Imprinting of Nucleotide and Monosaccharide Recognition Sites in

Synthesis and Applications of Boronate Affinity Materials: From Class ..... Bhim Bali Prasad , Indu Pandey , Amrita Srivastava , Deepak Kumar , Mahavi...
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Anal. Chem. 2002, 74, 702-712

Imprinting of Nucleotide and Monosaccharide Recognition Sites in Acrylamidephenylboronic Acid-Acrylamide Copolymer Membranes Associated with Electronic Transducers Nesim Sallacan, Maya Zayats, Tatyana Bourenko, Andrei B. Kharitonov, and Itamar Willner*

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Molecular recognition sites for the nucleotides adenosine 5′-monophosphate (1), guanosine 5′-monophosphate (2), cytosine 5′-monophosphate (3), and uridine 5′-monophosphate (4) are imprinted in an acrylamide-acrylamidephenylboronic acid copolymer (5) membrane. The imprinted membranes are assembled on piezoelectric Au quartz crystals or Au electrodes via electropolymerization or on the gate surface of an ISFET device by radical polymerization. The imprinted membranes reveal selectivity toward the imprinted nucleotide, and the association of the respective nucleotides with the recognition sites is transduced by the following: (i) microgravimetric, quartz crystal microbalance (QCM) measurements; (ii) Faradaic impedance analyses, and (iii) potentiometric responses of the ISFET devices. While the microgravimetric QCM measurements reflect the swelling of the polymers upon the association of the nucleotides with the recognition sites, the ISFET response is due to the charging of the polymer membrane as a result of the formation of the nucleotide-boronate complex. The selective detection of the nucleotides may lead to new DNA/RNA sequencing methods. Also, specific recognition sites for β-D(+)glucose (6), D(+)-galactose (7), and β-D(-)-fructose (8) were imprinted in an acrylamide-acrylamidephenylboronic acid copolymer (5) membrane associated with an ISFET device. Selective sensing of the respective monosaccharides is accomplished in the presence of the imprinted membrane-functionalized ISFET devices. Imprinting of molecular recognition sites in organic or inorganic polymers has been a subject of extensive research efforts in the last two decades.1,2 Two general strategies have been * Corresponding author: (tel) 972-2-6585272; (fax) 972-2-6527715; (e-mail) [email protected]. (1) (a) Haupt, K. Analyst 2001, 126, 747-756. (b) Sellergren, B. Trends Anal. Chem. 1997, 16, 310-320. (c) Lanza, F.; Sellergren, B. Anal. Chem. 1999, 71, 2092-2096. (d) Whitcombe, M. J.; Vulfson, E. N. Adv. Mater. 2001, 13, 467-478. (e) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 24952504. (2) (a) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (b) Bru ¨ ggemann, O.; Haupt, K.; Ye, L.; Yilmaz, E.; Mosbach, K. J. Chromatogr., A 2000, 889, 15-24. (c) Katz, A.; Davis, M. E. Nature (London) 2000, 403, 286-289. (d) Glad, M.; Norrlo ¨w, D.; Sellergren, B.; Siegbahn, N.; Mosbach, K. J. Chromatogr. 1985, 347, 11-15. (e) Panasyuk, T. L.; Mirsky, V. M.; Piletsky, S. A.; Wolfbeis, O. S. Anal. Chem. 1999, 71, 4609-4613.

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suggested to generate the imprinted sites. One approach3 involves the polymerization of monomers that include complementary functions to the imprinted substrate such as H-bonds, electrostatic interactions, and π-donor-acceptor interactions. The resulting polymer includes imprinted contours of the substrate, and by washing off of the template molecule, an imprinted recognition site is generated. The second approach includes the covalent attachment of polymerizable groups to the substrate4 or the coordination of the substrate to monomer units,5 followed by the copolymerization of these units into rigidified matrixes. Cleavage of the polymer-linked substrate units yields the imprinted recognition sites. Imprinted sites with structural6 and chiral7 selectivities were prepared, and the matrixes with synthetically incorporated recognition sites were used as sensing interfaces8 for chromatographic separations9 and for catalyzed and selective chemical transformations.10 The imprinting of polymers for sensing applications is particularly attractive since it enables us to design tailored specific sensing interfaces. This approach suffers, however, from (3) (a) Mosbach, K.; Ramstro ¨m, O. Bio/technology 1996, 14, 163-170. (b) Klein, J. U.; Whitcombe, M. J.; Mulholland, F.; Vulfson, E. N. Angew. Chem. 1999, 111, 2100-2103; Angew. Chem., Int. Ed. 1999, 38, 2057-2060. (c) Yilmaz, E.; Mosbach, K.; Haupt, K. Anal. Commun. 1999, 71, 285-287. (d) Sellergren, B.; Shea, K. J. J. Chromatogr., A 1995, 690, 29-39. (e) Lee, C. W.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 2857-2863. (4) (a) Wulff, G.; Gross, T.; Scho¨nfeld, R. Angew. Chem., Int. Ed. 1997, 36, 1962-1964. (b) Piletsky, S. A.; Piletskaya, E. V.; Chen, B.; Karim, K.; Weston, D.; Barrett, G.; Lowe, P.; Turner, A. P. F. Anal. Chem. 2000, 72, 43814385. (5) (a) Malik, S.; Johnson, R. D.; Arnold, F. H. J. Am. Chem. Soc. 1994, 116, 8902-8903. (c) Hart, B. R.; Shea, K. J. J. Am. Chem. Soc. 2001, 123, 20722073. (6) (a) Matsui, J.; Nicholls, I. A.; Takeuchi, T. Tetrahedron: Asymmetry 1996, 7, 1357-1361. (b) Ramstro¨m, O.; Nicholls, I. A.; Mosbach, K. Tetrahedron: Asymmetry 1994, 5, 649-656. (c) Schweitz, L.; Andersson, L. I.; Nilsson, S. Anal. Chem. 1997, 69, 1179-1183. (7) (a) Nilsson, K. G. I.; Lindell, J.; Norrlo ¨w, O.; Sellergren, B. J. Chromatogr., A 1994, 680, 57-61. (b) Vallano, P. T.; Remcho, V. T. J. Chromatogr., A 2000, 887, 125-135. (8) (a) Makote, R.; Collinson, M. M. Chem. Mater. 1998, 10, 2440-2445. (b) Kempe, M. Anal. Chem. 1996, 68, 1948-1953. (c) Mayes, A. G.; Andersson, L. I.; Mosbach, K. Anal. Biochem. 1994, 222, 483-488. (d) Kriz, D.; Berggren-Kriz, C.; Andersson, L. I.; Mosbach, K. Anal. Chem. 1994, 66, 2636-2639. (9) (a) Lin, J. M.; Nakagama, T.; Uchiyama, K.; Hobo, T. Chromatographia 1996, 43, 585-591. (b) Schweitz, L.; Spe´gel, P.; Nilsson, S. Analyst 2000, 125, 1899-1901. (10) (a) Brunkan, N. M.; Gagne, M. R. J. Am. Chem. Soc. 2000, 122, 62176225. (b) Whitcombe, M. J.; Alexander, C.; Vulfson, E. N. Synlett 2000, 911-923. 10.1021/ac0109873 CCC: $22.00

© 2002 American Chemical Society Published on Web 01/08/2002

two basic limitations: (i) The imprinted polymers are usually thick and include a low density of recognition sites. This introduces diffusion barriers for the association of the analyzed substrate with the imprinted sites, resulting in slow response times and moderate sensitivities. (ii) To develop electronic sensor devices, it is essential to communicate the chemical recognition event with electronic transducers. The macroscopic dimensions of the polymers usually prohibit the electronic communication between the recognition site and the transducer. Not surprisingly, most of the sensor devices based on imprinted polymers are either optical and include chromogenic markers11 or involve microgravimetric analysis of the bound substrate using piezoelectric crystals (quartz crystal microbalance, QCM).12 Recently, the generation of imprinted recognition sites in monolayer or thin-film assemblies was suggested to eliminate diffusion barriers of the analyzed substrate and to improve the electrical contact between the recognition site and electronic transducers.13 Also, imprinted TiO2 thin films assembled on Al2O3 gate interfaces of ion-sensitive field-effect transistors (ISFETs) were used in the specific analysis of 4-chlorophenoxyacetic acid or 2,4-dichlorophenoxyacetic acid14 and for the chiroselective analysis of different chiral carboxylic acids.15 The specific analysis of nucleotides is interesting for the development of rapid sequencing methods for DNA/RNA. Boronic acid ligands bind vicinal diols strongly and reversibly, eq 1. This

property was used to develop optical sensors for sugars16 or gelating materials that undergo sol-gel transition upon binding of sugars.17 Boronic acid acrylamide copolymeric hydrogels were recently employed as sensing matrixes for glucose.18 The degree of swelling of the hydrogel upon the binding of the sugar was (11) (a) Wang, W.; Gao, S. H.; Wang, B. H. Org. Lett. 1999, 1, 1209-1212. (b) Liao, Y.; Wang, W.; Wang, B. H. Bioorg. Chem. 1999, 27, 463-476. (12) (a) Haupt, K.; Noworyta, K.; Kutner, W. Anal. Commun. 1999, 36, 391393. (b) Ji, H. S.; McNiven, S.; Ikebukuro, K.; Karube, I. Anal. Chim. Acta 1999, 390, 93-100. (c) Liang, C. D.; Peng, H.; Bao, X. Y.; Nie, L. H.; Yao, S. Z. Analyst 1999, 124, 1781-1785. (d) Lee, S. W.; Ichinose, I.; Kunitake, T. Chem. Lett. 1998, 12, 1193-1194. (e) Kugimiya, A.; Takeuchi, T. Electroanalysis 1999, 11, 1158-1160. (f) Malitesta, C.; Losito, I.; Zambonin, P. G. Anal. Chem. 1999, 71, 1366-1370. (g) Dickert, F. L.; Forth, P.; Lieberzeit, P.; Tortschanoff, M. Fresenius' J. Anal. Chem. 1998, 360, 759762. (13) (a) Lahav, M.; Katz, E.; Doron, A.; Patolsky, F.; Willner, I. J. Am. Chem. Soc. 1999, 121, 862-863. (b) Mirsky, V. M.; Hirsch, T.; Piletsky, S. A.; Wolfbeis, O. S. Angew. Chem., Int. Ed. 1999, 38, 1108-1110. (14) Lahav, M.; Kharitonov, A. B.; Katz, O.; Kunitake, T.; Willner, I. Anal. Chem. 2001, 73, 720-723. (15) Lahav, M.; Kharitonov, A.; Willner, I. Chem. Eur. J. 2001, 7, 3992-3997. (16) (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Nature 1995, 374, 345-347. (b) Yoon, J.; Czarnik, A. W. J. Am. Chem. Soc. 1992, 114, 58745875. (c) Deng, G.; James, T. D.; Shinkai, S. J. Am. Chem. Soc. 1994, 116, 4567-4572. (17) James, T. D.; Murata, K.; Harada, T.; Ueda, K.; Shinkai, S. Chem. Lett. 1994, 273-276.

used for the quantitative analysis of glucose using surface plasmon resonance (SPR), microgravimetric measurements (QCM), and Faradaic impedance spectroscopy. Similarly, cross-linked polymers composed of 2-hydroxyethylmethacrylate and 4-vinylphenylboronic acid were used as functional hydrogels for the binding of nucleotides.19 Also, layered systems composed of the polyanionic acrylic acid/acrylamide boronic acid and the cationic poly(dimethyldiallylammonium chloride) were used to analyze adenosine 5′-monophosphate (AMP) and adenosine 5′-triphosphate (ATP) using a quartz crystal microbalance.20 The boronic acid ligand was often used to imprint molecular recognition sites in polymers for the specific binding21 and separation22 of sugars. For example, imprinted sites for phenyl R-D-mannopyranoside were generated in an acrylic acid-4-vinylphenylboronic acid copolymer.23 Only a few reports attempted to develop imprinted polymers for nucleotides. Ethylene glycol dimethacrylate-methacrylic acid copolymers were used to imprint the pyrimidine/purine bases appearing in nucleic acids by applying hydrogen bonds as recognition interactions.24 Functional fluorophore-labeled imprinted polymers for the cyclic adenosine 3′,5′-monophosphate (cAMP) were prepared, and the fluorescence quenching of the label by the bound nucleotide was used to probe cAMP.25 A further attempt to imprint molecular recognition sites for AMPs included the use of a layered array composed of an anionic acrylic acidacrylamide phenylboronic acid layer, acting as a template for AMP and a cationic polymer layer that caps the template layer with the bound AMP.26 In none of these studies, however, was the specific and selective binding of nucleotides demonstrated. In the present study, we describe the imprinting of specific recognition sites in an acrylamide-acrylamidephenylboronic acid cross-linked copolymer for the following nucleotides; adenosine 5′-monophosphate (AMP, 1), guanosine 5′-monophosphate (GMP, 2), cytosine 5′-monophosphate (CMP, 3), and uridine 5′-monophosphate (UMP, 4) (see Chart 1). We also describe the imprint of specific recognition sites for β-D(+)-glucose (6), D(+)-galactose (7), and β-D(-)-fructose (8) (see Chart 2) in the same copolymer matrix. We assemble the imprinted polymers on Au wire electrodes, piezoelectric Au quartz crystals, or ISFETs. We discuss the use of an electrochemical method (Faradaic impedance spectroscopy), the microgravimetric quartz crystal microbalance analyses, and ISFET measurements as transduction means for the nucleotide and saccharide sensing events. EXPERIMENTAL SECTION Materials. The acrylic monomer functionalized with phenylboronic acid, m-acrylamidephenylboronic acid, was synthesized (18) Gabai, R.; Sallacan, N.; Chegel, V.; Bourenko, T.; Katz, E.; Willner, I. J. Phys. Chem. B 2001, 105, 8196-8202. (19) O ¨ zdemir, A.; Tuncel, A. J. Appl. Polym. Sci. 2000, 78, 268-277. (20) Kanekiyo, Y.; Sano, M.; Iguchi, R.; Shinkai, S. J. Polym. Sci. A 2000, 38, 1302-1310. (21) (a) Wulff, G.; Schmidt, H.; Witt, H.; Zentel, R. Angew. Chem., Int. Ed. Engl. 1994, 33, 188-191. (b) Wulff, G.; Gimpel, J. Macromol. Chem. 1982, 183, 2469-2477. (22) (a) Wulff, G.; Schauhoff, S. J. Org. Chem. 1991, 56, 395-400. (b) Wulff, G.; Haarer, J. Macromol. Chem. 1991, 192, 1329-1338. (23) Wulff, G.; Poll, H. G.; Minarik, M. J. Liq. Chromatogr. 1986, 9, 385-405. (24) Spivak, D. A.; Shea, K. J. Macromolecules 1998, 31, 2160-2165. (25) Turkewitch, P.; Wandelt, B.; Darling, G. D.; Powell, W. S. Anal. Chem. 1998, 70, 2025-2030. (26) Kanekiyo, Y.; Ono, Y.; Inone, K.; Sano, M.; Shinkai, S. J. Chem. Soc., Perkin Trans. 2 1999, 557-561.

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Chart 1. Chemical Structures of Nucleotides

Chart 2. Chemical Structures of Monosaccharides

according to the published procedure.27 Acrylamide, N,N′-methylenebisacrylamide, N,N,N′,N′-tetramethylethylenediamine, nucleotide sodium salts of AMP, CMP, and UMP were purchased from Aldrich, and GMP was from Fluka. Ammonium persulfate and the monosaccharides β-D(+)-glucose, D(+)-galactose, and βD(-)-fructose, were purchased from Sigma. All other chemicals were obtained from Aldrich and were used as supplied. Ultrapure water from Serapur PRO90CN was used throughout all the experiments. Preparation of the Polymer-Modified Electrodes. Nucleotide-imprinted acrylamide-acrylamidephenylboronic acid copolymer (5) films were electrogenerated on an electrode surface (an Au quartz crystal for QCM measurements or an Au wire electrode for Faradaic impedance spectroscopy). The electropolymerization was performed in an aqueous solution composed of m-acrylamidephenylboronic acid (0.2 M), acrylamide (1.8 M), N,N′-methylenebisacrylamide (0.04 or 0.2 M), and ZnCl2 (0.2 M) upon cycling the potential between 0.1 and -1.4 V with a scan rate of 50 mV‚s-1, and waiting for 20 s at -1.4 V (trapezoid sweep). After the performance of 4 or 10 cycles of the polymerization on Au quartz crystals or Au wire electrodes, the electrodes were soaked in a 0.02 M solution of the respective nucleotide for 20 min. This procedure was repeated 4 times until a total of 16 or 40 polymerization cycles for the Au quartz crystals or Au wire electrodes and 4 exposures to the respective nucleotide solution were performed, respectively. The electrodes were finally washed with 0.1 M HCl solution, ethanol, and distilled water. This procedure led to the preparation of the respective nucleotideimprinted copolymer 5 film-functionalized Au electrode. Preparation of the Polymer-Modified ISFETs. For the preparation of the imprinted polymer membranes for the nucleotides or monosaccharides on the ISFET devices, a mixture consisting of acrylamide (1.82 M), m-acrylamidephenylboronic acid (0.18 M), N,N′-methylenebisacryalmide (0.04 or 0.18 M), and the respective substrate (nucleotide or monosaccharide) (0.1 M) was dissolved in 1 mL of a freshly prepared solution of phosphate (27) Kitano, S.; Koyama, Y.; Kataoka, K.; Okano, T.; Sakurai, Y. J. Controlled Release 1992, 19, 162-170.

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saline buffer (PBS; 0.01 M sodium phosphate + NaCl (0.14 M), pH 7.0). The mixture was slightly heated until complete dissolution of the different components was accomplished. The initiators of the polymerization, ammonium persulfate (0.22 M, 0.1 mL) and N,N,N′,N′′-tetramethylenediamine (0.25 mL, 10% v/v), were added to the monomer solution just before placing a 0.1-µL drop of the mixture on the gate interface. The resulting modified chip was dried in air for at least 3 h. The ISFET was then rinsed with an NH3 solution (1% v/v, for 2 min) to eliminate the substrate from the membrane formed. This procedure results in the formation of the imprinted membrane for the respective substrate. The resulting functionalized chip was then thoroughly rinsed with PBS and used in the analysis. Measurements. Faradaic Impedance Spectroscopy. The measurements were conducted in a standard three-electrode electrochemical cell using the polymer-functionalized gold wire electrodes (d ) 0.5 mm, ∼0.2 cm2 geometrical area, roughness factor ∼1.2-1.5) as working electrode. The Au wire electrodes were cleaned prior to the immobilization of the polymer films by boiling in 2 M solution of KOH for 2 h, rinsing with distilled water, followed by 5-min soaking in concentrated HNO3 and rinsing with 0.05 M HEPES buffer, pH 7.4. The cleaned electrodes were stored in concentrated H2SO4. A conventional three-electrode cell consisted of the modified Au wire electrode, a glassy carbon auxiliary electrode separated from the bulk sample solution by a glass frit, and a saturated calomel reference electrode (SCE) connected to the working volume solution by a Luggin capillary. All the potentials are reported versus the SCE reference. The cell was shielded by a grounded Faraday cage. The impedance measurements were carried out using an electrochemical impedance analyzer (EG&G, model 1025) and a potentiostat (EG&G, model 283) connected to a computer (EG&G Software Power Suite 1.03) in 0.05 M HEPES buffer, pH 7.4, as a background electrolyte. A 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture was used as a redox probe. The measurements were performed at a bias potential of 0.175 V equal to the formal potential of the redox probe used, in the frequency range of 100 mHz to 50 kHz, using an alternating voltage of 10 mV. The impedance spectra were plotted in the form of complex plane diagrams (Nyquist plots). The experimental spectra were simulated by electronic equivalent circuits using ZView Version 2.18 software (Scribner Associates, Inc.). Microgravimetric Measurements. A QCM analyzer (EG&G model QCA 917) and a homemade cell for the QCM Seiko electrodes were employed for the microgravimetric analyses. Quartz crystals (AT-cut with resonance frequency ∼9 MHz)

Scheme 1. Electrochemical Synthesis of the Acrylamide-Acrylamidephenylboronic Acid Copolymer (5) with Embedded Imprinted Sites for Nucleotidesa

a

The scheme exemplifies the imprint of AMP (1).

sandwiched between Au electrodes (geometrical area 0.2 ( 0.05 cm2; roughness factor corresponds to ∼3.5) were used. Modification of the Au quartz crystals with a polymer was performed by the same procedure outlined for the Au wire electrodes. After the film formation, the QCM electrode was taken from the monomer solution, washed thoroughly with distilled water, treated with 0.1 M HCl solution, then washed with ethanol and distilled water, and dried in an argon flow. The crystal frequency in air, fair, was measured. Potentiometric ISFET Measurements. ISFET devices with Al2O3 gate interface (20 × 700 µm2, IMT, Neuchaˆtel, Switzerland) were used in all the experiments. As a reference, a Ag/AgCl electrode was used. The chip functionalized with the respective molecularly imprinted film was immersed in the working cell filled with 0.8 mL of PBS and varying concentrations of the respective substrate (nucleotide or monosaccharide). The output signal between the source of the ISFET and the reference electrode was recorded using a semiconductor parameter analyzer (HP 4155B). Each measurement was conducted for 15 min with a time interval of 1 min. The system configuration enables the measurements of the source-gate voltage (Vgs), while the drain current (Id) remained constant (Id ) 100 µA). The difference between Vgs values for the ISFET modified with respective films, with and without embedded substrate, was plotted against the substrate concentration in the bulk solution. All the measurements were carried out at ambient temperature and without stirring, to simulate real conditions of possible future in vivo applications. Reproducibility of the measurements was (2 mV in a number of experiments (n ) 5). The lower detection limits were calculated according to IUPAC recommendations28 as the intercept of the two linear segments in the calibration curves.

Evaluation of the Polymer Film Thickness. The thickness of the m-acrylamidephenylboronate copolymer membrane on the gate interface was evaluated using the method of impedance spectroscopy on ISFET devices.29 The electronic circuit and instruments used to follow the impedance properties of the modified ISFETs as well as the conditions under which the experiments were carried out were described elsewhere.29a To determine the transconductance transfer functions, the output potential, Vout, at variable frequencies ranging from 1 Hz to 100 kHz, was related to the imaginary impedance, Zim. The values of the output potentials corresponding to Zim were normalized at 1 Hz to give the respective transfer functions. RESULTS AND DISCUSSION We have developed an electrochemical method to assemble phenylboronic acid copolymer 5 on Au wire electrodes or Au quartz crystals (Scheme 1). Since the complexes between the m-acrylamidephenylboronic acid and the respective nucleotides exhibit moderate solubilities in aqueous media, the imprinting procedure was performed on the electropolymerized film assembled on the conductive supports at time intervals of polymerization. A thin film (16 or 40 polymerization cycles for Au quartz crystals and Au wire electrodes, respectively) of cross-linked acrylamide-acrylamidephenylboronic acid copolymer 5 was assembled on the Au electrodes by the electropolymerization of the acrylamide and m-acrylamidephenylboronic acid and N,N′-methylenebisacrylamide as cross-linker in the presence of ZnCl2. At (28) Buck, R. P.; Lindner, E. Pure Appl. Chem. 1994, 66, 2527-2536. (29) (a) Kharitonov, A. B.; Wasserman, J.; Katz, E.; Willner, I. J. Phys. Chem. B 2001, 105, 4205-4213. (b) Zayats, M.; Kharitonov, A. B.; Katz, E.; Willner. I. Analyst 2001, 126, 652-657.

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repeated time intervals of polymerization, the resulting polymer was immersed in the respective nucleotide solution to form the complex between the polymer interface and the nucleotide. Electropolymerization followed by treatment with the respective nucleotide solution was continued, and every 4 electropolymerization cycles were followed by one step of interaction with the nucleotide solution for a total number of 16 or 40 electropolymerization cycles on the Au quartz crystals and Au wire electrodes, respectively. The electrogenerated Zn was washed off from the polymer, and the nucleotides were removed by washing with 0.1 M HCl solution, ethanol, and distilled water, to yield the imprinted sites. The imprinted polymer is expected to bind the nucleotide. The association of the hydrophilic nucleotide and the formation of the negatively charged boronate complex are expected to swell the hydrogel, similarly to the swelling of 5 upon the sensing of glucose.18 Thus, any physical technique that probes the swelling of the polymer could act as a transduction method for the association of nucleotides with the imprinted sites.18 We have employed microgravimetric quartz crystal microbalance measurements and Faradaic impedance spectroscopy to probe the association of the nucleotides with the polymers. The electrodes assembled on a quartz crystal are functionalized with copolymer 5 with embedded imprinted sites for the respective nucleotides. The binding of the respective nucleotides to the phenylboronic acid residues causes a mass change, ∆m, that is reflected in the crystal frequency. The change in the crystal frequency upon binding of the analyte molecules is given by the Sauerbray equation (eq 2), where fo is the fundamental frequency

∆f ) -[fo2/NFqA]∆m ) -Cf∆m

(2)

of the crystal, N the shear modulus of quartz (167 kHz‚cm), Fq the quartz density (2.648 g‚cm-3), and A the crystal area. As the substrate mass, which binds to the respectively imprinted copolymers, is governed by the analyte concentration in the sample, the changes in ∆f obtained upon the interaction of the functionalized Au quartz crystal with the imprinted and nonimprinted guest molecules serve as a measure of selectivity. Furthermore, the association of the nucleotides with the boronic acid ligands generates an anionic species, eq 1, resulting in the swelling of the polymer. The uptake of water in the swollen polymer would then alter the mass associated with the crystal and affect its frequency (provided that the measurement is performed in air).18 Faradaic impedance spectroscopy is an effective method to probe the features of surface-modified electrodes.30 The complex impedance is usually presented as a sum of the real, Zre, and imaginary, Zim, components that originate from the resistance and capacitance of the cell, respectively. The general electronic equivalent circuit (Randles and Ershler model30a) includes the ohmic resistance of the elecrolyte solution, Rsol, the Warburg impedance, Zw, originating from the diffusion of ions from the bulk electrolyte to the electrode interface, the double-layer capacitance, Cdl, and the electron-transfer resistance, Ret, that occurs in the (30) (a) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980. (b) Patolsky, F.; Filanovsky, B.; Katz, E.; Willner, I. J. Phys. Chem. B 1998, 102, 10359-10367. (c) Kharitonov, A. B.; Zayats, M.; Alfonta, L.; Katz, E.; Willner, I. Sens. Actuators, B 2001, 76, 203-210.

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Figure 1. Calibration curves corresponding to the frequency changes, ∆f, upon interaction of (a) AMP (1)-imprinted copolymer 5-functionalized Au quartz crystal and (b) nonimprinted film-functionalized Au quartz crystal with variable concentrations of 1. (Inset) Net frequency changes of the AMP-imprinted copolymer 5-functionalized Au quartz crystal (solid line) and nonimprinted copolymer 5-modified Au quartz crystal (dashed line) upon analysis of the different nucleotides at a concentration of 5 × 10-3 M. The points W correspond to the frequency of the polymer generated by washing off the associated nucleotide with water prior to the analysis of the subsequent nucleotide.

presence of a redox probe in the electrolyte solution. The values Cdl and Ret directly relate to the dielectric or insulating features at the electrode/electrolyte interface. A typical shape of a Faradaic impedance spectrum (Nyquist plot) includes a semicircle segment lying on the Zre axis followed by a straight line. This semicircle region, observed at higher frequencies, corresponds to the electron-transfer-limited process, whereas the linear part is characteristic of the lower frequencies and represents the diffusion-limited processes. The spectrum provides information on the electron-transfer kinetics and diffusional characteristics, and the semicircle diameter corresponds to the electron-transfer resistance, Ret. The swelling of the imprinted polymer upon the association of the nucleotide should facilitate the electron transfer between the electrode and the solubilized redox label and, thus, decrease the electron-transfer resistance at the electrode surface.18 The difference between Ret values obtained for the Au electrode functionalized with the respectively imprinted copolymer 5 film before and after its interaction with the imprinted and nonimprinted substrates could serve as a measure of its selectivity to the imprinted substrate. Figure 1, curve a, shows the frequency changes of the AMPimprinted polymer upon the interaction with different concentrations of 1. As the concentration of AMP increases, the frequency

of the crystal decreases as a result of swelling and the uptake of water. At concentrations higher than 5 × 10-3 M, the changes in the crystal frequency level off. This is consistent with the saturation of the polymer with the nucleotide. Figure 1, curve b, shows the frequency changes of the nonimprinted polymer interface upon interaction with different concentrations of 1. The nonimprinted polymer was assembled on the Au quartz crystal (or Au electrodes) by the electropolymerization of the monomers and cross-linker under conditions similar to that for the preparation of the imprinted polymer, but in the absence of the nucleotide(s). A substantially lower change in the crystal frequency, ∆f, is observed, implying a lower degree of swelling of the nonimprinted polymer or a lower affinity and capacity for the association of 1. Figure 1(inset), solid line, shows the absolute frequency changes of the AMP-imprinted polymer-functionalized Au quartz crystal upon the analysis of a constant concentration of the different nucleotides, 1-4. In this experiment, the sensing interface is interacted with AMP (5 mM), which results in a frequency change of ∼-450 Hz. The bound AMP was washed off to restore the initial frequency of the pure imprinted polymer interface and further treated with UMP, which yields a frequency change of only ∼-160 Hz. The 1-imprinted polymer film was then used to sense the other nucleotides by the regeneration of the sensing interface with a washing cycle and the analysis of the respective nucleotide. After the nonimprinted nucleotides were analyzed, AMP was reanalyzed to confirm that the binding capacity of the polymer toward the imprinted substrate is retained. It is evident that while the frequency change for AMP is in the range of -400 to -450 Hz, for the other nucleotides, the frequency changes are only -160 to -220 Hz. Figure 1 (inset), dashed line, shows the frequency changes of the nonimprinted polymer-functionalized Au quartz crystal upon analysis of the different nucleotides. Clearly, the association of the different nucleotides with the nonimprinted polymer yields almost identical frequency changes in the range of -100 to -200 Hz. Thus, the binding capacity, and the swelling, of the nonimprinted polymer toward the different nucleotides is almost identical. These results clearly indicate that the AMPimprinted polymer reveals selectivity toward the binding of 1. The other nucleotides, GMP, UMP, and CMP, reveal a binding affinity to the AMP-imprinted polymer similar to that of the nucleotides to the nonimprinted polymer. The 1-imprinted polymer reveals a high affinity for the association of the imprinted substrate, AMP. The low affinity of the nucleotides to the nonimprinted polymer is attributed to the random binding of the nucleotides to the boronic acid ligand. The enhanced binding of AMP to the imprinted polymer is attributed to the successful imprint of the molecular contours in the polymer. Scheme 1 outlines schematically the formation of the molecular-imprinted sites. The association of the nucleotide with the boronic acid ligand yields the basic template for the imprinting. Hydrogen bonds between the acrylamide monomers and the different functionalities on the nucleotide yield, upon polymerization, the structural contour and the anchoring H-bonding sites acting synergistically in the specific binding of the target nucleotide. The imprinting procedure of selective recognition sites for nucleotides in the polymer 5 appears to be general. Figure 2, curve a, shows the frequency changes of the GMP-imprinted Au quartz crystal upon the analysis of GMP. Figure 2, curve b, depicts

Figure 2. Calibration curves corresponding to the frequency changes, ∆f, upon interaction of (a) GMP (2)-imprinted copolymer 5 film-functionalized Au quartz crystal and (b) nonimprinted copolymer 5-functionalized Au quartz crystal with variable concentrations of 2. (Inset) Net frequency changes of the 2-imprinted copolymer 5-functionalized Au quartz crystal (solid line) and nonimprinted copolymer 5-modified Au quartz crystal (dashed line) upon sensing different nucleotides at a concentration of 1.7 × 10-2 M. The points W correspond to the frequency of the polymer generated by washing off of the associated nucleotide with water prior to the analysis of the subsequent nucleotide.

the frequency changes of the crystal upon the analysis of 2 by the nonimprinted polymer. As before, the binding affinity of the GMP-imprinted substrate toward the imprinted substrate is substantially higher than that of the nonimprinted polymer. Figure 2 (inset), solid line, shows the frequency changes upon the analysis of a constant concentration of the different nucleotides by the GMP-imprinted polymer. For comparison, the dashed line in Figure 2 (inset) shows the frequency changes of the nonimprinted polymer upon the analysis of the different nucleotides. The analysis of GMP (17 mM) by the GMP-imprinted polymer yields a frequency change of ∼-550 Hz while the association of the foreign nucleotides alters the frequency of the modified crystal by -190 to -250 Hz only. Similar results are observed upon the imprinting of the other two nucleotides, 3 and 4. (See Supporting Information.). (i) The binding capacity of the imprinted polymers to the imprinted substrates is substantially higher than to that of the nonimprinted polymer. (ii) The imprinted polymers reveal selectivity toward the binding of the imprinted nucleotide. For example, the association of CMP (0.5 mM) with the CMPimprinted polymer is accompanied by a frequency change of -540 Hz, while the binding of all other nucleotides yields a frequency change of only -160 to -240 Hz. In fact, the success of imprinting specific recognition sites for the different nucleotides in the acrylamidephenylboronic acidAnalytical Chemistry, Vol. 74, No. 3, February 1, 2002

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Figure 3. Net frequency changes of the CMP (3)-imprinted copolymer 5-functionalized Au quartz crystals that include imprinted membranes with different cross-linking degrees upon the analysis of the different nucleotides, 5 × 10-4 M. The monomer-to-cross-linker ratio corresponds to 50:1 (solid line) and 10:1 (dashed line).

acrylamide copolymer is surprising since the polymer exists as a hydrogel. The high content of water in the polymer and its structural flexibility would perturb the structural features of the cavities. The successful imprint of the molecular recognition cavities suggests that even in the hydrogel structure the cooperative effects of the boronic acid ligand and the anchoring hydrogen bond sites yield sufficiently rigid contours for the association of the respective nucleotides (cf. Scheme 1). This implies, however, that the quality of the imprinted sites would be controlled by the degree of cross-linking of the polymer matrix. Higher cross-linking of the polymer would rigidify the hydrogel and up to a certain degree of cross-linking, the recognition features of the molecular cavities should be improved through rigidification. Figure 3 exemplifies the frequency changes of the CMP-imprinted polymer film generated at two levels of cross-linking (monomer-crosslinker 50:1 (solid line) and 10:1 (dashed line)) upon the analysis of the different nucleotides. The polymer film with enhanced crosslinking reveals two features: (i) The frequency changes upon the association of the imprinted substrate are slightly higher than that in the presence of the polymer with the lower degree of crosslinking. This suggests that the affinity of the imprinted polymer with a higher degree of cross-linking to the imprinted substrate is enhanced. (ii) The frequency changes caused by the association of the foreign nucleotides with the polymer with a higher degree of cross-linking are lower than those observed in the presence of the polymer with the lower degree of cross-linking. These results suggest that the binding of the foreign nucleotides to the boronic acid ligand of the polymer backbone is inhibited at higher crosslinking. This may be explained by the rigidification of the polymer matrix to a degree that perturbs the association of the nonimprinted nucleotides with the boronic acid ligands. (For similar effects of cross-linking on the UMP-imprinted polymer see Supporting Information.) The swelling of the polymers upon the association of the nucleotides enhances the permeability of the electrolyte through the polymer matrixes. Thus, electron transfer to a solution 708 Analytical Chemistry, Vol. 74, No. 3, February 1, 2002

Figure 4. Faradaic impedance spectra of the AMP (1)-imprinted copolymer 5-functionalized Au electrode: (a) prior to treatment with nucleotides; (b) after interaction with 1; (c) upon interaction with GMP (2); (d) upon interaction with UMP (4). Concentration of the nucleotides corresponded to 10 mM. (Inset) Changes in the interfacial electron-transfer resistances as a result of the interaction of the 1-imprinted copolymer 5-functionalized Au electrode with the imprinted substrate, 1, and other nucleotides 2 and 4. The nucleotide associated with the imprinted membrane is washed off with the buffer solution prior to the performance of the subsequent nucleotide analysis (points a in the figure).

solubilized redox label should be facilitated upon the swelling of the polymers. That is, the interfacial electron-transfer resistance to a redox label, derived from Faradaic impedance measurements, should decrease upon the swelling of the polymer. Figure 4 shows the Faradaic impedance spectra of the AMP-imprinted polymerfunctionalized Au electrode prior to treatment with the nucleotides (curve a) and after interaction with 1 (curve b). The interfacial electron-transfer resistance decreases from 7.1 to 5.1 kΩ after the association of 1. The decrease in the interfacial resistance of the polymer after the binding of 1 is consistent with the swelling of the polymer. Curves c and d in Figure 4 show the impedance spectra of the polymer treated with either 2 or 4, respectively. The interfacial electron-transfer resistances are identical, Ret ) ∼6.5 kΩ. The decrease in the interfacial electron-transfer resistances of the AMP-imprinted polymer treated with GMP or UMP is substantially lower than that observed with AMP, implying a lower swelling degree of the polymers to the “foreign” nucleotides. Figure 4 (inset) shows the changes in the interfacial electrontransfer resistances of the AMP-imprinted polymer upon the sensing of the imprinted substrate, AMP, and the other nucleotides. Similar results are observed with the other nucleotides (See also Supporting Information.). For example, Figure 5 shows the Faradaic impedance spectra of the CMP-imprinted polymerfunctionalized electrode prior to treatment with nucleotides, curve a, after treatment with 3, curve b, and as a result of interaction with the foreign nucleotides, 1, 2, and 4, curves c, d, and e, respectively. While the association of the imprinted substrate results in a significant decrease in the interfacial electron-transfer

Figure 5. Faradaic impedance spectra of the CMP (3)-imprinted copolymer 5-functionalized Au electrode: (a) prior to the treatment with nucleotides; (b) after treatment with CMP (3); (c) upon interaction with AMP (1); (d) after interaction with GMP (2); (e) upon interaction with UMP (4). The concentration of each of the nucleotides corresponded to 10 mM. (Inset) Changes in the interfacial electrontransfer resistances as a result of the interaction of the 3-imprinted copolymer 5-functionalized Au wire electrode with the imprinted substrate, 3, and other nucleotides 1, 2, and 4. The nucleotide associated with the imprinted membrane is washed off with HEPES buffer solution prior to the performance of the subsequent nucleotde analysis (points a in the figure).

resistance, ∆Ret ) 4.6 kΩ, indicating a high degree of polymer swelling, the other bases lead to a substantially lower extent of decrease of the interfacial electron-transfer resistance, ∆Ret ) 2.2, 1.6, and 1.4 kΩ for 1, 2. and 4, respectively (see Figure 5 (inset)). The changes in the interfacial electron-transfer resistances of the nonimprinted polymer-functionalized electrode yield an almost similar, and relatively small, change in the interfacial electrontransfer resistance upon interaction with the different nucleotides, ∆Ret ) 0.5 ( 0.2 kΩ. These results indicate that the nonimprinted polymer exhibits a moderate and similar affinity to the different nucleotides that results in a small degree of swelling. The Faradaic impedance spectroscopy results complement nicely the microgravimetric quartz crystal microbalance analyses. It is evident that the imprinting of the respective nucleotide contour into the polymer yields a functional polymer of high binding affinity and, thus, enhanced selectivity to the imprinted nucleotide. A further electronic means to transduce the association of the nucleotides with the imprinted sites involves the application of ISFET devices. The drain current of the ISFET device is switched on at a certain gate potential by the application of the appropriate potential between the source and gate electrodes. Alteration of the gate charge by a chemical modification or chemical recognition event alters the gate potential and perturbs the drain current. Thus, in order to retain the constant current flow, the gate-source voltage must be adjusted to compensate the potential changes on the gate. Recently, inorganic TiO2 films with imprinted sites

Figure 6. Gate-source potential changes of the ISFET functionalized with the β-D(+)-glucose (6)-imprinted copolymer 5 (monomer/ cross-linker ratio 50:1) upon the sensing of (a) different concentrations of 6; (b, c) varying concentrations of galactose (7) and fructose (8), respectively; (d) response of the ISFET functionalized with the nonimprinted copolymer 5 membrane to varying concentrations of 6; (e) gate-source potential change of the ISFET functionalized with the 6-imprinted copolymer 5 (monomer/cross-linker ratio 10:1) upon sensing of different concentrations of 6.

for chloroaromatics were assembled on the ISFET gate surfaces,14 and the ISFET device was employed for the selective sensing of 4-chlorophenoxyacetic acid or 2,4-dichlorophenoxyacetic acid. Similarly, chiral recognition sites were imprinted in a TiO2 film associated with ISFET devices, and the systems were employed for the chiroselective sensing of the imprinted substrates.15 The association of cis-diols with the boronic acid ligand charges the polymer, eq 1. This suggests that the assembly of a boronic acid polymer, and specifically an imprinted boronic acid polymer, on the gate surface of the ISFET could yield a functional interface that controls the gate potential upon binding of the host substrate to the polymer matrix (charging of the polymer). To prove the ability to sense substrates by the boronic acid copolymer on the ISFET device, initial experiments were performed using glucose, galactose, and fructose as imprinted substrates. The copolymer 5 was deposited on the gate surface of the ISFET device in the presence of glucose, fructose, or galactose as imprints. The imprinted saccharides were washed off from the copolymer membrane with 1% NH3 to yield the molecular contours of the respective saccharides in the membrane. Figure 6, curve a, shows the gate-source potential, Vgs, of the glucose-imprinted polymerfunctionalized ISFET, upon the analysis of different concentrations of glucose. It is evident that glucose is sensed by the functionalized device in the concentration range of 1 × 10-6-8 × 10-4 M, with a sensitivity of 18.0 mV‚decade-1 and a lower detection limit corresponding to 8 × 10-7 M. Curves b and c of Figure 6 show the analysis of 7 and 8 by the glucose-imprinted polymer film. Clearly, the glucose-imprinted polymer reveals specificity, and the foreign sugars are differentiated by the functional polymer membrane. Also, the imprinted polymer reveals a substantially higher affinity for glucose, as compared to the nonimprinted membrane, leading to the enhanced sensitivity of the imprinted Analytical Chemistry, Vol. 74, No. 3, February 1, 2002

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polymer-functionalized device. Figure 6, curve d, shows the ISFET response upon the analysis of glucose by the nonimprinted polymer membrane. The response of the device is substantially lower, and the sensitivity for the detection of glucose by the nonimprinted polymer membrane in the entire concentration range of 1 × 10-6-1 × 10-2 M corresponds to 5.0 mV‚decade-1. Curves a and e of Figure 6 show the responses of the glucoseimprinted polymer-functionalized ISFET devices, where the polymer membranes are cross-linked using a monomer/cross-linker ratio of 50:1 and 10:1, respectively. It is seen that, as the crosslinking degree increases, the sensitivity of the functional device is slightly elevated to 21.0 mV‚decade-1. This is consistent with our hypothesis that enhanced cross-linking leads to rigidified molecular contours of improved recognition properties. Similar results are observed upon the imprinting of the other monosaccharides in the phenylboronic acid acrylamide-acrylamide copolymer (see Supporting Information). The galactose- and fructoseimprinted polymers reveal specificity for the imprinted substrates, and the nonimprinted substrates are not recognized by the polymers. Interestingly, the imprinted polymers reveal a lower affinity for the foreign, nonimprinted, saccharides than the nonimprinted polymer. This might originate from the rigidification of the polymer matrix by the imprinted molecular contours, a process that perturbs the association of the foreign monosaccharides with the boronic acid ligands. It should be noted that the sensitivity of the imprinted polymer-functionalized ISFET devices increases from 18.0 to 56.2 mV‚decade-1 in the order glucose < galactose < fructose. This is explained by the corresponding stability constants, log Ka, of the complexes formed between boronic acid and the saccharides: 2.04, 2.44, and 3.64 for 6-8, respectively. The acrylamide-acrylamidephenylboronic acid copolymer was also employed as a matrix to imprint molecular recognition sites for the nucleotides and for the analysis of the nucleotides by the ISFET devices. The polymer was in situ polymerized in the presence of the respective nucleotide 1, 2, 3, or 4 on the gate interface of the ISFET. The nucleotides were washed off with 1% NH3 to yield the specific imprinted sites in the polymer membrane. The formation of the boronic acid-ribose unit complex provides the primary template for the generation of the imprinted cavity. The secondary association of acrylamide polymer units with the purine or pyrimidine bases or phosphate sites through hydrogen bonds provides the mechanism for rigidifying the molecular recognition contour in the polymer and for the orientation of the respective functional groups for binding of the substrate. Figure 7A, curve a, shows the gate-source potential of the ISFET device functionalized with the copolymer 5 membrane that includes imprinted sites for AMP, upon the sensing of variable concentrations of AMP. The substrate is detected in the concentration range of 3.0 × 10-5-5 × 10-3 M with a sensitivity that corresponds to 14.0 mV/decade and a detection limit of 1.5 × 10-5 M. Curves b-d of Figure 7A show the responses of the AMPimprinted ISFET device upon the sensing of GMP, CMP, and UMP, respectively. Clearly, the responses of the device to the nonimprinted substrates are substantially lower. For example, GMP and CMP are detected only at concentrations higher than 10-3 M with the poor sensitivity of 2 and 4 mV/decade. Figure 7A, curve e, shows the response of a nonimprinted acrylamide710 Analytical Chemistry, Vol. 74, No. 3, February 1, 2002

Figure 7. (A) Gate-source potential changes of the ISFET functionalized with the AMP (1)-imprinted copolymer 5 film upon the analysis of (a) varying concentrations of imprinted substrate 1; (bd) different concentrations of nonimprinted substrates GMP (2), CMP (3), and UMP (4), respectively; (e) response of the nonimprinted copolymer 5-functionalized ISFET to varying concentrations of 1. (B) Gate-source potential changes of the ISFET functionalized with the 4-imprinted copolymer 5 film upon the analysis of (a) varying concentrations of imprinted substrate 4; (b-d) different concentrations of the nonimprinted nucleotides 1, 2, and 3, respectively; (e) response of the nonimprinted copolymer-functionalized ISFET to varying concentrations of 4.

acrylamidephenylboronic acid membrane in the presence of AMP. Clearly the polymer membrane sensitivity is only 5 mV/decade in the concentration range of 5 × 10-4-1 × 10-2 M. Similar results are observed with the GMP-, CMP-, and UMP-imprinted membranes on the ISFET device (see Supporting Information). The GMP-imprinted membrane associated with the ISFET device allows the sensing of 2 in the concentration range of 4 × 10-5-2 × 10-3 M with a sensitivity of 17 mV/decade and a detection limit of 1.5 × 10-5 M. The response of the GMP-imprinted membrane to 1, 3, and 4 is very low. The CMP-imprinted membrane associated with the ISFET device reveals selectivity toward the

detection of 3. The functionalized device enables the sensing of 3 in the concentration range of 2 × 10-6-5 × 10-4 M with a sensitivity of 17 mV/decade and a detection limit that corresponds to 8 × 10-7 M. The other nucleotides, 1, 2, and 4, yield only very low responses in the presence of the CMP-imprinted membrane, implying low affinity of these nucleotides to the imprinted membrane. Similarly, Figure 7B, curve a, shows the analysis of 4 by the UMP-imprinted film associated with the ISFET device. The imprinted substrate is sensed in the concentration range of 1.0 × 10-5-5.0 × 10-3 M, with a sensitivity that corresponds to 27.0 mV/decade and a lower detection limit of 2.0 × 10-5 M. As before, the imprinted polymer reveals sensing selectivity and the other nucleotides, 1-3, are differentiated by the sensing interface (curves b-d, respectively). This set of experiments applying the nucleotide-imprinted polymers in analyzing the respective imprinted substrates, and the complementary control experiments, reveal some important conclusions on the activity and selectivity of the functional imprinted polymer membranes: (i) Imprinting of molecular recognition sites in the polymer membranes enhances the detection limits and the sensitivities of the sensing interfaces. This is probably due to the higher binding affinities of the imprinted membranes for the imprinted substrates. (ii) The imprinted polymer membranes reveal structural selectivity toward the sensing of the imprinted substrates. This suggests that the imprinting process leads to well-defined molecular contours that include the boronic acid ligand and H-bonding anchoring sites in appropriately aligned positions in the tailored cavities. In contrast to the previous nucleotide-imprinted polymer systems that employ microgravimetric QCM or Faradaic impedance spectroscopy as transduction means, where a high level of response of the foreign nucleotide or of the nonimprinted interface was detected, the ISFET devices with the imprinted polymer membranes reveal high specificity and low noise responses for the foreign nucleotides. Similarly, the nonimprinted polymer membranes on the ISFET devices reveal minute response signals to the nucleotides. This difference probably originates from the different mechanisms leading to the transduced signals. The microgravimetric QCM or Faradaic impedance signals originate from the swelling of the membranes upon the association of the nucleotides, whereas the ISFET response is attributed to the charge of the polymer membrane. The low-capacity binding of the foreign nucleotides to the imprinted polymers or the binding of the nucleotides to the nonimprinted membranes is sufficient to alter the swelling degree of the polymer, leading to the microgravimetric or impedance responses. This low-capacity binding is presumably insufficient to alter the gate potential and to yield the ISFET response. An attempt was made to characterize the sensing interface and the imprinted sites in the film. The film thickness was determined by the use of impedance spectroscopy measurements on the modified gate interface.31 The impedance features of the gate interface are controlled by the chemical composition of the (31) (a) Antonisse, M. M. G.; Snellink-Rue¨l, B. H. M.; Lughtenberg, R. J. W.; Engbersen, J. F. J.; van den Berg, A.; Reinhoudt, D. N. Anal. Chem. 2000, 72, 343-348. (b) Lughtenberg, R. J. W.; Egberink, R. J. M.; van den Berg, A.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Electroanal. Chem. 1998, 452, 69-86.

Figure 8. Transconductance curves at variable frequencies for (a) bare ISFET device and (b) acrylamide-acrylamidephenylboronic acid copolymer film-functionalized ISFET device. Using the corresponding time constants, τ1 and τ2 and eqs 3-5, the film thickness was estimated to be 360 ( 10 Å.

modified gate.32 In recent studies,15,29 we suggested impedance spectroscopy measurements on a chemically functionalized gate as a method to evaluate the thickness of chemically assembled thin films on the gate surface. According to this method, the transconductance functions of the systems are recorded at variable applied frequencies, and the time constants, τ1, eq 3, and τ2, eq 4,

τ1 ) Rmem(Cmem + Cox) ≈ RmemCox

(3)

τ2 ) RmemCmem

(4)

are extracted from the curves (cf. Figure 8). Using these time constants, the resistance of the polymer membrane, Rmem, and its capacitance, Cmem, were calculated. The value Cox corresponds to the capacitance of the Al2O3 gate, on which the polymer film is assembled. From these values, the polymer film thickness δ was determined using eq 5, where o is the dielectric constant of the

Cmem ) omemA/δmem

(5)

vacuum (o ) 8.85 × 10-12 F‚m-1), mem is the dielectric constant of the membrane (mem ) 3.0), and A (1.4 × 10-8 µm2) is the gate area. The film thickness was calculated to be 360 ( 10 Å. CONCLUSIONS The present study has addressed the imprinting of molecular recognition sites for nucleotides and monosaccharides in an acrylamide-acrylamidephenylboronic acid cross-linked polymer membrane. We have used microgravimetric quartz crystal mi(32) (a) Souteyrand, E.; Cloarec, J. P.; Martin, J. R.; Wilson, C.; Lawrence, I.; Mikkelsen, S.; Lawrence, M. F. J. Phys. Chem. B 1997, 101, 2980-2985. (b) Cloarec, J. P.; Martin, J. R.; Polychronakos, C.; Lawrence, I.; Lawrence, M. F.; Souteyrand, E. Sens. Actuators, B 1999, 58, 394-398. (c) Prasad, B.; Lal, R. Meas. Sci. Technol. 1999, 10, 1097-1104. (d) Chovelon, J. M.; Jaffrezic-Renault, N.; Gros, Y.; Fombon, J. J.; Pedone, D. Sens. Actuators, B 1991, 3, 43-50.

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crobalance measurements, Faradaic impedance spectroscopy, and ion-sensitive field-effect transistor devices to probe and transduce the association of the respective substrates with the imprinted membranes. The study revealed that the imprinting of the molecular recognition sites in the polymer leads to several unique features of the functional polymer: (i) The detection limit and sensitivity of the imprinted polymers for the imprinted substrates are enhanced as compared to the detection limits and sensitivities of the corresponding nonimprinted polymers. This implies that the binding affinities of the substrates to the respective imprinted sites in the polymers are enhanced. (ii) The imprinted polymers reveal selectivity to the imprinted substrates. The selectivity is enhanced by increasing the cross-linking degree of the polymer upon imprinting. This is explained by the higher rigidity of the imprinted sites at higher degrees of cross-linking. The specificity of the imprinted sites in the organic polymer is attributed to the cooperative binding interactions between the imprinted substrate and the polymer consisting of the boronic acid ligand as the ligating site for the monosaccharides or the nucleotides ribose units and complementary H-bonds between the acrylamide units and the monosaccharides or nucleotides. The multisite binding cavity generates a rigid structural contour for the binding of the imprinted saccharides or nucleotides. The sensing of the nucleotides by the microgravimetric QCM analyses and the electrochemical detection of the nucleotides, using Faradaic impedance spectroscopy, differ mechanistically from the analyses employing the functional ISFET devices. While the microgravimetric and electrochemical detection means are based on the swelling of the functional imprinted membranes, the ISFET analyses of the nucleotides (or monosaccharides) are based on the alteration of the membrane charge by the formation of complexes with the boronate ligand. This difference leads to different sensitivity and

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different response times of the functional sensing devices. Since the swelling process is slow, the response times of the microgravimetric measurements is ∼10-12 min. The response time of the ISFET device is substantially faster, ∼5 min. Since observable swelling requires substantial loading of the membranes with the analyzed nucleotides, the microgravimetric and electrochemical (impedance) analyses of the nucleotides is ∼102-103 less sensitive than the detection of the nucleotides by the ISFET devices. The successful selective analysis of the nucleotides suggests, however, that future applications of the functional polymer devices could include the sequencing of nucleic acids. ACKNOWLEDGMENT This research is supported by The Israel Ministry of Science as an Infrastructure Project in Material Science. I.W. acknowledges the Max Planck Research Award for International Cooperation. SUPPORTING INFORMATION AVAILABLE Microgravimetric analyses of the different nucleotides by GMPand UMP-imprinted membranes associated with Au quartz crystals; analyses of the different nucleotides by the GMP- or UMPimprinted membranes associated with electrodes and using Faradaic impedance spectroscopy; analyses of the different saccharides or nucleotides by D(+)-galactose-, fructose-, GMP-, and CMP-imprinted membranes associated with ISFET devices. This material is available free of charge via the Internet at http:// pubs.acs.org.

Received for review September 10, 2001. Accepted November 12, 2001. AC0109873