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Despite advances made in the field, rapid detection methods for the human pathogen Candida albicans are still missing. In this regard, bio-electrochemical systems including electrochemical sensors and biosensors satisfy the increasing demand for rapid, reliable, and direct microbial analyses. In this study, the bioelectrochemical characteristics of C. albicans were investigated for use in an analytical system that determines the viability of the organisms. The electrochemical responses of viable and non-viable cells of C. albicans and Saccharomyces cerevisiae were monitored. Cyclic voltammograms (CV) showed an irreversible oxidation peak at about 750 mV that accounts for viable cells. The peak current increased at viable cell numbers ranging from 3 × 10(5) to 1.6 × 10(7)cells/ml, indicating that the amount of viable cells can be accurately quantified. To elucidate the underlying electron transfer processes, the influence of electron transfer chain (ETC) - inhibitors on the electrochemical behavior of the two organisms were investigated. Inhibition of the function of classical respiratory chain (CRC) led to a decrease in the electrochemical response, whereas the oxidation current increased when the alternative oxidase (AOX) pathway was blocked by salicylhydroxamic acid (SHA). Blocking the AOX pathway improved the electrochemical performance, suggesting an involvement in the CRC, with cytochrome c oxidase (COX) as a relevant protein complex. Mutants, in which components of COX were deleted, showed a lower electro-activity than the wild-type strain. Particularly, deletion of subunit COX5a almost completely abolished the electrochemical signal. We believe that this work can be utilized for the development of early detection assays and opens the door for new technological developments in the field of C. albicans.

Glucose oxidase (GOD) was immobilized on screen-printed platinum electrodes by entrapment in a screen printable paste polymerized by irradiation with UV-light. The influences of different additives, in particular polymers and graphite, on the sensitivity and stability of the sensor and the permeability of the enzyme layer for a possible electrochemical interferent were investigated. The chosen additives were Gafquat 755N, poly-L-lysine, bovine serum albumin (BSA), sodium dodecylsulfate (SDS), polyethylene glycol (PEG), Nafion and graphite. All additives led to increases of glucose signals, i.e. improved the sensitivity of glucose detection with Gafquat 755N, poly-L-lysine, SDS and graphite showing the strongest influences with increases by a factor 4, 6.5, 5 and 10, respectively. Ascorbic acid was used as a model interferent showing the influence of the enzyme layer composition on the selectivity of the sensor. The addition of Gafquat 755N or poly-L-lysine led to higher signals not only for glucose, but also for ascorbic acid. SDS addition already reduced the influence of ascorbic acid, which was almost completely eliminated when Nafion (5%) and PEG (10%) were added. A comparable beneficial effect on the selectivity of the sensors was also observed for the addition of 0.5% graphite. Thus, the enzyme electrodes with PEG, Nafion or graphite as additives in the enzyme layer were applied to glucose determinations in food samples and samples obtained from E. coli cultivations.

Atomic force microscopy (AFM) and an optical grating coupler system were used to improve the understanding of the biosensing layer on a Ta(2)O(5)-light-guiding surface. Exemplary, we investigated the immobilization of the protein avidin, the subsequent binding of biotinylated oligonucleotides and hybridization of a complementary 12-mer. The AFM measurements revealed the height of approximately 1.6 nm for a single avidin molecule, while the thickness of the avidin layer on the biosensor surface seemed to be 2.8-3.0 nm. This result lead to the conclusion that the protein was not forming a simple monolayer. However, the thickness of the avidin layer could not be determined directly, but only after shifting of protein by the tip of the AFM leading to grooves of 1 micro m(2) and approximately 3 nm depth. As the height of oxide particles forming the waveguide surface was also in the range of 1.5 nm, the depth of these grooves could also be a result of the deposition of proteins on top of the oxide particles. This was consistent with the increased roughness of the surface after protein binding. Thus, investigations with the grating coupler were used to determine quantitatively the amount of immobilized avidin. On a biotinylated surface the amount of immobilized avidin lead to the assumption of a complete monolayer, whereas simple adsorption proved to be less efficient. A binding ratio of 1:1.3 for avidin and a biotinylated oligonucleotide was achieved. Up to 83% of the bound single strand were accessible for a subsequent hybridization reaction with a 12-mer. These results supported the model of avidin being deposited mainly on top of the oxide particles leading to the picture of a 'rough' complete protein monolayer, which was postulated from the AFM investigations.

Various techniques for the fabrication of thick film biosensors are described. With electrodes printed on conventional thick film substrates, glucose is measured via the formation of hydrogen peroxide using glucose oxidase. Additional diffusion barriers extend the linear range of the sensor up to 15 m m . Using a new, flexible, unfired substrate material sensors with a three-dimensional structure can be made. The resulting cavities can be filled with carbon paste. Hence, the mediator technique and the use of dehydrogenases was applied to the fabrication of thick film biosensors. The linear range, sensitivity and stability of these sensors is strongly dependent on preparation methods such as the immobilization technique (mediated electrode) and the amount of cofactor and enzyme. Without any diffusion barrier the linear range is limited to about 1 m m for the sensor based on the dehydrogenase.