chiral additive, the electrophoretic mobility of the enantiomer-aptamer complex is assumed to be very close to that of the free aptamer (µApt) since an aptamer molecule is heavy and highly negatively charged.9 Therefore, the following can be assumed: ∆µ ∝ (µf - µApt). This implies that the aptamer chiral selectors are particularly well-suited for the CE enantioseparation of cationic species (or neutral analytes using the driving force of the electroosmotic flow).12 In this context, it has been demonstrated that enantioselective aptamers are valuable CE chiral additives for both the separation enantiomers of the arginine target9 and the design of competitive enantioselective CE assays.10,11 In addition, the significant electrophoretic mobility difference between free enantiomers and the aptamer chiral selector allowed performing easily the partial-filling CE mode in order to eliminate the UV detection interferences due to the oligonucleotides.9-11 On the other hand, a major limitation in the use of aptamers as CE chiral selectors is expected for the analysis of anionic targets, especially for monocharged species. In such case, the enantioseparation would be difficult to achieve since the (µf - µApt) difference is assumed to be small. Furthermore, the elimination of the detection interferences due to the aptamer (4) Gong, M.; Wehmeyer, K. R.; Limbach, P. A.; Heineman, W. R. Electrophoresis 2007, 28, 837. (5) German, I.; Buchanan, D. D.; Kennedy, R. T. Anal. Chem. 1998, 70, 4540. (6) Haes, A. J.; Giordano, B. C.; Collins, G. E. Anal. Chem. 2006, 78, 3758. (7) Zhang, H.; Li, X. F.; Le, X. C. J. Am. Chem. Soc. 2008, 130, 34. (8) Li, T.; Li, B.; Dong, S. Chem. Eur. J. 2007, 13, 6718. (9) Ruta, J.; Ravelet, C.; Grosset, C.; Fize, J.; Ravel, A.; Villet, A.; Peyrin, E. Anal. Chem. 2006, 78, 3032. (10) Ruta, J.; Ravelet, C.; Baussanne, I.; Dećout, J. L.; Peyrin, E. Anal. Chem. 2007, 79, 4716. (11) Ruta, J.; Ravelet, C.; Baussanne, I.; Fize, J.; Dećout, J. L.; Peyrin, E. J. Sep. Sci. 2008, 31, 2239. (12) Vespalec, R.; Bocek, P. Chem. Rev. 2000, 100, 375.

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plug is expected to be virtually impossible in a partial-filling mode. The objective of this work was to establish the broad applicability of aptamers as CE chiral selectors by evaluating the feasibility to extend their use to the separation of anionic solute enantiomers. The strategy employed was based on the specific modulation of the electrophoretic mobility of the aptamer chiral selector. It is well-known that the electrophoretic mobility of nucleic acids can be strongly reduced through the covalent linkage of a large and uncharged polymer, which is also called drag-tag.13 Such approach has been successfully employed to perform endlabeled free-solution electrophoresis for the separation of singlestranded DNA fragments.14,15 More recently, it has been also demonstrated that the transient association of peptide nucleic acids (PNA) amphiphile-modified DNA fragments to nonionic micelles was able to shift efficiently their electrophoretic mobility.16 Therefore, it was hypothesized that the design of an aptamermodified micellar electrokinetic chromatography (MEKC) mode where nonionic micelles act as a pseudostationary phase and a hydrophobic group-tagged aptamer partitions into the uncharged micellar phase could alter significantly the mobility of the aptamer zone and thus promote the enantioseparation of anionic species. Several SELEX (systematic evolution of ligands by exponential enrichment) experiments have reported the isolation of aptamer sequences directed against small anionic chiral compounds of interest.17-22 As a studied model system, it was chosen to test the ability of the aptamer-modified MEKC mode to separate the enantiomers of the adenosine monophosphate (AMP). The used DNA aptamer, previously employed to separate the adenosine enantiomers,23,24 is also able to bind the adenine nucleotides with a significant micromolar affinity.25 Such system constituted a wellsuited model since the mobility of the AMP nucleotide was identical to that of the 37-base DNA aptamer under the electrophoretic conditions of the study (see below). In addition, analogues of nucleotides and nucleosides form an interesting group of drugs for combating certain viral diseases.26,27 It has been also shown that, in some cases, L-nucleotides possess better antiviral properties than their corresponding D-isomers.28 Furthermore, important papers have focused on the usefulness of short L-nucleic acid (13) McCormick, L. C.; Slater, G. W. Electrophoresis 2007, 28, 674. (14) Won, J. I.; Meagher, R. J.; Barron, A. E. Electrophoresis 2005, 26, 2138. (15) Meagher, R. J.; Won, J. I.; Coyne, J. A.; Lin, J.; Barron, A. E. Anal. Chem. 2008, 80, 2842. (16) Grosser, S. T.; Savard, J. M.; Schneider, J. W. Anal. Chem. 2007, 79, 9513. (17) Huizenga, D. E.; Szostak, J. W. Biochemistry 1995, 34, 656. (18) Burke, D. H.; Hoffman, D. C. Biochemistry 1998, 37, 4653. (19) Kato, T.; Yano, K.; Ikebukuro, K.; Karube, I. Nucleic Acids Res. 2000, 28, 1963. (20) Sazani, P. L.; Larralde, R.; Szostak, J. W. J. Am. Chem. Soc. 2004, 126, 8370. (21) Anderson, P. C.; Mecozzi, S. Nucleic Acids Res. 2005, 33, 6992. (22) Carothers, J.; Oestreich, S. C.; Szostak, J. W. J. Am. Chem. Soc. 2006, 128, 7929. (23) Michaud, M.; Jourdan, E.; Ravelet, C.; Villet, A.; Ravel, A.; Grosset, C.; Peyrin, E. Anal. Chem. 2004, 76, 1015. (24) Ruta, J.; Ravelet, C.; De´sire´, J.; Dećout, J. L.; Peyrin, E. Anal. Bioanal. Chem. 2008, 390, 1051. (25) Deng, Q.; German, I.; Buchanan, D.; Kennedy, R. T. Anal. Chem. 2001, 73, 5415. (26) Zemlicka, J. Pharmacol. Ther. 2000, 85, 251. (27) Lyer, R. P.; Padmanabhan, S.; Zfang, G.; Morrey, J. D.; Korba, B. E. Curr. Opin. Pharmacol. 2005, 5, 520. (28) Gondeau, C.; Chaloin, L.; Varga, A.; Roy, B.; Lallemand, P.; Pe´rigaud, C.; Barman, T.; Vas, M.; Lionne, C. Biochemistry 2008, 47, 3462.

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ligands for the development and identification of new potential drugs.29,30 Therefore, several papers have been recently reported for the chiral separation of nucleotide and nucleoside derivatives.31-34 The developed aptamer-modified MEKC mode used nonionic micelles as pseudostationary phase and a cholesteryl-tagged DNA aptamer as chiral selector (Chol-Apt). The cholesteryl moiety was selected as hydrophobic tag of the aptamer because it promotes efficiently the interaction of DNA strands with hydrophobic surfaces or liposomes, without alteration of their hybridization properties.35,36 In addition, cholesteryl-modified oligonucleotides can be easily synthesized and are commercially available. The effects of the different electrophoretic operating conditions (concentration and nature of the nonionic surfactant, preincubation of Chol-Apt and surfactant, capillary temperature, and applied voltage) on the migration behavior of the AMP enantiomers in the aptamer-modified MEKC mode were evaluated. Such new methodology was also extended to the enantioseparation of other adenine nucleotides, i.e., adenosine diphosphate (ADP) and adenosine triphosphate (ATP). EXPERIMENTAL METHODS Chemicals. Natural β-D-adenosine 5′-monophosphate (DAMP), β-D-adenosine 5′-diphosphate (D-ADP), and β-D-adenosine 5′-triphosphate (D-ATP) nucleotides were obtained from SigmaAldrich (Saint-Quentin, France). The mirror image of these nucleotides, i.e., β-L-adenosine 5′-monophosphate (L-AMP), β-L-adenosine 5′-diphosphate (L-ADP), and β-L-adenosine 5′-triphosphate (L-ATP), were synthesized, purified, and characterized as previously described by He et al.37 Poly(oxyethylene(20)) sorbitan monolaurate (Tween 20) was supplied by Seppic (Paris, France). Poly(oxyethylene(23)) lauryl ether (Brij 35), and poly(oxyethylene(9)) dodecyl ether (Thesit) were purchased from SigmaAldrich. KCl and MgCl2 were supplied by Prolabo (Paris, France) and Panreac Quimica (Barcelona, Spain), respectively. Water was obtained from a Purite Still Plus water purification system (Thame, U.K.) fitted with a reverse osmosis cartridge. The cholesteryl-tetraethylene glycol-5′-modified DNA aptamer (Chol-Apt; used sequence: 5′-ATTATACCTGGGGGAGTATTGCGGAGGAAGGTATAAT) was synthesized and HPLC-purified by Eurogentec (Angers, France). The identity of the modified oligonucleotide was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Capillary Electrophoresis Experiments. An Agilent capillary electrophoresis system (Agilent Technologies, Waldbronn, Ger(29) Denekas, T.; Tro ¨ltzsch, M.; Vater, A.; Klussmann, S.; Messlinger, K. Br. J. Pharmacol. 2006, 148, 536. (30) Williams, K. P.; Liu, X. H.; Schumacher, T. N. M.; Lin, H. Y.; Ausiello, D. A.; Kim, P. S.; Bartel, D. P. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11285. (31) Bolyan, D.; Ganzler, K. Biomed. Chromatogr. 1998, 12, 179. (32) Lipka, E.; Daniel, C.; Vaccher, M. P.; Glaçon, V.; Ewing, D.; Mackenziev, G.; Len, C.; Bonte, J. P.; Vaccher, C. Electrophoresis 2004, 25, 444. (33) Cass, Q. B.; Watanabe, C. S.; Rabi, J. A.; Bottari, P. Q.; Costa, M. R.; Nascimento, R. M.; Cruz, J. E.; Ronald, R. C. J. Pharm. Biomed. Anal. 2003, 33, 581. (34) Magora, A.; Abu-Lafi, S.; Levin, S. J Chromatogr., A 2000, 866, 183. (35) Erkan, Y.; Czolkos, I.; Jesorka, A.; Wilhelmsson, L. M.; Orwar, O. Langmuir 2007, 23, 5259. (36) Banchelli, M.; Betti, F.; Berti, D.; Caminati, G.; Bombelli, F. B.; Brown, T.; Wilhelmsson, L. M.; Nordeń, B.; Baglioni, P. J. Phys. Chem. B 2008, 112, 10942. (37) He, J.; Roy, B.; Pe´rigaud, C.; Kashlan, O. B.; Cooperman, B. S. FEBS J. 2005, 272, 1236.

many) equipped with a diode array detector (DAD) and an Agilent Chemstation software was used throughout. A 50 µm inner diameter (i.d.) and 363 µm outer diameter (o.d.) poly(vinyl alcohol) (PVA)-coated capillary with extended light path (total and effective lengths of 64.5 and 56 cm, respectively) was employed (Agilent Technologies). The running buffer (20 mM phosphate buffer, 5 mM KCl, 5 mM MgCl2, pH 7.0) was prepared and daily degassed using an ultrasonic bath. The experiments were performed in the presence of KCl and MgCl2 in order to operate under conditions similar to those used for the in vitro selection of the DNA aptamer.17 Furthermore, a recent report has established the preponderant role of the Mg2+ cations on the aptamer-based adenosine enantioseparation.24 The solutions of analyte racemates (330 µM of each enantiomer, unless otherwise stated) and surfactants were prepared in the running buffer. All solutions were filtered prior to use through 0.20 µm pore size membranes. The Chol-Apt stock solution was prepared in water and stored at -20 °C. The working Chol-Apt solution was obtained by dilution of the filtered stock solution to a final concentration of 200 µM in the running buffer. Prior to the first utilization, the working Chol-Apt solution was renaturated by heating at 80 °C for 5 min and left to stand at room temperature for 30 min. The PVA-coated capillary was conditioned at the beginning of the day and rinsed between runs first with water then with the running buffer in both cases for 5 min at 8 bar. All experiments were carried out in anionic mode (anode at the outlet and cathode at the inlet). The CE conditions were as follows: detection wavelength, 260 nm; applied voltage from -15 to -30 kV; capillary cassette temperature from 10 to 40 °C. In the partial-filling aptamer-modified MEKC mode, the capillary was first filled with the surfactant solution (1000 mbar for 2.5 min). The hydrodynamic injection of the 200 µM Chol-Apt plug was then performed at 1000 mbar for 0.05 min, unless otherwise stated. It is well-known that the volume effectively introduced into the capillary is dependent on the experimental conditions including the capillary dimensions, the solution viscosity, and the capillary temperature. A solution of running buffer (containing AMP as UV marker) was pumped into the surfactant-prefilled capillary at 1000 mbar until the species reached the detection window. The plug length was estimated from the elution time of the front of the zone. For example, the hydrodynamic injection at 1000 mbar for 0.05 min corresponded to an introduced volume of ∼70 nL for a 2.5 mM Tween 20 concentration and ∼65 nL for a 25 mM concentration (at a capillary temperature of 30 °C). Injection of the analyte samples was performed hydrodynamically at 50 mbar for 3-6 s in triplicate. During the run, the capillary ends were kept in the running buffer. The observed mobility (µ) of the injected species was calculated using the equation µ ) lL/tV, where l is the effective length and L the total length of the capillary, t is the species migration time, and V is the applied voltage. In the MEKC mode, the viscosity correction factor was estimated to account for the analyte mobility changes due to the surfactant solution viscosity and the determination of the viscosity-corrected mobility. This was attained using the hydrodynamic method where the AMP-enriched running buffer or the surfactant-containing running buffer was forced to pass through the capillary under a 1000 mbar

pressure. The time required to push the solutions past the detector was measured. The viscosity correction factor was then determined from the ratio of the measured times at a given surfactant concentration and at zero surfactant present in the buffer. The selectivity factor (R) was calculated according to R ) tD/tL. The efficiency (N) was estimated as follows: N ) 5.54(t/w50)2 where w50 is the peak width at half-height. The resolution (Rs) was determined using the following expression: Rs ) [1.18(tD tL)]/(w50(D) + w50(L)). Under aptamer-modified MEKC conditions (capillary temperature of 40 °C, applied voltage of -20 kV), the intra- and interday repeatability of the AMP enantiomer mobility was studied (n ) 6). A good repeatability was obtained since the relative standard deviation (RSD) of the enantiomer mobility was less than 2%. The RSD of the selectivity was inferior to 1%. RESULTS AND DISCUSSION Principle of the Aptamer-Modified MEKC Mode. The aptamer-modified MEKC methodology developed in this work focuses on the specific shift of the aptamer mobility in the presence of a nonionic micellar phase. The pseudostationary phase is assumed to slow down the Chol-Apt chiral selector through the strong interaction, mediated by the hydrophobic effect, between its lipophilic cholesteryl tag and the micelle core. The general principle is illustrated in Figure 1. A PVA-coated capillary is prefilled with the running buffer containing the nonionic surfactant. With the use of a partial-filling procedure, the Chol-Apt plug is then introduced into the capillary before the racemate sample injection. When the electric field is applied, both the aptamer and the AMP enantiomers are expected to migrate toward the anodic end while the nonionic pseudostationary phase is assumed to remain quasi-immobile due to the suppression of the electroosmotic flow through the use of a PVA-coated capillary. Thus, the mobility of Chol-Apt is expected to be strongly reduced in direct relation to its partitioning into the micellar pseudophase, allowing the AMP enantiomers to pass through the micelleanchored aptamer zone and interact stereospecifically with the chiral selector. AMP Enantioseparation by Aptamer-Modified MEKC. The migration behavior of both AMP and Chol-Apt was first investigated in surfactant-free solution at a capillary temperature of 30 °C and an applied voltage of -25 kV (Figure 2, parts a and b). The migration of Chol-Apt resulted in the occurrence of two partially separated electrophoretic peaks, corresponding very likely to the folded and unfolded forms of the oligonucleotide. A similar CE behavior has been previously reported for the G-quartet forming antithrombin DNA aptamer.2,38 The observed mobility of the unfolded form of Chol-Apt and AMP were identical (-2.38 ± 0.01 × 10-4 vs -2.39 ± 0.04 × 10-4 cm2 V-1 s-1), whereas the mobility of the folded aptamer was higher due to its compacted three-dimensional (3D) structure (-2.74 ± 0.01 × 10-4 cm2 V-1 s-1). Obviously, the introduction of a 200 µM Chol-Apt plug in a partial-filling CE format resulted in a significant detection interference of the oligonucleotide zone for the nonresolved AMP racemate. In order to evaluate the potential interactions between the nonionic pseudostationary phase and the AMP enantiomers, the (38) Szilagyi, A.; Bonn, G. K.; Guttman, A. J. Chromatogr., A 2007, 1161, 15.


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Figure 1. Principle of the aptamer-modified MEKC mode. (a) A PVA-coated capillary is prefilled with three different plugs, (i) a plug containing a quasi-immobile nonionic micellar phase, (ii) a plug containing the cholesteryl-tagged aptamer, and (iii) a plug containing the AMP sample. (b) When the electric field is applied, the mobility of Chol-Apt is strongly reduced in direct relation to its partitioning into the micellar pseudophase so that the AMP enantiomers pass through the micelle-anchored aptamer zone. (c) The stereoselective interaction between AMP and the micelle-interacting aptamer zone led to the chiral separation. Arrows indicate the migration direction of the interacting species when the electric field is applied, and their respective lengths represent the expected velocity magnitude.

nucleotide racemate was also injected using the MEKC mode. A 25 mM concentration of Tween 20, one of the more common nonionic surfactant used in MEKC39 (critical micelle concentration (cmc) and aggregation number are ∼50 µM and ∼30, respectively),40 was employed to form the pseudostationary phase. In such case, the chiral selector plug was not introduced into the capillary. In the conditions of capillary temperature and voltage described above, the AMP racemate, which was not resolved, migrated with a slightly shifted velocity (Figure 2c) mainly due to the increasing viscosity effects (viscosity-corrected mobility: -2.27 ± 0.02 × 10-4 cm2 V-1 s-1). This indicates that the nonionic Tween 20 micelles were roughly inert toward the studied analyte. Subsequent experiments were carried out using the aptamermodified MEKC mode (capillary temperature of 30 °C, applied voltage of -25 kV). The capillary was prefilled with a Tween 20 surfactant solution of various concentrations (from 2.5 to 50 mM) followed by the introduction of a 200 µM Chol-Apt plug. The resulting electropherograms are reported in Figure 2d-h. The presence of a 2.5 mM Tween 20 solution in the capillary was sufficient to provide the chiral discrimination of AMP: the L-enantiomer peak was clearly visualized, whereas the D-enantiomer, comigrating with the aptamer, cannot be detected (Figure (39) Silva, M. Electrophoresis 2007, 28, 174. (40) Velev, O. D.; Gurkov, T. D.; Ivanov, I. B.; Borwankar, R. P. Phys. Rev. Lett. 1995, 75, 264.

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2d). The peak observed at a migration time of about 9 min corresponded likely to a small fraction of Chol-Apt which did not bind to the pseudostationary phase.16 As expected from the selection procedure,17 the “natural” D-enantiomer interacted more strongly with the chiral selector than the L-enantiomer, explaining the enantiomer migration behavior. Such data demonstrate that (i) the micellar phase affected strongly and specifically the migration behavior of Chol-Apt (viscosity-corrected mobility: -1.11 ± 0.04 × 10-4 and -0.75 ± 0.03 × 10-4 cm2 V-1 s-1 for the two aptamer forms), allowing the interaction of AMP with the chiral selector zone, (ii) the transient anchoring of Chol-Apt into the nonionic micelles did not impair the D-AMP binding ability of the aptamer,17 and (iii) the DNA aptamer displayed enantioselective properties not only for adenosine23 but also for the AMP nucleotide. As the surfactant concentration increased, the separation window, delimitated by the unbound aptamer fraction peak and the micelle-interacting aptamer zone, was greatly enlarged due to the greater association of Chol-Apt with the pseudostationary phase (Figure 2e-h). The increase in the medium viscosity with increasing surfactant concentration was assumed to also affect the separation window width through the reduction of the Chol-Apt plug length effectively introduced in the capillary (see the Experimental Methods). Nevertheless, the AMP racemate was baseline-resolved, without detection interferences, over the entire 5-50 mM surfactant concentration

Figure 2. Electropherograms for the analysis of the AMP racemate (a and c-h) and Chol-Apt (b) under free-surfactant CE, MEKC, or aptamermodified MEKC conditions. Running buffer: 20 mM phosphate buffer, 5 mM KCl, 5 mM MgCl2, pH 7.0. Capillary: 50 µm i.d. PVA-coated silica capillary (total and effective lengths of 64.5 and 56 cm, respectively). Applied voltage: -25 kV. Capillary temperature: 30 °C. Detection wavelength: 260 nm. (a and b) Free-surfactant CE: sample injection, 50 mbar for 6 s (AMP) (a) or 3 s (Chol-Apt) (b). (c) MEKC: sample injection, 50 mbar for 6 s (AMP). Concentration of the Tween 20 surfactant solution in the capillary: 25 mM. (d-h) Aptamer-modified MEKC: sample injection, 50 mbar for 6 s (AMP). Concentration of the Tween 20 surfactant solution in the capillary: 2.5 (d), 5 (e), 25 (f), 35 (g), and 50 mM (h). Separation plug length: 200 µM Chol-Apt solution applied at 1000 mbar for 0.05 min. The peak marked by an asterisk corresponds to the unbound fraction of Chol-Apt, and the arrow indicates the beginning of the micelle-interacting aptamer zone.

range (Figure 2e-h). The migration time of L-AMP in the aptamermodified MEKC was close to that obtained under MEKC conditions using the same Tween 20 concentration (compare Figure 2, parts c and f). These data indicate that the nontarget enantiomer interacted weakly with the aptamer. In contrast, it has been previously shown by liquid chromatography that L-adenosine displayed a significant affinity toward the same oligonucleotidic chiral selector.23,24 Such difference could be explained by the presence of an electrostatic repulsion mechanism between the phosphate moiety of L-AMP and the aptamer phosphate groups

at or near the binding site. As shown in Table 1, a slight decrease in the selectivity was observed between 5 and 25 mM, whereas the R value did not change over the 25-50 mM range of Tween 20 concentrations. In the partial-filling technique, the separation process occurs only inside the effective chiral selector plug.9,41 For a low surfactant concentration, the residence time of AMP in the micelle-anchored aptamer zone was important (see the front of the active chiral selector zone in Figure 2e), improving enantioselective interactions between the analytes and the chiral (41) Amini, A.; Pettersson, C.; Westerlund, D. Electrophoresis 1997, 18, 950.


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Table 1. Selectivity Factor, Efficiency, and Resolution for AMP Enantiomers under Different Conditions of Aptamer-Modified MEKCa

surfactant concnc 2.5 mM 5 mM 25 mM 35 mM 50 mM applied voltagee -15 kV -20 kV -25 kV -30 kV capillary temperaturef 10 °C 20 °C 30 °C 40 °C surfactant typeg Tween 20 Brij 35 Thesit

selectivity factorb

NLb

nad 1.12 1.09 1.09 1.09

27 000 29 000 112 000 44 000 50 000

na 2900 4400 3000 2400

na 2.24 2.41 1.80 1.73

1.14 1.13 1.09 1.08

81 000 69 000 112 000 123 000

2400 2800 4400 7000

2.60 2.48 2.41 2.43

na 1.22 1.09 1.04

69 000 113 000 112 000 63 000

na 1100 4400 21 000

na 2.77 2.41 1.66

1.13 1.11 1.13

69 000 60 000 49 000

2800 2200 2700

2.48 1.98 2.47

NDb

resolutionb

a Running buffer: 20 mM phosphate buffer, 5 mM KCl, 5 mM MgCl2, pH 7.0. PVA-coated silica capillary. Detection wavelength: 260 nm. b RSD of the selectivity, efficiency, and resolution was typically less than 1%, 15%, and 5%, respectively. c Surfactant type, Tween 20; applied voltage, -25 kV; capillary temperature, 30 °C. d na: not available; D-AMP not detected. e Surfactant type, Tween 20; surfactant concentration, 25 mM; capillary temperature, 30 °C. f Surfactant type, Tween 20; surfactant concentration, 25 mM; applied voltage, -25 kV. g Surfactant concentration, 25 mM; applied voltage, -20 kV; capillary temperature, 30 °C.

selector. On the other hand, as the surfactant concentration increased, the aptamer mobility was diminished with favorable consequences on the enantioseparation (see the introduction section). Thus, these opposing effects could almost counterbalance each other, explaining the weak selectivity variation over the surfactant concentration range. Such behavior is of interest as it allowed modulating the separation window width without significant alteration of the selectivity factor. In terms of efficiency, the theoretical plate number values for the L-enantiomer appeared to be largely higher (by a factor 10-20) than those retrieved for the D-enantiomer for all the surfactant concentrations (Table 1). Such weak efficiency for D-AMP was probably dependent on the slow mass transfer kinetics determined by the strong interaction between the aptamer and the target enantiomer. These selectivity and efficiency data were associated with a significant diminution of the resolution for the higher 35-50 mM Tween 20 concentrations. As an alternative to the introduction of a “pure” Chol-Apt zone, a preincubated mixture of Chol-Apt (200 µM) and Tween 20 (25 mM) was also tested as a separation plug in the aptamer-modified MEKC method (capillary temperature of 30 °C, applied voltage of -25 kV). The preincubation allowed the full partitioning of the Chol-Apt to the micelles prior to the beginning of the run and was then responsible for the increase in the effective separation plug length. As a consequence, the chiral discrimination process was improved (data not shown). However, the “pure” on-capillary mixing mode where the binding of Chol-Apt to the pseudostationary phase occurred solely during its migration in the 1174


capillary (Figure 1) appeared to be much more flexible and simpler from a practical point of view. Therefore, this latter mode was adopted for further investigations. The influence of the applied voltage variation (from -15 to -30 kV) on the separation of the AMP enantiomers was investigated for a 25 mM Tween 20 concentration and a 30 °C capillary temperature. Figure 3a and Table 1 show the resulting electropherograms and the electrophoretic parameters, respectively. The voltage decrease was responsible for a weak improvement of selectivity. A low electric field led to a velocity reduction for the different species interacting in the capillary. This favored the reaction time for both the association of Chol-Apt with the micellar phase and the stereoselective binding of AMP to the chiral selector. The resolution was found to be weakly affected over the voltage range due to the concomitant decrease in the efficiency performances when the voltage diminished (Table 1). The effects of the capillary temperature variation (from 10 to 40 °C) on the AMP nucleotide enantioseparation were also evaluated (applied voltage, -25 kV; Tween 20 concentration, 25 mM). The obtained electropherograms and the electrophoretic data are gathered in Figure 3b and Table 1, respectively. The selectivity was greatly enhanced when the temperature decreased. Moreover, the D-enantiomer was not detected at 10 °C due to a too long migration time. Such behavior was probably dependent, in part, on enthalpically driven interactions between D-AMP and Chol-Apt. This was consistent with previous data reported with this DNA aptamer.23 The temperature increase could also alter the transient attachment of Chol-Apt with the micellar phase as well as modify the structural properties of the micelles and the contact time between AMP enantiomers and the effective chiral separation zone. The D-AMP efficiency was significantly improved with the temperature increasing, due likely to the faster mass transfer kinetics between the target and the aptamer. The temperature increase resulted in a significant reduction of the resolution due to the decrease in the selectivity factor (Table 1). Finally, the role of the nature of the surfactant on the AMP chiral separation was evaluated. Two other nonionic micelleforming poly(oxyethylene) surfactants (Thesit and Brij 35) were tested as an alternative to the Tween 20 micellar phase. They differ principally in the type and size of the hydrophilic headgroup. This corresponds to 9 and 23 ethylene oxide residues for Thesit and Brij 35, respectively, versus the 20 ethylene oxide residue linked to a sorbitan moiety for the Tween 20 surfactant. The cmc and aggregation number values are ∼100 µM and ∼100, respectively, for Thesit and ∼40-100 µM and ∼40, respectively, for Brij 35.42-44 Figure 3c and Table 1 show the data obtained for a surfactant concentration of 25 mM, under 30 °C capillary temperature and -20 kV voltage conditions. The selectivity factor varied weakly with the type of the surfactant. This indicates that the nature of the used surfactant did not impact profoundly on the enantioseparation. Similar efficiency and resolution were found for the Tween 20 and Thesit pseudostationary phases; the use of the Brij 35 micellar phase resulted in a weaker resolution. The major difference was related to the width of the separation window. The Tween 20 and Thesit pseudostationary phases provided equivalent (42) Hink, M. A.; van Hoek, A.; Visser, A. J. W. G. Langmuir 1999, 15, 992. (43) Kelepouris, L.; Blanchard, G. J. J. Phys. Chem. B 2002, 106, 6600. (44) Toth, G.; Madarasz, A. Langmuir 2006, 22, 590.

Figure 3. Effects of applied voltage (a), capillary temperature (b), and surfactant nature (c) on the AMP enantioseparation under aptamermodified MEKC conditions. Running buffer: 20 mM phosphate buffer, 5 mM KCl, 5 mM MgCl2, pH 7.0. Capillary: 50 µm i.d. PVA-coated silica capillary (total and effective lengths of 64.5 and 56 cm, respectively). Surfactant concentration: 25 mM. Separation plug length: 200 µM Chol-Apt solution applied at 1000 mbar for 0.05 min. Detection wavelength: 260 nm. Sample injection: 50 mbar for 6 s.

separation windows (compare Figure 3, parts a and c). On the other hand, the use of the Brij 35 surfactant was responsible for a notable enlargement of the separation window width. Enantioseparation of Three Adenine Nucleotides (AMP, ADP, and ATP) by Aptamer-Modified MEKC. In order to extend the applicability of the methodology, the aptamer-modified MEKC was subsequently tested toward anionic species of lower mobility than that of aptameric chiral selector. Both ADP and ATP nucleotides were employed as model racemates. Due to the diand triphosphate moieties, ADP and ATP should migrate with a higher electrophoretic mobility than AMP in free-solution CE. However, the observed mobility of the two nucleotides was found to be largely inferior to that obtained for the AMP nucleotide and

Chol-Apt in the surfactant-free running buffer (-2.02 ± 0.01 × 10-4 cm2 V-1 s-1 for ATP and -1.45 ± 0.01 × 10-4 cm2 V-1 s-1 for ADP; capillary temperature of 30 °C, applied voltage of -25 kV). It is well-known that divalent cations form strong complexes with ADP and ATP through their binding to the phosphate groups, whereas the AMP-cation complex is much less stable. As previously reported,45 the mobility of ADP and AMP was strongly shifted as a consequence of their complexation with Mg2+ present in the running buffer. Therefore, it was necessary to adopt electrophoretic conditions providing a large separation window in order to accomplish the chiral (45) Cahours, X.; Morin, P.; Dreux, M. Chromatographia 1998, 48, 739.


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Figure 4. Electropherograms for the chiral separation of AMP, ADP, and ATP under aptamer-modified MEKC conditions, using Brij 35 as micelle-forming surfactant. Running buffer: 20 mM phosphate buffer, 5 mM KCl, 5 mM MgCl2, pH 7.0. Capillary: 50 µm i.d. PVA-coated silica capillary (total and effective lengths of 64.5 and 56 cm, respectively). Surfactant concentration: 35 mM. Separation plug length: 200 µM Chol-Apt solution applied at 50 mbar for 40 s. Detection wavelength: 260 nm. Sample injection: 50 mbar for 6 s (AMP, ADP, and ATP, 220 µM each). (a) Applied voltage: -18 kV. Capillary temperature: 35 °C. (b) Applied voltage: -30 kV. Capillary temperature: 25 °C.

analysis of these compounds. The objective was to separate the enantiomers of the three nucleotides (AMP, ADP, and ATP) in a single run. In this context, a high 35 mM Brij 35 surfactant concentration was selected and the Chol-Apt plug length introduced in the capillary was reduced to about 45 nL. The applied voltage and the capillary temperature were -18 kV and 35 °C, respectively. As shown in Figure 4a, the three racemates were completely resolved without any detection interference, even for the last-migrating D-ADP enantiomer. Such result demonstrates that (i) the aptamer chiral selector displayed broad enantioselective properties toward the adenine nucleotides and (ii) the aptamer-modified MEKC mode appeared to be also useful for anionic species which are characterized by lower electrophoretic mobility than that of the oligonucleotidic selector. In order to reduce the analysis time, the three racemates were also injected at a higher voltage (-30 kV), under lower capillary temperature conditions (25 °C), in order to increase the selectivity. The chiral resolution of the three nucleotides was also achieved under such conditions (Figure 4b). To the best of our knowledge, this is the first example of the enantioseparation of several nucleotides of same base. CONCLUSION In summary, the present work reports a new enantioselective aptamer-based CE mode where an aptamer-modified pseudo-

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stationary phase is created during the run by an on-capillary mixing approach and serves as the active chiral discriminating zone in a partial-filling format. Such an approach allows extending the use of the CE aptamer to enantioseparation of an anionic target which displays an electrophoretic mobility identical to that of the selector. The separation window width, selectivity, resolution, and analysis time can be efficiently modulated by varying some critical electrophoretic factors such as the concentration and the nature of the surfactant, the applied voltage, or the capillary temperature. For example, through the enlargement of the separation window, the enantioseparation of species of lower mobility than that of the aptameric chiral selector is easily attainable. Moreover, such aptamer-modified MEKC mode could be also find applications in the achiral analysis field for the development of affinity CE assays10 dedicated to small negatively charged analytes or for the separation of small anionic targets and analogues on the basis of their respective binding affinity for the aptamer ligand.

Received for review November 18, 2008. Accepted December 15, 2008. AC802443J

Aptamer-Modified Micellar Electrokinetic Chromatography for the

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