## 3 Results and Discussion
### Validation of PDMS-Based Microdevices  
Final application of the PDMS-based microdevices presented in this study is the specific capture of low amounts of microRNAs as biomarkers of oncological pathologies in the context of liquid biopsy. Since the concentration of such circulating biomarkers is quite low, a polymerase chain reaction amplification step is required. The adsorbed miRNAs were directly reverse-transcribed on-chip, then releasing cDNA molecules in solution. This solution was collected to feed the DNA amplification step, using standard tubes and equipment. A panel of microRNAs often referred as biomarkers of NSCLC [39; 40] was selected to validate the microdevices. Either microRNA-20a or microRNA-222 or even microRNA-320 were spiked in water or in human plasma added with lysis buffer and subjected to purification via adsorption on the functionalized microdevices. After the washing step with water to remove the unwanted material possibly adsorbed on channel walls, the proper reverse transcription mix was inserted in each microdevice and the reaction was allowed to proceed directly on-chip on a thermal cycler equipped with a flat thermal block, as previously described [19]. Finally, the solution containing the proper cDNAs was used to feed the RT-qPCR reactions (Figure 9). For all the tested conditions, a decrease in the threshold cycles (Ct) is observed when the amount of spiked microRNA increases. Interestingly, the same amount of spiked microRNA gave different results when spiked in water or in plasma, with a better Ct in plasma. A possible explanation of this phenomenon relies on the passivation effect due to proteins present in plasma but, obviously, absent when the microRNA is dissolved in water. In other words, the components of a biological sample not only do not adversely compete with microRNA molecules for the adsorption sites, but favor instead their better interaction with the channel surfaces. Same amount of functional sites are initially available on channel surfaces for the two conditions, but in water more anchorage free sites could capture in a constrained conformation the microRNA with  
Figure 8: Adsorption of synthetic microRNA on Chip B. The titration of efficiency in capturing microRNAs with time is measured by fluxing miR-1246-TAMRA dissolved in human plasma added with lysis buffer at constant concentration. Fluorescence signal is the mean of maximum signal measured in consecutive coils, while error bars refer to standard error. A linear fit is also shown (dotted line; R = 0.999). respect to that in plasma. As a result, this microRNA could be not completely available for the enzyme in the reverse transcription step, yielding a lower amount of cDNA molecules to be amplified via RT-qPCR. Moreover, components of the reverse transcription mix as well as the just-copied cDNA molecules could also be captured on non-passivated chip surfaces. Both in water or in human plasma, the two types of microdevices are able to capture different amounts of synthetic microRNAs, which are directly reverse-transcribed on chip and amplified in standard conditions. As shown in Figure 9, three different microRNAs associated with non-small cell lung cancer were successfully detected at different starting concentrations in spiked water or plasma, confirming no preferences in the adsorption of different sequences. Since synthetic microRNAs were spiked in water or plasma in known concentrations, we treated the data of all three microRNA as similar and fitted all data together (dotted lines in Figure 9). The resulting fit is a straight line (in semi-log graphs) both for miR spiked in water and in plasma confirming the linearity between microRNA incubated and adsorbed on surfaces. The good performances of microdevices were further tested by detecting microRNAs which are naturally present also in human plasma collected from a healthy donor, even if at lower concentrations.