Allan Glargaard Hansen

Interfacial electrochemical electron transfer in biology – Towards the level of the single molecule

Physical electrochemistry has undergone a remarkable evolution over the last few decades, integrating advanced techniques and theory from solid state and surface physics. Single‐crystal electrode surfaces have been a core notion, opening for scanning tunnelling microscopy directly in aqueous electrolyte (in situ STM). Interfacial electrochemistry of metalloproteins is presently going through a similar transition. Electrochemical surfaces with thiol‐based promoter molecular monolayers (SAMs) as biomolecular electrochemical environments and the biomolecules themselves have been mapped with unprecedented resolution, opening a new area of single‐molecule bioelectrochemistry. We consider first in situ STM of small redox molecules, followed by in situ STM of thiol‐based SAMs as molecular views of bioelectrochemical environments. We then address electron transfer metalloproteins, and multi‐centre metalloenzymes including applied single‐biomolecular perspectives based on metalloprotein/metallic nanoparticle hybrids.

A silicon-on-insulator vertical nanogap device for electrical transport measurements in aqueous electrolyte solution

A novel concept for metal electrodes with few 10 nm separation for electrical conductance measurements in an aqueous electrolyte environment is presented. Silicon-on-insulator (SOI) material with 10 nm buried silicon dioxide serves as a base substrate for the formation of SOI plateau structures which, after recess-etching the thin oxide layer, thermal oxidation and subsequent metal thin film evaporation, feature vertically oriented nanogap electrodes at their exposed sidewalls. During fabrication only standard silicon process technology without any high-resolution nanolithographic techniques is employed. The vertical concept allows an array-like parallel processing of many individual devices on the same substrate chip. As analysed by cross-sectional TEM analysis the devices exhibit a well-defined material layer architecture, determined by the chosen material thicknesses and process parameters. To investigate the device in aqueous solution, we passivated the sample surface by a polymer layer, leaving a micrometre-size fluid access window to the nanogap region only. First current–voltage characteristics of a 65 nm gap device measured in 60 mM buffer solution reveal excellent electrical isolation behaviour which suggests applications in the field of biomolecular electronics in a natural environment.

Electron transfer behaviour of biological macromolecules towards the single-molecule level

Redox metalloproteins immobilized on metallic surfaces in contact with aqueous biological media are important in many areas of pure and applied sciences. Redox metalloprotein films are currently being addressed by new approaches where biotechnology including modified and synthetic proteins is combined with state-of-the-art physical electrochemistry with emphasis on single-crystal, atomically planar electrode surfaces, in situ scanning tunnelling microscopy (STM) and other surface techniques. These approaches have brought bioelectrochemistry important steps forward towards the nanoscale and single-molecule levels. We discuss here these advances with reference to two specific redox metalloproteins, the blue single-copper protein Pseudomonas aeruginosa azurin and the single-haem protein Saccharomyces cerevisiae yeast cytochrome c, and a short oligonucleotide. Both proteins can be immobilized on Au(111) by chemisorption via exposed sulfur-containing residues. Voltammetric, interfacial capacitance, x-ray photoelectron spectroscopy and microcantilever sensor data, together with in situ STM with single-molecule resolution, all point to a coherent view of monolayer organization with protein electron transfer (ET) function retained. In situ STM can also address the microscopic mechanisms for electron tunnelling through the biomolecules and offers novel notions such as coherent multi-ET between the substrate and tip via the molecular redox levels. This differs in important respects from electrochemical ET at a single metal/electrolyte interface. Similar data for a short oligonucleotide immobilized on Au(111) show that oligonucleotides can be characterized with comparable detail, with novel perspectives for addressing DNA electronic conduction mechanisms and for biological screening towards the single-molecule level.

Voltammetry and Electrocatalysis of Achromobacter Xylosoxidans Copper Nitrite Reductase on Functionalized Au(111)-Electrode Surfaces

A long-standing issue in protein film voltammetry (PFV), particularly electrocatalytic voltammetry of redox enzyme monolayers, is the variability of protein adsorption modes, reflected in distributions of catalytic activity of the adsorbed protein/enzyme molecules. Use of well-defined, atomically planar electrode surfaces is a step towards the resolution of this central issue. We report here the voltammetry of copper nitrite reductase (CNiR, Achromobacter xylosoxidans) on Au(111)-electrode surfaces modified by monolayers of a broad variety of thiol-based linker molecules. These represent positively charged and electrostatically neutral, hydrophobic and hydrophilic, aliphatic and aromatic, and variable-length micro-environments, as well as their combinations. Optimal conditions for enzyme function seems to be a combination of hydrophobic and hydrophilic surface linker properties, which can lead to close to complete non-catalytic monolayer interfacial electron transfer function and electrocatalysis with activity approaching enzyme activity in homogeneous solution. Thiophenol (combined hydrophobic stacking and interdispersed water molecules), 4-methyl-thiophenol (hydrophobic and water molecules), and 3- and 4-aminothiophenol (hydrophilic, hydrophobic) offer the overall most efficient micro-environments. Subtle differences with even small structural linker differences, however, lead to widely different electrocatalytic properties, strikingly illuminated by the ω-mercaptoamines. CuNiR thus shows highly efficient, close to ideal reversible electrocatalytic voltammetry on cysteamine-covered Au(111)-electrode surfaces, most likely due to two cysteamine orientations previously disclosed by in situ scanning tunnelling microscopy. Such a dual orientation exposes both a hydrophobic and a positively charged, hydrophilic surface feature. In contrast, the higher cysteamine homologues expose only the hydrophilic component with no electrocatalytic activity on these surfaces. These results offer a basis for rational surface design in forthcoming biological electrocatalysis useful both fundamentally and in novel biosensor technology.

Electron transfer and redox metalloenzyme catalysis at the single-molecule level

Voltammetry based on single-crystal, atomically-planar metal electrodes is novel in bioelectrochemistry. Together with in situ scanning tunneling microscopy (STM) directly in aqueous buffer, single-crystal voltammetry has disclosed new detail in molecular adsorption and interfacial electron transfer (ET). Image interpretation requires, however, theoretical support, as STM represents both electronic and topographic features. Molecules with accessible redox levels offer other insight into electron tunneling mechanisms, addressed in detail for ET metalloproteins. We present here in situ STM of the blue redox metalloenzyme copper nitrite reductase (Achromobacter xylosoxidans, Ax CuNiR) on Au(111) electrode surfaces modified by a self-assembled cysteamine monolayer. Ax CuNiR displays strong nitrite reduction waves in this environment. Ax CuNiR/cysteamine/Au(111) surfaces were imaged at KNO2 concentrations where most of the enzyme is in the enzyme-substrate bound state. Molecular resolution for both cysteamine/Au(111) and Ax CuNiR/cysteamine/Au(111) electrode surfaces was achieved. The enzyme coverage is about 1.5 × 10−13 mol cm−2, which is low compared with an ideal close-packed monolayer. The adlayer behaves as an assembly of individual molecules, reflected in distributions of molecular appearance, although a number of molecules do show the triangular shape of the trimeric Ax CuNiR structure. The apparent average molecular height is about 11 A. This suggests that details of electronic structures and larger assemblies are needed to disentangle enzyme mechanisms at the single-molecule level.

In situ scanning probe microscopy and new perspectives in analytical chemistry

Abstract The resolution of scanning tunnelling microscopy (STM) and other scanning probe microscopies is unprecedented but the techniques are fraught with limitations as analytical tools. These limitations and their relationship to the physical mechanisms of image contrast are first discussed. Some new options based on in situ STM, which hold prospects for molecular- and mesoscopic-scale analytical chemistry, are then reviewed. They are illustrated by metallic electro-crystallisation and -dissolution, and in situ STM spectroscopy of large redox molecules. The biophysically oriented analytical options of in situ atomic force microscopy, and analytical chemical perspectives for the new microcantilever sensor techniques are also discussed.

Self-Assembly of Biomolecules on Electrode Surfaces; Oligonucleotides, Amino Acids, and Proteins toward the Single-Molecule Level

Publisher Summary This chapter presents an overview of some recent experimental and theoretical studies in a “bottom-up” fashion. The approach is from building blocks to higher organized systems that is, from mononucleotides to DNA fragments and from amino acids to proteins organized on single crystal surfaces. It provides a brief overview of molecular tunneling mechanisms in scanning tunneling microscopy (STM) and in situ STM and describes the features of the central substrate electrode systems mostly used, the single-crystal Au (1 1 1) surface. It overviews data for mono- and oligonucleotide monolayers and offers some views on electronic conduction mechanisms across adlayers of DNA-based molecules. It describes the electrochemical and in situ STM studies of amino acids and redox metalloproteins. The studies of artificial proteins on Au (1 1 1) have been presented. The approaches discussed offer ways of circumventing the “noise” problems by mapping precisely conditions where structural “coherence” can be expected to apply, with concomitant minimization of fluctuational effects on chemical and electronic function.

Nanoscale and single-molecule interfacial electron transfer

Electrochemical science and technology in the 21st century have reached high levels of sophistication. A fundamental quantum mechanical theoretical frame for interfacial electrochemical electron transfer (ET) was introduced by Revaz Dogonadze. This frame has remained for four decades as a basis for comprehensive later theoretical work and data interpretation in many areas of chemistry, electrochemistry, and biology. We discuss here some new areas of theoretical electrochemical ET science, with focus on nanoscale electrochemical and bioelectrochemical sciences. Particular attention is given to in situ scanning tunneling microscopy (STM) and single-electron tunneling (SET, or Coulomb blockade) in electrochemical. systems directly in aqueous electrolyte solution and at room temperature. We illustrate the new theoretical formalism and its perspectives by recent cases of electrochemical SET, negative differential resistance patterns, and by ET dynamics of organized assemblies of biological macromolecules, such as redox metalloproteins and oligonucleotides on single-crystal Au(111)-electrode surfaces.

Organized monolayers of biological macromolecules on Au(111) surfaces

Single-crystal electrochemistry and scanning tunneling microscopy directly in aqueous electrolyte solution (in situ STM) are established in physical electrochemistry but new in studies of adsorption and interfacial electrochemistry of biological macromolecules. These high-resolution techniques have now been applied comprehensively to proteins and other biomolecules in recent studies, discussed in this report. Focus is on three systems. The first one is a pair of amino acids, cysteine and cystine. These are strongly adsorbed via thiolate and disulfide, respectively, with identical reductive desorption and in situ STM patterns. Long-range lateral order can be imaged to molecular resolution. The amino acids are also reference molecules for the blue single-copper protein Pseudomonas aeruginosa azurin. This protein assembles in two well-defined orientations. One applies on bare Au(111) to which the protein is linked via its surface disulfide group. This orients the copper center away from the electrode surface. The other mode is by hydrophobic interactions with variable-length alkanethiols self-assembled on Au(111). In this mode the copper center is directed towards the surface. Adsorption and long-range electron tunneling in both modes have been characterized in detail using different electrochemical and spectroscopic techniques, as well as STM. Other data show that penta-(A–T) oligonucleotide adsorbed via a covalently bound thiol linker also displays reductive desorption and in situ STM to molecular resolution. The three systems thus appear to open new perspectives for broader use of high-resolution electrochemical techniques in comprehensive investigations of large biological molecules.

Self-assembled monolayer of a peroxo-bridged dinuclear cobalt(III) complex on Au(111)

Densely packed Self-Assembled Monolayers (SAMs) of a peroxide-bridged dicobalt complex, [Co2(O2)(bpbp)(O2CCH2CH2S)]2+, 3, (bpbp- = 2,6-bis((N,N-bis-(2-picolyl)amino)-methyl)-4-tert-butylphenolato) have been prepared on atomically planar Au(111) surfaces. Surface voltammetric and interfacial capacitance data, along with electrochemical scanning tunnelling microscopy (in situ STM) imaging, support the formation of a densely packed adlayer of 3 attached via a gold-thiolate bond. In solution, the disulfide linked precursor for 3 reversibly binds dioxygen with high affinity. Electrochemical measurements show that the redox potential of the O22-/O2*- couple of the monolayer of 3 is cathodically shifted by nearly 500 mV compared to the precursor in solution. This is attributed to the close proximity of the O2 binding site to the gold surface. Since the redox potential of the O22-/O2*- couple reflects tentatively the binding affinity of O2 to the deoxygenated CoII2 binding site, the potential of the O22-/O2*- couple of the SAM of 3 suggests a much higher affinity towards O2 compared to the solution precursor.