Enhanced Electronic Conductance across Bacteriorhodopsin, Induced

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Enhanced Electronic Conductance across Bacteriorhodopsin, Induced by Coupling to Pt Nanoparticles Izhar Ron,†,§ Noga Friedman,‡ Mordechai Sheves,*,‡ and David Cahen*,† †

Departments of Materials and Interfaces and ‡Organic Chemistry, Weizmann Institute of Science, POB 26, Rehovot 76100, Israel

ABSTRACT Studying solid-state electronic conductance of biological molecules requires interfacing the biomolecules with electronic conductors without altering the molecules. To this end, we developed and present here a simple, solution-based approach of conjugating Bacteriorhodopsin (bR)-containing membranes with metallic clusters. Our approach is based on selective electroless deposition of Pt nanoparticles on suspended membrane fragments through chemical interaction of the Pt precursor with the protein's residues. Optical absorption measurements show that the membranes retain their photoactivity after this procedure. The result of the Pt deposition is best shown by conductive probe atomic force microscopy mapping of electronic current transport across such soft biological layers, which allows reproducible microscopic electrical characterization of the electronic conductance of the resulting junctions. The maps show that chemical contact between the protein and the deposited electrode yields better electronic coupling than a physical contact, demonstrating that also with biomolecules, the type and method of deposition of the electrical contact are critical to the behavior of the resulting junctions. SECTION Electron Transport, Optical and Electronic Devices, Hard Matter

coupling and increases long-range electron transfer (ET) efficiency. We reported recently selective electroless deposition of Ag and Pt clusters on solid-supported Bacteriorhodopsin (bR)containing monolayers.25 Our approach was selective in the sense that metal particles were deposited only on bR and not onto the surrounding substrate. In addition, metal binding occurred only on one side of the membrane pigment, that is, binding of the metal precursor was to a specific group at one of the protein's surfaces. Particularly with Pt, ELD was demonstrated both in situ, namely, on membranes that were already adsorbed to a surface, and ex situ, namely, on membranes suspended in a liquid. Here, we describe how the method for selective ex situ ELD of Pt nanoparticles on bR allows characterization of the protein's photoactivity and spectral properties after contacting. We examine the resultant conjugates for their electrical properties, that is, the conductivity of the protein sandwiched between the underlying substrate and the metallic nanoparticle, and show that they display altered (enhanced) performance in this configuration. Figure 1a shows schematically the preparation of bR-Pt conjugates, prepared by ELD on suspended membranes.

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olid-state electronic transport through biological molecules is of interest by itself and to assess potential future use of biomolecules in electronics.1-11 In addition to the chemical aspects of interfacing biomolecules with solid, electronically conducting inorganic substrates, such as the binding chemistry of the molecules to a given surface and the methodology to characterize the shape and functionality of the molecules, two main issues are important from the physical point of view, • how to electrically connect, or “wire”, these biomolecules. • their electronic conductance. A variety of approaches was used to make electronically conducting contacts to organic molecules in general and to biomolecules in particular.12 An approach that was applied to several biomolecular systems is electroless deposition (ELD).13-18 This method is attractive because it offers a wet chemical approach to make electronic electrical contact, as well as selectivity, which can be used to exploit the biomolecules as templates for growing the contact (metal) on the molecules. Another approach is based on binding metal nanoparticles to the surface-adsorbed biomolecules19-23 and using them as electrodes. In this configuration, a nanometer-sized scanning probe contacts the biomolecule via this mediating metal cluster to connect the system to the outside world. Electrochemical studies using this configuration24 showed that interfacing a biomolecule with Au nanoparticles improves electronic

r 2010 American Chemical Society

Received Date: August 2, 2010 Accepted Date: September 22, 2010 Published on Web Date: October 05, 2010

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DOI: 10.1021/jz101072s |J. Phys. Chem. Lett. 2010, 1, 3072–3077

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complex binds to amine groups of lysine residues at the cytoplasmic side.25 Figure 1b shows the difference spectra taken at different times during incubation of bR with the Pt complex, after the addition of the reducing agent (dimethylamineborane, DMAB), and after washing off excess reaction materials. Reduction of the Pt complexes to metallic particles, as well as washing of the protein-Pt conjugate by centrifugation, does not alter the bR absorption maximum, and the conjugates remain intact, as judged by the accumulation of the M intermediate following irradiation. The bR-Pt conjugates can be physically adsorbed on several substrates. Figure 2a shows a scanning electron microscope (SEM) image of bR-Pt adsorbed on Si. Pt particles were deposited on the two membrane fragments, but the Pt particles are observed more clearly on one of them. It is likely that the two fragments are adsorbed with opposite orientations (the left one is adsorbed with the Pt particles facing down, toward the substrate). This is another indication that Pt binds selectively to only one side of the membranes. Figure 2b shows an atomic force microscope (AFM) image of the bR-Pt conjugates adsorbed on a Au surface. We used the latter configuration for current measurements of the bR-Pt conjugates, using conductive probe atomic force microscopy (CP-AFM). Unlike most previous CP-AFM studies on proteins that focus on current-voltage (I-V) measurements at fixed locations, we scan our samples to acquire current maps so as to get reproducible results (cf. also Figure 1 in ref 8 and Figure 8 in ref 25, reproduced here as Figure 2c, d). The Pt-coupled membrane fragments, adsorbed on a bare Au surface, were scanned in contact mode under a constant applied bias. In contrast to previous CP-AFM measurements on bare bR membranes on the Au surface, which showed that no current could be detected that can be attributed to the membrane pigments in this configuration under such voltages,8,29 bR-Pt conjugates do show conducting hot spots in the CP-AFM current map. The current that flows through these spots depends reversibly on the bias voltage (Figure 2e). Scanning in the “interleave” mode, that is, scanning sequentially each line in the image under a different bias voltage (1.5 V line scan followed by a 2.5 V scan and vice versa in another acquired image) shows this reversibility, which indicates that no electrical breakdown occurred in these junctions. Furthermore, the conductivity through these spots was not altered by scanning in the reverse direction, indicating strong and stable contact between the particles and the membrane (Figure 3). Figure 4 shows representative current profiles under varying bias voltage (Figure 4a) and line profiles of current versus topography (Figure 4b). The difference in current magnitude between the bare Au surface and the conductive spots, corresponding to the position of Pt nanoparticles, can be seen (Figure 4a). We ascribe this difference to the protein barrier. The evolution of the current through the “hot spots” (formed by the presence of Pt nanoparticles) from 1.5 to 2.5 V indicates that currents through bare Au areas will reach saturation at lower bias voltage, explaining why in Figure 2e, currents through bare Au and bR-Pt hot spots seem to be of similar magnitude, while, in fact, in Figure 2d the bare Au current magnitude is higher.

Figure 1. (a) Schematic representation of Pt growth on bRcontaining membranes. DMAB stands for dimethylamine borane. (b) Difference spectra, showing the change in bR absorbance under yellow light illumination (>550 nm), with increasing incubation time with Pt ions.

Monitoring the binding of the Pt complexes (PtCl42-) relies on the optical properties of bR. Using UV-visible absorption enabled us to draw two important conclusions (Figure 1b). (1) The absorption maximum of bR (λmax = 568 nm) did not change throughout the ELD process, indicating that the protein maintains its native conformation to a great extent. (2) The protein remains photoactive, and the particles are likely to grow on its cytoplasmic side. We can conclude this from a difference spectrum, showing the effect of irradiation with yellow light (>550 nm). Optical absorption is used here to detect the evolution of Pt binding to bR as reflected by the growing difference spectra, in response to yellow light, as reaction time is increased. The yellow light-induced difference spectrum is a measure of accumulation of the M intermediate of the bR photocycle (λmax = 412 nm). The photochemically induced M intermediate triggers a proton release from the extracellular side, followed by a reprotonation process that takes place from the cytoplasmic side.26 Alterations in the cytoplasmic side as a result of, for example, binding of agents to groups at this side, are thought to slow down the reprotonation process, which can be reflected in a slower M state decay.27,28 Previously, we proposed that the Pt


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Figure 2. (a) SEM image of bR-Pt conjugates on a Si surface. (b) AFM image (1.7 μm 1.7 μm, z scale = 12 nm) of bR-Pt conjugates on a Au surface. (c-e) Conducting probe AFM current images (700 nm 700 nm, z scale = 500 nA) of (c) bR under 1 V, (d) bR-Pt under 1 V, and (e) bR-Pt under 1.5 V. Some examples of individual Au grains are marked with red circles. (a-d) are reproduced with permission from ref 25 ( Wiley-VCH Verlag GmbH & Co. KGaA).

Figure 3. Height (a,d) and current (b,c;e,f) AFM images acquired under 1.5 (a-c) and 2.5 V (d-f) constant bias. Arrows show the direction of scanning. Each line in (d-f) was acquired sequentially after the corresponding line in (a-c) using the Interleave mode.

Comparing topographic and current line sections, we find that the current is not a result of pinholes (Figure 4b). While current peaks are observed almost exclusively in positions corresponding to the presence of Pt particles (and not in topographically lower regions, which may indicate pinholes), there are also such positions of particles where a current peak


is not observed. To interpret this observation, we refer to features shown in Figures 2 and 3. Figure 2 indicates that the current drops over the grain boundaries of the Au substrate (see red circles, Figure 2c-e). An interesting feature related to the orientation of the adsorbed membranes is demonstrated in Figure 3. One of the membrane fragments shows conduction

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Figure 4. (a) Current profiles taken over a line crossing the bR-Pt membrane surface and a bare Au surface (extracted from Figure 3e), taken under bias voltages of 1.5 (black, top) and 2.5 V (red, bottom). Current hot spots corresponding to the presence of Pt nanoparticles on top of the membrane surface are indicated. (b) Height (black) and current (red) profiles, taken from the same line over the bR-Pt membrane surface as acquired in Figure 2e.

Figure 5. Schemes of configurations that do not (a) and that do (b) allow current enhancement in Au/br-Pt/AFM tip junctions. Junctions formed over the grain boundaries of the Au surface may introduce a barrier in the form of a free space gap and of a surface with low conductivity.

AFM tip and the Pt particle and (2) direct contact between the membrane and a conducting area of the underlying Au substrate. In summary, we presented a simple method to interface bR with Pt nanoparticles and to monitor this process by following the optical pigment's properties. This method allows for creating metal-protein-metal nanoparticle junctions by simple physical adsorption of the conjugate onto a conductive substrate. Reproducible CP-AFM characterization, made possible by current mapping, indicates that part of the junctions show increased conductivity with respect to junctions formed by the AFM tip contacting a bare bR membrane (without a metal nanoparticle). Further experiments on a smoother metallic surface accompanied by full I-V measurements may help quantify the contribution of the nanoparticles to the overall electronic conductivity through the proteins in this configuration.

spots (red (middle left) contour), while another one (green (left bottom) contour) does not (Figure 3e). We previously observed that while Pt binds only to one side of the membranes, such one-sided binding occurs on one and the same side of all membrane fragments. Therefore, the orientation of adsorption of the membranes on the substrate determines if current enhancement will take place. To explain this phenomenon, we refer to the scheme in Figure 5. If the Pt particles face the substrate, the AFM tip does not sense the particles, and therefore, no current enhancement is detected (Figure 5a). In contrast, tip-membrane coupling is stronger if Pt particles face the tip and current enhancement is seen in membrane fragments that are characterized by Pt particles on the membrane surface away from the Au substrate (Figure 5b). Once a bR-Pt conjugate is adsorbed on the Au substrate, with the particles facing the AFM tip, localized junctions, where a Pt particle is positioned on a membrane area that is on top of a grain boundary, can be formed (marked by circles in Figure 5b). The lack of current enhancement in such a configuration may indicate that enhancement occurs only if two conditions are fulfilled, (1) good coupling between the


EXPERIMENTAL SECTION Electroless Deposition of Pt on bR Membranes in Solution. Pt nanoparticles (polydispersed, 5-15 nm in diameter) were grown on bR membranes in suspension by adding 100 μL of a

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2 mM aged solution of K2PtCl4 to 2 mL of O.D. = 0.4 (∼6 μM) suspension of wild-type bR for 24 h, followed by the addition of 20 μL of 10 mM DMAB and stirring for 1 min. Immediately after this step, the mixture was centrifuged three times for 10 min (@14 000 rpm) and washed with water to remove excess reagents. Adsorption of the resulting conjugates on Si or Au surfaces was done through physisorption by immersing the substrates in the bR-Pt NP conjugate suspension for 1-2 h. CP-AFM was performed on wild-type bR membranes and on bR-Pt NPs conjugates, physisorbed on a clean Au surface under ambient conditions (23 °C, 50% R.H.). Under these conditions, the membranes are essentially dry, with only the tightly bound water molecules and a water meniscus at the membrane surface. The granular Au film was prepared by e-beam evaporation of a 100 nm layer of Au on a 3-aminopropyltrimethoxysilane (APTMS)-terminated glass substrate (as an adhesion layer). Current Maps with CP-AFM. These experiments were done using a Nanoscope V Multimode (Veeco, U.S.A.) with the extended TUNA module for low-current measurements and Pt-Ir-coated tips (Nanosensors, Switzerland). The sample was glued on a stab, and Ag paste was spread on the corner and connected between the sample surface and the conducting stab. Imaging was carried out in contact mode, and the current was acquired at each pixel of the scan simultaneously. The applied bias voltage was varied by changing the sample DC bias in between scans. When the bias voltage dependence was measured, images were acquired in the “interleave” mode, where each line was scanned two times, successively, with a different voltage applied at each time. This way, two current maps were constructed simultaneously, each under a different bias voltage. Line profiles were extracted using WSxM software.30

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AUTHOR INFORMATION (13)

Corresponding Author: *To whom correspondence should be addressed. E-mail: david.cahen@ weizmann.ac.il.

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Present Addresses:

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§

Department of Physical Chemistry, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. (16)

ACKNOWLEDGMENT We thank S.R. Cohen for help with the AFM experiments, A. Biller for fruitful discussions, the Nancy and Stephen Grand Center for Sensors and Security, the Ilse Katz Center for Materials Research, and the Kimmelman center for Biomolecular Structure and Assembly for support. M.S. holds the Katzir-Makineni professorial chair in chemistry. D.C. holds the Rowland and Sylvia Schaefer Chair in Energy Research.

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Enhanced Electronic Conductance across Bacteriorhodopsin, Induced

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