Prestazioni della tecnica di transfezione selettiva di cellule di mammifero in adesione tramite strutture integrate su silicio

The NaChip project (IST-2001-38915) aims to the realization of a non-invasive system for monitoring the electrical activity of neurons culture. Such device is a silicon microchip containing EOSFET (Electrolyte Oxide Silicon Field Effect) transistors, through which it is possible to record the electrical activity from nervous cells. This device has an elevated signal/noise ratio, an intrinsic characteristic of the instrument. To improve the relationship of the signal/noise ratio, this job focuses on the improvement of the cell-transistor connection, through the accumulation of sodium channels at the neuron-to-substrate interface. We have used B1 and B2 auxiliary subunits of Nav1.2 channel, that, interacting through their extracellular domain with particular proteins, respectively contactin 1 (CNT1) and tenascin C (TNC), can carry the accumulation of the sodium channel in the cell/transistor interface. Through the creation of fluorescent fusion proteins for B1 and B2, it has been possible to analyze the effect of the overexpression of the chimeras in E18 rat hippocampal neurons, and their role in the sorting of the channel in the presence of substrate coatings. Such analysis has been carried out by means of TIRF microscopy, that allows the observation of the adhesion zone of the cell with an elevated signal/noise ratio. This has brought us to the discovery of an unexpected effect of B2 in the considerable increaseing of the dendritic branching, thus allowing to take advantage of such characteristic in order to improve the electrical coupling connection cell/transistor and to increase the signal/noise ratio. The necessity of a selective transfection of a single neuron over the recording zone, has carried to the development of a innovative and not using the stimulation site EOSC (Electrolyte Oxide programmed technique Semiconductor Capacitor). This site can been used for ectroporation of a limited number of cells in adhesion, allowing to put inside the cell various types of molecules as it will be explained in the present job.