Noncompetitive Fluorescence Polarization Aptamer-based Assay for

Jul 24, 2009 - (Apt-T), previously selected by Vianini et al.,24 was first employed as a model functional nucleic acid. This aptamer has been successf...
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Anal. Chem. 2009, 81, 7468–7473

Noncompetitive Fluorescence Polarization Aptamer-based Assay for Small Molecule Detection Josephine Ruta, Sandrine Perrier, Corinne Ravelet, Jennifer Fize, and Eric Peyrin* De´partement de Pharmacochimie Mole´culaire UMR 5063, Institut de Chimie Mole´culaire de Grenoble FR 2607, CNRS-Universite´ Grenoble I (Joseph Fourier), 38041 Grenoble cedex 9, France In this paper, a new fluorescence polarization (FP) assay strategy is described reporting the first demonstration of a noncompetitive FP technique dedicated to the small molecule sensing. This approach was based on the unique induced-fit binding mechanism of nucleic acid aptamers which was exploited to convert the small target binding event into a detectable fluorescence anisotropy signal. An anti-L-tyrosinamide DNA aptamer, labeled by a single fluorescent dye at its extremity, was employed as a model functional nucleic acid probe. The DNA conformational change generated by the L-tyrosinamide binding was able to induce a significant increase in the fluorescence anisotropy signal. The method allowed enantioselective sensing of tyrosinamide and analysis in practical samples. The methodology was also applied to the L-argininamide detection, suggesting the potential generalizability of the direct FP-based strategy. Such aptamer-based assay appeared to be a sensitive analytical system of remarkable simplicity and ease of use. Because of its homogeneous format, speed, accuracy, and automated high-throughput capability, fluorescence polarization (FP) constitutes one of the most employed techniques for the routine analysis of small molecules in a variety of application fields including clinical, food, and environmental areas.1 At the present time, the FP-based analysis of small molecules relies on a competitive format. This approach is based on the increase in the fluorescence anisotropy of a small fluorescent-labeled analyte (tracer) when bound by a specific molecular recognition element (MRE). In the presence of free (unlabeled) analyte, the competition with the tracer for the binding sites of the MRE leads to a decrease in the polarization signal, allowing the analyte quantification. Antibodies are classically employed as MREs to perform FPbased immunoassays.2-5 More recently, both molecular imprinted polymers6 and nucleic acid aptamers7,8 have been also successfully exploited for the small species FP sensing. To note, the use of * To whom correspondence should be addressed. E-mail: eric.peyrin@ ujf-grenoble.fr. (1) Smith, D. S.; Eremin, S. A. Anal. Bioanal. Chem. 2008, 391, 1499–1507. (2) Eremin, S. A.; Gallacher, G.; Lotey, H.; Smith, D. S.; Landon, J. Clin. Chem. 1987, 33, 1903–1906. (3) Mannaert, E.; Daenens, P. Analyst 1996, 121, 857–861. (4) Maragos, C. M.; Plattner, R. D. J. Agric. Food Chem. 2002, 50, 1827–1832. (5) Sanchez Martinez, M. L.; Aguilar Caballos, M. P.; Gomez Hens, A. Anal. Chem. 2007, 79, 7424–7430.

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aptamers has also opened up the possibility to carry out direct FP assays for the detection of macromolecular targets such as proteins.9-12 The aim of this paper was to develop a new assay strategy for the design of a noncompetitive FP method dedicated to the small molecule analysis. Noncompetitive assays present numerous advantages over the competitive ones. They do not require the synthesis of fluorescent analogues as tracers, are typically more sensitive and rapid, and provide a larger linear range.13 The present approach was based on the unique conformational flexibility and adaptability features of nucleic acid aptamers. It is well established that, upon small molecule binding, aptamers commonly fold from a flexible, disordered structure into a structured conformation.14 Such target-dependent adaptive transition has recently appeared of great importance in biosensing applications. Various noncompetitive assays, employing in most cases fluorescence intensity or electrochemical detection, have exploited the induced-fit binding mechanism for the small target determination.15,16 We hypothesized that an aptamer, labeled by a single fluorescent dye at its extremity, could be used as a direct small molecule FP sensing system through the translation of the molecular recognition event into a detectable fluorescence anisotropy response. For labeled biomacromolecules, FP measurements typically reflect the contribution from (i) the local motional freedom of the fluorescent reporter and (ii) the global rotational diffusion of the entire species.17-19 Numerous NMR and molecular modeling studies have shown that the structural reorganization of aptamers, mediated by the small target complexation, can imply base rearrangements, sugar-phosphate backbone distortions, (6) Hunt, C. L.; Pasetto, P.; Ansell, R. J.; Haupt, K. Chem. Commun. 2006, 1754–1756. (7) Wang, Y.; Killian, J.; Hamasaki, K.; Rando, R. R. Biochemistry 1996, 35, 12338–12346. (8) Cruz-Aguado, J. A.; Penner, G. Anal. Chem. 2008, 80, 8853–8855. (9) Potyrailo, R. A.; Conrad, R. C.; Ellington, A. D.; Hieftje, G. M. Anal. Chem. 1998, 70, 3419–3425. (10) Fang, X.; Cao, Z.; Beck, T.; Tan, W. Anal. Chem. 2001, 73, 5252–5757. (11) Gokulrangan, G.; Unruh, J. R.; Holub, D. F.; Ingram, B.; Johnson, C. K.; Wilson, G. S. Anal. Chem. 2005, 77, 1963–1970. (12) Fu, H.; Guthrie, J. W.; Le, X. C. Electrophoresis 2006, 27, 433–441. (13) Hafner, F. T.; Kautz, R. A.; Iverson, B. L.; Tim, R. C.; Karger, B. L. Anal. Chem. 2000, 72, 5779–5786. (14) Hermann, T.; Patel, D. J. Science 2000, 287, 820–825. (15) Mok, W.; Li, Y. Sensors 2008, 8, 7050–7084. (16) Tombelli, S.; Minunni, M.; Mascini, M. Biosens. Bioelectron. 2005, 20, 2424– 2434. (17) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer Science: New York, 2006; Chapter 10. 10.1021/ac9014512 CCC: $40.75  2009 American Chemical Society Published on Web 07/24/2009

Figure 1. Schematic representation of the noncompetitive FP strategy for the design of the small molecule aptasensor. Double arrows represent the possible local and global motional contributions to the variation of the fluorescence anisotropy signal.

structural motif reorientations, three-dimensional topology changes, and gyration radius variations.20-23 In this context, it was expected that the target binding could affect the fluorescence anisotropy of the aptamer probe via two mechanisms (Figure 1). The first one could originate from the fluctuation of the structural dynamics within the reporter microenvironment, that is, a local mechanism. The second one could be related to the alteration of the overall size and/or shape of the aptamer, that is, a global mechanism. To test the feasibility of this strategy and establish the proofof-concept, an anti-L-tyrosinamide (L-Tym) 49-mer DNA aptamer (Apt-T), previously selected by Vianini et al.,24 was first employed as a model functional nucleic acid. This aptamer has been successfully used to develop fluorescence intensity-25 and chromatographic-based26 L-Tym assays. The effects of the nature (Fluorescein, Texas Red) and the attachment position (5′- or 3′end) of the fluorescent reporter as well as the length of the linker arm between the aptamer and the fluorophore (9- and 15-atom tether) on the variation of the fluorescence anisotropy signal were evaluated. Under optimized operating conditions, the FP titration curves of the Apt probes with increasing L-Tym concentration were established. The assay performances and the possibility to work under realistic conditions were subsequently analyzed to investigate the practical applicability of the analytical system. To evaluate the potential generalizability of such direct FP-based approach, the methodology was also applied to the L-argininamide (L-Arm) sensing using, as recognition unit, a 24-mer DNA aptamer (Apt-A) identified by Harada and Frankel.27 EXPERIMENTAL METHODS Chemicals and Apparatus. L-tyrosinamide, L-argininamide, L-tyrosine, L-phenylalanine, L-arginine, L-lysine, and Tris(hydroxym(18) Lee, B. J.; Barch, M.; Castner, E. W., Jr.; Volker, J.; Breslauer, K. J. Biochemistry 2007, 46, 10756–10766. (19) Benecky, M. J.; Kolvenbach, C. G.; Wine, R. W.; DiOrio, J. P.; Mosesson, M. Biochemistry 1990, 29, 3082–3091. (20) Lin, C. H.; Patel, D. J. Chem. Biol. 1997, 4, 817–832. (21) Schneider, C.; Su ¨ hnel, J. Biopolymers 1999, 50, 287–302. (22) Buck, J.; Fu ¨ rtig, B.; Noeske, J.; Wo ¨hnert, J.; Schwalbe, H. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15699–15704. (23) Bishop, G. R.; Ren, J.; Polander, B. C.; Jeanfreau, B. D.; Trent, J. O.; Chaires, J. B. Biophys. Chem. 2007, 126, 165–175. (24) Vianini, E.; Palumbo, M.; Gatto, B. Bioorg. Med. Chem. 2001, 9, 2543– 2548. (25) Merino, E. J.; Weeks, K. M. J. Am. Chem. Soc. 2005, 127, 12766–12767. (26) Michaud, M.; Jourdan, E.; Ravelet, C.; Villet, A.; Ravel, A.; Grosset, C.; Peyrin, E. Anal. Chem. 2004, 76, 1015–1020. (27) Harada, K.; Frankel, A. D. EMBO J. 1995, 14, 5798–5811.

ethyl)aminomethane were obtained from Sigma Aldrich (SaintQuentin, France). D-tyrosinamide was purchased from Millegen (Toulouse, France). NaCl and MgCl2 were obtained from ChimiePlus laboratoires (Bruye`res de Pouilly, 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. All the Fluorescein (F) or Texas Red (TR) labeled DNA aptamers were synthesized and HPLC-purified by Eurogentec (Angers, France). The identity of the modified oligonucleotides was confirmed by MALDI-TOF mass spectrometry. The sequence of aptamers and the chemical structure of linker arms used were as follows: Apt-T: 5′-AATTCGCTAGCTGGAGCTTGGATTGATGTGGTGTGTGAGTGCGGTGCCC-3′. Apt-A: 5′-GATCGAAACGTAGCGCCTTCGATC-3′. 15-atom linker: TR-SO2-NH-(CH2)5-CO-NH-(CH2)6-Apt; F-NHCS-NH-(CH2)6-O-PO2-O-(CH2)3-Apt. 9-atom linker: F-NH-CS-NH-(CH2)6-Apt. Fluorescence anisotropy readings were taken on a Tecan Infinite F500 microplate reader (Ma¨nnedorf, Switzerland) using black, 96-well Greiner Bio-One microplates (ref: 675086, Courtaboeuf, France). Excitation was set at 485 ± 20 nm or 585 ± 20 nm and emission was collected with 535 ± 25 nm or 635 ± 30 nm bandpass filters in relation to the nature of the fluorescent dyes. Methods. Unless otherwise stated, the binding buffer for the L-Tym sensor consisted of 5 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2. The binding buffer conditions for the L-Arm sensing were 5 mM Tris-HCl, pH ) 7.5, 5 mM NaCl. The different aptamer solutions were prepared in water and stored at -20 °C. The working aptamer solutions were obtained by adequate dilution of the stock solution in 1.25× concentrated binding buffer. Prior to the first utilization, the working solutions were heated at 80 °C for 5 min and left to stand at room temperature for 30 min. The analyte solutions were prepared in water. All solutions were filtered prior to use through 0.45 µm membranes. To construct the titration curves, the aptamer and analyte solutions were mixed into the individual wells (final volume ) 100 µL) at room temperature (unless otherwise stated). Blank wells of the microplate received 100 µL of the binding buffer. Various aptamer probe concentrations were initially evaluated (final concentration from 1 to 30 nM) to optimize the experimental conditions. The 10 nM concentration constituted the best choice, allowing both Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

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the probe detection with a fluorescence intensity signal-to-noise ratio >10 and optimal performances of the photomultiplier tube (gain for fluorescence intensity >60 according to the manufacturer’s instructions). All experiments were done in triplicate. The microplate was immediately placed into the microplate reader for the measurement. No anisotropy change was observed after further incubation time at room temperature for 30 and 60 min, demonstrating that the target-Apt binding reaction occurred rapidly. For additional experiments at low temperature, mixed aptamer-analyte samples were incubated in a cold room (4 °C) for a 30 min period. These samples were then transferred into the individual wells in the cold room, and the fluorescence anisotropy measurement was immediately performed. Further incubation for a 60 min period at 4 °C did not change significantly the fluorescence anisotropy signal. Human urine samples from 10 healthy volunteers of our laboratory were pooled and employed to test the L-Tym assay performances under biological conditions. Samples were filtered through 0.45 µm membranes, 5-fold diluted in water spiked with the target (final concentrations ) 500, 750, and 1000 nM). Three replicate samples, mixed with the aptamer probe containing 1.25× concentrated binding buffer (final concentration ) 10 nM), were analyzed as described above. Parameter Determination. The anisotropy (r) was calculated by the instrument software; as classically reported17

r)

Ivv - GIvh Ivv + 2GIvh

(1)

Ivv and Ivh are the vertically and horizontally polarized components of the emission after excitation by vertically polarized light. The instrumental correction factor G was determined from standard solutions according to the manufacturer’s instructions. For a 1:1 stoichiometry, the measured anisotropy r can be linked to the apparent dissociation constant Kd via the following relation:28

r)

rf Kd + r bc Kd + c

(2)

where rf is the anisotropy in absence of target, rb the anisotropy of maximally target-associated aptamer, and c the concentration of free target. For a limiting Apt probe concentration, the total concentration of target (cT) in the reaction system approximates the free target concentration c.28 The nonlinear regression of the r versus c (≈ cT) plots, where rb and Kd constituted the adjustable parameters, was achieved using the Table curve 2D software (Systat Software Gmbh, Erkrath, Germany). RESULTS AND DISCUSSION Design of the Direct FP Aptamer-Based Small Molecule Assay. It is well documented that FP measurements for dye-DNA conjuguates are largely dependent on the labeling strategy, that is, the nature and the position of the fluorescent reporters, as well as the length of the linker arm between the dye and the (28) Wilson, G. M. Reviews in Fluorescence; Springer Science: New York, 2005; Chapter 10.

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Figure 2. Titration curves of various Apt-T probes with increasing L-Tym concentrations (triplicate experiments). ∆r ) r - rf where rf is the fluorescence anisotropy in absence of analyte. Probe concentration ) 10 nM. Binding buffer conditions: 5 mM Tris-HCl, pH ) 7.5, 10 mM MgCl2, 50 mM NaCl.

oligonucleotide.11,18,29-33 Thus, these different experimental factors were first considered in the design of the direct L-Tym aptamer-based assay. Both Fluorescein (F) and Texas Red (TR) molecules, two of the most commonly fluorophores used as oligonucleotide tags, were attached to the 3′-end of the anti-L-Tym Apt via a 15-atom tether (3′-F-15-Apt-T and 3′-TR-15-Apt-T). The titration curves of the two aptamer probes with increasing L-Tym concentration (from 20 nM to 10 µM) were established under binding buffer conditions. As shown in Figure 2, the addition of the cognate ligand led to a significant increase in the fluorescence anisotropy for the two probes, revealing that the binding event can be effectively converted into a detectable FP response. As the size of L-Tym is negligible (Mw ) 180) compared to that of the aptamer (Mw ∼15000), the signal enhancement cannot be attributed to the increase in the molecular weight of the complex relatively to the unbound aptamer probe. It was therefore suggested that the FP response resulted from the target-induced conformational change mechanism which could imply (i) the restriction of the local motional freedom of the reporter, (ii) the increase in the volume and/or the modification of the shape of the aptamer, or (iii) a combination of both local and global processes. This is consistent with the recent findings of Wang et al. who have found that changes in the molecular conformation in the form of DNA wrapping of proteins substantially increased the fluorescence anisotropy signal.34 Over the entire target concentration range, the 3′-F-15-Apt-T probe exhibited a greater fluorescence anisotropy change than (29) Unruh, J. R.; Gokulrangan, G.; Lushington, G. H.; Johnson, C. K.; Wilson, G. S. Biophys. J. 2005, 88, 3455–3465. (30) Juskowiak, B.; Galezowska, E.; Zawadzka, A.; Gluszynska, A.; Takenaka, S. Spectrochim. Acta A 2006, 64, 835–843. (31) Kumke, M. U.; Li, G.; McGown, L. B.; Walker, G. T.; Linn, C. P. Anal. Chem. 1995, 67, 3945–3951. (32) Kumke, M. U.; Shu, L.; McGown, L. B.; Walker, G. T.; Pitner, J. B.; Linn, C. P. Anal. Chem. 1997, 69, 500–506. (33) Fogg, J. M.; Kvaratskhelia, M.; White, M. F.; Lilley, D. M. J. Mol. Biol. 2001, 313, 751–764. (34) Wang, H.; Lu, M.; Tang, M. S.; Van Houten, B.; Ross, J. B. A.; Weinfeld, M.; Le, X. C. Proc. Natl. Acad. Sci. U.S.A. 2009, DOI: 10.1073/ pnas.0902281106.

the 3′-TR-15-Apt-T probe (Figure 2). Such behavior could be attributed to the specific physicochemical properties of the two dyes. The rotation of the F moiety in a end-labeled DNA is largely uncoupled from the global motion of the molecule.11,29-32 Timeresolved anisotropy experiments have shown that only a very small portion of the anisotropy decay is caused by the overall rotation of the oligonucleotide.29,31 This is due to the negative charge on the F label which leads to electrostatic repulsion by the phosphate groups that keeps the dye away from DNA.33 On the other hand, TR in a dye-DNA conjugate exhibits a higher degree of coupling to the whole macromolecule as the result of strong van der Waals and electrostatic interactions between the positively charged dye and the DNA molecule.29 Moreover, in the absence of a target, the fluorescence anisotropy values varied from ∼0.08 for the 3′F-15-Apt-T probe to ∼0.19 for the 3′-TR-15-Apt-T probe in binding buffer conditions. Using the estimation procedure previously described,11 the expected r value for a 49-mer DNA oligonucleotide was about 0.22. These data confirm that the F label displayed a great level of local mobility while the TR label was more significantly coupled to the global motion of the aptamer. Therefore, the F and TR reporters were assumed to experience differently the local and/or global changes of the DNA structural dynamics so that a different magnitude of the fluorescence anisotropy change was observed for the two probes when reacted with L-Tym. The effects of the position of the label on the FP response were subsequently investigated. The F and TR dyes were attached to the 5′-end of the anti-L-Tym aptamer, using the same 15- atom linkers (5′-F-15-Apt-T and 5′-TR-15-Apt-T). In the absence of L-Tym, the fluorescence anisotropy values for 5′-labeled Apt-T were similar to those obtained for 3′-labeled Apt-T. Such result shows that the label position did not affect the fluorescence anisotropy signal of uncomplexed Apt-T probes. The titration curves of the two probes with increasing target concentrations were then constructed (Figure 2). A weak fluorescence anisotropy variation was observed for both 5′-F-15-Apt-T and 5′-TR-15-Apt-T. This indicates that the dye location (3′- versus 5′-extremity) played an essential role in the signal generation. A transduction mechanism based only on the alteration of the global rotational diffusion of the aptamer should be roughly independent of the fluorescent reporter position.18,35 It is therefore suggested that the FP assay response was related, at least in part, to changes of the fluorophore local motion. The reasoning for this observation could be that the targetinduced conformational change of the functional nucleic acid was responsible for distinct local structure constraints in the vicinity of the two DNA extremities, leading to a larger restriction of the dye motional freedom for the 3′-labeled Apt-T probes relative to that for the 5′-labeled Apt-T probes. The influence of the linker length on the fluorescence anisotropy signal of the F-Apt-T probes was also evaluated. A 9-atom spacer arm was employed to link the F dye to the 3′- and 5′- ends of the oligonucleotidic recognition unit (3′-F-9-Apt-T and 5′-F-9Apt-T) and the titration curves of the two probes were established. Similar FP responses were observed for the F-9-Apt-T and F-15(35) Nishimoto, E.; Yamashita, S.; Szabo, A. G.; Imoto, T. Biochemistry 1998, 37, 5599–5607.

Figure 3. Fluorescence anisotropy change (∆r ) r - rf) of the 3′F-9-Apt-T probe (L-Tym concentration ) 1.5 µM) under (a) various MgCl2 concentration (50 mM NaCl) and (b) NaCl concentration (10 mM MgCl2) conditions (triplicate experiments). rf is the fluorescence anisotropy in the absence of analyte. Probe concentration ) 10 nM. Binding buffer conditions: 5 mM Tris-HCl, pH ) 7.5.

Apt-T probes (see for comparison Figures 2 and 4), revealing that the 9- or 15-atom linker arm length had a minimal effect, if any, on the generation of the fluorescence anisotropy signal. Finally, the influence of the binding buffer cation concentration on the FP response was studied. Titration curves for the 3′-F-9Apt-T probe were constructed under various salt amounts in the assay medium. For a fixed NaCl concentration (50 mM), the fluorescence anisotropy change upon target binding was found to be highly dependent on the MgCl2 concentration. No significant FP response was observed in absence of MgCl2, the optimal signal being attained over the 10-15 mM MgCl2 concentration range (Figure 3). This is consistent with a previous study which has shown that the Mg2+ cation plays an essential role in the formation of the Apt-T active conformation.36 For a fixed MgCl2 concentration of 10 mM, the assay signal was optimal for the 50 mM NaCl concentration (Figure 3). The weaker fluorescence anisotropy change observed for lower and higher NaCl concentrations could originate from the effects of the ionic strength on both the DNA stabilization37 and the electrostatic interactions between L-Tym and Apt-T.36 The nonlinear fitting analysis of the fluorescence anisotropy data was performed to determine the L-Tym affinity for the (36) Lin, P. H.; Yen, S. L.; Lin, M. S.; Chang, Y.; Louis, S. R.; Higuchi, A.; Chen, W. Y. J. Phys. Chem. B 2008, 112, 6665–6673. (37) Owczarzy, R.; Moreira, B. G.; You, Y.; Behlke, M. A.; Walder, J. A. Biochemistry 2008, 47, 5336–5353.

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Figure 4. Titration curves of the 3′-F-9-Apt-T probe with increasing cognate (L-Tym) and non-cognate (D-Tym, L-Tyr, L-Phe) ligand concentrations and enantioselective sensing of Tym (100 nM of L-Tym in the presence of 10 µM of D-Tym, black circle). Triplicate experiments. ∆r ) r - rf where rf is the fluorescence anisotropy in absence of analyte. Probe concentration ) 10 nM. Binding buffer conditions: 5 mM Tris-HCl, pH ) 7.5, 10 mM MgCl2, 50 mM NaCl.

aptameric recognition element (see experimental section). To provide a reliable estimation, the dissociation constants (Kd) were calculated from the titration curves which exhibited the most important FP response, that is, using the 3′-F-Apt-T probes. Assuming a 1:1 stoichiometry, very close Kd values of 1.7 ± 0.1 µM (for 3′-F-9-Apt-T) and 2.2 ± 0.3 µM (for 3′-F-15-Apt-T) were obtained. Models with multiple aptamer binding sites for the target28 did not improve the fit of data. These binding constant data were in excellent agreement with the dissociation constant value of ∼2 µM determined recently by isothermal titration calorimetry (ITC) under similar binding buffer conditions.36 Aptamer-Based Assay Performances. The fluorescence anisotropy changes obtained for the most efficient 3′-F-Apt-T probes were in the same order of magnitude than those retrieved with the previously described competitive FP aptamer-based assays dedicated to the small molecule determination7,8 (Figures 2 and 4). For the 3′-F-9-Apt-T probe, the detection limit, based on a signal-to-noise ratio >3, was found to be equal to 200 nM. This compares favorably with the previously reported fluorescence intensity-based assay for the same species.25 The L-Tym assay displayed linearity up to 1.5 µM (R2 > 0.97). As shown in Figure 4, the assay sensitivity can be further improved by incubating the samples at 4 °C during 30 min before the fluorescence anisotropy measurements (see experimental section). In such conditions, the detection limit was reduced to 100 nM with a linear range up to 750 nM (R2 > 0.97). It is, however, of interest to notify that the direct FP aptamer-based protein assays display typically a lower detection limit (because of the nanomolar Kd values) and a larger dynamic linear range.10,11 The FP assay selectivity was subsequently investigated. As reported in Figure 4, the 3′-F-9-Apt-T probe was able to discriminate against closely related compounds, that is, L-tyrosine and L-phenylalanine. This is in accordance with previous selectivity data.24 In addition, aptamers are known to display, in most cases, 7472

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high chiral recognition properties and different types of enantioselective aptamer-based assays have been reported.26,38-41 In this context, titration curves of the 3′-F-9-Apt-T probe with increasing D-enantiomer concentrations were established (at room temperature and after sample incubation at 4 °C). D-Tym did not cause any significant response over the tested concentration range (Figure 4), indicating that the cross-reactivity of the aptameric receptor with the non-target enantiomer was negligible. This is consistent with results obtained by enantioselective chromatographic analysis.26 The L-Tym assay was also performed in the presence of high D-Tym concentration (10 µM) in a 4 °C preincubated sample. It was found that 100 nM of target enantiomer can be detected in the presence of 10 µM of nontarget enantiomer (Figure 4). This shows that the present method allowed the detection of an enantiomeric impurity down to approximately 1% in a non-racemic sample. Finally, the performances of the aptasensor in biological environment were evaluated. 5-fold diluted human urine samples were spiked with known amounts of target (see experimental section). Satisfactory recoveries ranging from 92% to 110% were obtained using the 3′-F-Apt-9-T probe (Table 1), demonstrating the practical applicability of such analytical system. Generalizability of the Strategy. The ability of the present strategy to function with another type of small target-aptamer system was investigated using, as molecular recognition element, a DNA aptamer (Apt-A) selected toward L-argininamide (L-Arm).27 In contrast with the L-Tym aptamer for which the secondary structure is not identified,24 the L-Arm aptamer is structurally well (38) Turney, K.; Drake, T. J.; Smith, J. E.; Tan, W.; Harrison, W. W. Rapid Commun. Mass Spectrom. 2004, 18, 2367–2374. (39) Ruta, J.; Ravelet, C.; Baussanne, I.; Decout, J. L.; Peyrin, E. Anal. Chem. 2007, 79, 4716–4719. (40) Shoji, A.; Kuwahara, M.; Ozaki, H.; Sawai, H. J. Am. Chem. Soc. 2007, 129, 1456–1464. (41) Ruta, J.; Perrier, S.; Ravelet, C.; Roy, B.; Perigaud, C.; Peyrin, E. Anal. Chem. 2009, 81, 1169–1176.

Table 1. Recovery and Relative Standard Deviation (RSD) of L-Tym-Spiked Five-Fold Diluted Human Urine Samples by Direct FP Aptamer-Based Assay Using the 3′-F-Apt-9-T Probe spiked concentration (nM)

recovery

RSD

500 750 1000

92% 95% 110%

6.4% 7.2% 3.5%

characterized, comprising a 7-base-pair stem and a 10-residue hairpin loop.23,27 Apt-A was labeled with the F dye at its 3′- and 5′-ends (using a 9-atom tether) to design the 3′-F-9-Apt-A and 5′F-9-Apt-A probes. Titration curves of the two probes with L-Arm concentration increasing (from 10 µM to 1 mM) were established (Figure 5). For both probes, a significant fluorescence anisotropy increase was obtained over the target concentration range; 3′-F9-Apt-A was responsible for a slightly greater FP response than 5′-F-9-Apt-A. In addition, the aptamer-based assay was able to discriminate successfully against analogues such as L-arginine and

(Figure 5), in accordance with previous results.27 The dissociation constant for the L-Arm-Apt-A complex was estimated using the nonlinear fitting analysis of the fluorescence anisotropy data of the probes. The Kd values ranged from 166 ± 15 µM (for 5′-F-9-Apt-A) to 213 ± 15 µM (for 3′-F-9-Apt-A), identical to those calculated by ITC, circular dichroism, and differential scanning calorimetry (Kd in the ∼150-200 µM range).23 All these data suggest that the present direct FP-based methodology can be potentially generalizable. L-lysine

CONCLUSION In summary, we have described a sensitive aptamer-based assay which is able to detect the target binding through a new FP strategy. To the best of our knowledge, this is the first demonstration of a non-competitive FP assay dedicated to the small molecule detection. The induced-fit mechanism of aptamers has been previously exploited for the development of various kinds of homogeneous, direct fluorescence intensity-based small molecule aptamer-based assays.15,16 These involve the integration of fluorophore transducing elements in the aptamer, the insertion of communication modules, or the design of aptamer beacons. In comparison, the present method is much simpler, easier to use, and less expensive as it only requires attaching a single fluorescent dye to an aptamer extremity. In addition, the ratiometric nature of the FP technique allows reducing some drawbacks of the fluorescence intensity-based assays, such as bleaching and nonuniform emission of the fluorophore.9 Further experiments will be performed to investigate in detail the transduction mechanism and the critical factors involved in the sensor sensitivity. ACKNOWLEDGMENT This work was supported by grants from the “SEST-Micraptox” n°2007-013-01 ANR program. We thank G. Mary for his assistance.

Figure 5. Titration curves of the F-9-Apt-A probes with increasing cognate (L-Arm) and non-cognate (L-Lys, L-Arg) ligand concentrations (triplicate experiments). ∆r ) r - rf where rf is the fluorescence anisotropy in the absence of analyte. Probe concentration ) 10 nM. Binding buffer conditions: 5 mM Tris-HCl, pH ) 7.5, 5 mM NaCl.

Received for review July 2, 2009. Accepted July 6, 2009. AC9014512

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