## 2 Materials and Methods
### On-Chip microRNAs Functional Assay and Real-Time PCR  
To explore the microdevices performances, a functional assay with synthetic miRNA-1246-TAMRA was setup. Briefly, the fluorescently-labelled miR-1246 spiked either in water or human plasma, was inserted in the microdevice, normally at 10 \(\upmu\)L/min flow rate. After a wash with water at 1 \(\upmu\)L/min for 10-15 min, the adsorbed microRNA was imaged either by fluorescence or confocal microscopy. For the fluorescence microscopy imaging, a Leica DMLA microscope (Leica Microsystems, Wetzlar, Germany), equipped with a mercury lamp and fluorescence filter L5 (Leica Microsystems, Wetzlar, Germany) was employed. All microdevices were observed with a 2.5\(\times\) magnification objective and measured with a cooled CCD camera (DFC 420C, Leica Microsystems, Wetzlar, Germany). To acquire images covering the whole chip, the microscope motorized stage was controlled through a BeanShell script running in MicroManager (version 1.4.23, micro-manager.org), acquiring 54 tiles for each image. The reconstruction of images as well as all the analyses were performed with the Fiji software [23] and Fiji custom macros. The confocal imaging was performed with a Leica SP5-II confocal microscope (Leica Instruments, Wetzlar, Germany), equipped with a helium/neon (543 nm) laser. Samples were observed utilizing a 20\(\times\) objective, while to help the microRNAs visualization on walls, microdevice channels were cut at 30\({}^{\circ}\). To acquire the TAMRA signal, fluorophores were excited with the helium/neon laser, using an emission detector wavelength range from 570 to 660 nm. On-chip miRNA purification and detection was validated with synthetic miRNAs dissolved in water or in blood plasma. Purification of synthetic microRNAs was performed by spiking syntheticmicroRNAs ranging from 0.1 pg to 1 ng either in water or in human plasma mixed in a 1:1 ratio with lysis buffer (APL1 buffer, Qiagen, Germantown, MD, USA). To obtain human plasma, blood samples were collected from healthy donors into EDTA-treated collection tubes. For plasma preparation, fresh blood was centrifuged at 700 g for 10 min to pellet the cell fraction. The supernatant was then collected avoiding contact with the buffy coat. Before using, plasma samples were centrifuged at 1000 g for 5 min at 4 \({}^{\circ}\)C to pellet the debris. The proper amount of synthetic microRNA diluted either in water or in plasma was inserted in the microdevice at room temperature with the microsyringe at 10 \(\upmu\)L/min for a suitable time (10-20 min). During this step, the microRNA is adsorbed on the amino-silanized surfaces of microdevice channels. To remove the excess of microRNAs, a wash with water was performed at 1 \(\upmu\)L/min for 10-15 min. For the on-chip analysis of miRNAs, a two-steps protocol based on reverse transcription coupled with real-time PCR was employed. Reverse transcription (RT) was performed directly on-chip on the miRNAs adsorbed by the PDMS surface by adding the RT master mix at 10 \(\upmu\)L/min into the microdevice. TaqMan(r) MicroRNA Reverse Transcription Kit included 100 mM dNTPs (with dTTP), MultiScribe(tm) Reverse Transcriptase (50 U/\(\upmu\)L), 10\(\times\) Reverse Transcription Buffer, RNase Inhibitor (20 U/\(\upmu\)L), 20\(\times\) miR-specific RT primers. The reaction was carried out for 30 min at 16 \({}^{\circ}\)C, 30 min at 42 \({}^{\circ}\)C, and 5 min at 85 \({}^{\circ}\)C on a XP Thermal cycler (Bioer Technology Co., Ltd.) equipped with a flat thermal block. Once synthetized, cDNA was subsequently recovered from the microdevice with the help of microsyringes and amplified with TaqMan(r) MicroRNA Assay--has-miR-specific in standard conditions. An amount of 20 \(\upmu\)L of real-time PCR mix were composed of TaqMan(r) Small RNA Assay (20\(\times\)), TaqMan(r) Universal PCR Master Mix II (2\(\times\)), nuclease-free water and cDNA, following the manufacturer's instructions.