Wiring of Redox Enzymes on Three Dimensional Self-Assembled

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Wiring of Redox Enzymes on Three Dimensional Self-Assembled Molecular Scaffold Marco Frasconi,†,||,^ Arnon Heyman,‡,|| Izhar Medalsy,§ Danny Porath,§ Franco Mazzei,*,† and Oded Shoseyov*,‡ †

Department of Chemistry and Drug Technology, “Sapienza” University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, and the Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences and Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel § Institute of Chemistry and Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel ‡

bS Supporting Information ABSTRACT: The integration of biological molecules and nanoscale components provides a fertile basis for the construction of hybrid materials of synergic properties and functions. Stable protein 1 (SP1), a highly stable ring shaped protein, was recently used to display different functional domains, to bind nanoparticles (NPs), and to spontaneously form two and threedimensional structures. Here we show an approach to wire redox enzymes on this self-assembled protein
nanoparticle hybrid. Those hybrids are genetically engineered SP1s, displaying glucose oxidase (GOx) enzymes tethered to the protein inner pore. Moreover, the Au-NP
protein hybrids self-assembled to multiple enzymatic layers on the surface. By wiring the redox enzymes to the electrode, we present an active structure for the bioelectrocatalytic oxidation of glucose. This system demonstrates for the first time a three-dimensional assembly of multiple catalytic modules on a protein scaffold with an efficient electrical wiring of the enzyme units on an electrode surface, thus implementing a hybrid electrically active unit for nanobioelectronic applications.

’ INTRODUCTION The organization of molecular components in nanoscale structures on surfaces is a challenging topic in modern science. Biomolecules and polymers in general are capable of selfassembling into a wide diversity of structures with distinct nanoscale architectures.1,2 Proteins in particular are attractive candidates for this purpose due to their well-defined structure and the feasibility of structural modification by genetic engineering.3 The combination of biological molecules and novel nanomaterial components is of great importance in the process of developing new nanoscale devices for future biological, medical, and bioelectronic applications.4,5 The chemical modification of enzymes with redox-relay groups,6 the immobilization of enzymes in redox polymers,7 and the conjugation of biomolecules to metal nanoparticles (NPs)8 and carbon-based nanomaterials9 were reported as means to establish electrical communication between redox proteins and electrodes. Recently, the spatial organization of redox-active biomolecules on DNA scaffolds was reported as a means to program biocatalytic transformations at the electrode surface.10 We have previously presented an enzymatic NP based on a protein molecular scaffold displaying hundreds of enzymes in one catalytic unit.11 Here we report how this system can be assembled using the boiling stable SP1 protein onto an electrode surface, thus fabricating a well r 2011 American Chemical Society

connected enzymatic multilayer. SP1 is a highly stable protein,12 that acts as a molecular scaffold to tether NPs13 and protein domains,11 and selectively and controllably binds to surfaces.14 The ability of this protein scaffold to self-assemble into threedimensional nanostructures, combined with the precision to connect to NPs and enzymes, provides a promising approach for the self-organization of composite nanostructures. In contrast to other enzyme hybrid structures, such as the conjugation of enzymes with metal-NPs or carbon nanotubes,9 the system is based on mild immobilization conditions (i.e., protein matrix) and can generate laterally nanostructured arrays with high local concentration of enzyme on the electrode surface. Indeed, the immobilization of biological molecules on carbon nanoscaffolds, such as carbon nanotubes, is limited by the fact that their closed shell does not allow for a high degree of functionalization, since adsorption or covalent immobilization can be achieved only at the functionalized end of the opened tubes. SP1 can be genetically engineered to present 12 surrounding glucose oxidase (GOx) monomers: this GOx-SP1 variant was expressed and purified from Escherichia coli. An active enzymatic Received: June 1, 2011 Revised: August 20, 2011 Published: September 06, 2011 12606

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Figure 1. (A) Schematic representation of the assembly of GOx-Cys-SP1 Au NP hybrid structure on the Au surface by means of dithiol bridging units. (B) AFM topographic image of the GOx-SP1 multilayer on the gold surface and corresponding topographic cross sections.

structure was then self-assembled, and hundreds of active enzymes clinging around the SP1 were imaged by transmission electron microscopy (TEM), forming a catalytic particle.11a The next challenge was to harness the catalytic reaction of GOx in order to achieve an efficient electron transfer communication between the enzyme active site and electrode surface. Herein, Au-NPs were inserted to the inner pore of GOx-SP1 modified with a Cys amino acid; this enabled the wiring of the enzymes, fused to the protein scaffold, with the electrode surface. We demonstrate that the biocatalytic oxidation of glucose charges the gold nanostructure in the SP1 inner pore; this charging process was followed by SPR spectroscopy and electrochemical measurements.

’ EXPERIMENTAL SECTION Chemicals and Materials. General reagents were purchased from Sigma-Aldrich and used without further purification. Citrate stabilized bare gold nanoparticles (diameter 2 nm) were obtained from Ted Pella Inc. (Redding, CA, product no. 15701). All aqueous solutions were prepared using deionized water (specific resistivity g 18.2 MΩ cm) obtained from a Direct-Q 3 UV apparatus (Millipore, France). Bacterial Strain and Culture Conditions. Escherichia coli strain DH5α was used for cloning, and E. coli strain BL21 (DE3) was used for expression. All bacteria were grown in Luria
Bertani (LB) media, at 37 C on a rotary shaker at 250 rpm. When grown for protein expression, 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added to the media at 0.8 O.D., and the bacteria were grown for a further 4 h. Vector Construction. The SP1 mutant incorporating GOx and Cys tags (named GOx-Cys-SP1) was constructed as follows: Plasmid pEGLS [GOx-L-SP1 in pET29a-vector, Novagen (Madison, WI)] previously described11 was digested with Hind3 and XhoI, and the truncated vector retrieved. In parallel, another vector (M43CSP1) was digested with the same enzymes to yield part of the mutated SP1 gene.11 The two products were ligated using T4 DNA ligase (Fermentas International Inc. Canada) to yield the final GOx-Cys-SP1 gene in pET29a-vector. Protein Purification and Refolding. The protein was purified and refolded as described previously.11 GOx activity was measured using the starch/KI assay as described previously.15 Au Nanoparticle Binding. GOx-Cys-SP1 protein at concentration of 0.1 mg mL
1 was incubated overnight with unconjugated gold

nanoparticles (Ted Pella, Inc., Redding, CA, product no. 15701) in 100 mM sodium phosphate buffer pH 6.0. Residual Au nanoparticles were removed using a 30 kDa cutoff ultrafiltration device (Viva Science AG, Hannover, Germany) with repeated washing. Atomic Force Microscopy (AFM) Imaging. The AFM measurements were performed with a Dulcinea AFMsystem (NanoTec Electronica, Madrid, Spain) under ambient conditions. A Multi75B soft tapping mode AFM tip (Budget Sensor, Sofia, Bulgaria) was used, with a nominal spring constant of ∼3 N/m, resonance frequency of ∼65 kHz, and tip apex radius < 25 nm. The tip
sample interaction was minimized by using soft AFM tips and low driving amplitudes.

In Situ Electrochemical Surface Plasmon Resonance (SPR) Experiments. The SPR Kretschmann-type spectrometer, Autolab Esprit (Eco Chemie, Utrecht, The Netherlands), with an LED light source with an emission wave of 670 nm and a prism refraction index of 1.52, was used to perform the optical measurements of the SPR angle. The voltammetric experiments were performed with a μAutolab electrochemical analyzer (Eco Chemie, Utrecht, The Netherlands) in a three-electrode setup. Glass supported gold substrates for SPR spectroscopy and electrochemistry, composed of a gold sensing surface (thickness of 50 nm) deposited onto a glass microscope slide with titanium adhesion layer (1.5 nm), were obtained from Xantec Bioanalytics (Muenster, Germany). Auxiliary Pt and reference Ag/AgCl/KClsat electrodes were parts of the cell. The gold substrate at the prism mounted against the Teflon cell with use of an O-ring was used as a working electrode (0.085 cm2 geometrical area, roughness factor ca. 1.2). The gold substrates were extensively cleaned in a freshly prepared piranha solution (3:1 H2SO4 98%/H2O2 30%). After 1 h, the gold surfaces were thoroughly rinsed with ethanol, dried in a stream of nitrogen gas, and modified by the thiol by the interaction with ethanolic solutions of the respective thiol, fixed total thiol content of 10 mM, for 12 h, followed by rinsing of the surfaces with ethanol and water.

’ RESULTS AND DISCUSSION Assembly of GOx-Cys-SP1 Au NP on SAM-Modified Gold Surface. Cysteine amino acids were integrated to the SP1 at

position 43, facing the protein inner pore. The new mutant, incorporating both GOx and thiol groups (named GOx-CysSP1), enabled the binding of 2 nm Au NPs in SP1’s inner pore (forming a hybrid nanostructure). Effective binding of Au NPs to 12607

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Langmuir mutants of SP1 proteins was demonstrated and characterized by AFM, EFM, and TEM.13b A mixed self-assembled monolayer (SAM) of biphenyl-4, 40 -dithiol, as bridging unit, and 3-mercapto-1-propanol, as diluent hydroxyl-terminated thiol (Figure 1A), was formed on an ultraflat gold surface,16 followed by absorption of 2 nm Au NPs as a junction layer. The GOx-Cys-SP1 was then assembled, from a diluted solution, on the functionalized surface and characterized by AFM. In Figure 1B, a single monolayer of the GOx-Cys-SP1 Au NP hybrid is observed with a height of 8 nm, corresponding to the 6 nm high GOx-Cys-SP1 plus 2 nm Au NP. Multiple topographic cross sections revealed also multilayer assembly of GOx-Cys-SP1 Au NP hybrids up to four 8 nm high layers. To obtain more information about the adsorption kinetics, the assembly of GOx-Cys-SP1 on the modified surface was monitored by SPR spectroscopy (Supporting Information, Figure S1). A densely packed GOx-Cys-SP1 Au NP hybrid monolayer on the gold surface was obtained using a molar ratio of dithiol/ 3-mercapto-1-propanol that corresponds to ca. 1:12. Taking into account the GOx-Cys-SP1 average diameter of ca. 50 nm, a highly packed monolayer of GOx-Cys-SP1 exhibits a surface coverage corresponding to ca. 8.3  10
14 mol cm
2. Given that one SP1 dodecamer holds six GOx dimer units, a densely packed GOx-Cys-SP1 monolayer yields a surface coverage of GOx corresponding to ca. 5  10
13 mol cm
2. In a control experiment, no immobilization of the GOx-Cys-SP1 was observed on a SAM prior the modification with Au NPs. Evidence that the GOx enzyme is catalytically active on to the electrode surface was obtained by the activation of the biocatalytic functions of the enzyme, in the presence of ferrocenemethanol as a diffusional electron mediator. Indeed, electrocatalytic anodic currents were observed in the presence of glucose, and as the concentration of glucose increased, the catalytic currents were intensified (Supporting Information, Figure S2). Assembly of GOx-Cys-SP1 Au NP Multilayer. One of the advantages of using SP1 as a molecular scaffold is its selfassembled properties.11 Thus, the GOx-Cys-SP1 Au NP monolayer bound to the modified gold surface can be used as a “template layer” to further create protein
Au NPs layers on the surface, as demonstrated in Figure 1B. Furthermore, the kinetic analysis, obtained after 400 min incubation of the hybrid monolayer electrode in the presence of GOx-Cys-SP1 Au-NP in solution, confirmed the assembling of a multilayer hybrid system with a dissociation binding constant (KD) of 0.4 μM (Supporting Information, Figure S3). Compared with the hybrid monolayer, an 18-fold enhancement of the signal intensity was obtained, indicating a spontaneous assembly of a multilayer structure on the surface. The activity of the enzyme units on the multilayer SP1 scaffold, immobilized onto the electrode surface, was confirmed by the electrocatalytic anodic current observed in the presence of glucose and the diffusional redox mediator ferrocenemethanol (Supporting Information, Figure S4). The amperometric responses of the GOx-Cys-SP1 Au NP multilayermodified electrode are up to 19-fold higher than the current response generated by the GOx-Cys-SP1 Au NP monolayerfunctionalized electrode (Supporting Information, Figure S5). This higher current observed with the multilayer structure, as compared to the monolayer-functionalized electrode, can be ascribed to the higher content of enzyme units in the 3D protein architecture. Biocatalytic Growth of Au NPs in the SP1 Scaffold. The electrical wiring of the enzyme with the Au NP and the

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Figure 2. Cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of glucose by the GOx-Cys-SP1 Au NP multilayer hybrid system bridged to the Au electrodes by the biphenyl-4,40 -dithiol, following the enlargement of the particles by the glucose/O2/ AuCl4reaction mixture at different time intervals: (a) 0 min, (b) 5 min, (c) 10 min, (d) 20 min, (e) 40 min, (f) 60 min, and (g) 80 min. The measurements were performed in a 0.1 M phosphate buffer solution, pH 7.0, under N2, for a glucose concentration of 20 mM. Potential scan rate 10 mV s
1. Inset: Dependence of the electrocatalytic currents generated by the enlarged Au NPs at different time intervals. The catalytic currents were measured at E = 0.500 V vs Ag/AgCl.

connectivity within the Au NPs in the multilayer structure is a crucial point on the way to harness the full enzymatic potential and achieve a functional conductive GOx multilayer. Cyclic voltammetry was used to investigate the link between redox chemistry and the catalytic activity of the hybrid system that was self-assembled on the gold surface. Only a slight bioelectrocatalytic current was, however, generated by the hybrid multilayer in presence of glucose. To improve the electrical communication, we used biocatalytic deposition of gold in the protein inner pore, using the NPs as nucleation sites. The biocatalytic enlargement of Au NPs would generate a connection of the enzyme to the Au NPs and between the Au NPs in the GOx-Cys-SP1 multilayer. As recently reported, different enzymes can be employed as biocatalysts to synthesize metal NPs17,18 and other materials, such as semiconductors NPs (e.g., CdS, PbS) or ferrocyanidebased NPs.19 For example, the enzyme glucose oxidase catalyzes the oxidation of glucose by oxygen to yield gluconic acid and H2O2; the latter product can be used to reduce AuCl4- in the presence of Au NP seeds, and to enlarge the metallic NPs.17 The enlargement of the Au NPs in the GOx-Cys-SP1 was performed by the GOx catalyzed oxidation of glucose (15 mM) in an air saturated solution that includes 0.2 mM AuCl4- and 2 mM cetyltrimethylammonium chloride (CTAC). The biocatalytic oxidation of glucose proceeds through the reduction of O2 to H2O2; the H2O2 acts as a reducing agent to reduce AuCl4- on the Au NPs, resulting in the enlargement of the Au NP seeds. The biocatalytic growth of Au NPs on the GOx-Cys-SP1 Au NPs multienzyme hybrid structure, probed in situ by the SPR shift of the surface, results in a significant change in the angle position of the minimum reflectance and a noticeable broadening of the curve (Supporting Information, Figure S6). As the contact time with glucose/O2/ AuCl4- reaction mixture increases, the 12608

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Figure 3. SPR spectra of the SP1-Cys-GOx multilayer hybrid system, after the enlargement of Au NPs, bridged to the gold surface by the selfassembled monolayer of biphenyl-4,40 -dithiol in the presence of various concentrations of glucose (mM): 0, 1, 2, 4, 10, 20, 40, and 95. Inset: SPR spectra corresponding to the SP1-Cys-GOx multilayer hybrid system, before the enlargement of Au NPs, bridged to the gold surface by the self-assembled monolayer of biphenyl-4,40 -dithiol upon the addition of various concentrations of glucose. The measurements were performed in pH 7.0 phosphate buffer 0.1 M.

shift of the SPR curve becomes larger, attaining the largest plasmon shift for a contact time close to 40 min. For comparison, the biocatalytic growth of Au NPs was also performed for the GOx-Cys-SP1 Au NPs monolayer functionalized electrode and the SPR shift was recorded (Supporting Information, Figure S7). The changes of the SPR signal also increase as the contact time is elevated; however, those changes are minute compared to those obtained for the multilayer hybrid structure. Figure 2 depicts the bioelectrocatalytic current generated with contact time upon the treatment of the hybrid system with glucose and AuCl4-, for time intervals of enlargement corresponding to 5, 10, 20, 40, 60, and 80 min. The enlargement of the Au NPs results in an enhancement of the bioelectrocatalytic current (Figure 2, inset). The catalytic anodic current values increase with the growth time interval attaining a plateau at 40 min, which confirms that the electrical communication between the enzyme and the Au NPs (electrical wiring) and between the Au NPs of GOx-Cys-SP1 multilayer (electrical conductivity) was achieved. Control experiments showed no increase in the biocatalytic current in the absence of AuCl4- in the reaction mixture, and that the added glucose was essential to enlarge the Au NPs bound in the central core of GOx-Cys-SP1. The bioelectrocatalytic current exhibits an increase with the concentration of glucose added to the metallization reaction mixture. This means that a minimum concentration of glucose and thus of H2O2 is required for the reaction to occur. Furthermore, the biocatalytic growth of the Au NPs is responsible for the connection of GOx-Cys-SP1 Au NP multilayer.

Figure 4. (A) Plasmon angle shifts induced upon addition of different concentrations of glucose to the following: SP1-Cys-GOx multilayer hybrid system, after the enlargement of Au NPs, bridged to the Ausupport by the self-assembled monolayer of 1,9-nonanedithiol (a), and biphenyl-4,40 -dithiol (b); SP1-Cys-GOx multilayer hybrid system, before the enlargement of Au NPs, bridged to the gold surface by 1, 9-nonanedithiol (c), and biphenyl-4,40 -dithiol (d); native GOx immobilized with glutaraldehyde, as cross-linking agent, on Au NPs bridged to the gold surfaces by 1,9-nonanedithiol before (e) and after (f) the biocatalytic enlargement of the NPs. Inset: The relative changes of the plasmon angle upon the addition of various concentrations of glucose to the SP1-Cys-GOx multilayer hybrid system after the enlargement of Au NPs bridged to the gold surface with 1,9-nonanedithiol linker (closed symbol) and biphenyl-4,40 -dithiol linker (open symbol). (B) Relative changes of the plasmon angle upon the addition of various concentrations of glucose to the SP1-Cys-GOx multilayer (a) and GOx-Cys-SP1 monolayer (b) hybrid system, after the enlargement of Au NPs, bridged to the gold electrode with 1,9-nonanedithiol linker.

Biocatalytic Activity of SP1-Cys-GOx Multilayer Hybrid System Probe by SPR. The biocatalytic properties of the

GOx-Cys-SP1 multilayer hybrid structure, after the enlargement of Au NPs, were probed by means of SPR spectroscopy. The shift of the plasmon peak position as a result of the metal 12609

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Figure 5. (A) Cyclic voltammograms corresponding to the biocatalyzed oxidation of glucose by the GOx-Cys-SP1 multilayer hybrid system bridged to the gold electrode by the biphenyl-4,40 -dithiol, in the presence of different glucose concentrations: (a) 0 mM, (b) 5 mM, (c) 10 mM, (d) 20 mM, (d) 40 mM, (f) 80 mM, and (g) 160 mM. Potential scan rate 10 mV s
1. (B) Comparison of the catalytic currents obtained at variable concentration of glucose to the following: GOx-Cys-SP1 multilayer hybrid system (closed symbol) and GOx-Cys-SP1 monolayer hybrid system (open symbol), after biocatalytic enlargement of Au NPs. (C) Cyclic voltammograms corresponding to the biocatalyzed oxidation of glucose by the GOx-Cys-SP1 multilayer hybrid system, linked to the gold electrode, in the presence of (a) 0 mM glucose under N2, (b) 20 mM glucose under N2, (c) 20 mM glucose under O2, and (d) 20 mM glucose and 1.5 mM ascorbic acid under N2. (D) Square wave voltammetry of gold electrode modified with GOx-Cys-SP1 Au NP monolayer before (a) and after (c) the biocatalytic enlargement of Au NPs; GOx-Cys-SP1 multilayer hybrid system before (b) and after (d) the biocatalytic enlargement of Au NPs. The experiments were performed in phosphate buffer 0.1 M pH 7.0, potential amplitude 150 mV, frequencies 300 Hz.

nanostructures charging, including NPs, nanowires, and nanoplates, on a gold surface was demonstrated in numerous studied.19,20 Figure 3 shows the SPR spectra of the GOx-CysSP1 multilayer hybrid system bridged to the gold electrode by the self-assembled monolayer of biphenyl-4,40 -dithiol before the addition of glucose, and after the addition of different concentrations of glucose in the range 1
95 mM. The plasmon angle θ (angle of minimum reflectance) shifts to higher values, and the change in the plasmon angle reaches a saturated shift that corresponds to Δθ = 0.36 at glucose concentrations higher than 95 mM (Figure 4A, curve b). Figure 3, inset, shows the SPR spectra corresponding to the GOx-Cys-SP1 multilayer hybrid system, before the enlargement of Au NPs, in the presence of different concentrations of glucose. The addition of glucose leads to a shift in the plasmon angle, but this shift is smaller than those observed for the GOx-Cys-SP1 hybrid system after enlargement of Au NPs (Figure 4A, curve d).

To elucidate the mechanism leading to the shift in the plasmon angle, the GOx-Cys-SP1 was assembled on gold surfaces modified with different dithiol bringing units (Figure 4A). Curve a shows the plasmon angle shifts of GOx-Cys-SP1 multilayer hybrid system, after the enlargement of Au NPs, bridged to the gold surface by the self-assembled monolayer of 1,9-nonanedithiol, upon addition of different concentrations of glucose. Curve b shows the θ changes of the analogous system, that is bridged with the biphenyl-4,40 -dithiol. Curves c and d correspond to the changes in the plasmon angle of the Au surface modified with the GOx-Cys-SP1 Au NPs multilayer bound to a 1,9-nonanedithiol linker and biphenyl-4,40 -dithiol linker upon the addition of different concentrations of glucose, respectively. A comparable θ change was observed after immobilization of native GOx with glutaraldehyde, as cross-linking agent, on Au NPs modified surfaces before and after the biocatalytic enlargement of the NPs, curve e and f, respectively, that overlaps with curves c and d. 12610

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Langmuir While these small increases in the θ values observed for systems c
f are attributed to the alteration of the refractive index of the media upon addition of glucose (linear dependence with the concentration of glucose), the substantially higher changes in the values of the plasmon angle shifts for the connected layers of GOx-Cys-SP1 (curve a and b) are attributed to the charging of the Au nanostructure in the central core of the protein by the biocatalytic process of electrically connected GOx (logarithmic dependence on the concentration of glucose). These spectral shifts of localized surface plasmon (LSP) band position are attributed to the changes in the plasma frequency, caused by the increase in charge density, resulting from the charging of the enlarged Au NPs. Since the localized plasmon of enlarged Au NPs immobilized on the interface is coupled to the bulk gold surface plasmon, the charging of the enlarged Au NPs would alter not only the LSP band position but also the coupling between the nanostructure and the surface plasmon wave, resulting in the change of the SPR spectrum.20,21 The dithiol monolayer between the enlarged Au NPs and the Au surface provides both the supporting layer and the tunneling barrier for the electron transfer from the nanostructure to the bulk Au surface, leading to the storage of electrons on the enlarged NPs. Thus, the charging of the nanostructures by the biocatalytic process and the discharging of the Au NPs by tunneling of the electrons to the bulk Au surface leads to a steady state equilibrium charging of the Au NPs. Figure 4A, inset, shows the relative changes of the plasmon angle derived from the charging of the Au NPs in a system consisting of connected hybrid layers of GOx-Cys-SP1 bridged to the Au-support with the 1,9-nonanedithiol monolayer (curve a) and with the biphenyl-4,40 -dithiol linker (curve b). These curves were obtained by subtracting the changes in the plasmon angles originating from the alteration of the bulk solution refractive index at variable concentrations of glucose (Figure 4A, curves c and d), from the total plasmon shifts observed in a system consisting of GOx-Cys-SP1 Au NP hybrids system linked to the Au surface by the monolayer 1, 9-nonanedithiol or biphenyl-4,40 -dithiol (Figure 4A, curves a and b respectively). The changes of the plasmon angles increase with the concentration of glucose and tend to saturate at a glucose concentration of ca. 95 mM. The changes in the θ values are more pronounced for the system with 1,9-nonanedithiol linker than for those obtained with the biphenyl-4,40 -dithiol linker. Thus, a fully conjugated dithiol bridge, such as biphenyl-4,40 -dithiol, exhibits a relatively low tunneling barrier, whereas 1,9-nonanedithiol reveals a high tunneling barrier for the electron transfer from the Au NPs to the Au surface. For comparison, the calibration curve corresponding to the biocatalytic activity of GOx-Cys-SP1 Au NPs monolayer functionalized electrode, after Au NPs enlargement, is shown in Figure 4B, curve b. Evidently, the SPR angle shift of the multilayer structure is significantly higher than the monolayer GOxCys-SP1 modified surface, consistent with the presence of multiple enzymatic layers on the surface. Bioelectrochemical Properties of SP1-Cys-GOx Multilayer Hybrid System. The bioelectrocatalytic current (measured at 0.500 V vs Ag/AgCl) generated by the connected hybrid layers of GOx-Cys-SP1 assembled on electrode surface by the fully conjugated dithiol bridges (biphenyl-4,40 -dithiol) showed a linear relation with the content of glucose in the concentration range 0.5
100 mM (Figure 5A). The anodic current density saturates at a concentration of 140 mM with a saturation current density of Isat = 2565 μA cm
2 (Figure 5B). The catalytic anodic current is observed at an applied potential close to Eap > 0.200 V

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(vs Ag/AgCl) that is positive in comparison with the redox potential of the redox active site of the GOx.22 This overpotential might originate from the limitation for the transfer of electrons from the GOx to the metal wire in the SP1 cavity and from the metal wire through the dithiol bridge to the bulk Au electrode. The bridge-controlled electron transfer is supported by examining the overpotential required to achieve a certain anodic electrocatalytic current density by the integrated electrode with different dithiol bringing units. The fully conjugated 4,40 -biphenyldithiol was compared with 1,9-nonanedithiol linker. The current densities derived from the Tafel equation for 4,40 -biphenyldithiol and 1, 9-nonanedithiol correspond to 21 and 6 mA cm
2, respectively. Thus, the fully conjugated dithiol bridge, biphenyl-4,40 -dithiol, exhibits a relatively low tunneling barrier, whereas 1,9-nonanedithiol reveals a high tunneling barrier for the electron transfer from the Au nanostructure to the Au surface, in agreement with the conclusion in the previous section. As a further support, the bioelectrocatalytic current of GOxCys-SP1 Au NPs monolayer functionalized electrode, after NPs enlargement, was recorded in the presence of different concentrations of glucose (Supporting Information, Figure S8). The amperometric response increases with the concentration of glucose and reaches saturation at a concentration of 50 mM with a saturation current density of Isat = 110 μA cm
2 (Supporting Information, Figure S8, inset). This value was 23-fold lower than the current density generated by the integrated GOx-Cys-SP1 Au NPs multilayer hybrid system, after NPs enlargement (Figure 5B). The success to enhance the sensitivity of the glucose detection by the latter system is attributed to the presence of several enzymatic layers on the surface. Furthermore, it should be noted that, in control experiments where GOx-Cys-SP1 not functionalized with Au NP was covalently linked to a mercaptoundecanoic acid monolayer modified electrode, no bioelectrocatalytic current was detected, although the enzyme existsted in an active state on the electrode (confirmed by the diffusional redox mediator ferrocenemethanol). Similar results were obtained after immobilization of native GOx with glutaraldehyde, as cross-linking agent, on Au NPs modified surfaces before and after the biocatalytic enlargement of the NPs, although also in this experiment the enzyme was in an active state. Although there is no direct evidence for the complete organization of Au NPs in pillars all over the surface, the formed organization is sufficient for efficient electronic communication between multiple catalytic domains and the electrode, and for functional activity. The electrochemical experiments indicate that the enlarged Au NPs in the SP1’s cavity act as an electron relay for the electrical wiring of the enzyme redox-active center. Further, the biocatalytic growth of Au NPs generates a connection between the Au NPs of multiple layers of GOx-Cys-SP1, allowing an efficient electronic communication between several enzyme layers and the electrode surface. The oxidation of glucose by FAD dependent GOx is known to be affected by oxygen. In addition, nonspecific oxidizable interfering compounds for biosensing of glucose could significantly contribute to the resulting anodic current. Cyclic voltammograms of the connected hybrid layers of GOx-Cys-SP1 assembled on the electrode surface were performed for the bioelectrocatalytic oxidation of glucose (20 mM) in the absence and in presence of O2. A small decrease of anodic current in the presence of O2 was observed (Figure 5C, curve d). Furthermore, the cyclic voltammograms of the GOx-Cys-SP1 Au NP in the presence of glucose is unperturbed by an oxidizable interference such as ascorbic acid (Figure 5C). 12611

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’ CONCLUSIONS In the present study, we have successfully employed engineered thiols at the center of GOx-SP1 as vehicle for nanoscale organizing of Au NPs. The ability of SP1 to combine extreme stability, multidisplay of active enzymes, specific encapsulation of NPs, and spontaneous assembly of complex multilayer structures are successfully combined here to make a unique molecular scaffold. In the demonstrated concept, which can be extended to other enzymatic systems, a protein is utilized as a molecular scaffold to connect several enzymatic units onto an electrode surface. The tailored protein design enabled the assembly of a three-dimensional functional structure, thus fabricating multiple enzymatic layers on the surface. The biocatalytic optimization of the electrical contact by the biocatalytic growth of the Au NPs generates an integrated electrically wired system, thereby enhancing the electron transfer between the enzyme and the enlarged Au NPs in the central core of GOx-Cys-SP1 bridged to the gold surface by a dithiol monolayer. Indeed, the highly efficient charge transfer through the GOx-Cys-SP1 multilayer provides a hybrid electrically active unit for nanobioelectronic applications. ’ ASSOCIATED CONTENT

bS

Supporting Information. ICP-MS analysis of Au content in the hybrid structure, SPR sensorgrams corresponding to the immobilization of GOx-Cys-SP1 on the surface, SPR curves corresponding to the biocatalytic enlargement of Au NPs in the GOx-Cys-SP1 Au NP multilayer and monolayer functionalized electrode, cyclic voltammograms of GOx-Cys-SP1 Au NP multilayer and monolayer functionalized electrode with glucose and electron diffusional redox mediator, and the biocatalyzed oxidation of glucose by the GOx-Cys-SP1 monolayer system. This

material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (O.S.); [email protected] (F.M.). Present Addresses ^

Department of Chemistry, Northwestern University, 2145 Sheridan Road Evanston, Illinois 60208-3113, United States. Author Contributions

)

The protein assembly was investigated in electrochemical studies to evaluate the redox properties of the obtained hybrid systems: because of its high sensitivity, square wave voltammetry (SWV) was used for this purpose. From the SWV study, no peak response was observed for the GOx-Cys-SP1 Au NP monolayer and for the multilayer hybrid structure before the biocatalytic enlargement of Au NPs (Figure 5D, curves a and b, respectively). In this configuration, the GOx active site lacks electrical communication with the Au NP in the SP1 cavity. SWV of the hybrid monolayer system after Au NPs enlargement showed a single peak at E =
0.410 V vs Ag/AgCl (pH = 7.0) (Figure 5D, curve c); the obtained formal potential is more reducing than the oneelectron potentials reported for GOx-FAD22 and can be explained by a different reaction of the cofactor, such as its twoelectron reduction/oxidation. The formal potential reported for our system is in agreement with the value reported by Xiao et al. (E =
0.45 V, pH = 7.0) for a GOx monolayer in which electrons were relayed by 1.4 nm diameter Au NPs between FAD/FADH2 and a gold electrode.8a A more negative formal potential was observed for deglycosylated GOx adsorbed on a glassy carbon electrode, due to a strong interaction of the enzyme with the vitreous carbon.23 A good electrical communication in the connected hybrid layers of GOx-Cys-SP1 was evidenced from the high faradic current (Figure 5D, curve d), that is 18-fold higher than the responses generated by the hybrid monolayer.

ARTICLE

These authors contributed equally to this work.

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