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Nanotemplating for Two-Dimensional Molecular Imprinting Raluca Voicu,*,† Karim Faid,† Abdiaziz A. Farah,† Farid Bensebaa,‡ Raluca Barjovanu,† Christophe Py,† and Ye Tao† Institute for Microstructural Sciences and Institute of Chemical Processing and EnVironmental Technology, National Research Council Canada, Ottawa, Ontario, K1A 0R6 Canada ReceiVed December 8, 2006. In Final Form: March 1, 2007 A new 2D molecular imprinting technique based on nanotemplating and soft-lithography techniques is reported. This technique allows the creation of target-specific synthetic recognition sites on different substrates using a uniquely oriented and immobilized template and the attachment of a molecularly imprinted polymer on a substrate. The molecularly imprinted polymer was characterized by AFM, fluorescence microscopy, and ATR-FTIR. We evaluated the rebinding ability of the sites with theophylline (the target molecule). The selectivity of the molecularly imprinted polymer was determined for the theophylline-caffeine couple. The molecularly imprinted polymer exhibited selectivity for theophylline, as revealed by competitive rebinding experiments. Fluorescence microscopy experiments provided complementary proof of the selectivity of the molecularly imprinted polymer surfaces toward theophylline. These selective molecularly imprinted polymers have the potential for chemical sensor applications. Because of its 2D nature, this novel chemical sensor technology can be integrated with many existing high-sensitivity multichannel detection technologies.
Introduction Molecular imprinting is a technique used to create recognition sites for a template molecule in a polymeric matrix. These artificially generated recognition sites are complementary in shape, size, and functionality with respect to the template and favor the rebinding of template molecules to other compounds with similar structures.1,2 For a comprehensive review of molecular imprinting, see Alexander et al.3 Molecularly imprinted polymers (MIPs) have already been successfully used for mimicking natural receptors and for the synthesis of polymers with high affinity sites for drugs, small analytes, peptides, and proteins.4-7 MIPs are very attractive because of their stability, ease of preparation, and potential low cost.8-10 The classical bulk molecular imprinting methodology presents some drawbacks related to the heterogeneity of the shape, orientation, and affinity of the recognition sites and hindered access to the desired selective cavities. To address some of these shortcomings, Yilmaz et al. immobilized molecular templates on a solid support (silica microspheres) prior to polymerization.11 Imprinting a polymer with binding sites situated at the surface exhibits several advantages: the sites are more accessible, the mass transfer is faster, and the binding kinetics is more rapid. Thus, surface molecular imprinting is very attractive for sensor * Corresponding author. E-mail:
[email protected]. † Institute for Microstructural Sciences, National Research Council Canada. ‡ Institute of Chemical Processing and Environmental Technology, National Research Council Canada. (1) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9-14. (2) Wulff, G.; Haarer, J. Makromol. Chem. 1991, 192, 1329-1338. (3) Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O’Mahony, J.; Whitcombe, M. J. J. Mol. Recognit. 2006, 19, 106-180. (4) Vlatakis, G.; Andersson, L. I.; Mosbach, K. Nature 1993, 361, 645-647. (5) Wulff, G. Chem. ReV. 2002, 102, 1-27. (6) Haupt, K. Anal. Chem. 2003, 75, 376A-383A. (7) Zimmerman, S. C.; Lemcoff, N. G. Chem. Commun. 2004, 5-14. (8) Zimmerman, S. C.; Wendland, M. S.; Rakow, N. A.; Zharov, I.; Suslick, K. S. Nature 2002, 418, 399-403. (9) Graham, A. L.; Carlson, C. A.; Edmiston, P. L. Anal. Chem. 2002, 74, 458-467. (10) Liu, J. Q.; Wulff, G. Angew. Chem., Int. Ed. 2004, 43, 1287-1290. (11) Yilmaz, E.; Haupt, K.; Mosbach, K. Angew. Chem., Int. Ed. 2000, 39, 2115-2118.
applications.12 MIP recognition surfaces have already been prepared using various soft lithography techniques.13-15 There are reports about the creation of template cavities on different surfaces such as gold substrates,16-19 synthetic membranes,20-22 nanowires,23 and silica.24 The incorporation of large molecules, such as polypeptides,25,26 proteins,15b,27 bacteria,28 and yeast cells,13a,29 as templates for sensitive coatings on mass-sensitive or planar waveguides has been described in the literature. These MIPs were synthesized with only one template at a time, excluding the use of MIPs from applications where multianalyte detection is required. We developed a novel 2D molecular imprinting methodology to overcome some of the limitations of the conventional 3D (12) Hillberg, A. L.; Brain, K. R.; Allender, C. J. AdV. Drug DeliVery ReV. 2005, 57, 1875-1889. (13) (a) Hayden, O.; Podlipna, D.; Chen, X.; Krassnig, S.; Leidl, A.; Dickert, F. L. Mater. Sci. Eng., C 2006, 26, 924-928. (b) Dickert, F. L.; Lieberzeit, P.; Miarecka, S. G.; Mann, K. J.; Hayden, O.; Palfinger, C. Biosens. Bioelectron. 2004, 20, 1040-1044. (14) Lieberzeit, P. A.; Gazda-Miarecka, S.; Halikias, K.; Schirk, C.; Kauling, J.; Dickert, F. L. Sens. Actuators, B 2005, 111-112, 259-263. (15) (a) Chi Huang, H.; Lin, C. I.; Joseph, A. K.; Der Lee, Y. J. Chromatogr., A 2004, 1027, 263-268. (b) Chou, P. C.; Rick, J.; Chou, T. C. Anal. Chim. Acta 2005, 542, 20-25. (16) Piletsky, S. A.; Piletskaya, E. V.; Sergeyeva, T. A.; Panasyuk, T. L.; El’skaya, A. V. Sens. Actuators, B 1999, 60, 216-220. (17) Chailapakul, O.; Crooks, R. M. Langmuir 1993, 9, 884-888. (18) Piacham, T.; Josell, Å.; Arwin, H.; Prachayasittikul, V.; Ye, L. Anal. Chim. Acta 2005, 536, 191-196. (19) Wei, X.; Li, X.; Husson, S. M. Biomacromolecules 2005, 6, 1113-1121. (20) Piletsky, S. A.; Matuschewski, H.; Schedler, U. Wilpert, A.; Piletska, E. V.; Thiele, T. A.; Ulbricht, M. Macromolecules 2000, 33, 3092-3098. (21) Han, M.; Kane, R.; Goto, M.; Belfort, G. Macromolecules 2003, 36, 4472-4477. (22) Araki, K.; Maruyama, T.; Kamiya, N.; Goto, M. J. Chromatogr., B 2005, 818, 141-145. (23) Yang, H.-H.; Z, S.-Q.; Tan, F.; Zhuang, Z.-X.; Wang, X.-R. J. Am. Chem. Soc. 2005, 127, 1378-1379. (24) Shiomi, T.; Matsui, M.; Mizukami, F.; Sakaguchi, K. Biomaterials 2005, 26, 5564-5571. (25) Rachkov, A.; Minoura, N. Biochem. Biophys. Acta 2001, 1544, 255-266. (26) Andersson, L. I.; Muller, R.; Vlatakis, G.; Mosbach, K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4788-4792. (27) a) Shi, H.; Tsai, W.-B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398, 593-597. (28) Aherne, A.; Alexander, C.; Payne, M. J.; Perez, N.; Vulfson, E. N. J. Am. Chem. Soc. 1996, 118, 8771-8772. (29) Dickert, F. L.; Hayden, O.; Halikias, K. P. Analyst 2001, 126, 766-771.
10.1021/la063562q CCC: $37.00 © 2007 American Chemical Society Published on Web 04/04/2007
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Figure 1. Schematic illustration of the principle of 2D molecular imprinting. (a) Amination of the PDMS stamp; (b) attachment of the carboxylic derivative of theophylline on the aminated stamp; (c) inking with the recognition monomer (MAA); (d) rinsing and inking with a mixture of anchoring monomer (TEVS), radical initiator (BPO) and cross-linking agent (DVB); (e) Stamping on Si/SiO2 or a PDMS substrate and polymerization; (f) removal of the stamp and rebinding of theophylline; and (g) rebinding of fluorophore labeled-theophylline.
molecular imprinting techniques. Two-dimensional molecular imprinting is based on nanotemplating through microcontact printing. This methodology incorporates the advantages of oriented immobilization of the template and the attachment of the MIP on a substrate surface. Thus, the recognition sites are homogeneous on the surfaces, and they can be probed by surface characterization techniques. This methodology has potential in applications where multiple molecular templates can be immobilized, allowing multianalyte detection at the same time. The principle of 2D molecular imprinting is depicted in Figure 1. A stamp is modified via the attachment of a monolayer of the template molecule. The modified stamp is first brought into contact with the recognition monomer and then with the anchoring monomer. The first monomer provides the recognition groups that will interact with the template molecule, and the second one offers the groups able to anchor the whole assembly to the substrate. Next, the modified stamp is pressed against the substrate of choice, and the assembly is then polymerized. The patterned molecularly imprinted polymer (MIP) will be formed on the substrate (Si/SiO2 or PDMS). The ultimate goal of this approach is to apply direct detection techniques for the rebinding of the template molecule to the MIP. To prove the concept of this 2D molecular imprinting methodology, we have chosen theophylline as a model system using the attachment chemistry described previously by Mosbach.11 We assessed the rebinding ability of the MIP and tested its selectivity by comparing the attachment of theophylline with that of caffeine, which is very similar (Figure 7). We demonstrated the reproducibility of the results and the stability of the MIP, which is remarkable when compared to the same parameters for other MIPs obtained by microcontact printing.15b Among the several advantages of this new methodol-
ogy, one can mention the directionality of the template due to its binding to the stamp and the attachment of the MIP to the substrate. Methods Materials. Poly(dimethylsiloxane) (PDMS) was obtained from Dow Corning (Sylgard 184 kit) in the form of a silicone elastomer base and a curing agent. The Si/SiO2 wafers were from Silicon Quest International (SQI), and they consist of 3000 Å thermal oxide grown on n-type Si (100). Hydrogen peroxide (H2O2, 30%) and reagent-grade sulfuric acid (H2SO4) were purchased from Fisher Scientific. 3-Aminopropylmethyldiethoxysilane (APMDES) was obtained from Gelest. Inc. N,N′-Diisopropylcarbodiimide (99%), 8-(3-carboxypropyl)-1,3-dimethylxanthine (theophylline-8-butanoic acid), trifluoroacetic acid 99%, methacrylic acid (MAA), divinylbenzene (DVB), triethoxyvinylsilane (TEVS), theophylline (Th), and caffeine (Caff) were purchased from Aldrich and used as received. The dapoxyl(2-aminoethyl)sulfonamide (Dap) fluorescent dye was acquired from Molecular Probes. Benzylperoxide (BPO) was recrystallized from methanol and vacuum dried. Absolute ethanol (EtOH) was obtained from Commercial Alcohols Inc., methanol was obtained from Aldrich, and the isopropanol was of VLSI grade from GEM Microelectronics Materials. Instrumentation. ATR-FTIR spectra were recorded using an HATR (horizontal attenuated total reflectance) accessory from SpectraTech installed in a Nicolet Magna-IR 550 spectrometer (1000 scans at 4 cm-1 resolution). Contact angle measurements were undertaken with a FTA200 dynamic contact angle analyzer from Folio Instruments Inc. (First Ten Angstroms). XPS data obtained with Kratos Axis ultra (Kratos, Manchester, U.K.) were recorded in two different modes: spectroscopy and imaging. In the spectroscopy mode, a hemispherical analyzer and
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Figure 2. Attachment of the carboxyl derivative of theophylline to the PDMS stamp.
Figure 4. AFM image of the 25 µm lines of the patterned molecularly imprinted polymer on Si/SiO2.
Figure 3. ATR-FTIR spectra of a hydrophilic PDMS surface (bottom) and a PDMS substrate with attached theophylline. an eight-channel detector were used. The sample was fixed on a bar holder. Multipoint analysis was allowed because the bar holder itself was mounted on an automated sample stage. The size of the analyzed area was about 1 mm2. Survey and high-resolution XPS spectra were collected using 160 and 40 eV pass energies, respectively. The pressure in the analyzer chamber was around 10-9 Torr. An electron flood gun was used to neutralize the charge during the experiment. Binding energies (BE) were referenced to the carbon 1s peak at 285.0 eV. A Nanoscope IIIa AFM (Digital Instruments) was used to analyze the topography of the patterned MIP surface. Images were acquired in tapping mode. The measurements were performed in air at 27 °C using a Si cantilever with a spring constant of ca. 20-100 N/m, a tip radius of 5-10 nm, and a resonance frequency of about 300 kHz. A Nikon Eclipse 80i fluorescence microscope was used to image the samples with excitation light at 365 nm and emission light detected
at wavelengths longer than 420 nm. Images were taken with 5 and 20× magnification. Surface Chemistry: Modification of Surfaces for the Attachment of the Target (Theophylline). Substrate. Micropatterned PDMS substrates were obtained by replicating a mold with line features of 10, 25, 50, and 100 µm width and 10 µm depth, patterned by standard photolithography techniques using an SU-8 negative photoresist purchased from MicroChem Corp. PDMS substrates were obtained by thermal polymerization (100 °C for 2 h) of a 10/1 mixture of elastomer/curing agent of Sylgard 184. Cured PDMS surfaces were subsequently washed in a Soxhlet setup using hexanol for 3 h and rinsed in acetone and ethanol, followed by sonication in an EtOH/H2O (2/1) mixture and drying in an oven. Surfaces were rendered hydrophilic by creating -OH groups on the surface in an oxygen plasma reactor for 1 min at 2 × 10-1 mbar and 100 W rf power. PDMS-OH surfaces were stored in MilliQ DI water prior to further modification in order to preserve their hydrophilic character. The silicon wafers, each with a 3000 Å silicon dioxide top layer, were cleaned in piranha solution (3/1 H2SO4/H2O2, 30%) for 5 min, rinsed copiously with DI water, and dried with nitrogen. Amination. PDMS-OH substrates were immersed overnight in a 100 mM solution of APMDES in absolute ethanol. The amino-
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Figure 7. ATR-FTIR spectra of MIP after rebinding in mixed Th/ Caff solutions (0.1 M total concentration) with different Th/Caff ratios: (a) 10/90, (b) 20/80, (c) 30/70, and (d) 50/50. Figure 5. Images obtained by fluorescence microscopy of (a) the molecularly imprinted polymer and (b) the molecularly imprinted polymer after rebinding in labeled theophylline (DapTh).
Figure 6. ATR-FTIR spectra of (a) MIP on PDMS, (b) MIP on PDMS after rebinding in theophylline, and (c) MIP on PDMS after rebinding in caffeine. terminated substrates (PDMS-NH2) were rinsed thoroughly with ethanol and dried with a nitrogen gun. Theophylline (Target Molecule) Attachment. PDMS-NH2 substrates were dipped overnight in a 10 mL absolute ethanol solution of 83 mM theophylline-8-butanoic acid and 320 mM N,N′diisopropylcarbodiimide. Theophylline-modified substrates were copiously rinsed with ethanol and immersed in an ethanol solution of 104 mM trifluoroacetic acid and 287 mM N,N′-diisopropylcarbodiimide for 2 h in order to consume any of the amino groups remaining unreacted during the theophylline step. Contact Patterning. Microcontact Printing: Inking and Stamping. MAA was used to ink the stamp (a theophylline-modified PDMS substrate). Stamps were covered for 30 min with 0.1 mL of MAA, rinsed with 25 mL of EtOH (to remove excess MAA), and dried in a nitrogen stream. Theophylline-modified PDMS stamps were inked for another 30 min with 0.1 mL of a mixture of TEVS, DVB, and BPO. After being blown dry with nitrogen, the stamps were placed in contact with a clean Si/SiO2 surface or PDMS surface in order
to perform a surface polymerization reaction and the transfer of the nanotemplated recognition cavities. Thermal copolymerization reactions were performed at 70 °C under a nitrogen atmosphere for 3 h. The patterned Si/SiO2 or PDMS surfaces were stored in EtOH and blown dry with nitrogen before use. The MIP lines created on Si/SiO2 or PDMS surfaces bear the recognition cavities capable of rebinding the theophylline molecule. Rebinding of the Theophylline. Dapoxyl-labeled fluorescent theophylline (DapTh) has an emission at 576 nm and was used to trace the rebinding process. The procedure for labeling theophylline will be described elsewhere.30 Patterned MIPs on the Si/SiO2 surface were immersed overnight in a 0.26 mM solution of the labeled theophylline. Surfaces were rinsed thoroughly with ethanol, methanol, and isopropanol to remove any excess DapTh. Fluorescence microscopy was used to image the surface after copiously rinsing the surface with ethanol, methanol, and isopropanol. Rebinding SelectiVity of Theophylline and Caffeine. Competitive Rebinding. Patterned MIPs on PDMS were immersed overnight in mixed solutions of theophylline and caffeine with different Th/Caff ratios: 10/90, 20/80, 30/70, and 50/50. The total concentration of theophylline and caffeine was 100 mM. The samples were thoroughly rinsed with ethanol and dried with a nitrogen gun. They were characterized by ATR-FTIR. Subsequent Rebinding. The patterned MIP on Si/SiO2 bearing the recognition sites for theophylline were formed and immersed overnight in theophylline and caffeine solutions, respectively. After rinsing, the samples were immersed overnight in the DapTh solution and then thoroughly rinsed with ethanol to remove any physisorbed molecules and imaged by fluorescence microscopy. The selectivity of the MIP for theophylline over caffeine was proven by comparing the fluorescence intensities determined from the profile lines of the two samples.
Results and Discussion Attachment of the Theophylline to the PDMS Stamp. The attachment of theophylline to the PDMS stamp presents several advantages. First and foremost, it offers the possibility of using (30) Farah, A.; Voicu, R.; Bensebaa, F.; Py, C.; Barjovanu, R.; Faid, K. To be submitted for publication.
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microcontact printing to create patterned surfaces with molecularly imprinted polymer. It also ensures the directionality of theophylline molecules at the surface of the modified stamp through covalent bonds. This approach plays an important role in the self-assembly of the recognition groups around the monolayer of theophylline molecules and in the subsequent formation of the recognition sites. To the best of our knowledge, this is the first report concerning the characterization of theophylline attachment to the PDMS surface. The binding of theophyline to the PDMS surface is based on silane and carbodiimidization chemistry. PDMS stamps were initially treated in an air plasma reactor to create -OH groups at the surface. The surface wettability was tested using contact angle measurements. Contact angle values of 100-105° were measured on a flat PDMS substrate indicating a hydrophobic surface. Upon plasma treatment, lower contact angle values (1517°) were obtained, corresponding to a hydrophilic surface. Each value presented is the average of at least six measurements on different spots on the surface. The -OH groups at the surface reacted readily with APMDES. XPS confirmed the occurrence of the -NH2 groups at the surface of the stamp. The highresolution XPS spectrum of N 1s exhibited the characteristic signature of N in the -NH2 group, with a binding energy of 400.0 eV (Figure 2). The contact angle measurements have also confirmed that the reaction has occurred. Indeed, a contact angle of 66.7° was obtained, indicating a more hydrophobic surface after the introduction of the aminosilane molecules. Furthermore, ellipsometry measurements have also revealed a thickness of 7 Å for these aminated PDMS surfaces, which is equivalent to that of the monolayer regime. A carboxylic derivative of theophylline was attached to the -NH2-terminated stamp in the presence of the carbodiimide. The occurrence of the amide link between the theophylline and aminosilane on the surface was proven by XPS. The highresolution spectra of N 1s and C 1s indicate the signatures of N in the NH group at 399.0 eV and C in the CO group at 288.5 eV (Figure 2). The presence of polar groups such as the carbonyl functionality of theophylline resulted in the more hydrophilic character of the functionalized surface, as indicated by the decrease in contact angle values from 66.7 to 50.6°. ATR-FTIR measurements further provide unambiguous evidence for the oriented immobilization of theophylline on the aminated surface: The ATR-FTIR spectrum exhibits peaks that are characteristics of CO groups at 1709 and 1662 cm-1 and NH groups at 1550 cm-1 from the amide bond formation as depicted in Figure 3. Two-Dimensional Molecular Imprinting. Our 2D molecular imprinting methodology is based on microcontact printing. Among the different soft lithographic patterning techniques, microcontact printing has proven to be the most versatile method for surface modification approaches in materials and life science applications.31,32 As a first step, the theophylline-modified PDMS stamp was inked with MAA monomer, which possesses the carboxylic acid functionality and hence the recognition sites of the MIP. This step is important because it allows the self-assembly of the MAA molecules around the oriented theophylline groups through noncovalent interactions (hydrogen bonds) between its carboxylic groups and theophylline. These assemblies formed the recognition sites, which were used later to rebind theophylline. The theophylline-modified PDMS stamp inked with MAA monomer was rinsed with EtOH to eliminate the excess MAA (31) Burdinski, D.; Saalmink, M.; van den Berg, J. P. W. G.; van der Marel, C. Angew. Chem., Int. Ed. 2006, 45, 4355-4358. (32) Gates, B. D.; Xu, Q.; Love, J. C.; Wolfe, D. B.; Whitesides, G. M. Annu. ReV. Mater. Res. 2004, 34, 339-372.
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from the stamp surface. Tests performed by ATR-FTIR for this step indicated that the rinse with 25 mL EtOH does not eliminate the MAA. It may also be possible that some swelling occurs during the inking step, with some of the MAA being adsorbed by the PDMS, to be released later in the vicinity of the templates when the mixture containing DVB is stamped on the final substrate. However, experiments to determine the imprinting effect and the MIP selectivity (vide infra) indicate that the MAA is locked during polymerization in the recognition sites after its transfer from the surface of the stamp. In the second step, the stamp was inked with a mixture of TEVS, DVB, and BPO. The TEVS acted as an anchoring monomersthe silane groups bound to either the PDMS or Si/ SiO2 substrate and the double bond copolymerized with the MAA. The DVB was used as a cross-linker to cement the overall structure. BPO was the radical initiator triggering the polymerization reaction. After being dried, the double-inked stamp was pressed against either the air-plasma-treated PDMS or the piranha-cleaned Si/SiO2 substrate. The recognition sites were locked in through polymerization and transferred onto the PDMS or Si/SiO2 substrate. After the stamp was peeled off, the patterned MIP was obtained on the substrate. Either PDMS or Si/SiO2 substrates were used, depending on the characterization technique employed. There were no differences between the MIP obtained on the PDMS or Si/SiO2 substrates. The lines of MIP formed corresponded to the tops of the stamp grooves that were in conformal contact with the substrate during the polymerization. There was no material transferred from the bottom of the grooves, which are 10 µm above the substrate. The patterned MIP on a Si/SiO2 substrate was characterized by AFM. Figure 4 illustrates the AFM image obtained for the 25 µm lines. The lines of MIP formed on the Si/SiO2 substrate were clearly observed with the width of the lines corresponding exactly to the dimensions of the grooves of the PDMS stamp. The thickness of the lines is 31.6 nm. AFM proves that the polymer lines are formed on the Si/ SiO2 substrate. This is the first example of a patterned MIP obtained through microcontact printing and rapid surface polymerization (3 h compared to the 17 h necessary for polymerization in other MIP systems15b). Rebinding with Theophylline. Two experiments were designed to test the MIP’s rebinding capability with theophylline molecules. In the first experiment, DapTh was used as the target and beacon molecule. The patterned MIP on the Si/SiO2 substrate was first imaged by fluorescence microscopy (Figure 5a). After overnight immersion in DapTh and a thorough rinse, the MIP was imaged again by fluorescence microscopy (Figure 5b). As illustrated by Figure 5, before rebinding the MIP does not exhibit any fluorescence signal, whereas after rebinding in DapTh the MIP presents fluorescence in the range of wavelengths corresponding to the emission of the dye. Figure 5b clearly indicates that the pattern obtained by fluorescence is similar to the geometrical pattern observed by AFM, which is proof that theophylline rebinds in the recognition sites. The theophylline rebound to the polymer’s recognition sites through hydrogen bonds with the carboxylic groups of the MIP acting as recognition groups. In the second experiment, instead of using a fluorophoretagged target molecule, theophylline was used directly to assess the rebinding capability of the MIP. The MIP on PDMS was immersed in a theophylline solution, and the sample was characterized by ATR-FTIR before and after rebinding in theophylline solution. The spectra are presented in Figure 6. The MIP spectrum (Figure 6a) presents the signature of the carboxylic
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groups involved in hydrogen bonds through the peaks at 3340 and at 1710 cm-1 as OH (hydroxyl) and CdO (carbonyl) stretching vibrations, respectively. The theophylline exhibits a characteristic peak at 1665 cm-1. The absorption bands in the 1700-1720 cm-1 region are assigned to the CdO stretching vibration. Other bands in the 1600-1665 cm-1 region are assigned to the CdN stretching modes in the purine ring system.33 Experiments performed with NIP (non-imprinted polymer) confirm that theophylline rebinding on the MIP is due to an imprint effect. The NIP was obtained under the same conditions as for the MIP but without theophylline as a template. The NIP rebound an order of magnitude less theophylline than the MIP, proving the importance of the recognition sites imprinted on the surface of the polymer (Supporting Information). Selectivity. Caffeine is the best candidate11 to use in testing the selectivity of the 2D MIP because it has a very similar structure to theophylline; they differ only by a methyl group (Figure 7). This set the bar very high for the selectivity test. When the MIP was immersed in a caffeine solution, a peak at 1656 cm-1, which is characteristic of caffeine,33 was observed in the ATR-FTIR spectrum (Figure 6c). This indicates that the MIP prepared using theophylline as the template molecule rebound theophylline and caffeine to a similar extent if the rebinding process took place in a theophylline-only solution or a caffeine-only solution. The structures of the two molecules are so similar that there is insufficient structural difference to block caffeine sterically from rebinding in theophylline recognition sites. However, because theophylline forms an extra hydrogen bond with the recognition sites when compared with caffeine, its rebinding to the MIP will be thermodynamically more favorable. Therefore, the selectivity would become more evident in a competitive rebinding process (i.e., the MIP is expected to rebind more theophylline in a mixture of theophylline and caffeine). The MIP samples on PDMS imprinted for theophylline were submitted to a series of competitive rebinding tests. The samples were immersed in mixed solutions of theophylline and caffeine with different ratios (Th/ Caff ) 10/90, 20/80, 30/70, and 50/50). After a thorough rinse, the MIPs were characterized by ATR-FTIR as a direct detection method to demonstrate the selectivity of the MIP toward theophylline. The spectra are illustrated in Figure 7 and they are normalized to the CdO peak at 1709 cm-1. The theophylline has a characteristic peak at 1664 cm-1, whereas caffeine has one at 1656 cm-1. Besides the main peak at 1664 cm-1, a shoulder at 1656 cm-1 is also observed, indicating the presence of rebound caffeine. Even if both theophylline and caffeine are present, theophylline is definitely rebound in a greater proportion because the peak at 1664 cm-1 is predominant. Only when the caffeine concentration rises to 90% does the caffeine peak at 1656 cm-1 become predominant in the spectrum. It seems that the very high caffeine concentration in the solution increases the probability of the caffeine molecules to rebind with the MIP. The spectra indicate without any doubt that the theophylline-imprinted polymer exhibits preferential rebinding to theophylline and demonstrates selectivity toward theophylline. The existence of the imprinted sites and the MIP selectivity toward theophylline demonstrate that the MAA is locked during the polymerization in the recognition sites. These are proof that the MAA molecules are incorporated into the MIP as a result of their transfer from the surface of the stamp, minimizing the effect of possible swelling of the stamp during the MAA inking step. (33) Nafisi, S.; Shamloo, D. S.; Mohajerani, N.; Omidi, A. J. Mol. Struct. 2002, 608, 1-7.
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Figure 8. Fluorescence images of (a) MIP/Th/DapTh and (b) MIP/ Caff/DapTh samples.
The reproducibility of the results and the stability of the MIP were also tested. Several MIPs were formed and tested in the competitive rebinding experiments, and the results were reproducible (93% rate of success over 14 trials). The stability of the MIP was tested by using the same MIP in several binding/ extraction/rebinding cycles. Extractions were performed by leaving the samples in EtOH overnight. The interactions between bound theophylline and ethanol have to overcome the hydrogen bonds between the carboxylic groups from the MAA in the MIP recognition sites and the bound theophylline. The shift in equilibrium toward the unbound theophylline is achieved by the long immersion step in ethanol. The ATR-FTIR spectra of the MIP after the extraction indicated that theophylline was removed. Up to four cycles of binding/extraction/rebinding were used with no sign of degradation. The MIP obtained by this novel 2D molecular imprinting methodology exhibits remarkable stability when compared with other MIP15b systems. Subsequent Rebinding Experiments. A complementary experiment was set up to demonstrate further the selectivity of the theophylline-imprinted polymer. Two samples of patterned MIP on SiO2 were first immersed in theophylline and caffeine. These two samples were then immersed into a DapTh solution and imaged by fluorescence microscopy. The fluorescence images from MIP/Th/DapTh and MIP/Caff/DapTh samples are shown in Figure 8. The main point of this experiment is to test the availability of the recognition sites for DapTh after being first immersed in either theophylline or caffeine. As illustrated in Figure 9, the intensity profile of the fluorescence signal for MIP/Th/DapTh is weaker than that for MIP/Caff/DapTh. This further supports the MIP selectivity toward theophylline. When MIP is immersed in theophylline solution, the recognition sites are occupied in a larger number than in caffeine solution as a result of the higher affinity of MIP for theophylline than for caffeine. As a consequence, DapTh was bound to fewer recognition sites in the MIP/Th/DapTh series than in the MIP/Caff/DapTh series. Under these conditions, it is normal to observe a weaker fluorescence signal for the MIP/ Th/DapTh series than for the MIP/Caff/DapTh series, as illustrated
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lines in Figure 9. The ratio indicates that there are 6.5 times fewer recognition sites available for DapTh in the MIP/Th/DapTh series than in the MIP/Caff/DapTh series. These tests confirmed the recognition and preferential rebinding of theophylline by MIP.
Conclusions
Figure 9. Profile lines for (a) MIP/Th/DapTh and (b) MIP/Caff/ DapTh samples.
in Figure 8. These sets of experiments were repeated for four different samples ,and the results were very similar, proving the reproducibility of the MIP selectivity. During this step, there might be free exchange between Th and Caff and DapTh as a result of equilibrium binding. Th is more strongly bound to the MIP sites than the Caff (an extra hydrogen bond), so Th is not displaced. The binding of Th is thermodynamically more favorable than the binding of Caff. The size of the DapTh molecule (structure depicted in Supporting Information) would suggest a lower probability for this process when compared to the direct binding of DapTh to the sites available on the surface. The profile lines obtained from the fluorescence images are used as an indication of the selectivity by calculating the ratio between the two fluorescence intensities. The intensities are calculated as differences between the average maximum and average minimum for the two samples, as illustrated by the dashed
We presented a novel and versatile nanotemplating methodology allowing the rapid creation of synthetic recognition sites by 2D molecular imprinting. The proof-of-concept experiments have demonstrated that MIP can not only be formed and transferred onto different substrates but also is able to rebind the target molecule. Several cycles of binding/extraction/rebinding of the template proved the high stability of MIP. The selectivity between theophylline and caffeine has been proven through competitive rebinding experiments and fluorescence microscopy. This is the first successful example of combining microcontact printing with molecular imprinting technology to generate high-resolution arrays of specific molecular recognition sites on well-defined substrate surfaces. Acknowledgment. This work was partially funded by a grant from CRTI Canada (CBRN Research and Technology Initiative). We thank M. Beaulieu for AFM pictures and P. L’Ecuyer, K. Tufa, and D. Kingston for the XPS measurements. Supporting Information Available: ATR-FTIR characterization of the non-imprinted polymer and the non-imprinted polymer after binding in theophylline and caffeine. The structure of labeled theophylline is included. This material is available free of charge via the Internet at http://pubs.acs.org. LA063562Q
