Biosensors and bioelectrochemistry

This review describes recent developments in the field of biosensors and bioelectrochemistry. Nanoparticles have been used to improve sensor performance and to develop biosensors based on new detection principles. Their use has extended into all areas of biosensor and bioelectrochemistry research. Other active areas of biosensor development include DNA sensing, immunosensing, direct electron transfer between an electrode and a redox protein or enzyme, and in vivo sensors.     

Copper and iron metalloproteins

The most important oxidases and all oxygen carriers are copper and/or iron metalloproteins. Unique metabolic devices have evolved to utilize these essential metals effectively.     

Carbon-nanotube-modified electrodes for the direct bioelectrochemistry of pseudoazurin

The bioelectrochemistry of the blue copper protein, pseudoazurin, at glassy carbon and platinum electrodes that were modified with single-wall carbon nanotubes (SWNTs) was investigated by multiple scan rate cyclic voltammetry. The protein showed reversible electrochemical behavior at both bare glassy carbon electrodes (GCEs) and SWNT-modified GCEs (SWNT|GCEs); however, direct electrochemistry was not observed at any of the platinum electrodes. The effect of the carbon nanotubes at the GCE was to amplify the current response 1000-fold (nA at bare GCE to μA at SWNT|GCE), increase the apparent diffusion coefficient D _app of the solution-borne protein by three orders of magnitude, from 1.35 × 10⁻¹¹ at bare GCE to 7.06 × 10⁻⁸ cm² s-1 at SWNT|GCE, and increase the heterogeneous electron transfer rate constant k _s threefold, from 1.7 × 10⁻² cm s⁻¹ at bare GCE to 5.3 × 10⁻² cm s⁻¹ at SWNT|GCE. Pseudoazurin was also found to spontaneously adsorb onto the nanotube-modified GCE surface. Well-resolved voltammograms indicating quasi-reversible faradaic responses were obtained for the adsorbed protein in phosphate buffer, with I _pc and I _pa values now greater than corresponding values for solution-borne pseudoazurin at SWNT|GCEs and with significantly reduced ΔE _p values. The largest electron transfer rate constant of 1.7 × 10⁻¹ cm s⁻¹ was achieved with adsorbed pseudoazurin at the SWNT|GCE surface in deaerated buffer solution consistent with its presumed role in anaerobic respiration of some bacteria.     

Control of electron transfer in metalloproteins

The role of protein structure in the control of electron transfer in metalloproteins is briefly discussed, with reference to existing theoretical models and available three dimensional information.     

Metalloproteomics, metalloproteomes, and the annotation of metalloproteins

Metalloproteomics includes approaches that address the expression of metalloproteins and their changes in biological time and space. Metalloproteomes are investigated by a combination of approaches. Experimental approaches include structural genomics, which provides insights into the architecture of metal-binding sites in metalloproteins and establishes ligand signatures from the types and spacings of the metal ligands in the protein sequence. Theoretical approaches employ these ligand signatures as templates for homology searches in sequence databases. In this way, the number of metalloproteins in the iron, copper, and zinc metalloproteomes in various phyla of life has been estimated. Yet, manganese metalloproteomes remain poorly defined. Metals have catalytic and structural functions in proteins. However, additional functions have evolved. Proteins that control metal homeostasis and proteins that are metal-regulated bind metal ions transiently and are generally not accounted for in estimates from bioinformatics. Thus, metalloproteomes are dynamic and likely to be larger than present estimates suggest. This account discusses the assignment of transition metals in metalloproteins and the ensuing issues facing analytical chemists and structural and computational biologists. Biological and chemical selectivities render metal selection by metalloproteins either more stringent or less stringent depending on the metal homeostatic system of the organism, the subcellular location of the protein, and environmental factors. Failure to recognize the principles of metal utilization has led to assigning the wrong metal in metalloproteins and has missed some of the regulatory functions of transition metal ions.     

Conception of contact potential differences in the bioelectrochemistry phenomena concerning cellsin Vitro

Summary The contact potential difference is presented in relation to the electrokinetic potential and the surface potentials of the cells.     

Molecular dynamics simulations of metalloproteins

Molecular dynamics simulations are now commonly applied to metalloproteins, despite the challenges introduced by the presence of metal ions. Force field parameters are nowadays available also for these 'exotic' atoms and several biological systems have been successfully studied. Some of the most relevant results and methodological advancements are reviewed.     

NMR structures of paramagnetic metalloproteins

Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+ -binding proteins and for Ca2+ -binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.     

Structural control of electron-transfer properties in metalloproteins

Summary The factors that control long-range electron transfer between two redox centers in a protein are summarized. Rack-induced bonding in blue copper proteins is described. The protein conformation forces the Cu(II) ion into a distorted geometry, lying at least 70 kJ mol^–1 above the preferred square-planar geometry in energy. The distortion has the effect that the structural change associated with electron transfer is minimal and thus the reorganization energy small. Variations in back bonding are suggested to modulate the reduction potentials of blue proteins without any change in the energy of the charge-transfer transitions. In proton pumps there must be a structural control of the electron transfer rates (electron gating) and model studies suggest that this is best achieved by variations in the reorganization energy.