## 3 Results and Discussion
### On-Chip Polysome Capture and RNA Purification  
Given the promising results reported in the previous paragraphs, a proof-of-concept for an integrated system was assessed to directly capture polysome from cell lysates and extract mRNA from them. The functionalization protocol set-up on silicon plane surfaces was thus applied to a silicon/Pyrex microdevice (Figure S2) as a proof-of-concept for an integrated system devoted to both polysome capture from cell lysates and mRNA extraction from the immobilized polysomes. To compare microdevice results with protocols set up on plane samples, the same conditions were implemented on-chip, in particular prepurified polysomes were employed. Polysomes were immobilized on the negatively-functionalized surfaces of the microdevice and, after a washing step, the captured mRNA was released. Based on results described in the previous paragraph and after testing the different silanization protocols on microdevices (see Section 2.2.2), the functionalization with triCAS was performed on three microdevices. In parallel, three additional nonfunctionalized microdevices were used as a control. First, a prepurified polysomal fraction was inserted in the microdevices and incubated for 1 h at 4 \({}^{\circ}\)C to allow adsorption of polysomes in conditions similar to those used for plane surfaces. Next, the unbound material was collected, and an elution step combining a thermal treatment and a change in saline concentration was then performed. These steps favor both the detachment of mRNA from polysomes and its subsequent elution. Both the unbound material and the eluted RNAs were stained with RiboGreen and quantified with a spectrofluorimeter, while RNAs quality and integrity was checked with capillary electrophoresis (Figure 3). The functionalized microdevices confirm their good performances with respect to the untreated microdevices. In fact, the quantity of unbound material (Figure 3, panel A, black bars) measured in the first case is negligible with respect to the unbound material obtained from untreated microdevices. Moreover, the fraction of RNA eluted from functionalized microdevices is clearly more abundant than RNA eluted from the untreated microdevices (Figure 3, panel A, gray bars). The adsorption and elution efficiencies reflect independently the performances of the microdevices, concerning both polysome adsorption and RNA release steps, respectively. Both parameters indicate that the silanized microdevice show slightly better performances. The RNA captured via polysome adsorption amounted to the 88% in the silanized microdevice and to the 70% in the untreated microdevice, while the \begin{table}
\begin{tabular}{c c} \hline \hline
**Surface** & **Polysome Density (\(\times\) 10\({}^{8}\)/cm\({}^{2}\))** \\ \hline TG-SO & \(7\pm 2\) \\ \hline triCAS & \(15\pm 3\) \\ \hline CST & \(12\pm 3\) \\ \hline MCOOH gold & \(19\pm 5\) \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Number of polysomes adsorbed on plasma-treated TG-SO and functionalized TG-SO surfaces. Silanizations are described in Section 2.2.2. The result obtained with negatively-charged gold surfaces is shown for comparison. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & & **(nm, Center/Width)** & & & \\ \hline
**Surface** & **H1** & **H2** & **W1** & **W2** & **L1** & **L2** \\ \hline TG-SO & 6.5/2.6 & 14.7/3.4 & 29.3/9.4 & 64.8/4.6 & 35.7/2.7 & 85.5/16.3 \\ \hline triCAS & 3.9/0.4 & 10.7/2.2 & 25.8/12.2 & 72.8/12.9 & 35.2/12.3 & 100.8/18.3 \\ \hline CST & 4.8/1.7 & 7.3/2.2 & 22.8/9.8 & 62.0/14.0 & 30.1/11.1 & 82.4/30.1 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Height (H1, H2) and lateral size values (width—W1, W2; length—L1, L2) obtained form the Gaussian fits of the morphological value distributions of polysomes deposited on bare and functionalized thermally grown silicon oxide (center and width of Gaussian fits are shown). Data and fits are reported in Figure S9.