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Tryptophan Repressor-Binding Proteins from Escherichia coli and Archaeoglobus fulgidus as New Catalysts for 1,4-Dihydronicotinamide Adenine Dinucleotide-Dependent Amperometric Biosensors and Biofuel Cells Muhammad Nadeem Zafar,† Federico Tasca,† Lo Gorton, Eric V. Patridge,‡ James G. Ferry,‡ and Gilbert No¨ll*,†,§ Department of Analytical Chemistry/Biochemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, Department of Biochemistry and Molecular Biology, Eberly College of Science, Pennsylvania State University, 205 South Frear Laboratory, University Park, Pennsylvania, 16802-4500, and University of Siegen, Organic Chemistry 1, Adolf-Reichwein-Strasse 2, D-57068 Siegen, Germany The tryptophan (W) repressor-binding proteins (WrbA) from Echerichia coli (EcWrbA) and Archaeoglobus fulgidus (AfWrbA) were investigated for possible use in 1,4dihydronicotinamide adenine dinucleotide (NADH) dependent amperometric biosensors and biofuel cells. EcWrbA and AfWrbA are oligomeric flavoproteins binding one flavin mononucleotide (FMN) per monomer and belonging to a new family of NAD(P)H:quinone oxidoreductases (NQOs). The enzymes were covalently linked to a low potential Os redox polymer onto graphite in the presence of single-walled carbon nanotube (SWCNT) preparations of varying average lengths. The performance of the enzyme modified electrodes for NADH oxidation was strongly depending on the average length of the applied SWCNTs. By blending the Os redox polymer with SWCNTs, the electrocatalytic current could be increased up to a factor of 5. Results obtained for AfWrbA modified electrodes were better than those for EcWrbA. For NADH detection, a linear range between 5 µM and 1 mM, a lower limit of detection of 3 µM, and a sensitivity of 56.5 nA µM-1 cm-2 could be reached. Additionally spectroelectrochemical measurements were carried out in order to determine the midpoint potentials of the enzymes (-115 mV vs NHE for EcWrbA and -100 mV vs NHE for AfWrbA pH 7.0). Furthermore, an AfWrbA modified electrode was used as an anode in combination with a Pt black cathode as a biofuel cell prototype. To catalyze the oxidation of 1,4-dihydronicotinamide adenine dinucleotide (NADH) is an important task in bioanalytical chemistry, because NAD+-dependent dehydrogenases, which catalyze the oxidation of specific substrates with concomitant reduction * To whom correspondence should be addressed. Gilbert No ¨ll, University of Siegen, Organic Chemistry 1, Adolf-Reichwein-Str. 2, D-57068 Siegen, Germany. Phone: +49 (0)271 740 4360. Fax: +49 (0)271 740 3270. E-mail:
[email protected]. † Lund University. ‡ Pennsylvania State University. § University of Siegen.
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of NAD+ to NADH, constitute the largest family of redox enzymes known today.1 In principle, a variety of amperometric biosensors can be realized by employing these enzymes. The electrons gained from substrate oxidation are transferred to NAD+ and have to be detected at the electrode in terms of NADH. Unfortunately, the direct oxidation of NADH at bare electrodes requires a large overpotential and suffers from pronounced irreversibility.1-3 Therefore a second catalytic process becomes necessary. One strategy to mediate the electron transfer (ET) from NAD+-dependent enzymes to electrodes involves the covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer on gold followed by a covalent linkage of N6-(2-aminoethyl)-NAD+.2 Within this monolayer, the ET from the attached NADH to the gold surface is mediated by PQQ. NAD+-dependent dehydrogenases such as lactate dehydrogenase can be assembled on top of the NAD+ containing layer.2 An advantage of this method is that NAD+ is immobilized at the surface, and a constant level of the NAD+/ NADH redox couple in the bulk solution is not required. On the other hand, this strategy is limited to enzyme monolayers at the electrode. Alternatively, nitrocompounds immobilized on nanostructured electrodes have been shown to catalyze the oxidation of NADH at low overpotentials.4,5 Furthermore, different types of carbon nanotubes (sometimes after further modification or combination with redox mediators) were found to catalyze the oxidation of NADH quite efficiently.6-19 Another possibility is the combination of NAD+-dependent dehydroge(1) Gorton, L.; Domı´nguez, E. In Encyclopedia of Electrochemistry, Vol. 9, Biochemistry; Wilson, G. S., Ed.; Wiley-VCH: Weinheim, 2002; pp 67143. (2) Bardea, A.; Katz, E.; Bu ¨ ckmann, A. F.; Willner, I. J. Am. Chem. Soc. 1997, 119, 9114–9119. (3) Barton, S. C.; Gallaway, J.; Atanassov, P. Chem. Rev. 2004, 104, 4867– 4886. (4) Mano, N.; Kuhn, A. Biosens. Bioelectron. 2001, 16, 653–660. (5) Mano, N.; Thienpont, A.; Kuhn, A. Electrochem. Commun. 2001, 3, 585– 589. (6) Li, X.; Zhou, H.; Yu, P.; Su, L.; Ohsaka, T.; Mao, L. Electrochem. Commun. 2008, 10, 851–854. (7) Luz, R. d. C. S.; Damos, F. S.; Tanaka, A. A.; Kubota, L. T.; Gushikem, Y. Electrochim. Acta 2008, 53, 4706–4714. 10.1021/ac900365n CCC: $40.75 2009 American Chemical Society Published on Web 04/13/2009
nases with NAD(P)H:quinone oxidoreductases (NQOs) such as diaphorase, a dimeric flavin adenin dinucleotide (FAD) containing protein, which oxidize NAD(P)H with concomitant reduction of quinones or other redox mediators.20-23 On the basis of glucose dehydrogenase and diaphorase, a glucose biosensor was developed,20 and by combination of diaphorase with alcohol, aldehyde, and formate dehydrogenase, a biofuel cell capable of oxidizing methanol completely to CO2 and H2O has been presented.21 In the latter case, benzylviologen was used as a redox mediator in order to catalyze ET from diaphorase to the anode. In our previous work we have shown that diaphorase can be covalently linked to a low potential Os redox polymer, which transfers the electrons gained from NADH oxidation to the electrode.24,25 When the Os polymer was blended with oxidatively shortened and length-separated singlewalled carbon nanotubes (SWCNTs), the catalytic current for NADH oxidation could be increased by a factor of 5.24 In this contribution, we report on the performance of the tryptophan (W) repressor-binding protein (WrbA) from Echerichia coli (EcWrbA) and Archaeoglobus fulgidus (AfWrbA) with respect to applications in biosensors and biofuel cells.26-29 WrbA is an oligomeric flavoprotein that binds one flavin mononucleotide FMN per monomer. The molecular mass of monomeric EcWrbA is 21 kDa and that of AfWrbA is 22 kDa.26 EcWrbA was discovered in 1993, when it was copurified with the tryptophan repressor (TrpR).30 For a long time, the function of WrbA was not known. With respect to its redox properties, a role in oxidative stress defense was implicated.31 In line with these findings, WrbA has been recently identified as a new family of NQOs.26,28 Since for (8) Manso, J.; Mena, M. L.; Yanez-Sedeno, P.; Pingarron, J. M. Electrochim. Acta 2008, 53, 4007–4012. (9) Radoi, A.; Compagnone, D.; Valcarcel, M. A.; Placidi, P.; Materazzi, S.; Moscone, D.; Palleschi, G. Electrochim. Acta 2008, 53, 2161–2169. (10) Du, P.; Liu, S.; Wu, P.; Cai, C. Electrochim. Acta 2007, 53, 1811–1823. (11) Chakraborty, S.; Retna Raj, C. Electrochem. Commun. 2007, 9, 1323–1330. (12) Huang, M.; Jiang, H.; Qu, X.; Xu, Z.; Wang, Y.; Dong, S. Chem. Commun. 2005, 5560–5562. (13) Zhang, M.; Gorski, W. J. Am. Chem. Soc. 2005, 127, 2058–2059. (14) Valentini, F.; Salis, A.; Curulli, A.; Palleschi, G. Anal. Chem. 2004, 76, 3244– 3248. (15) Zhu, L.; Zhai, J.; Yang, R.; Tian, C.; Guo, L. Biosens. Bioelectron. 2007, 22, 2768–2773. (16) Raj, C. R.; Chakraborty, S. Biosens. Bioelectron. 2006, 22, 700–706. (17) Wang, J. Electroanalysis 2005, 17, 7–14. (18) Yan, Y.-M.; Yehezkeli, O.; Willner, I. Chem.sEur. J. 2007, 13, 10168–10175. (19) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1181–1218. (20) Antiochia, R.; Gorton, L. Biosens. Bioelectron. 2007, 22, 2611–2617. (21) Palmore, G. T. R.; Bertschy, H.; Bergens, S. H.; Whitesides, G. M. J. Electroanal. Chem. 1998, 443, 155–161. (22) Gros, P.; Comtat, M. Biosens. Bioelectron. 2004, 20, 204–210. (23) Montagne, M.; Durliat, H.; Comtat, M. Anal. Chim. Acta 1993, 278, 25– 33. (24) Tasca, F.; Gorton, L.; Wagner, J. B.; No ¨ll, G. Biosens. Bioelectron. 2008, 24, 272–278. (25) Nikitina, O.; Shleev, S.; Gayda, G.; Demkiv, O.; Gonchar, M.; Gorton, L.; Csoeregi, E.; Nistor, M. Sens. Actuators, B 2007, B125, 1–9. (26) Patridge, E. V.; Ferry, J. G. J. Bacteriol. 2006, 188, 3498–3506. (27) Andrade, S. L. A.; Patridge, E. V.; Ferry, J. G.; Einsle, O. J. Bacteriol. 2007, 189, 9101–9107. (28) Carey, J.; Brynda, J.; Wolfova, J.; Grandori, R.; Gustavsson, T.; Ettrich, R.; Smatanova, I. K. Protein Sci. 2007, 16, 2301–2305. (29) Wolfova, J.; Brynda, J.; Mesters, J. R.; Carey, J.; Grandori, R.; Smatanova, I. K. Mater. Struct. Chem., Biol., Phys. Technol. 2008, 15, 55–57. (30) Yang, W.; Ni, L.; Somerville, R. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5796–5800. (31) Natalello, A.; Doglia, S. M.; Carey, J.; Grandori, R. Biochemistry 2007, 46, 543–553.
EcWrbA and AfWrbA catalytic activity toward oxidation of NADH has been reported,26 it is of interest to study these enzymes with respect to applications in NAD+/NADH-dependent amperometric biosensors and biofuel cells. Similar to diaphorase, EcWrbA and AfWrbA can be covalently linked to a low potential Os redox polymer. The performance of both enzymes in the presence of different types of SWCNTs was compared. Furthermore, spectroelectrochemical measurements were carried out in order to determine the redox potentials of the enzymes. EXPERIMENTAL PART Chemicals and Materials. EcWrbA and AfWrbA were prepared as described elsewhere.26 Water was purified in a Milli-Q water purification system (Millipore, Bedford, MA). Poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) was obtained from Aldrich (http:// www.sigmaaldrich.com). Poly(vinylpyridine)-[osmium-(N,N′-methylated-2,2′-biimidalzole)3]2+/3+ was synthesized as reported elsewhere.32 Single-walled carbon nanotubes (SWCNTs) were purchased from Nanocyl, Sambreville, Belgium. Triton X-100 and controlled pore glass (CPG 3000 Å) were both from Fluka (Buchs, Switzerland). Spectrographic graphite electrodes and homemade pyrolytic graphite electrodes were used. Pyrolytic graphite (PG) was obtained as a gift from Mr. Robert Pulley, Minerals Technologies (mineralstech.com). Spectrographic graphite electrodes were from Ringsdorff Werke GmbH, Bonn, Germany, (type RW001, 3.05 mm diameter and 13% porosity http://www.sglcarbon.com). All solutions used for immobilization were prepared in Milli-Q water (Millipore, Bedford, MA), and the NADH used as a substrate was dissolved in 0.1 M MOPS buffer solutions. For flow injection measurements, the working buffer solutions were degassed before use to avoid air bubbles in the flow system. For voltammetric measurements (unless otherwise stated), argon was purged through the solutions for some minutes prior to the experiments. Flow injection measurements were performed with a flow-through amperometric cell of the wall-jet type33 at an applied potential of +290 mV vs NHE. The carrier flow was maintained at a constant flow rate of 1 mL min-1 by a peristaltic pump. The injection loop volume was 50 µL. The dispersion factor34 of the system was 1.04 at this flow rate. Voltammetric measurements were performed with an EG&G potentiostat/galvanostat model 273 A or an Autolab potentiostat/galvanostat PGSTAT30 (Eco Chemie, Utrecht, The Netherlands) using modified electrodes as the working electrode, a saturated calomel reference electrode (SCE), and a platinum foil counter electrode. All potentials discussed in the main part are referred to the normal hydrogen electrode (NHE). The current densities were calculated with respect to the geometric electrode area. The spectroelectrochemical setup used in this work has been described elsewhere.35 As working electrode, a gold capillary cell with an optical path length of 1 cm was applied. The extinction coefficients of the enzymes were ε450 ) 11.6 mM-1 cm-1 for EcWrbA and ε457 ) 14.0 mM-1 cm-1 for AfWrbA.26 As redox (32) Mao, F.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2003, 125, 4951–4957. (33) Appelqvist, R.; Marko-Varga, G.; Gorton, L.; Torstensson, A.; Johansson, G. Anal. Chim. Acta 1985, 169, 237–247. (34) Ruzicka, J.; Hansen, E. H. In Flow Injection Analysis, 2nd ed.; Winefordner, J. D., Ed.; John Wiley & Sons: New York, 1988; pp 23-26. (35) Bistolas, N.; Christenson, A.; Ruzgas, T.; Jung, C.; Scheller, F. W.; Wollenberger, U. Biochem. Biophys. Res. Commun. 2004, 314, 810–816.
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mediators,36-38 2,6-dichloroindophenol, phenazine methosulfate, methylene blue, resazurin, 2-OH-1,4-naphthoquinone, anthraquinone 2,6-disulfonate, safranine T, diquat, 1,1′-bis(hydroxyethyl)-4,4′bipyridyl dichloride, and 1,1′-propylene-2,2′-bipyridylium dibromide were applied. Electrode Preparation and Equipment. Spectrographic graphite electrodes were polished as reported previously.39 In this work, 0.7 mg of the different SWCNT preparations was dissolved in 1 mL of Milli-Q water and sonicated overnight. Then 5 µL of dispersion was placed on the top of the polished electrode and spread evenly using a microsyringe tip. Next, 2 µL of the osmium redox polymer (10 mg/mL in Milli-Q water) was mixed with 5 µL of SWCNT solution. Following this, 5 µL of enzyme (0.1 mg/ mL in Milli-Q water) was added to the mixture. Finally 1 µL of PEGDGE (1 mg/mL) was added. The electrode was then allowed to dry and placed overnight at 4 °C in a water saturated atmosphere for the complete cross-linking reaction to occur. Electrodes not including SWCNTs were prepared as described above but without any addition of the SWCNT dispersions. Oxidative Shortening and Length Separation of SWCNTs. In order to perform the oxidative shortening, a suspension of 200 mg of SWCNTs and a mixture of 6 mL of sulfuric acid and 2 mL of nitric acid (98% and 70%, respectively) was sonicated for 12 h at 40 °C. The solution was then adjusted to pH 7 by adding a solution of NaOH. Thereafter the solvent was removed by centrifugation. To remove amorphous carbon, a suspension of the SWCNTs in Piranha solution with a 4:1 ratio of sulfuric acid (98%) and hydrogen peroxide (30 wt %) (note, this solution has to be treated with great care) was sonicated at 70 °C for 2 h. The solution was then again neutralized with NaOH and centrifuged. Next, the SWCNTs were dissolved in a 1 wt % Triton X-100 Milli-Q water solution and stabilized by sonication overnight. The dispersion was length separated by size exclusion chromatography using a column filled with controlled pore glass (CPG 3000 Å). Milli-Q water was used as the eluent. A total of 60 fractions of equal volume (15 mL) containing SWCNTs were collected. Cryogenic Transmission Electron Microscopy (CryoTEM). Specimens for electron microscopy were prepared in a controlled environment vitrification system (CEVS) to ensure stable temperature and to avoid loss of solution during sample preparation. The specimens were prepared as thin liquid films,
