"Electronic" interfaces with enzymes have been rather crude in terms of achieving communication at the interface with redox proteins. The glucose biosensor was 'invented' 30 years ago, but provides little insight in terms of achieving better communication with enzymes. This paper will look at the use of protein engineering to explore and optimize the charge transfer between an enzyme and an electrode. It considers whether a greater understanding of the enzyme structure will aid our future design capability in enzyme biosensors.

According to Marcus’ theory electron transfer (ET) rates are expected to drop by 104 when the distance between an electron donor and acceptor is increased from 8 to 17Å. The crystal structure for GOx was solved and published in 1992: at 17Å, which is the distance estimated from the redox centre in glucose oxidase to the outside of the enzyme, it should still be in range to predict electron transfer (ET) and produce a visible current yet such non-selective “short-circuit” electron transfer has not been reported to be efficient and, indeed, is not typical of the normal modus operandii between redox proteins in the cell. Instead, proteins appear to be insulated against ‘short-circuit’ or ‘promiscuous’ charge transfer and ET is initiated indirectly by ‘third parties’: ie the enzyme’s substrate and cosubstrate, that exclusively enter the active site (thereby insuring specificity) to perform a highly selective redox turnover.
-What prevents the promiscuous redox activity?
-Are directly ‘wired proteins’ fundamentally ‘forbidden’?
A fundamental approach investigating how protein engineering and site directed mutagenesis (SDM) can be used to enhance the connectivity between the electron transport pathway in a protein and an electrode has revealed some interesting and potentially key information.

Mobile diffusing redox mediators used to 'shuttle connect' with redox enzymes in classical second-generation biosensors, show dependence on diffusion and kinetic properties of the non-bonded mediator. ET with a bonded external redox centre has been suboptimal. Nevertheless, the perceived prizes for achieving this connectivity are significant, if we could understand how to approach the design and engineering of such enzymes. Enzyme based molecular structures could in principle, ultimately also be designed to exhibit semi- conductive properties, to hold a charge or behave like switches or memory, although to date much of the focus on molecular switching has used ordered redox terminated self-assembled layers on metal electrodes.

GOx belongs to a family of flavoproteins; it is a dimeric protein with MW = 160 kDa when glycosylated. There are circa 80 different flavoproteins containing Flavin adenine dinucleotide (FAD) or Flavin mononucleotide (FMN). This paper will show fast electrode kinetics of the FAD redox group within the enzyme for the first time and we follow the interaction between FADH and O2. Clearly, the ability to achieve such outstanding electrochemistry in a mutant enzyme is enticing, especially since the enzyme-electrode remains responsive to glucose, even using an applied oxidation potential <0V vs Ag/AgCl to measure FADH oxidation.

"Proteomics: High Throughput Protein Analysis based on Mass Spectrometry"
Catherine Fenselau, University of Maryland

The proteome is composed of all the proteins in a cell or organism at any given time. Proteomic strategies are designed to provide automated high throughput analysis of these large protein mixtures. This talk will be presented from an analytical chemist’s point of view. What are the analytical objectives, the analytical figure of merit, and the obstacles in the application of mass spectrometry and bioinformatics to the automated identification of peptides and proteins?

In many applications, changes in proteins in disease are studied by quantitative comparison of protein abundances. Different approaches to such comparisons will be illustrated in comparisons of drug-susceptible and drug-resistant cancer cells, including differential stable isotope labeling by metabolic incorporation, chemical alkylation or enzyme catalysis. Isotope-free methods for global comparisons include 2D gel electrophoresis and comparison of HPLC peak areas, while targeted label-free quantitation can be provided by multiple reaction monitoring.

Proteomic strategies have, thus far, been based on digestion of proteins with trypsin and analysis of tryptic peptides by LC-MSMS. Arguments will be presented for working with heavier proteolytic peptides, e.g., in the 3000 to 15,000 Da range. We have recently realized this possibility, using LC-MS/MS measurements with a high resolution mass analyzer and a new (commercial) computer program. Comparisons will be offered between a trypsin-based workflow and one that cleaves at aspartic acids, based on the number of peptides identified, the number of proteins represented, and coverage of proteins in a ribosomal proteome from human cancer cells.

Eric Bakker, Roland De Marco, Pengchao Si, Ewa Grygolowicz-Pawlak
Curtin University of Technology, Perth, Australia.

The detection of ionic species is an important cornerstone of clinical diagnostics and environmental analysis. While potentiometric techniques have been successfully established for this purpose, the field has more recently seen important improvements. This talk will outline principles and progress in ultra-trace level potentiometric sensing, and focus on the development of biosensor principles based on such transducers.

Knowledge of the underlying membrane chemistry has also helped to further develop voltammetric ion transfer sensing principles based on related materials. Recent progress has focused on a multilayer materials platform to realize thin layer voltammetric sensing films. This is accomplished with nanoscale films that are deposited by electropolymerization and spin coating. It is shown that intrinsically anion responsive conducting polymers can be used to detect cationic species by overlaying them with a sensing membrane containing a calculated excess of cation-exchanger. The electrochemically triggered incorporation of the cation-exchanger from the conducting polymer into the sensing membrane during reduction results in extraction of cations from the sample. This has implications for stripping voltammetry, chemical separation and detection based on mild non-redoxactive ion extraction processes based on environmentally benign materials.