Micro electrochemical sensors and PCR systems: cellular and molecular tools for wine yeast analysis

Nowadays, exciting bioanalytical microsystems are currently receiving increasing attention in biology since they can comply with the considerable demand for reliable, sensitive and low-cost analysis tools. Small reagents volumes, low power consumption, portability, fast analysis, high throughput and systems integration are the key aspects that make these systems more and more appealing within both the academic and industrial communities. In the last years, many microdevices were developed for a wide range of biological applications, particularly dedicated to cellu-lar or molecular analysis. Many efforts were devoted to the realization of Cell-Based Biosensors (CBBs) to monitor the dynamic behaviour of cell cultures for pharmacological screening and basic research. Other researchers focused their interests in the development of so-called Lab-on-a-Chip (LOC) systems for DNA analysis mostly applied to clinical diagnosis. This thesis deals with the investigation of two miniaturized devices – a cell-based biosensor and a DNA amplification system – for the cellular and molecular analysis of wine yeasts, respectively. The first device consists of integrated electrochemical sensors – Ion-Sensitive Field-Effect Transistor (ISFET), impedimetric and temperature sensors – for the real time evaluation of pH and cell settling of yeasts under batch culture conditions. The assessment of yeast performance and robustness has been focused on ethanol tolerance, as it is one of the main stress factors acting in wine, and thus, one of the major causes of stuck fermentations. A good agreement between extracellular acidification and cell growth trends at different ethanol concentration has been demonstrated, significantly reducing the time of the traditional assays. Moreover, resistivity measurements have shown the possibility to follow progressive settling of the cell suspension. Concerning the second system, a Polymerase Chain Reaction (PCR) microdevice has been biologically validated by successfully amplifying yeast genomic DNA fragments. Additionally, the outcome of PCR has been positively assessed with diluted samples and boiled yeast cultures, demonstrating the possibility to skip the time-consuming purification process for potential LOC applications with very little or no pre-PCR sample manipulations. The encouraging results from both microsystems have demonstrated their suitability for wine yeast analysis, aimed at quality improvements of the winemaking process