## 3 Results and Discussion
### From PDMS Planar Surfaces to Spiral-Shaped Microdevices  
The setup of a functional assay on PDMS planar surfaces as well as the characterization of spiral-shaped microdevices were detailed in the previous paragraphs. These results, however, cannot be directly utilized to effectively functionalize the microdevices, therefore further experiments were undertaken. In particular, protocols had to be adapted to the automated microdevice manipulation. Firstly, the silanization protocol was arranged in order to provide the best performances in terms of microRNAs adsorption. The silanization mixture, AS, was injected in the microdevice, which was next immersed in water at 60 \({}^{\circ}\)C for different periods. A time spanning from few minutes to one hour was employed to functionalize the microdevices (Figure 5, Panel (a)), while all other functionalization conditions were kept constant. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sample** & **O 1s (\%)** & **C 1s (\%)** & **Si 2p (\%)** \\ \hline Channel A & 29.4 & 51.7 & 18.6 \\ Chip A bulk & 29.7 & 51.5 & 18.5 \\ Channel B & 29.4 & 49.9 & 20.7 \\ Chip B bulk & 29.9 & 50.1 & 20.0 \\ Planar PDMS & 28.8 & 50.7 & 20.5 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: XPS relative quantification (%) of O, C, and Si detected with a 0\({}^{\circ}\) take-off angle on inner (Channel A or B) and outer surfaces (Chip A or B bulk) of microdevices channels and on PDMS planar surfaces. The standard error does not exceed the 1-2% of the reported value. Figure 4: X-ray photoelectron spectroscopy (XPS) survey spectra taken at 0\({}^{\circ}\) take-off angle. Spectra were taken inside microdevices channels (curves named Channel A and Channel B) and outside channels, i.e., on freshly cut microdevice surfaces (A bulk and B bulk). A planar PDMS surface was also measured for comparison (planar PDMS). Even at short silanization time, a good adsorption of microRNA was observed, allowing selecting 5 min of silanization as the standard condition for following experiments. This conclusion was reinforced by measuring the unbound microRNA collected from every microdevice, at the spectrophotometer. In addition, in this case, the amount of unbound material from microdevices silanized for 5 min was comparable with those of microdevices silanized for 40 or even for 60 min (Figure S2). Once functionalized, the microdevice is ready to capture microRNAs. Therefore, the next optimization concerned the flux of microRNA insertion, which should be fast enough to allow the adsorption on the microdevice surfaces but not too fast impeding the effective contact of microRNAs with the positively charged surfaces. The same concentration of fluorescent microRNA was inserted in the silanized microdevices (Figure 5, Panel (b)) with two different flow rates for a sufficient time to deliver 600 ng of total microRNA. Both the fluxes gave quite similar results in terms of microRNA adsorption for both the microdevices types (A and B), therefore 10 \(\upmu\)L/min was selected as optimal, reducing the insertion time and speeding up the duration of the assay. Finally, a washing step is needed to remove the unbound material. The microdevice was flowed with water, whose pH is slightly acidic (see Section 3.1) and therefore the surface charges are not  
Figure 5: Optimization of protocols from PDMS planar surfaces to spiral microdevices. Fluorescent miRNAs were used to assess functionalization parameters. Panel (**a**): optimization of silanization time. The microdevice is functionalized with a mixture of 0.1% APTMS and 0.9% PEG-s silane (named AS) in immersion at 60 \({}^{\circ}\)C for different times. An amount of 600 ng of fluorescent microRNAs diluted in water are then absorbed to microdevice AS surfaces and the resulting fluorescence is reported as a function of the silanization time. Panel (**b**): optimization of the flux speed during microRNA insertion. 3 ng/uL miR-1246-TAMRA was inserted either at 2 \(\upmu\)L/min x 100 min or 10 \(\upmu\)L/min x 20 min.