## 3 Results and Discussion
### Setup of the Optimal Conditions for microRNAs Adsorption on PDMS Planar Surfaces  
PDMS planar surfaces were selected to mimic the surfaces of microdevice channels with the aim of studying the best pH conditions for microRNAs capture and release. The interaction between primary amines coating surfaces and nucleic acid is indeed well documented [26; 27]. Since the main driving force of this phenomenon relies on the electrostatic interaction, a change in the pH of nucleic acid solutions could affect their adsorption. As a first step in the setup of the optimal working conditions, an investigation on the dependence of microRNA adsorption on pH was therefore undertaken. Moreover, considering the potential use of this microdevice aimed at biomarker detection, the synthetic miR-1246 was selected because of its suggested potential role as biomarker for several cancer types [28; 29], including the non-small cell lung cancer (NSCLC) [30]. Synthetic miR-1246 conjugated with the fluorescence dye TAMRA was diluted in buffers with pH ranging from 4 to 9, while the ionic strength was kept constant at 20 mM to minimize the possible interference of counter-ions or ionic bridges. The fluorescent microRNA was incubated on the silanized PDMS surfaces. After adsorption and washing, the surfaces were imaged with a fluorescence microscope. The adsorption of microRNAs was higher at pH around 5, but a quite good adsorption was observed also at neutral pH (Figure 2, Panel (a)), confirming our previous findings related to the behavior of amino-silanized surfaces [27]. Interestingly, the highest adsorption (fluorescence signal \(=26.4\times 10^{3}\pm 180\)) was obtained when the microRNA was diluted in water (i.e., very low ionic strength, pH slightly acidic). This behavior could be easily explained considering the net charge of the interacting molecules. In fact, RNA molecules are negatively charged under physiological conditions, due to the presence of charged phosphate groups along the backbone of the nucleic acid. Under the same conditions, the majority of the amino groups are in their protonated forms, as discussed below, so the APTMS exposes a positive surface, prone to attract negative molecules. Instead, at basic pH the amino groups are mostly in their un-protonated form, thus the APTMS surface becomes neutral. Moreover, the presence of PEG-s molecules helps the uniform distribution of positive charges, as demonstrated by our group [19; 31]. Once adsorbed, the release of microRNA was also investigated as a function of pH. The pK of a typical organic primary amine in solution ranges around 10-11 units [32]; but, when molecules carrying primary amines are covalently bound to a surface, the pK of such amines could be lowered until around 6.5 on gold surfaces [33], and around 4 on silicon oxide [34]. To verify to which extent the amine groups attached to PDMS surfaces (amino-silanized PDMS surfaces object of this study) are influenced by such effect, three pH conditions were selected for the releasing step, i.e., pH 5, 7 and 9 (Figure 2, Panel (b)). As expected, the worst result is obtained for elution at pH 5, but also elution at pH 7 resulted scarce at every adsorption pH tested. On the contrary, microRNA release was almost complete when the elution step occurred at pH 9, i.e., well over the above reported values of surface-bound amines pK. We consider then that the lowering of the pK value of amines attached to PDMS, if ever happens, is negligible in our conditions. [PERSON] et al. [35] suggest indeed that the pK shift may be connected to the presence of a hydrophobic environment surrounding the amine groups. Our formulation, embedding APTES with PEG molecules, may mitigate this effect. Notably, elution  
Figure 2: MicroRNA adsorption (Panel (**a**)) and elution (Panel (**b**)) from flat polydimethylsiloxane (PDMS) surfaces as a function of pH. Panel (**a**): 10 ng of synthetic miR-1246, conjugated with the florescent dye TAMRA, were incubated on the silanized surfaces at different pH.