Biocatalytic Implant of Pt Nanoclusters into Glucose Oxidase: A

Sep 10, 2010 - Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. ‡ Faculty of Biotechnology and Food Engineering,...
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Biocatalytic Implant of Pt Nanoclusters into Glucose Oxidase: A Method to Electrically Wire the Enzyme and to Transform It from an Oxidase to a Hydrogenase Omer Yehezkeli,† Sara Raichlin,† Ran Tel-Vered,† Ellina Kesselman,‡ Dganit Danino,‡ and Itamar Willner*,† †

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, and ‡Faculty of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel

ABSTRACT The enzyme glucose oxidase (GOx) reduces, in the presence of glucose and under anaerobic conditions, PtCl62- to Pt nanoclusters (NCs) that are implanted in the protein. The assembly of the Pt NC/GOx hybrid on a dithiol monolayer yields an electrically contacted enzyme electrode, and the bioelectrocatalytic oxidation of glucose is activated (turnover rate ca. 2780 ( 70 s-1). The Pt NC/GOx hybrid is also used, under anaerobic conditions, as a biocatalyst for H2 evolution. SECTION Nanoparticles and Nanostructures

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he electrical contacting of redox enzymes is one of the most fundamental challenges in bioelectrochemistry and provides the basis for developing amperometric biosensors and biofuel cells.1-4 To achieve effective electrical communication between the enzyme redox center and electrodes, the structural alignment of the redox site close to the electrode is desirable, and the positioning of a relay unit between the redox center and the electrode for mediated electron transfer is required. Accordingly, the reconstitution of apo-proteins on cofactor-modified electrodes was employed as a versatile method to align redox enzymes on electrodes.5-7 Furthermore, the reconstitution of apo-enzymes on cofactorfunctionalized Au nanoparticles (NPs) was used to align the enzymes on a conductive surface, with the Au NPs acting as relay units that mediate the electron transfer between the redox centers and the electrode.8,9 Whereas this method proved to be an effective means to electrically wire redox enzymes with electrodes, the need to modify the NPs with the respective cofactor and to reconstitute the apo-enzyme on the cofactor turned the method into an art with limited practical utility. The use of enzymes for the biocatalytic growth of metallic NPs was extensively developed in the past few years, and the products of different biocatalyzed transformations were used to reduce metal salts for growing NPs.10-13 Flavoenzymes, for example, oxidize substrates while generating reduced flavin adenine dinucleotide, FADH2. The oxidation of the FADH2 by O2 regenerates the FAD cofactor while generating H2O2. In an oxygen-free environment, the reduced cofactor FADH2 is a powerful reducing agent that may thermodynamically reduce different metal salts to metallic nanoclusters at the redox center of the enzyme. For example, the reduction potential of FADH2 embedded in glucose oxidase, E° = -0.48 V versus SCE at pH 7.4,14 can be used to reduce platinum salts into Pt nanoclusters (NCs). Such implanted NCs may facilitate

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the assembly of the protein on the electrode and act as charge transport mediators that electrically contact the enzyme with the electrode. Here we report on the biocatalytic growth of Pt NCs in glucose oxidase, GOx. We demonstrate the assembly of the Pt NC-functionalized GOx on electrodes using dithiol linkers and the electrical contacting of GOx toward the bioelectrocatalyzed oxidation of glucose. Furthermore, we find that the Pt NC-functionalized GOx acts as a hydrogenase, and under anaerobic conditions, leads to the evolution of hydrogen while oxidizing glucose. GOx (5 mg mL-1) was treated with 20 mM glucose in the presence of 5 mM PtCl62- in a phosphate buffer solution (50 mM, pH 7.4) under argon for a time interval of 1 h, and the resulting Pt NC-modified protein was separated using an Amicon Ultra 50KD filter. Cryo-TEM measurements indicated that Pt nanoclusters exhibiting sizes of 3-5 nm were generated in the protein. (See Figure S1 of the Supporting Information.) The Pt NC-functionalized GOx was then immobilized on the electrode (Scheme 1, Path I). A Au electrode was modified with a monolayer of 1,4-benzene dithiol, and the Pt NC-functionalized GOx was linked to the surface by the binding of the free thiol groups to the Pt NCs. The surface coverage of the Pt NC/GOx hybrid on the electrode was then probed by spectroscopically assaying the GOx activity associated with the electrode,15 and it corresponded to ca. 3.5  10-13 mol 3 cm-2 (see also the Supporting Information). Figure 1 shows the cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of different concentrations of glucose by the Pt NC/GOx hybrid-modified Au electrode. Electrocatalytic anodic currents at an onset potential of Received Date: August 12, 2010 Accepted Date: August 31, 2010 Published on Web Date: September 10, 2010

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Figure 2. Cyclic voltammograms corresponding to the bioelectrocatalytic oxidation of glucose by a Au electrode modified with: (a) The Pt NC-implanted GOx. (b) GOx covalently linked to a monolayer of Pt NPs. (c) A monolayer of Pt NPs. The measurements were performed in a phosphate buffer (0.1 M, pH 7.4) that contained 40 mM glucose. Scan rate: 5 mV s-1. Measurements were performed under N2.

Figure 1. Cyclic voltammograms corresponding to the bioelectrocatalytic oxidation of glucose by the Pt NC/GOx hybrid monolayermodified Au electrode. The measurements were performed in a phosphate buffer solution (0.1 M, pH 7.4) that contained: (a) 0, (b) 20, (c) 40, (d) 60, (e) 80, (f ) 100, (g) 120, and (h) 140 mM glucose. Scan rate: 5 mV s-1. Inset: calibration curve for the bioelectrocatalytic currents at E = 0.3 V versus SCE associated with the different glucose concentrations. All measurements were performed under N2.

is shown in Figure 1 (inset). Knowing the surface coverage of the enzyme and the saturation current, the turnover rate of electrons between the redox center of GOx and the electrode was estimated to be ket = 2780 ( 70 s-1. Realizing that the exchange rate of electrons between the redox center of GOx and its native acceptor (O2) is 650 s-1,16 we conclude that implanting the Pt NCs into the protein results in an effectively electrically contacted GOx. It should be noted that the onset potential (0.1 V vs SCE) for the bioelectrocatalytic oxidation of glucose is substantially more positive than the thermodynamic redox potential of the FAD/FADH2 center. This is attributed to an overpotential required to transfer the electrons from the redox centers to the electrode by means of the Pt NCs. A similar phenomenon was observed for the electrical contacting of other metal NPs/enzyme hybrids with electrodes.3 Several control experiments demonstrated the significance of implanting the Pt NCs into the protein for establishing the resulting enhanced electrical contacting of the enzyme with the electrode (Figure 2). In one experiment, Pt NPs capped with mercaptopropionic acid and mercaptoethane sulfonic acid were linked to the 1,4-benzene dithiol monolayer associated with the Au electrode, and GOx was covalently linked to the Pt NPs (using 10 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 10 mM N-hydroxysuccinimide (NHS) in phosphate buffer (50 mM, pH 7.4) for 2 h). Although this enzyme electrode revealed a biocatalytic activity, as reflected by the spectroscopic assay indicating a GOx surface coverage of 4.4  10-13 mol cm-2, the enzyme lacked an electrical contact with the electrode, which did not lead to the bioelectrocatalytic oxidation of glucose (Figure 2, curve b). Similarly, Pt NPs capped with mercaptoethane sulfonic acid/mercaptopropionic acid (1 mg mL-1) were reacted with a benzene dithiol-functionalized Au electrode and did not exhibit any electrocatalytic functions toward the oxidation of glucose (curve c). These results indicate that the incorporation of the Pt nanoclusters into the protein, in a close proximity to the active site, is essential to electrically contact the enzyme with the electrode. The effective electrical contacting of the Pt NC/enzyme hybrid with the Au surface leads to an O2-insensitive enzyme

Scheme 1. Schematic Presentation for the Enzymatic Implantation of a Pt Nanocluster at the Redox-Active Center of Glucose Oxidasea

a Pt NC/GOx hybrid is, then, used for: (Path I) Wiring the hybrid to a Au electrode in a monolayer configuration and (Path II) Generation of H2 through the biocatalytic oxidation of glucose.

ca. E = 0.1 V versus SCE are observed. It should be noted that in the absence of glucose no electrocatalytic anodic currents are observed (curve a), implying that the anodic currents are due to the bioelectrocatalytic oxidation of glucose. As the concentration of glucose is increased, the anodic currents are intensified, and they level off to a saturation value at a glucose concentration of ca. 140 mM. The resulting calibration curve

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that the Pt NC/GOx system acts as a hydrogenase that facilitates H2 evolution. The study paves the way to electrically contact other redox enzymes with electrodes and introduces an approach to apply metal NCs/enzyme hybrids for hydrogenation processes.

SUPPORTING INFORMATION AVAILABLE A Cryo-TEM image of the Pt NC/GOx hybrid, the determination of the content of the enzymatically active Pt NC/GOx hybrid on the electrode, and experimental procedures are detailed. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. E-mail: willnea@ vms.huji.ac.il. Tel: þ972-2-6585272. Fax: þ972-2-6527715.

Figure 3. Time-dependent generation of H2 using the Pt nanocluster-implanted GOx. Measurements were performed in an Ar-purged phosphate buffer solution (25 mM, pH 6.0) that contained 0.8 μM of the modified GOx and 100 mM glucose.

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Willner, I.; Yan, Y. M.; Willner, B.; Tel-Vered, R. Integrated Enzyme-Based Biofuel Cells - A Review. Fuel Cells 2009, 9, 7–24. (2) Barton, S. C.; Gallaway, J.; Atanassov, P. Enzymatic Biofuel Cells for Implantable and Microscale Devices. Chem. Rev. 2004, 104, 4867–4886. (3) Wang, J. Electrochemical Glucose Biosensors. Chem. Rev. 2008, 108, 814–825. (4) Heller, A. Electron-Conducting Redox Hydrogels: Design, Characteristics and Synthesis. Curr. Opin. Chem. Biol. 2006, 10, 664–672. (5) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G. L.; Buckmann, A. F.; Heller, A. Electrical Wiring of Glucose Oxidase by Reconstitution of FAD-Modified Monolayers Assembled onto Au-Electrodes. J. Am. Chem. Soc. 1996, 118, 10321–10322. (6) Fruk, L.; Kuo, C. H.; Torres, E.; Niemeyer, C. M. Apoenzyme Reconstitution as a Chemical Tool for Structural Enzymology and Biotechnology. Angew. Chem., Int. Ed. 2009, 48, 1550– 1574. (7) Liu, J. Q.; Chou, A.; Rahmat, W.; Paddon-Row, M. N.; Gooding, J. J. Achieving Direct Electrical Connection to Glucose Oxidase using Aligned Single Walled Carbon Nanotube Arrays. Electroanalysis 2005, 17, 38–46. (8) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. ”Plugging into Enzymes”: Nanowiring of Redox Enzymes by a Gold Nanoparticle. Science 2003, 299, 1877–1881. (9) Zayats, M.; Katz, E.; Baron, R.; Willner, I. Reconstitution of Apo-Glucose Dehydrogenase on Pyrroloquinoline QuinoneFunctionalized Au Nanoparticles Yields an Electrically Contacted Biocatalyst. J. Am. Chem. Soc. 2005, 127, 12400– 12406. (10) Baron, R.; Zayats, M.; Willner, I. Dopamine-, L-DOPA-, Adrenalineand Noradrenaline-Induced Growth of Au-Nanoparticles: Assays for the Detection of Neurotransmitters and of Tyrosinase Activity. Anal. Chem. 2005, 77, 1566–1571. (11) Shankar, S. S.; Rai, A.; Ankamwar, B.; Singh, A.; Ahmad, A.; Sastry, M. Biological Synthesis of Triangular Gold Nanoprisms. Nat. Mater. 2004, 3, 482–488. (12) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Biocatalytic Growth of Au Nanoparticles: From Mechanistic Aspects to Biosensors Design. Nano Lett. 2005, 5, 21–25.

electrode for the bioelectrocatalytic oxidation of glucose, and the cyclic voltammograms generated by the enzyme electrode are similar in the absence or presence of oxygen. Also, the rapid electron exchange between the enzyme and the Au surface yields a selective electrode, and the contribution of the nonspecific oxidation of the interferants ascorbic or uric acids (each 0.1 mM) to the amperometric responses of glucose (5 mM) oxidation is minute (e6%). The Pt NC/GOx hybrid represents an enzyme of new functions, and it acts as a hydrogenase that drives H2 evolution. The redox potential of the reduced FADH2 cofactor, formed upon the oxidation of glucose, is thermodynamically adequate to reduce protons to hydrogen. In the absence of oxygen, the vicinity of the Pt NCs to the reduced cofactor allows the electron transfer from FADH2 to the Pt NCs, and their charging activates the Pt-catalyzed H2 evolution (Scheme 1, path II). Figure 3 shows the timedependent generation of H2 in the system under anaerobic conditions (GC detection using a MS 5 Å column, L = 2 m; Ar carrier gas at a flow rate of 10 mL min-1; column temperature, 50 °C; injector and TC detector temperatures, 120 °C). The H2-evolution experiments were conducted at pH 6.0 to thermodynamically favor the evolution of H2 (E°0 = 0.36 V). Control experiments revealed that a mixture of Pt NPs (diameter ca. 3 nm) and GOx in solution did not lead to any H2 evolution. Also, the introduction of N,N0 -dimethyl-4,40 -bipyridinium (MV2þ) to the Pt NC-free GOx in the presence of the Pt NPs in solution resulted in the effective evolution of H2. That is, the reduction of MV2þ by the FADH2 yields MVþ 3 ,17 which mediates the generation of H2 at the Pt NPs. These results indicate that the presence of the Pt NCs in close contact with the reduced cofactor is essential to evolve H2. In conclusion, the present Letter demonstrates the biocatalytic growth of Pt NCs in close proximity to the FAD redox center of GOx. The metallic NCs enabled the electrical contacting of the enzyme with the electrode and the activation of its bioelectrocatalytic functions. Moreover, we demonstrated

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(13) (14)

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Willner, I.; Baron, R.; Willner, B. Growing Metal Nanoparticles by Enzymes. Adv. Mater. 2006, 18, 1109–1120. Gorton, L.; Johansson, G. Cyclic Voltammetry of FAD Adsorbed on Graphite, Glassy-Carbon, Platinum and Gold Electrodes. J. Electroanal. Chem. 1980, 113, 151–158. The activity of the Pt NC/GOx is ca. 10% lower than that of native GOx (similar protein content). This might be due to the partial denaturation of the enzyme by the Pt NCs or the partial Pt NC-catalyzed decomposition of H2O2, the product that assays the enzyme activity. Thus, the reported coverage might be up to 10% lower than the actual coverage. Bourdillon, C.; Demaille, C.; Gueris, J.; Moiroux, J.; Saveant, J. A Fully Active Monolayer Enzyme Electrode Derivatized by Antigen-Antibody Attachment. J. Am. Chem. Soc. 1993, 115, 12264–12269. Katz, E.; Sheeney-Haj-Ichia, L.; Willner, I. Electrical Contacting of Glucose Oxidase in a Redox-Active Rotaxane Configuration. Angew. Chem., Int. Ed. 2004, 43, 3292–3300.

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