Gold Nanoparticle Assisted Assembly of a Heme Protein for

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J. Phys. Chem. C 2007, 111, 6124-6132

Gold Nanoparticle Assisted Assembly of a Heme Protein for Enhancement of Long-Range Interfacial Electron Transfer Palle S. Jensen,† Qijin Chi,*,† Flemming B. Grumsen,‡ Jose´ M. Abad,§ Andy Horsewell,‡ David J. Schiffrin,§ and Jens Ulstrup*,† Departments of Chemistry and of Manufacturing Engineering and Management and NanoDTU, Technical UniVersity of Denmark, DK-2800 Kgs. Lyngby, Denmark, and Center for Nanoscale Science and Chemistry Department, UniVersity of LiVerpool, LiVerpool L69 7ZD, United Kingdom ReceiVed: December 8, 2006; In Final Form: February 19, 2007

Interfacial electron transfer (ET) of biological macromolecules such as metalloproteins is the key process in bioelectrochemistry, enzymatic electrocatalysis, artificial ET chains, single-molecule electronic amplification and rectification, and other phenomena associated with the area of bioelectronics. A key challenge in molecular bioelectronics is to improve the efficiency of long-range charge transfer. The present work shows that this can be achieved by nanoparticle (NP) assisted assembly of cytochrome c (cyt c) on macroscopic singlecrystalline electrode surfaces. We present the synthesis and characterization of water-soluble gold nanoparticles (AuNPs) with core diameter 3-4 nm and their application for the enhancement of long-range interfacial ET of a heme protein. Gold nanoparticles were electrostatically conjugated with cyt c to form nanoparticleprotein hybrid ET systems with well-defined stoichiometry. The systems were investigated in homogeneous solution and at liquid/solid interface. Conjugation of cyt c results in a small but consistent broadening of the nanoparticle plasmon band. This phenomenon can be explained in terms of long-range electronic interactions between the gold nanoparticle and the protein molecule. When the nanoparticle-protein conjugates are assembled on Au(111) surfaces, long-range interfacial ET across a physical distance of over 50 Å via the nanoparticle becomes feasible. Moreover, significant enhancement of the interfacial ET rate by more than an order of magnitude compared with that of cyt c in the absence of AuNPs is observed. AuNPs appear to serve as excellent ET relays, most likely by facilitating the electronic coupling between the protein redox center and the electrode surface.

Introduction Synthesis and characterization methods of nanoparticles composed of a variety of materials such as metals, metal oxides, semiconductors, and polymers have been vigorously developed over the past two decades.1-7 This has paved the way for multifarious use of nanoparticles as versatile building blocks in nanoscale science and technology.8,9 Specific functionalization of nanoparticles with biological molecules to assemble bioinorganic hybrid nanostructures holds particular and growing interest for applications ranging from fundamental approaches to biological processes to the construction of biosensing devices.10-13 Such types of hybrid conjugates are expected to display the combined advantages of high specificity from the biological components and unique electronic/optical properties from the nanoparticles. Research on gold nanoparticles has attracted considerable attention mainly due to the unique properties of gold6 and to the rapid development of thiol-gold self-assembly chemistry.14 Gold nanoparticles can now be prepared in a controlled manner by the introduction of organothiolate ligands,2,15-17 a strategy * To whom correspondence should be addressed. (Q.C.) E-mail: [email protected]. Phone: +45 45252352. Fax: +45 45883136. † Department of Chemistry and NanoDTU, Technical University of Denmark. ‡ Department of Manufacturing Engineering and Management, Technical University of Denmark. § University of Liverpool.

that offers significant advantages for the preparation of small particles (99%, Sigma-Aldrich), NaBH4 (>99.9%, Sigma-Aldrich), tetraoctylammonium bromide ((TOA)Br; >98%, Fluka), DL-6,8-thioctic acid (TA; >99%, Sigma-Aldrich), 11-mercaptoundecanoic acid (MUA; >95%, Sigma-Aldrich), 1-hexanethiol (>98%, Sigma), cysteamine (>98%, Fluka), 1-propanethiol (>98%, Sigma), NaHCO3 (>97%, Fluka), H2SO4 (suprapure, Merck), NaOH (suprapure, Merck), acetone (>98%, Sigma-Aldrich), toluene (>99.9%, Sigma-Aldrich), tetrahydrofuran (THF; >99.9%, Aldrich), ethanol (suprapure, Merck), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methionine (EDC; SigmaAldrich), N-hydroxysuccinimide (NHS; >97%, Sigma-Aldrich), hydroxylamine hydrochloride (>98%, Fluka), K2HPO4 (suprapure, Merck), KH2PO4 (suprapure, Merck), and N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES; >99.5%, Sigma). Other reagents were at least of analytical grade. Horse heart cyt c (type VI, Sigma) was further purified using HPLC equipped with a cation exchange column (source 30S) and the protein concentration determined by UV-vis absorption spectroscopy. Synthesis of Water-Soluble Gold Nanoparticles. Gold nanoparticles (AuNPs) were synthesized according to the BrustSchiffrin method by either the one-step2 or two-step19 procedure. In the one-step procedure, HAuCl4 was first transferred from an aqueous solution to the organic phase (toluene) by (TOA)Br and then reduced by addition of NaBH4 in the presence of the desired thiol ligand(s). In the two-step procedure, AuNPs were also formed in the two-phase system but in the absence of the thiol ligand. The stabilizing thiol ligand was added to form SAMs on the AuNP shell by replacing (TOA)Br. For the details see refs 2 and 19. AuNPs capped with different thiols were synthesized (schematically represented in Figure 1) and dispersed in 20 mM HEPES (pH 10). To determine the molar absorption coefficient for TA-AuNPs (1), the product was dried in vacuum to yield a powdery material. Preparation of Cyt c-AuNP Conjugates. Cyt c-AuNP conjugates were prepared by incubating a mixture of cyt c and AuNPs with excess cyt c in HEPES (20 mM, pH 7.5) overnight at 4 °C. This reaction mixture was purified by ultrafiltration to remove free cyt c in an Amicon cell, model 8010m with a Diaflo YM-30 membrane that allows elution of free cyt c but retains the conjugate. Ultrafiltration was repeated until complete removal of free cyt c, as verified spectrophotometrically by a constant absorbance ratio between 410 and 520 nm (A410/A520) in the filtrate. The assembly of cyt c-AuNP conjugates on Au(111) surfaces was achieved by a stepwise procedure (Figure S1 in the

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Figure 2. Schematic illustration of the molecular assembly of cyt c on Au(111) surfaces: (A) three-dimensional structure of cyt c with the surface charge distribution indicated in blue for positive charge and red for negative charge, (B) cyt c-nanoparticle hybrid structure on the Au(111) surface, and (C) reference system without gold nanoparticles. For simplicity, the capping ligands on the AuNPs are mostly omitted. Not drawn to scale.

Supporting Information). The Au(111) surface was first modified with a SAM consisting of cysteamine and 1-propanethiol prepared from ethanol containing 0.5 mM cysteamine and 5.0 mM 1-propanethiol. A two-dimensional (2D) array of AuNPs was then prepared by immersing the modified Au(111) electrodes overnight (ca. 20 h) in a solution of AuNPs (∼0.5 µM, concentration based on nanoparticles), EDC (0.5 mM), and NHS (0.5 mM) in HEPES buffer (20 mM, pH 7.5). The electrodes were rinsed with HEPES buffer and Milli-Q water (18.2 MΩ). To recover the carboxyl group termination on the AuNPs, the electrodes were further incubated in 20 mM hydroxylamine hydrochloride containing HEPES solution (pH 7.5) for several hours, followed by thorough rinsing with Milli-Q water. Finally, the electrodes were immersed in cyt c solution (30-40 µM) at 4 °C overnight to assemble the protein on the AuNPs (Figure 2). Instrumentation and Measurements. UV-vis measurements were conducted using an 8453 spectrophotometer (HewlettPackard) with subtraction from blank solutions. A JEOL JEM3000F high-resolution transmission electron microscope was used for the characterization of the morphology and size distribution of the AuNPs. The samples were prepared by evaporation of a drop of nanoparticle solution on carbon films supported by standard copper grids. Mean particle sizes and standard deviations were determined from measurements performed on at least 230 AuNPs. Electrochemical measurements were carried out using an Autolab system (Eco Chemie, The Netherlands) controlled by the general-purpose electrochemical system (GPES) at room temperature (23 ( 2 °C). A three-electrode system consisting of a platinum coiled wire as the counter electrode (CE), a reversible hydrogen electrode (RHE) as the reference electrode (RE), and a Au(111) working electrode (WE) was used with the WE in a hanging-meniscus configuration.30a The RHE was checked against a saturated calomel electrode (SCE) after each measurement, and the potentials are reported versus this reference. Purified argon (Chrompack, 5 N) was used to purge dioxygen from the solutions before the measurements, and a

gas stream was maintained over the solution during the measurements. STM images were recorded in constant-current mode using a PicoSPM system (Molecular Imaging Co.) equipped with a bipotentiostat for potential control of both the substrate and tip. In situ imaging under electrochemical control was conducted in a home-designed cell with a three-electrode system similar to that used for normal electrochemical measurements.30 STM tips were prepared from either tungsten or Pt/Ir wires (φ ) 0.25 mm) by electrochemical etching and insulated with apiezon wax to reduce or eliminate Faradaic currents.30a Results and Discussion Synthesis and Characterization of Water-Soluble AuNPs. The synthesis of water-soluble AuNPs using carboxyl group terminated thiols as capping ligands is relatively straightforward and with high reproducibility. The different types of AuNPs employed (Figure 1) displayed long-term stability when dispersed in HEPES buffer (pH g 10), although the stability is significantly less at low pH (e6).19 The morphology and size distribution of nanoparticles were evaluated from high-resolution transmission electron microscopy (HRTEM). As an example, Figure 3 shows a TEM image (Figure 3A) and the sizedistribution histogram (Figure 3C) for TA-capped AuNPs. Images were chosen for particle size analysis from the regions of low particle density in which particle overlap was minimized. Similar observations were obtained for the other types of AuNPs with an average core diameter between 3 and 4 nm (Table S1 in the Supporting Information). The dominant morphology of all types of AuNPs was cuboctahedral, with single-crystalline facets on the nanoparticle surface. The periodic lattice of the facets is predominantly a (111) type of structure, as visualized in HRTEM images for single nanoparticles (Figure 3B). The inset in Figure 3B shows the fast Fourier transform of the lattice of single-crystalline facets on the AuNPs, confirming the zone axis in the HREM images to be the (111) structure. STM imaging was used to further characterize the AuNPs. TA-AuNPs were chosen as representative and deposited

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Figure 3. HRTEM characterization of TA-AuNPs: (A, B) TEM micrographs, (C) size distribution histogram. The inset in (B) shows the fast Fourier transform of the surface lattice of single-crystalline facets on TA-AuNPs.

Figure 4. STM images of TA-AuNPs physically deposited on Au(111) surfaces. Constant-current images recorded in air with It ) 0.2 nA and Vb ) 0.45 V. Scan areas: 100 nm × 100 nm (A), 20 nm × 20 nm (B).

physically on Au(111) surfaces. The physical deposition did not give a monolayer-level uniform distribution of AuNPs on the surface due to aggregation occurring during solvent evaporation, but individual nanoparticles are well resolved in the STM images (Figure 4). These measurements show that the average size of TA-AuNPs is 5-6 nm within a narrow deviation. This size is larger than that measured from TEM (Figure 3). However, the latter displays only the core size, while STM images include the ligand (TA) monolayer. Since the dimension of the TA monolayer is close to 1 nm, the size of TA-AuNPs measured from STM is thus consistent with the TEM analysis. A similar agreement was reported by Stimming and co-workers.31

Similarly to TEM, the surface plasmon band in the UV-vis spectra has been used commonly for characterization of metallic NPs. The collective oscillations of conduction electrons within the particles enable scattering and absorption of light at a specific frequency, giving a deep color to NP solutions. The surface plasmon feature is often emphasized, but the spectra actually represent combined scattering and absorption, i.e., extinction. In the present case, the apparent absorption maximum is approximately 520 nm for all the NP solutions as expected for AuNPs in this size range (Figure S2 in the Supporting Information). Compared to the (TOA)Br-stabilized precursors, the thiol-capped AuNPs exhibit a slight red shift (ca. 3-5 nm)

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Jensen et al. would be the origin of the band shift and broadening effects observed. Interfacial ET from the excited Au plasmon to the bound cyt c could shorten the lifetime of the plasmon excited state, leading to a corresponding bandwidth increase. As a result, the bandwidth increase in this case offers a way to estimate the rate constant for interfacial ET from the excited plasmon state to the bound (oxidized) cyt c. Assuming that the rate constant (kET) is equivalent to the inverse lifetime shortening (1/∆t), then from Heisenberg’s uncertainty principle and the relation between energy and frequency, the rate constant is given by

kET e 2πc∆ν

Figure 5. UV-vis spectra of cytochrome c-TA-AuNP conjugates (solid curves) and TA-AuNPs (dotted lines) in 20 mM HEPES buffer (pH 7.5).

in the maximum absorption, similar to that found previously.19 The apparent molar extinction coefficient of TA-AuNPs was calculated to be 2.2 × 106 M-1 cm-1.32 This value accords well with the expectation for small AuNPs.33 Electronic Interaction between Cyt c and the Nanoparticle. The AuNP-cyt c conjugates are formed by electrostatic interaction between the carboxyl groups on the AuNP and the lysine residues around the heme center in cyt c. The conjugates are stable up to an ionic strength of approximately 100 mM. The UV-vis spectra of pure TA-AuNPs and of the bioconjugates are compared in Figure 5. The Lorentzian-shaped plasmon band from the nanoparticles and the Soret band from cyt c are clearly resolved (solid curve, Figure 5). After unbound cyt c had been removed completely by purification, the ratio of the two largely equally sized TA-AuNP and cyt c moieties (both 3-4 nm) was estimated to be close to 1:1. The UV-vis spectra also show that the conjugation of cyt c causes a small but consistent red shift (∼5 nm) in the plasmon band (peak 1 in the solid curve of Figure 5), whereas the Soret band (peak 2) position remains virtually the same as for free cyt c in the same buffer. The plasmon band is also broadened by at least 5 nm, which corresponds to an increase of 160 cm-1 in the bandwidth on a frequency scale or to an energy difference of ∼20 meV. Broadening of Lorentzian electronic transitions in molecules caused by excited-state lifetime effects is a wellknown band shape determinant,34 for example, caused by radiationless electronic relaxation. In some cases this feature dominates entirely over solvation or molecular mode broadening otherwise normal in molecular transitions.35 The plasmon band shift and broadening caused by bioconjugation are fundamentally of interest, but the precise origin in most cases remains an open issue. Three possible factors that can cause the plasmon band shift or/and broadening of TA-AuNPs are (a) aggregation of nanoparticles upon bioconjugation, (b) changes in local dielectric environments, and (c) electronic communication between the redox center in cyt c and the nanoparticle core shell. The electrostatic conjugation of cyt c, however, did not cause any detectable aggregation of the nanoparticles. Since the nanoparticle core shell is separated from cyt c by approximately the length of a TA molecule (∼1 nm) and TA-capped AuNPs are highly hydrophilic in the buffer used (pH g 7.5), the conjugation is unlikely to cause significant changes in the local dielectric environment of the nanoparticle, although this cannot be entirely excluded. It is suggested instead that electronic interactions

(1)

where c is the speed of light and ∆ν is the bandwidth change in frequency units. A value of kET e 3 × 1013 s-1 is obtained for the change in bandwidth observed, implying that the ET reaction could be a very fast process. Such a value seems, at first sight, too high, but the following observations are appropriate: (1) The value is an upper limit. (2) The rate constant refers to excited-state interfacial electron transfer from the NP to cyt c; the process therefore has a significantly higher driving force and is expected to be much faster than in the ground state. (3) The rate constant represents averaged contributions from a large number of transitions from individual electronic levels in the NP, just as in normal interfacial electrochemical ET. (4) The value does not exceed but is close to the effective nuclear vibrational frequency in aqueous solution; this value is the upper limit for electron-transfer processes in the adiabatic limit. In short, interfacial ET between the NP core and the bound cyt c emerges as an attractive interpretation for the origin of the observed NP plasmon band shift and broadening, and the kinetic parameters extracted on the basis of this assumption are physically meaningful. Two-Dimensional Arrays of AuNPs on Au(111) Surfaces. The construction of molecular electronic devices requires two key steps: (a) organizing molecules and/or materials into nanoscale structures and (b) interfacing the nanostructures with macroscopically addressable components such as metal and semiconductor electrodes. Preparation of two-dimensional (2D) arrays of the AuNPs on gold surfaces was accomplished by a stepwise procedure (Figure S1 in the Supporting Information). The Au(111) surface was first modified with a mixed SAM consisting of cysteamine and 1-propanethiol in which the latter served as a diluent, followed by carbodiimide-mediated amidation to immobilize the AuNPs. The surface structural changes for each step of the procedure were probed by in situ STM. Figure 6 shows representative STM images of the electrode surface for different steps of the self-assembly. The mixed organic monolayer (Figure 6A) shows surface structural characteristics commonly observed for thiol SAMs on gold; i.e., the monolayer is composed of ordered domains and pinholes. High-resolution STM images can be obtained to image individual thiol molecules within the ordered domains as previously for pure cysteamine SAMs.36 The STM images further show that AuNPs formed close-packed adlayers with a higher level of order (Figure 6B,C) than the samples physically deposited on the electrode surface (Figure 4A). Control experiments further showed that no nanoparticles were found when the samples were prepared under the same conditions but in the absence of NHS and EDC. This is a clear indication that AuNPs were covalently linked to the electrode surface. The organization level was analyzed from the cross-sectional height profiles. The height differences between bound AuNPs determined by cross-sectional analyses, due to the conformational flexibility of the linker chain,

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Figure 6. STM images of various Au(111) surfaces in phosphate buffer (5 mM, pH 7.2) under electrochemical potential control: (A) cysteamine/ 1-propanethiol-modified Au(111), (B) 2D array of TA-capped AuNPs, (C) 2D array of MUA-capped AuNPs. Scan areas: 100 nm × 100 nm (A, B), 65 nm × 65 nm (C). It ) 0.15-0.4 nA, Vb ) -0.55-0.4 V, and Ew ) -0.09 to +0.16 V vs SCE.

Figure 7. Cyclic voltammograms of cyt c immobilized on the TA-AuNP (A, solid lines) and MUA-AuNP (B, solid lines) modified Au(111) electrodes in phosphate buffer (5 mM, pH 7.2). Black dotted lines show AuNP-modified Au(111) electrodes without cyt c for comparison. Scan rate 500 mV s-1.

are far smaller than the size of the AuNPs, indicating that a monolayer rather than a multilayer is formed. Long-Range Interfacial Electron Transfer through a AuNP Relay. The 2D array of AuNPs serves as a platform for further assembly of protein molecules. Cyt c is attached to the AuNP layer through the ligand molecules (Figure 2B). The bifunctional ligands employed, for example, thioctic acid, are good linkers for this application, since they provide specific covalent attachment to the nanoparticle and at the same time electrostatic binding to the heme group region of the redox protein. The expectation was that this arrangement could represent an efficient way to promote long-range protein interfacial electrochemical ET.23,24 The electrical linkage is further established through a short-chain molecular wire that covalently links the AuNPs to the Au(111) electrode surface (Figure 2B). Reversible voltammetry of the assembled cyt c at the potential expected for this protein 23a (solid curves, Figure 7) was observed. No faradic response was observed in the absence of either the protein (dotted curves, Figure 7) or the AuNPs. The linear dependence on the scan rate of the cathodic and anodic peak currents observed (data not shown) is typical for surfacecontrolled electrochemical processes, in accordance with the nature of the assembled systems (Figure 2). The surface population of protein molecules estimated from the Faradaic charge is equivalent to 20-30% of that for a close-packed cyt c monolayer. The organization is therefore at the submonolayer level, which reduces lateral interactions between the cyt c molecules and consequently overcomes significant interference with ET kinetics.

TABLE 1: Comparison of Interfacial ET Rate Constants of Cytochrome c Immobilized on Different Surface Systems system

AuNP size (nm)

distancea

kapp (s-1)

TA-AuNP/Au(111) C11-AuNP/Au(111) C11C6-AuNP/Au(111) TA/Au(111) C11/Au(111)

3.2 4.0 2.8

15 25 25 6 11

94 16 78 4 6

a The value is the gross distance from the redox center to the Au(111) electrode surface but not including AuNP, represented by the number (n) of CH2 units. For simplicity, the amide bond is counted as a methylene unit.

To obtain the apparent rate constants (kapp), a series of cyclic voltammograms (CVs) with different scan rates (e.g., 0.1-3 V s-1) for each system were collected. The data were analyzed on the basis of Laviron’s method,37 using the following equation as a simplified approach:

m)

RT kapp nF υ

(2)

where m is a parameter determined by the peak separation obtained from the CVs, n is the number of electrons transferred, υ is the scan rate, F is the Faraday constant, and the other symbols have their usual meaning. Thus, the rate constant can be obtained from the slope of the plot of m vs 1/υ. The rate constants for the different layers investigated are listed in Table 1. The inclusion of a gold nanoparticle within the molecular linkage results in an enhancement of the ET

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Figure 8. Theoretical modeling: (A) schematic representation of the two-step ET process in the system, (B) simulated cyclic voltammogram with k1 ) 90 s-1 and k2 ) 1000 s-1.

rate by a factor of 3-25 depending on the linkers employed. The strongest enhancement by over 1 order of magnitude is observed for the cyt c-TA-AuNP system. These results clearly demonstrate that the introduction of a nanoparticle within the molecular linker opens an efficient long-range ET channel between cyt c and the cysteamine-modified Au(111) electrode. There is a striking difference between fast interfacial ET of the cyt c-AuNP hybrid and the absence of a detectable ET response for cyt c alone. This observation indicates first that a good covalent and/or electrostatic ET contact is essential for facile long-range interfacial ET. An array of chemically attached AuNPs obviously represents such a kind of contact. Second, a significant enhancement in the ET rate constant is observed even though the length of the connection between the electrode and the heme group is increased by the inclusion of a gold nanoparticle within the molecular wire. Finally, the ET enhancement is not strongly related to the number of Au-S bonds connecting the molecular wire to the Au nanoparticle as seen by comparison of the TA-AuNP/Au(111) and C11C6-AuNP/ Au(111) entries in Table 1. The observed enhancement points to a significant change in mechanism of interfacial ET to cyt c in the presence and absence of AuNPs within the linker. We propose an electron hopping mechanism involving a two-step process, i.e., cyt c f AuNP and AuNP f Au(111) electrode, as a rationalization of these results. The process is described as two consecutive electrochemical steps equivalent to a hopping mechanism:38 k1

k2

-1

-2

protein {\ } AuNP {\ } electrode k k

(3)

where k1 and k-1 are the rate constants for interfacial ET between the AuNP and cyt c, i.e., oxidation and reduction of cyt c, respectively, and k2 and k-2 are similarly the rate constants for interfacial ET from the AuNP to the electrode and vice versa. This reaction sequence is analogous to that of interfacial ET for two-center proteins in an electrochemical configuration.39 Interfacial ET between an array of nanoparticles and an electrode can be very efficient.40 In the absence of electrochemically active cyt c, a large capacitive charging current from the AuNPs is observed (e.g., dotted curves, Figure 7). Thus, the linker between the gold electrode and the nanoparticles does not act as an insulating dielectric, and good electronic communication between the substrate and the gold nanoparticles is evident. A simulation approach to obtain the rate constant ratio of the two ET steps was introduced. The simulations were accomplished using a theoretical model previously established

and a homemade program based on MATHCAD PROFESSIONEL 2001.41 The program directly computes the cyclic voltammograms of the two-center AuNP-cyt c hybrid by numerical solution of the kinetic equations of the oxidized and reduced states of each center (Figure 8A). The simulated CV (Figure 8B) closest to the experimental observations (e.g., Figure 7A) can be achieved only by using a large k2:k1 ratio (g10:1), showing that the two-step process is determined primarily by electron transfer between the protein and the nanoparticle as the rate-limiting step. A comparison of the results for the assemblies using thioctic and undecanoic acids (Table 1) also shows that the enhancement of the ET rate due to the presence of a nanoparticle in the linker is strongly dependent on the length of the alkane chain joining the AuNP to the rest of the structure. The AuNP-induced notable interfacial ET rate enhancement can be ascribed to several factors: (1) The electric field between the electron acceptor and donor groups is inhomogeneous, and the shorter length of the thioctic acid junction may result in a higher local field across the molecular wire. (2) It is well-known that the chemical contact is an important factor in determining conductivity across molecular wires,42 and the involvement of two thiol-Au junctions as in the case of thioctic acid will most likely result in a better coupling of the nanoparticle to both the heme group and the metal electrode. (3) Better electronic contact between the covalently linked AuNP and the Au(111) surface than the electrostatic contact between the AuNP and the protein can be an important factor. (4) The most important factor is probably the stronger electronic coupling of cyt c to the AuNP than directly to the thiol-modified Au(111) electrode surface. The simulation of the voltammograms, along with the data shown, suggests that this is indeed a dominant factor for the enhancement of ET by the bridging nanoparticle. In summary, AuNPs appear to function as ET relays that facilitate electronic coupling between the protein and the Au(111) electrode surface through long-range electronic interactions. The more favorable electronic contacts are reflected primarily in improved electronic transmission coefficients for electron transfer. However, more precise quantification of these conceptually appealing considerations is complicated by the rate constant dependence on the electrochemical potential differences and the composite nature of the interfacial Au(111)/AuNP/ protein region. Conclusions Stoichiometric cyt c-AuNP hybrid systems have been synthesized and their interfacial ET features in homogeneous

Gold Nanoparticle Assisted Heme Protein Alignment solution and at a liquid/solid interface investigated. The hybrid ET systems were constructed with both water-soluble AuNPs and two-dimensional surface molecular architecture electrostatically linked to cyt c. Stepwise characterization was accomplished systematically using UV-vis spectroscopy, electrochemistry, and STM. Conjugation of cytochrome c results in a small but consistent broadening and shift of the nanoparticle plasmon band, most likely due to long-range electronic interactions between the two components of the conjugates. Most importantly, a notable AuNP-induced enhancement of the ET rate is observed. The introduction of AuNPs opens channels allowing long-range protein interfacial ET across a larger physical distance with significantly enhanced ET kinetics. The mechanistic origin of the enhancement is not fully understood, but from the electrochemical analysis combined with theoretical modeling it is concluded that the AuNPs serve as effective ET relays facilitating long-range ET between the protein and the Au(111) electrode surface. Since it is a key challenge in molecular bioelectronics to improve the efficiency of long-range protein interfacial ET, the present research adds clues for the design and optimization of redox protein based electronic devices where long-range charge transfer is essential. Acknowledgment. We thank Dr. Jingdong Zhang (Department of Chemistry, Technical University of Denmark) for assistance in the STM experiments and Prof. Alexander M. Kuznetsov (The Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences) for helpful discussions. Q.C. and J.U. acknowledge financial support from the Danish Research Council for Technology and Production Sciences (Contract No. 26-00-0034). P.S.J. acknowledges a Ph.D grant from the Lundbeck Foundation (Reference No. 1071067) and the NanoScience Center at the University of Copenhagen. J.M.A. acknowledges a postdoctoral fellowship from Fundacio´n Ramo´n Areces, Spain. D.J.S. and J.U. acknowledge support from the EU (Research Training Network SUSANA, Supermolecular Self-Assembled Interfacial Nanostructures, Contract No. HPRNCT-2002-00185). Supporting Information Available: Schematic illustration of a two-dimensional assembly of AuNPs on Au(111) surfaces (Figure S1), the size distribution analysis of AuNPs (Table S1), the UV-vis spectra of different types of AuNPs (Figure S2), and a high-scan-rate CV and a Laviron plot (Figure S3). This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Schmid, G. Chem. ReV. 1992, 92, 1709-1727. (2) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802. (3) Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. Chem. Soc., Chem. Commun. 1995, 1655-1656. (4) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (5) Rao, C. N. R.; Kulkarni, G. U.; Govindaraj, A.; Satishkumar, B. C.; Thomas, P. J. Pure Appl. Chem. 2000, 72, 21-33. (6) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293-346. (7) Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chem. ReV. 2005, 105, 1025-1102. (8) Rotello, V., Ed. Nanoparticles: Building Blocks for Nanotechnology; Springer: New York, 2004. (9) Ozin, G. A.; Arsenault, A. C. Nanochemistry: A Chemical Approach to Nanomaterials; RSC Publishing: London, 2005. (10) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (11) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (12) Tan, W.; Wang, K.; He, X.; Zhao, X. J.; Drake, T.; Wang, L.; Bagwe, R. P. Med. Res. ReV. 2004, 24, 621-638. (13) Rosi, N. L.; Mirkin, C. A. Chem. ReV. 2005, 105, 1547-1562.

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