## 3 Results and Discussion
### Functional Assay for the Characterization of Microdevices for microRNA Adsorption  
Once set up the working parameters for both microdevices, a functional assay aimed at verifying the potentialities of the two microdevices types was carried out. In the previous paragraph, all parameters were set up by measuring the fluorescence signal related to the presence of miR-1246-TAMRA with a wide-field fluorescence microscope. This method is excellent for a relative comparison of the different conditions applied to the microdevices, but results rather poor when a strict quantification is needed. In particular, the collected fluorescence signal is mostly related to the microRNA adsorbed on channels top and bottom surfaces, with a minor contribution from channels lateral surfaces. To check the uniformity of miRNA adsorption on surfaces, a confocal microscope was then used to collect three-dimensional images of the channels for the two kinds of microdevices. Channels of both microdevices were cut diagonally with an angle of about 30\({}^{\circ}\) after microRNA adsorption, to ease the imaging at the confocal microscope (Figure 6). From data reported in Figure 6, all microdevice surfaces can be considered as properly functionalized and then available for microRNAs capture. The presence of the fluorescent microRNA,  
Figure 6: Check for surface availability for microRNA adsorption. Panel (**a**): 1200 ng of miR-1246-TAMRA diluted in water are adsorbed on functionalized microdevice A. Panel (**b**): 600 ng of miR-1246-TAMRA diluted in water are adsorbed on functionalized microdevice B. After washes, the dry channels were cut diagonally with an angle of about 30\({}^{\circ}\), as depicted on top of each image (CAD representations), and imaged at the confocal microscope in z-stacking mode. Images are 3D reconstructions of microdevice channels. indeed, is homogenously distributed on all the visible surfaces, confirming the good working conditions previously set up. Next, we moved to consider the effect of the different total channel surface on the maximum loading capacity of the two kinds of microdevices. In Figure 7, an estimation of the total area covered by fluorescent miRNAs versus the quantity of miRNAs fluxed through the chip is reported. This titration confirmed that Microdevice B, which presents a higher total channel surface (S/V \(\approx\) 46.2 mm\({}^{-1}\)), adsorbs a higher amount of microRNA compared to Microdevice A (S/V \(\approx\) 18.0 mm\({}^{-1}\)). For this experiment all other parameters than concentration, i.e., flux rate, microRNA insertion time, flux and washing time, were kept constant. The insertion of 300 ng of pure microRNA saturated the capturing ability of Microdevice A, while a double amount was still well adsorbed by Microdevice B. The total area possibly occupied by microRNA was calculated as 356 mm\({}^{2}\) for Chip A and 460 mm\({}^{2}\) for Chip B. For Chip A, this value was reached with the adsorption of 300 ng of microRNA, while the double was still adsorbed by Chip B, reflecting the different S/V for the two microfluidic devices. The S/V of Chip B is indeed about the double than S/V of Chip A. The ability to capture pure microRNA was demonstrated for both microdevices. A further characterization of microdevice performances, more geared toward a possible use of these devices on field, was performed by introducing a constant concentration of fluorescent microRNA spiked in human plasma added with lysis buffer (Figure 8), for different times. The addition of lysis buffer is needed to favor the release of miRNA from vesicles encapsulation or protein complexes, in the perspective of microdevice use with clinically relevant samples. For this analysis, Chip B was selected as the more performant with respect to the Chip A (Figure 7). Comparing the fluorescence signal intensity that is detected on microdevices fluxed for different times, a linear increment is clearly visible as the fluxing time is increased, as expected.