NAD(P)H Cofactors by Imprinted Polymer

Molecularly Imprinted Membranes. Francesco Trotta , Miriam Biasizzo , Fabrizio Caldera. Membranes 2012 2 (3), 440-477 ...
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Anal. Chem. 2003, 75, 509-517

Analysis of NAD(P)+/NAD(P)H Cofactors by Imprinted Polymer Membranes Associated with Ion-Sensitive Field-Effect Transistor Devices and Au-Quartz Crystals Svetlana P. Pogorelova, Maya Zayats, Tatyana Bourenko, Andrei B. Kharitonov, Oleg Lioubashevski, Eugenii Katz, and Itamar Willner*

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Specific recognition sites for the NAD(P)+ and NAD(P)H cofactors are imprinted in a cross-linked acrylamideacrylamidophenylboronic acid copolymer membrane. The imprinted membranes, associated with pH-sensitive fieldeffect transistors (ISFETs) or Au-quartz piezoelectric crystals, enable the potentiometric or microgravimetric analysis of the oxidized NAD(P)+ cofactors and the reduced NAD(P)H cofactors, respectively. The NAD+- and NADP+-imprinted membranes associated with the ISFET allow the analysis of NAD+ and NADP+ with sensitivities that correspond to 15.0 and 18.0 mV‚decade-1 and detection limits of 4 × 10-7 and 2 × 10-7 M, respectively. The NADH- and NADPH-imprinted membranes associated with the ISFET device enable the analysis of NADH and NADPH with sensitivities that correspond to 24.2 and 21.8 mV‚decade-1 and lower detection limits that are 1 × 10-7 and 2 × 10-7 M, respectively. The ISFET devices functionalized with the NADH and NADPH membranes are employed in the analysis of the biocatalyzed oxidation of lactic acid and ethanol in the presence of lactate dehydrogenase and alcohol dehydrogenase, respectively. The quantitative analysis of the oxidized cofactors β-nicotinamide adenine dinucleotide (NAD+) and β-nicotinamide adenine dinucleotide phosphate (NADP+) and the respective reduced 1,4dihydro-β-nicotinamide adenine dinucleotide cofactors (NAD(P)H) are of utmost importance in developing biosensors involving NAD(P)+/NAD(P)H-dependent enzymes1 and in monitoring biocatalyzed transformations that involve NAD(P)+/NAD(P)H-dependent enzymes.2 The amperometric analysis of NAD(P)+ reveals irreversible electrochemical reduction accompanied by high overpotentials3 and resulting in a nonenzymatically active dimer.4 The * To whom correspondence should be addressed. Tel: 972-2-6585272. Fax: 972-2-6527715. E-mail: [email protected]. (1) (a) Bartlett, P. N.; Tebbutt, P.; Whitaker, R. G. Prog. React. Kinet. 1991, 16, 55-155. (b) Katz, E.; Shipway, A. N.; Willner, I. In Bioelectrochemistry; Wilson, G. S., Ed.; Encyclopedia of Electrochemistry 9; Wiley-VCH GmbH: Weinheim, Germany, 2002; Chapter 17, p 559. (2) Biade, A. E.; Bourdillon, C.; Laval, J. M.; Mairesse, G.; Moiroux, J. J. Am. Chem. Soc. 1992, 114, 893-897. (3) (a) Burnett, J. N.; Underwood, A. L. Biochemistry 1975, 5, 2060-2066. (b) Schmakel, C. O.; Santhanam, K. S. V.; Elving, P. J. J. Am. Chem. Soc. 1975, 97, 5083-5092. (4) Cunningham, A. J.; Underwood, A. L. Biochemistry 1967, 6, 266-271. 10.1021/ac020292h CCC: $25.00 Published on Web 12/20/2002

© 2003 American Chemical Society

NAD(P)+ cofactors were reduced electrocatalytically or bioelectrocatalytically in the presence of Rh complexes5 or in the presence of NAD(P)+-dependent enzymes (e.g., ferredoxin-NADP+ reductase, lipoamide dehydrogenase, formate dehydrogenase),6 respectively. Direct, nonmediated electrochemical reduction of NAD(P)+ was also achieved at modified electrodes, e.g., in the presence of an L-histidine-modified Ag electrode.7 Similarly, the direct oxidation of NAD(P)H is electrochemically irreversible and involves high overpotentials.8 The electrocatalyzed two-electron oxidation of NAD(P)H was extensively studied in the presence of different electrocatalysts,9 for example, o-quinones,10 phenazine, phenoxazine, and phenothiazine derivatives.11 There is no method, however, to selectively analyze NAD+ in the presence of NADP+ or alternatively to selectively analyze NADH and NADPH. Imprinting of molecular recognition sites in organic12 or inorganic polymers13 has been the subject of extensive research efforts. There are two general methods to generate imprinted sites in polymer membranes. One approach14 involves the polymerization of monomers that include a complementary function to the imprinted substrate such as H-bonds, electrostatic interactions, and π-donor-acceptor interactions. Polymerization of the mono(5) (a) Ruppert, R.; Herrmann, S.; Steckhan, E. Tetrahedron Lett. 1987, 28, 6583-6586. (b) Wienkamp, R.; Steckhan, E. Angew. Chem., Int. Ed. Engl. 1982, 21, 782-783. (c) Chardon-Noblat, S.; Cosnier, S.; Deronzier, A.; Vlachopoulos, N. J. Electroanal. Chem. 1993, 352, 213-228. (6) (a) De Lacey, A. L.; Bes, M. T.; Gomez-Moreno, C.; Fernandez, V. M. J. Electroanal. Chem. 1995, 390, 69-76. (b) Chao, S.; Wrighton, M. S. J. Am. Chem. Soc. 1987, 109, 5886-5888. (c) Bergel, A.; Comtat, M. Bioelectrochem. Bioenerg. 1992, 27, 495-500. (7) Long, Y.-T.; Chen, H.-Y. J. Electroanal. Chem. 1997, 440, 239-242. (8) (a) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 47, 1337-1343. (b) Samec, Z.; Elving, P. J. J. Electroanal. Chem. 1983, 144, 217-234. (9) (a) Katakis, I.; Dominguez, E. Mikrochim. Acta 1997, 126, 11-32. (b) Gorton, L.; Persson, B.; Hale, P. D.; Boguslavsky, L. I.; Karan, H. I.; Lee, H. S.; Skotheim, T. A.; Lan, H. L.; Okamoto, Y. In Biosensors and Chemical Sensors; Edelman, P. G.; Wang, J., Eds.; ACS Symposium Series 487; American Chemical Society: Washington, DC,1992; Chapter 6, pp 56-83. (10) (a) Jaegfeldt, H.; Kuwana, T.; Johansson, G. J. Am. Chem. Soc. 1983, 105, 1805-1814. (b) Miller, L. L.; Valentine, J. R. J. Am. Chem. Soc. 1988, 110, 3982-3989. (11) (a) Gorton, L. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1245-1258. (b) Persson, B.; Gorton, L. J. Electroanal. Chem. 1990, 292, 115-138. (c) Schlereth, D. D.; Katz, E.; Schmidt, H.-L. Electroanalysis 1995, 7, 46-54. (12) (a) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (b) Sellergren, B. Trends Anal. Chem. 1997, 16, 310-320. (c) Haupt, K. Analyst 2001, 126, 747-756. (d) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495-2504.

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mer-substrate complex, followed by the removal of the substrate molecules acting as a template for the polymerization, yields the imprinted sites in the polymer. The second approach involves the covalent attachment15 or coordination16 of the substrate to polymerizable monomer units, followed by the copolymerization of the functional monomers with other monomers, to yield rigidified polymer matrixes. Cleavage of the polymer-linked substrate units leads to the formation of the polymer with imprinted sites. Polymers with imprinted sites, revealing structural17 and chiral18 selectivities, have been prepared. The imprinted polymers were used as specific sensing interfaces,19 functional materials for chromatographic separations,20 and matrixes for selective and catalyzed chemical transformations.21 The use of imprinted polymers is particularly tempting for sensing applications since membranes with tailored recognition functions can be generated. A major difficulty encountered in the use of imprinted polymers as active components in sensor devices is the coupling of the sensing membrane with an electronic transducer. The imprinted organic polymer is usually relatively thick, and the recognition sites lack direct electrical contact with the transducer. Indeed, most of the sensor devices based on imprinted polymers are either optical, and include chromogenic markers,22 or involve the microgravimetric analysis of the bound substrate using piezoelectric crystals (quartz crystal microbalance, QCM).23 Recently, we reported on the functionalization of the gate surface of ion-sensitive field-effect transistors (ISFETs) with imprinted TiO2 films for the potentiometric stereoselective24 and chiroselective25 analysis of different carboxylic acids by the functional devices. (13) (a) Morihara, K. In Molecular and Ionic Recognition with Imprinted Polymers; Bartsch, R. A., Maeda, M., Eds.; ACS Symposium Series 703; American Chemical Society: Washsington, DC, 1998; pp 300-313. (b) Katz, A.; Davis, M. E. Nature 2000, 403, 286-289. (c) Ichinose, I.; Senzu, H.; Kunitake, T. Chem. Mater. 1997, 9, 1296-1298. (d) Lee, S.-W.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 2857-2863. (e) Zayats, M.; Lahav, M.; Kharitonov, A. B.; Willner, I. Tetrahedron 2002, 58, 815-824. (14) (a) Klein, J. U.; Whitcombe, M. J.; Mulholland, F.; Vulfson, E. N. Angew. Chem., Int. Ed. 1999, 38, 2057-2060. (b) Yilmaz, E.; Mosbach, K.; Haupt, K. Anal. Commun. 1999, 71, 285-287. (c) Sellergren, B.; Shea, K. J. J. Chromatogr., A 1995, 690, 29-39. (15) (a) Wulff, G.; Gross, T.; Scho¨nfeld, R. Angew. Chem., Int. Ed. Engl. 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, 4381-4385. (16) (a) Malik, S.; Johnson, R. D.; Arnold, F. H. J. Am. Chem. Soc. 1994, 116, 8902-8911. (b) Hart, B. R.; Shea, K. J. J. Am. Chem. Soc. 2001, 123, 20722073. (17) (a) Schweitz, L.; Andersson, L. I.; Nilsson, S. Anal. Chem. 1997, 69, 11791183. (b) Ramstro ¨m, O.; Nicholls, I. A.; Mosbach, K. Tetrahedron: Asymmetry 1994, 5, 649-656. (c) Matsui, J.; Nicholls, I. A.; Takeuchi, T. Tetrahedron: Asymmetry 1996, 7, 1357-1361. (18) (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. (19) (a) Makote, R.; Collinson, M. M. Chem. Mater. 1998, 10, 2440-2445. (b) Kriz, D.; Berggren-Kriz, C.; Andersson, L. I.; Mosbach, K. H. Anal. Chem. 1994, 66, 2636-2639. (20) (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. (21) (a) Whitcombe, M. J.; Alexander, C.; Vulfson, E. N. Synlett 2000, 911923. (b) Brunkan, N. M.; Gagne, M. R. J. Am. Chem. Soc. 2000, 122, 62176225. (22) (a) Wang, W.; Gao, S.; Wang, B. Org. Lett. 1999, 1, 1209-1212. (b) Liao, Y.; Wang, W.; Wang, B. Bioorg. Chem. 1999, 27, 463-476. (23) (a) Malitesta, C.; Losito, I.; Zambonin, P. G. Anal. Chem. 1999, 71, 13661370. (b) Lee, S. W.; Ichinose, I.; Kunitake, T. Chem. Lett. 1998, 12, 11931194. (c) Ji, H. S.; McNiven, S.; Ikebukuro, K.; Karube, I. Anal. Chim. Acta 1999, 390, 93-100.

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Boronic acid ligands bind strongly and reversibly vicinal diols, eq 1. This property has been employed to develop optical sensors

for sugars26 or gelating materials27 that undergo sol-gel transitions upon the binding of the sugar. Boronic acid acrylamide copolymers were employed as active matrixes for the sensing of glucose28 or nucleotides.29 Swelling of the hydrogel upon binding of the substrate could be followed by microgravimetric quartz crystal microbalance measurements, surface plasmon resonance experiments, or Faradaic impedance spectroscopy.28 The boronic acid ligand is often used to imprint molecular recognition sites in polymers for specific binding30 and separation31 of sugars. The use of boronic acid-based imprinted polymer membranes as active sensing interfaces on ISFET devices is specifically tempting since they include a built-in mechanism for controlling the gate potential as a result of binding of the substrate. That is, the binding of the substrate to the boronic acid ligand generates the boronate complex and releases protons, eq 1. This process is anticipated to control the potential of any oxide gate interface. Indeed, in a recent comprehensive report, an acrylamide-acrylamidophenylboronic acid copolymer was imprinted with specific recognition sites for different nucleotides or saccharides.29 The specific potentiometric analysis of the nucleotides or saccharides was accomplished on polymer-functionalized ISFET devices. Here we report on the imprint of specific recognition sites for NAD+, NADP+, NADH, and NADPH in acrylamide-acrylamidophenylboronic acid copolymer membranes associated with ISFET devices. We demonstrate the selective sensing of the respective cofactors, and the application of the functional devices to follow biocatalyzed transformations. One report32 has addressed the molecular imprinting of NAD+ cofactors, where silica particles were imprinted with prearranged recognition sites for a bis-NAD+ cofactor. EXPERIMENTAL SECTION Materials. The acrylic monomer functionalized with 3-aminophenylboronic acid, 3-acrylamidophenylboronic acid (1), was synthesized according to the published procedure.33 Acrylamide, (24) Lahav, M.; Kharitonov, A. B.; Katz, O.; Kunitake, T.; Willner, I. Anal. Chem. 2001, 73, 720-723. (25) Lahav, M.; Kharitonov, A. B.; Willner, I. Chem. Eur. J. 2001, 7, 39923997. (26) (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Nature 1995, 374, 345-347. (b) Deng, G.; James, T. D.; Shinkai, S. J. Am. Chem. Soc. 1994, 116, 4567-4572. (27) James, T. D.; Murata, K.; Harada, T.; Ueda, K.; Shinkai, S. Chem. Lett. 1994, 273-276. (28) Gabai, R.; Sallacan, N.; Chegel, V.; Bourenko, T.; Katz, E.; Willner, I. J. Phys. Chem. B 2001, 105, 8196-8202. (29) Sallacan, N.; Zayats, M.; Bourenko, T.; Kharitonov, A. B.; Willner, I. Anal. Chem. 2002, 74, 702-712. (30) (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. (31) (a) Wulff, G.; Schauhoff, S. J. Org. Chem. 1991, 56, 395-400. (b) Wulff, G.; Haarer, J. Macromol. Chem. 1991, 192, 1329-1338. (32) Johansson, A.; Mosbach, K.; Mansson, M. O. Eur. J. Biochem. 1995, 227, 551-555.

N,N′-methylenebisacrylamide, N,N,N′,N′-tetramethylethylenediamine, γ-methacryloxypropyltrimethoxysilane, 2,2-dimethoxy-2phenylacetophenone, sodium salts of β-NAD+, β-NADH, β-NADP+, and β-NADH, L(+)-lactic acid, and 2-hydroxyethyl methacrylate, as well as the enzymes lactate dehydrogenase (EC 1.1.1.27 from rabbit muscle, type XI) (LDH) and NADP+-dependent alcohol dehydrogenase (EC 1.1.1.2 from Thermoanaerobium brockii) (AlcDH) were purchased from Sigma and Aldrich. All other chemicals were obtained from Aldrich and were used as supplied. Ultrapure water from Serapur PRO90CN was used throughout the experiments. Preparation of the Polymer-Modified Electrodes. The Al2O3 ISFET devices (20 × 700 µm2, Neuchaˆtel, Switzerland) were preliminarily functionalized with γ-methacryloxypropyltrimethoxysilane (10% v/v solution in mixture composed of toluene and water (25:1)) by dropping 0.2 µL of the silane solution on the top of the gate. The modification was conducted in an oven (Eurotherm) at 90 °C for 4 h. The silylated chips were then thoroughly rinsed with toluene and a phosphate buffer solution (PBS, 0.1 M, pH 7.2) and then dried in air at room temperature for 30 min. Subsequently, a drop of the mixture composed of the monomer 2-hydroxyethyl methacrylate (HEMA) and initiator 2,2-dimethoxy2-phenylacetophenone (4% v/v) was positioned on the silylated gate surfaces and irradiated with a UV lamp for 5 min. This procedure enabled the covalent linking of the poly-HEMA layer to the silane-functionalized gate interface.34 The poly-HEMA layer enables strong adherence of the sensing acrylamide film and prevents peeling off of the sensing membrane from the gate interface. For preparation of the imprinted polymer membranes on the ISFET devices, a mixture of acrylamide (1.82 M), 3-acrylamidophenylboronic acid (0.1 M), N,N′-methylenebisacrylamide (0.04 M), and the respective substrates (NAD+, NADH, NADP+, or NADPH) (0.1 M) was dissolved in 0.5 mL of a freshly prepared solution of phosphate buffer (0.1 M, pH 7.2). The mixture was slightly heated until complete dissolution of the mixture components was accomplished. The initiators of the polymerization, ammonium persulfate (0.22 M, 0.05 mL) and N,N,N′,N′-tetramethylenediamine (10% v/v, 0.12 mL), were added to the monomer solution just before placing an 0.1-µL drop of the mixture on the prefunctionalized gate interface. The resulting modified chips were dried in air for at least 3 h. The ISFETs were then rinsed with an NH3 solution (1% v/v) to eliminate the substrate from the membrane formed until a stable output signal was obtained. This procedure results in the formation of the imprinted membrane for the respective substrate. The resulting functionalized chips were then thoroughly rinsed with PBS and used for the analyses. Rough Au electrodes associated with the quartz crystal adhere well to the acrylamide-acrylamidophenylboronic acid copolymer. Accordingly, the copolymer membrane was directly assembled on the gold electrode. The prepolymerization mixture, 10 µL, which included the respective imprinting substrate (0.05 M), was dropped on the gold electrode surface, and then 1 µL of the (33) Kitano, S.; Koyama, Y.; Kataoka, K.; Okano, T.; Sakurai, Y. J. Controlled Release 1992, 19, 162-170. (34) Reinhoudt, D. N.; Engbersen, J. F. J.; Brzo´zka, Z.; van der Vlekkert, H. H.; Honig, G. W. N.; Holterman, H. A. J.; Verkerk, U. H. Anal. Chem. 1994, 66, 3618-3623.

initiators, ammonium persulfate (0.22 M, 0.3 µL) and N,N,N′,N′tetramethylenediamine (10% v/v, 0.7 µL), was spread over the prepolymerization mixture. The resulting modified QCM electrodes were dried in air for 3 h and rinsed with 0.1 M HCl solution. To eliminate the template from the membrane, the electrodes were treated with a NH3 solution (1% v/v) until no shift in the crystal frequency was observed (∼10 min). Ellipsometry measurements, where the polymer is assembled similarly on an Au-coated glass surface, indicate that the film thickness is ∼18 ( 1 nm. The swelling of the film upon analyzing the different substrates leads to an increase of the film thickness to 22 ( 1 nm. The optical characterization of the samples was conducted on a variable-angle spectroscopic ellipsometer of rotating analyzer type (J.A. Woollam, USA, M-88). Measurements were carried out at room temperature at five angles of incidence in the range of 60-75°, (wavelength region 300-800 nm). The recorded spectra were analyzed using the WVASE32 software package using three-layer optical model and Bruggeman effective medium approximation. Measurements. ISFET Measurements. ISFET devices with an Al2O3 gate interface (20 × 700 µm2, IMT, Neuchaˆtel, Switzerland) were used in all the experiments. A Ag/AgCl electrode was used as a reference. The chip functionalized with the respective molecularly imprinted sensing membrane was immersed in the working cell filled with 0.8 mL of PBS (0.1 M, pH 7.2) and varying concentrations of the respective substrate. The output signal between the source of the ISFET and the reference electrode was recorded using a semiconductor parameter analyzer (HP 4155B). The system configuration enables the measurements of the source-gate voltage (Vgs), while the drain voltage (Vd) remained constant (Vd ) 1 V). The difference between Vgs values for the ISFETs modified with respective films, with and without embedded substrate, was plotted against the substrate concentrations in the bulk solutions. All the measurements were carried out for 5 min 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 detection limits were estimated according to IUPAC recommendations35 as the intercept of the two linear segments in the calibration curves. Microgravimetric QCM Measurements. A QCM analyzer (EG&G model QCA 917) and a homemade cell for the QCM Seiko electrodes were used for the microgravimetric analyses. Quartz crystals (AT-cut with a frequency of ∼9 MHz) sandwiched between Au electrodes (geometrical area 0.2 ( 0.05 cm2, roughness factor corresponds to ∼3.5) were used. The modified electrodes were dipped in the analyte solution for 1 min and flushed in a nitrogen flow, and then the crystal frequency in air, fair, was measured. To build the calibration curves, the difference between fair values before and after the reaction of the electrode interface with the template molecules was plotted versus the concentrations of the analyte solution. After each measurement, the electrode was rinsed with NH3 (1% v/v) solution until a stable signal was obtained. All the measurements were carried out in a box of constant humidity that enabled to minimize an influence from the change of air humidity on the measurement results. The box contained a certain volume of saturated KCl solution and allowed us to keep the air humidity within the box of 86%.36 (35) Buck, R. P.; Lindner, E. Pure Appl. Chem. 1994, 66, 2527-2536.

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Chart 1. Structures of NAD(P)+ and NAD(P)H Cofactors

RESULTS AND DISCUSSION Each of the oxidized cofactors NAD+ or NADP+ or the reduced cofactors NADH or NADPH (see Chart 1) were imprinted in a cross-linked acrylamide-acrylamidophenylboronic acid copolymer associated with the Al2O3 gate surface of the ISFET devices. The method for the generation of the imprinted membrane is depicted in Scheme 1. The primary step, Scheme 1A, involves the silanization of the Al2O3 surface with γ-methacryloxypropyltrimethoxysilane to yield a polymerizable monomer film. It was polymerized with HEMA in the presence of 2,2-dimethoxy-2-phenylacetophenone to generate a poly-HEMA film on the surface. This base film is essential for good adhesion of the secondary functional film on the gate. Imprinting of the respective cofactor in the functional polymer film is described in Scheme 1B, for NAD+. The polyHEMA-functionalized ISFET was allowed to react with acrylamide, 3-acrylamidophenylboronic acid, and the cross-linking agent N,N′methylenebisacrylamide, in the presence of NAD+ (or any of the other oxidized or reduced cofactors). The primary formation of the boronate complex between the ribose units of the cofactor, followed by the polymerization of the monomers and formation of complementary hydrophobic and, to some extent, electrostatic and H-bonds between the polymer groups and the functionalities of the cofactor units, leads to the formation of the cofactor contours in the polymer. The elimination of the template cofactor with 1% NH3 generates the imprinted sites for the respective cofactor. Figure 1A shows the responses of the ISFET device functionalized with the NAD+-imprinted polymer upon the sensing of various concentrations of NAD+, curve a. The imprinted cofactor (36) Short Chemistry Handbook; Kurilenko, O. D., Ed.; Naukova Dumka: Kiev, 1974; p 729.

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NAD+ is detected in the linear concentration range of 5 × 10-78 × 10-5 M with a sensitivity that corresponds to 15.0 mV‚decade-1 and a lower detection limit of 4 × 10-7 M. The structurally related reduced cofactor NADH is detected by the functionalized ISFET in a substantially narrower concentration range of 3 × 10-7-1 × 10-6 M, and a lower sensitivity of 6.0 mV‚decade-1, curve b. Figure 1A, curves c and d, shows the responses of the NAD+-imprinted ISFET device to variable concentrations of NADP+ and NADPH, respectively. Clearly, no response of the functionalized device to these cofactors is observed. Also, the ISFET device functionalized with the nonimprinted acrylamide-acrylamidophenylboronic acid copolymer is insensitive to NAD+ in the entire concentration range, Figure 1A, curve e. These results clearly indicate that no nonspecific binding of NAD+ to the polymer takes place, or at least it is so weak that it could not influence the outcome signal, and that the reduced cofactors NAD(P)H are differentiated from the oxidized cofactors. Selectivity is also observed toward the analysis of NAD+ as compared to NADP+. That is, the substrate NADP+ that includes a single phosphonate group attached to the NAD+ backbone is differentiated by the imprinted sites. Similar results are observed upon imprinting of NADP+ in the acrylamide-acrylamidophenylboronic acid membrane associated with the ISFET devices, Figure 1B. Figure 1B, curve a, shows the ISFET response to various concentrations of NADP+. The imprinted substrate NADP+ is analyzed in the narrower linear concentration range of 3 × 10-7-5 × 10-6 M with a sensitivity that corresponds to 18.0 mV‚decade-1. The NADP+-imprinted ISFET device reveals no response to the oxidized cofactor NAD+, which lacks the phosphate group tethered to the imprinted substrate NADP+, and the reduced form NADH, Figure 1B, curves c and d, respectively. The NADP+-imprinted device reveals

Scheme 1. (A) Primary Functionalization of the Gate Oxide Interface with a Poly-HEMA Adhesion Film and (B) Preparation of the Acrylamide-Acrylamidophenylboronic Acid Copolymer Sensing Film Consisting of Imprinted Sites for NAD(P)+/NAD(P)H Cofactorsa

a

The scheme exemplifies the imprinting of NAD+.

specificity in respect to the reduced cofactor NADPH, curve b. The sensitivity for the detection of NADPH by the NADP+imprinted ISFET device is only 3.0 mV‚decade-1 in the concentration range of 1 × 10-7-1 × 10-5 M, implying a substantially lower affinity of NADPH to NADP+-imprinted sites. Also, the ISFET device functionalized with the nonimprinted polymer membrane does not respond to NADP+, Figure 1B, curve e. The observed impressive selectivity needs some further discussion: The inefficient binding of the NADP+ to the NAD+-imprinted film may be attributed to the larger steric dimensions of NADP+ relative to the NAD+-imprinted contours. This explanation, however, would not stand for the lack of binding of NAD+ to the sterically larger NADP+-imprinted sites. To account for this unique specificity, we believe that, upon imprinting of any of the cofactors, a collective pattern of several H-bonds and boronate bonds between the polymer and the substrate is formed. These collective interactions

lead to the synergetic binding of the substrate in the respective site. Thus, although the dimensions of the NADP+ site may structurally accommodate the NAD+, the latter cofactor does not bind to the site due to the structural perturbation of the collective pattern of complementary H-bonds and boronate ligands. The reduced cofactors 1,4-dihydronicotinamide adenine dinucleotide and 1,4-dihydronicotinamide adenine dinucleotide phosphate were also imprinted in acrylamide-acrylamidophenylboronic acid membranes associated with the ISFET devices. Figure 2A, curve a, shows the responses of the NADH-imprinted ISFET device to variable concentrations of NADH. The imprinted substrate is sensed in the concentration range of 1 × 10-7-5 × 10-5 M, with an average sensitivity of 24.2 mV‚decade-1, and a lower detection limit that corresponds to 1 × 10-7 M. The ISFET device functionalized with the NADH-imprinted assembly reveals full differentiation toward the analysis of NADH over NAD+, Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

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Figure 1. (A) Gate-source voltage changes of the ISFET functionalized with NAD+-imprinted copolymer 1 upon sensing of (a-d) variable concentrations of NAD+, NADH, NADP+, and NADPH, respectively; (e) response of nonimprinted copolymer 1-functionalized ISFET device to variable concentrations of NAD+. (B) Gate-source voltage changes of the ISFET functionalized with NADP+-imprinted copolymer 1 upon sensing of (a-d) variable concentrations of NADP+, NADPH, NAD+, and NADH, respectively; (e) response of nonimprinted copolymer 1-functionalized ISFET device to variable concentrations of NADP+.

Figure 2. (A) Gate-source voltage changes of the ISFET functionalized with NADH-imprinted copolymer 1 upon sensing of (a-d) variable concentrations of NADH, NAD+, NADP+, and NADPH, respectively; (e) response of nonimprinted copolymer 1-functionalized ISFET to variable concentrations of NADH. (B) Gate-source voltage changes of the ISFET functionalized with NADPH-imprinted copolymer 1 upon sensing of variable concentrations of (a) NADPH, (b) NAD+, (c) NADH, and (d) NADP+; (e) response of nonimprinted copolymer 1-functionalized ISFET to variable concentrations of NADPH.

NADP+, and NADPH, and the functional ISFET is not responding to the substrates, curves b-d, respectively. Also, the ISFET modified with the nonimprinted membrane does not respond to NADH in the entire concentration range, Figure 2A, curve e, implying that nonspecific adsorption does not take place. Similar results are observed for the ISFET device functionalized with the NADPH-imprinted membrane, Figure 2B. The imprinted substrate NADPH is effectively analyzed in the concentration range of 3 × 10-7-1 × 10-4 M with the lower detection limit of 2 × 10-7 M and a sensitivity that corresponds to 21.8 mV‚decade-1, curve a. The functionalized devices reveal low responses to NAD+, NADH, and NADP+, curves b-d, respectively, and the ISFET functionalized with the non-

imprinted polymer membrane does not respond to NADPH, Figure 2B, curve c. These results clearly demonstrate the successful imprint of the specific recognition sites for the oxidized cofactor NAD+ or NADP+ or the reduced cofactor NADH or NADPH and the assembly of functional sensing devices for the substrates. The set of experiments enables us to define the following conclusions: (i) ISFET devices functionalized with nonimprinted acrylamide-acrylamidophenylboronic acid membranes do not respond to the oxidized cofactors, NAD(P)+, or the reduced cofactors, NAD(P)H. Thus, despite the fact that the membrane includes the boronic acid ligands, nonspecific binding of the oxidized or reduced cofactors is negligibly small under the experimental conditions used. Presumably, the cross-linking of the membrane,

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and the relatively large size of the cofactors, prohibit their binding to the boronic acid ligands. (ii) The ISFET devices functionalized with the oxidized cofactors NAD+ or NADP+ reveal only minute response to the reduced cofactors NAD(P)H. Similarly, imprint of the reduced cofactors NADH or NADPH in the sensing membranes yields specific interfaces that almost do not recognize the oxidized cofactors NAD(P)+. Thus, although the reduced and oxidized cofactors include similar functionalities, their specific sensing is accomplished by the imprinted membrane. While the oxidized cofactor NAD(P)+ includes a planar structure of the nicotinamide unit, the 1,4-dihydronicotinamide component exhibits a “boat” nonplanar structure.37 This difference in structural motifs is, presumably, sufficient to change the H-bonded contours of the imprinted sites to yield the resulting specificity. (iii) The observed selectivities of the functional ISFET devices within the oxidized pairs NAD+ and NADP+ or the reduced cofactors NADH and NADPH, upon the imprint of the respective substrates, is significant. A single phosphate group hinged to the ribose unit of NADP+/NADPH is enough to induce the binding selectivity in the respective imprinted membranes. That is, the additional H-bonds generated with the phosphate group, and the size of the tethered phosphate group, contribute to the structural functionalities for the generation of imprinted sites of well-defined binding contours and complementary functionalities for the association of the imprinted cofactors. In a previous study,29 we demonstrated that the binding of nucleotides to the nucleotide-imprinted acrylamide-acrylamidophenylboronic acid copolymers is associated with the swelling of the membranes. The swelling process was characterized by microgravimetric quartz crystal microbalance measurements. To support the concept of imprinting of specific recognition sites for the NAD(P)+/NAD(P)H cofactors in the acrylamide-acrylamidophenylboronic acid copolymer, we examined the swelling processes associated with binding of the imprinted cofactors to the imprinted sites by microgravimetric quartz crystal microbalance analyses. Figure 3A, curve a, shows the frequency changes of the NAD+-imprinted acrylamide-acrylamidophenylboronic acid copolymer, assembled on an Au-quartz crystal, upon interaction with variable concentrations of NAD+. In these experiments, the polymer is treated with aqueous solutions of NAD+, and the frequencies of the crystal are monitored in air. Thus, the frequency changes reflect mass changes originating from binding of the host substrate to the polymer and the uptake of water as a result of swelling. The mass changes as a result of the binding of the imprinted molecular substrates are usually low (-20 to -50 Hz) and depend on the amount of imprinted sites and the molecular weight of an analyte molecule. The observed frequency changes, ∼-900 Hz, thus originate mainly from the uptake of water accompanying the binding of NAD+. Figure 3A, curve b, shows the frequency changes of the crystal functionalized with the NAD+imprinted membrane upon the analysis of NADP+ in the same concentration range. Clearly, the responses of the functionalized electrode to NADP+ are substantially lower. This indicates lower binding affinity and a lower degree of swelling upon binding of the nonimprinted oxidized cofactor. Figure 3A, curves c and d, shows the frequency changes of the electrode functionalized with (37) (a) Wu, Y.-D.; Houk, K. N. J. Am. Chem. Soc. 1991, 113, 2353-2358. (b) Siiman, O.; Rivellini, R.; Patel, R. Inorg. Chem. 1988, 27, 3940-3949.

Figure 3. (A) Calibration curves corresponding to frequency changes, ∆f, upon interaction of NAD+-imprinted copolymer 1-functionalized Au-quartz crystal with variable concentrations of (a-d) NAD+, NADP+, NADH, and NADPH, respectively; (e) response of nonimprinted copolymer 1-functionalized Au-quartz crystal to variable concentrations of NAD+. (B) Calibration curves corresponding to frequency changes, ∆f, upon interaction of NADH-imprinted copolymer 1-functionalized Au-quartz crystal with variable concentrations of (a-d) NADH, NADPH, NAD+, and NADP+, respectively; (e) response of nonimprinted copolymer 1-functionalized Au-quartz crystal to variable concentrations of NADH.

the NAD+-imprinted membrane upon the interaction with NADH and NADPH, respectively, whereas Figure 3A, curve e, shows the frequency changes of the crystal modified with the nonimprinted membrane treated with NAD+. Clearly, the reduced cofactors NADH and NADPH are poorly sensed by the NAD+-imprinted functionalized quartz crystal with the frequency changes of -50 and -20 Hz, respectively. Also, the crystal modified with the nonimprinted membrane is insensitive to the oxidized cofactor, NAD+. Similar selectivity is observed upon imprinting of the NADP+ cofactor in the acrylamide-acrylamidophenylboronic acid copolymer associated with the Au-quartz crystal. The functionAnalytical Chemistry, Vol. 75, No. 3, February 1, 2003

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Figure 4. Changes in gate-source voltage of the ISFET functionalized with NADH-imprinted copolymer 1 upon following the oxidation of lactate to pyruvate; (a) in the presence of lactate dehydrogenase and NAD+, [NAD+] ) 1 × 10-2 M; [lactate] ) 1 × 10-2 M; [LDH] ) 1.0 × 10-7 g‚mL-1; (b) without lactate dehydrogenase.

Figure 5. Changes in gate-source voltage of the ISFET functionalized with NADPH-imprinted copolymer 1 upon following the oxidation of ethanol to acetaldehyde in the presence of NADP+ (0.02 M), alcohol dehydrogenase (1 × 10-5 g‚mL-1), and (a) 0.02, (b) 0.01, and (c) 0.002 M ethanol, and (d) in the presence of 0.02 M NADP+ and 0.02 M ethanol, but without added alcohol dehydrogenase.

alized crystal yields a high-frequency change, ∼-1100 Hz, in the presence of 1 × 10-4 M NADPH, while a substantially lower response is observed upon treatment with NAD+ (∼-400 Hz in the presence of 1 × 10-4 NAD+). The crystal modified with the NADP+-imprinted membrane is almost insensitive to the addition of NADH or NADPH, (the frequency changes correspond to -30 and -50 Hz, respectively). Analogous results are observed upon the imprint of the reduced cofactor NADH in the acrylamide-acrylamidophenylboronic acid membrane associated with the Au-quartz crystal. Figure 3B, curve a, shows the frequency changes of the Auquartz crystal functionalized with the NADH-imprinted cofactor upon the analysis of NADH. A frequency change of ∼-1750 Hz is observed upon the analysis of 1 × 10-4 M NADH. A substantially lower response of the NADH-imprinted functional crystal is observed upon its treatment with NADPH (∼-670 Hz at 1 × 10-4 M NADPH), Figure 3B, curve b. The NADH-imprinted membrane associated with the Au-quartz crystal is almost insensitive to the oxidized cofactors NAD+ and NADP+, Figure 3B, curves c and d, respectively, revealing the responses of -270 and -210 Hz, respectively. The analyses of the NAD(P)+ and NAD(P)H cofactors by the imprinted membranes associated with the ISFET devices or the piezoelectric quartz crystals are based on two different transduction means that originate from the association of the substrates to the imprinted sites. The transduction signal of the functional ISFET devices is a potentiometric output that originates from the generation of the boronate complex as a result of the binding of the substrate to the recognition sites and from the change in the local pH at the gate interface (see eq 1). The transduction signal of the functional piezoelectric crystals is a frequency change that reflects mass changes on the sensing interface originating from binding of the substrates and swelling of the polymer matrixes. The major accomplishment of these studies is, however, the assembly of specific functional devices for the detection of the

NAD(P)+ or NAD(P)H cofactors. These functional devices can then be applied to characterize biocatalyzed transformations. Figure 4 shows the analysis of the biocatalyzed oxidation of lactate to pyruvate in the presence of LDH (see reaction cycle in Figure 4, inset), using an ISFET device functionalized with the NADHimprinted membrane. Figure 4, curve a, shows the time-dependent formation of NADH in the process. Curve b shows the response of the device upon treatment of the functionalized ISFET in the absence of LDH. Clearly, no response is detected, indicating that the ISFET response originates from the biocatalyzed reduction of NAD+ to NADH. The association of the reduced cofactor to the imprinted sites controls the potential of the gate surface. Similarly, the ISFET device functionalized with the NADPHimprinted membrane was employed to characterize the biocatalyzed oxidation of ethanol in the presence of AlcDH. This specific enzyme is an NADPH-dependent alcohol dehydrogenase. Figure 5, curves a-c, shows the time-dependent accumulation of NADPH in three systems consisting of various concentrations of ethanol. As the concentration of ethanol is elevated, the formation of NADPH is faster. Figure 5, curve d, shows the response of the functionalized device in a system consisting of NADP+ and ethanol without added AlcDH. The absence of a response means that the device responds selectively to NADPH generated by the biocatalyzed process. A final point relates to the reproducibility and stability of the functional devices. Although no attempt was made to optimize the preparation of the functional ISFET devices, we found that, in a series of six ISFET devices for each of the imprinted cofactors, reproducibility of the sensor responses was found to be ∼90%. The resulting functional devices also reveal good long-term stabilities. We found that, within a period of 30 days, while performing an analysis every second day, the response of the devices decreased by only ∼10% without lack of selectivity. Between measurements, the modified ISFETs were kept dry at 5 °C. Also, upon continuous analyses of the substrates for 12 h, a

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decrease of ∼20% in the responses of the devices was observed. In this context, it is important to note that the base monolayer of poly-HEMA is important for stability of the resulting devices. This layer provides an adhesion interface for the functional imprinted membranes.

overcoming the difficulties encountered in the electrochemical sensing of these cofactors. The use of an intermediate poly-HEMA layer between the Al2O3 gate surface and a sensing acrylamideacrylamidophenylboronic acid membrane results in substantial improvement in the sensor lifetime.

CONCLUSIONS The present study has addressed the assembly of ISFET devices and microgravimetric Au-quartz crystal electrodes that include imprinted membranes for the specific sensing of the oxidized cofactors NAD+ and NADP+ and the reduced cofactors NADH and NADPH. This is an entirely new method for the analysis of NAD(P)+ or NAD(P)H cofactor, which enables

ACKNOWLEDGMENT This study is supported by The Israel Ministry of Science as an Infrastructure Project in Material Science. Received for review May 2, 2002. Accepted October 22, 2002. AC020292H

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