Mikala Grubb

Voltammetry of native and recombinant pseudomonas aeruginosa azurin on polycrystalline Au- and single-crystal Au(111)-surfaces modified by decanethiol monolayers

Abstract The native blue single-copper protein azurin ( Pseudomonas aeruginosa ) was recently shown to adsorb in close to monolayer coverage and well-defined stable orientations on alkanethiol monolayers self-assembled on Au(111)-surfaces. Adsorption is caused by hydrophobic interactions between the alkanethiol and the hydrophobic protein surface around the copper centre, orienting the latter towards the electrode surface in a way favourable for electron exchange. In this report we show that similar stable adsorption of functional azurin on polycrystalline electrodes can be achieved, represented by azurin adsorption on a decanethiol monolayer. This facilitates significantly the use of this approach to protein immobilization. Reversible monolayer voltammetry is observed for scan rates up to about 1 V s −1 . The peaks separate at higher rates. Equilibrium potentials and interfacial electron transfer rate constants are indistinguishable from those at single-crystal Au(111)-electrodes. The sensitivity of azurin monolayer voltammetry on self-assembled alkanethiols to hydrophobic interactions was also used to address possible voltammetric differences between native and recombinant azurin. Voltammetric patterns, equilibrium reduction potentials, and electrochemical rate constants were, however, indistinguishable for the two proteins.

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.

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.