A microfluidic device (1000-1005), comprising: a semiconductor body (2) having a first side (2a) and a second side (2b) opposite to one another, and housing, at the first side, a plurality of wells (4), having a first depth; an inlet region (30) forming an entrance point for a fluid to be supplied to the wells; a main channel (6a) fluidically connected to the inlet region, and having a second depth; and a plurality of secondary channels (6b) fluidically connecting the main channel to a respective well, and having a third depth. The first depth is higher than the second depth, which in turn is higher than the third depth. According to an aspect, the microfluidic device further comprises a cover layer (8), arranged above the first side of the semiconductor body, configured for sealing the wells and provided with at least a first valve hole (54) which extends through the cover layer and overlaps, at least partially, the secondary channels; and a flexible layer (14), arranged above the cover layer and provided with at least a protrusion (74) extending through the first valve hole towards the semiconductor body and overlapping, at least partially, the secondary channels, the flexible layer being configured such that, when a pressure is applied on it, the protrusion contacts the semiconductor body and enters the secondary channels thus fluidically isolating the wells from one another.