Lior Sepunaru

Proteins as Electronic Materials: Electron Transport through Solid-State Protein Monolayer Junctions

Electron transfer (ET) through proteins, a fundamental element of many biochemical reactions, is studied intensively in aqueous solutions. Over the past decade, attempts were made to integrate proteins into solid-state junctions in order to study their electronic conductance properties. Most such studies to date were conducted with one or very few molecules in the junction, using scanning probe techniques. Here we present the high-yield, reproducible preparation of large-area monolayer junctions, assembled on a Si platform, of proteins of three different families: azurin (Az), a blue-copper ET protein, bacteriorhodopsin (bR), a membrane protein-chromophore complex with a proton pumping function, and bovine serum albumin (BSA). We achieve highly reproducible electrical current measurements with these three types of monolayers using appropriate top electrodes. Notably, the current-voltage (I-V) measurements on such junctions show relatively minor differences between Az and bR, even though the latter lacks any known ET function. Electron Transport (ETp) across both Az and bR is much more efficient than across BSA, but even for the latter the measured currents are higher than those through a monolayer of organic, C18 alkyl chains that is about half as wide, therefore suggesting transport mechanism(s) different from the often considered coherent mechanism. Our results show that the employed proteins maintain their conformation under these conditions. The relatively efficient ETp through these proteins opens up possibilities for using such biomolecules as current-carrying elements in solid-state electronic devices.

Electronic Transport via Proteins

A central vision in molecular electronics is the creation of devices with functional molecular components that may provide unique properties. Proteins are attractive candidates for this purpose, as they have specific physical (optical, electrical) and chemical (selective binding, self-assembly) functions and offer a myriad of possibilities for (bio-)chemical modification. This Progress Report focuses on proteins as potential building components for future bioelectronic devices as they are quite efficient electronic conductors, compared with saturated organic molecules. The report addresses several questions: how general is this behavior; how does protein conduction compare with that of saturated and conjugated molecules; and what mechanisms enable efficient conduction across these large molecules? To answer these questions results of nanometer-scale and macroscopic electronic transport measurements across a range of organic molecules and proteins are compiled and analyzed, from single/few molecules to large molecular ensembles, and the influence of measurement methods on the results is considered. Generalizing, it is found that proteins conduct better than saturated molecules, and somewhat poorer than conjugated molecules. Significantly, the presence of cofactors (redox-active or conjugated) in the protein enhances their conduction, but without an obvious advantage for natural electron transfer proteins. Most likely, the conduction mechanisms are hopping (at higher temperatures) and tunneling (below ca. 150-200 K).

Solid-State Electron Transport across Azurin: From a Temperature-Independent to a Temperature-Activated Mechanism

The temperature dependence of current-voltage values of electron transport through proteins integrated into a solid-state junction has been investigated. These measurements were performed from 80 up to 400 K [above the denaturation temperature of azurin (Az)] using Si/Az/Au junctions that we have described previously. The current across the ∼3.5 nm thick Az junction was temperature-independent over the complete range. In marked contrast, for both Zn-substituted and apo-Az (i.e., Cu-depleted Az), thermally activated behavior was observed. These striking temperature-dependence differences are ascribed to the pivotal function of the Cu ion as a redox center in the solid-state electron transport process. Thus, while Cu enabled temperature-independent electron transport, upon its removal the polypeptide was capable only of supporting thermally activated transport.

Temperature and Force Dependence of Nanoscale Electron Transport via the Cu Protein Azurin

Solid-state electron transport (ETp) via a monolayer of immobilized azurin (Az) was examined by conducting probe atomic force microscopy (CP-AFM), as a function of both temperature (248-373K) and applied tip force (6-15 nN). At low forces, ETp via holo-Az (with Cu(2+)) is temperature-independent, but thermally activated via the Cu-depleted form of Az, apo-Az. While this observation agrees with those of macroscopic-scale measurements, we find that for holo-Az the mechanism of ETp at high temperatures changes upon an increase in the force applied by the tip to the proteins; namely, above 310 K and forces >6 nN ETp becomes thermally activated. This is in contrast to apo-Az, where increasing applied force causes only small monotonic increases in currents due to decreased electrode separation. The distinct ETp temperature dependence of holo- and apo-Az is assigned to a difference in structural response to pressure between the two protein forms. An important implication of these CP-AFM results (of measurements over a significant temperature range) is that for reliable ETp measurements on flexible macromolecules, such as proteins, the pressure applied during the measurements should be controlled or at least monitored.

Temperature-Dependent Solid-State Electron Transport through Bacteriorhodopsin: Experimental Evidence for Multiple Transport Paths through Proteins

Electron transport (ETp) across bacteriorhodopsin (bR), a natural proton pump protein, in the solid state (dry) monolayer configuration, was studied as a function of temperature. Transport changes from thermally activated at T > 200 K to temperature independent at <130 K, similar to what we have observed earlier for BSA and apo-azurin. The relatively large activation energy and high temperature stability leads to conditions where bR transports remarkably high current densities above room temperature. Severing the chemical bond between the protein and the retinal polyene only slightly affected the main electron transport via bR. Another thermally activated transport path opens upon retinal oxime production, instead of or in addition to the natural retinal. Transport through either or both of these paths occurs on a background of a general temperature-independent transport. These results lead us to propose a generalized mechanism for ETp across proteins, in which tunneling and hopping coexist and dominate in different temperature regimes.

Electronic Transport via Homopeptides: The Role of Side Chains and Secondary Structure.

Many novel applications in bioelectronics rely on the interaction between biomolecules and electronically conducting substrates. However, crucial knowledge about the relation between electronic transport via peptides and their amino-acid composition is still absent. Here, we report results of electronic transport measurements via several homopeptides as a function of their structural properties and temperature. We demonstrate that the conduction through the peptide depends on its length and secondary structure as well as on the nature of the constituent amino acid and charge of its residue. We support our experimental observations with high-level electronic structure calculations and suggest off-resonance tunneling as the dominant conduction mechanism via extended peptides. Our findings indicate that both peptide composition and structure can affect the efficiency of electronic transport across peptides.

Electrochemical Red Blood Cell Counting: One at a Time.

We demonstrate that the concentration of a red blood cell solution under physiological conditions can be determined by electrochemical voltammetry. The magnitude of the oxygen reduction currents produced at an edge-plane pyrolytic graphite electrode was diagnosed analytically at concentrations suitable for a point-of-care test device. The currents could be further enhanced when the solution of red blood cells was exposed to hydrogen peroxide. We show that the enhanced signal can be used to detect red blood cells at a single entity level. The method presented relies on the catalytic activity of red blood cells towards hydrogen peroxide and on surface-induced haemolysis. Each single cell activity is expressed as current spikes decaying within a few seconds back to the background current. The frequency of such current spikes is proportional to the concentration of cells in solution.

Tuning electronic transport via hepta-alanine peptides junction by tryptophan doping.

Significance Charge transport is a process that is central to redox reactions, enzyme catalysis, and electronics. Integrating biomolecules into electronic devices provides a route to using natural evolution in artificial technology. Charge transport across biomolecules depends on the molecular structure and molecule–electrode contacts. We show that “doping” (7-alanine) peptide-based devices by one tryptophan enhances transport ≥10-fold, regardless of tryptophan position, a result that can be described by superexchange-mediated tunneling through multiple sites. Remarkably, close physical proximity of tryptophan to a gold electrode further enhances transport, showing how crucial electronic coupling to the electrode is. Our results provide insight into charge transfer through biomolecules and offer a strategy for tailored bioelectronics by mix-and-match amino acid building blocks. Charge migration for electron transfer via the polypeptide matrix of proteins is a key process in biological energy conversion and signaling systems. It is sensitive to the sequence of amino acids composing the protein and, therefore, offers a tool for chemical control of charge transport across biomaterial-based devices. We designed a series of linear oligoalanine peptides with a single tryptophan substitution that acts as a “dopant,” introducing an energy level closer to the electrodes’ Fermi level than that of the alanine homopeptide. We investigated the solid-state electron transport (ETp) across a self-assembled monolayer of these peptides between gold contacts. The single tryptophan “doping” markedly increased the conductance of the peptide chain, especially when its location in the sequence is close to the electrodes. Combining inelastic tunneling spectroscopy, UV photoelectron spectroscopy, electronic structure calculations by advanced density-functional theory, and dc current–voltage analysis, the role of tryptophan in ETp is rationalized by charge tunneling across a heterogeneous energy barrier, via electronic states of alanine and tryptophan, and by relatively efficient direct coupling of tryptophan to a Au electrode. These results reveal a controlled way of modulating the electrical properties of molecular junctions by tailor-made “building block” peptides.

Picosecond electron transfer from photosynthetic reaction center protein to GaAs.

An extremely fast electron transfer through an electronically coupled junction between covalently bound oriented photosynthetic reaction center protein photosystem I (PS I) and n-GaAs was measured by time-resolved photoluminescence. It was found that the n-GaAs band edge luminescence intensity increased by a factor of 2, and the fast exponential decay constant was increased by a factor of 2.6 following the PS I self-assembly. We attribute this to picosecond electron transfer from the PS I to the n-GaAs surface states.

Catalase-Modified Carbon Electrodes: Persuading Oxygen To Accept Four Electrons Rather Than Two.

We successfully exploited the natural highly efficient activity of an enzyme (catalase) together with carbon electrodes to produce a hybrid electrode for oxygen reduction, very appropriate for energy transformation. Carbon electrodes, in principle, are cheap but poor oxygen reduction materials, because only two-electron reduction of oxygen occurs at low potentials, whereas four-electron reduction is key for energy-transformation technology. With the immobilization of catalase on the surface, the hydrogen peroxide produced electrochemically is decomposed back to oxygen by the enzyme; the enzyme natural activity on the surface regenerates oxygen, which is further reduced by the carbon electrode with no direct electron transfer between the enzyme and the electrode. Near full four-electron reduction of oxygen is realised on a carbon electrode, which is modified with ease by a commercially available enzyme. The value of such enzyme-modified electrode for energy-transformation devices is evident.