## 2 Materials and Methods
### PDMS Microdevices Fabrication  
The two types of microfluidic devices were fabricated as follows: an upper PDMS reaction chamber was obtained by mold-casting and subsequently bonded on a silicon support coated by a thin layer of PDMS. The reaction chamber was designed (Figure 1, Panels (a) and (b)) by a 3D computer aided design (CAD) software (Rhinoceros(r)) and molded over a spiral shaped template fabricated with SU-8 photolithography on a silicon wafer support (Figure 1, Panel (c)). In details, the first spiral device (from now called Chip A) was designed with a squared channel with section of 200 m wide and 200 m deep, with walls spacing width of 200 m. The second device (from now called Chip B) was defined by a rectangular cross section 60 m wide and 200 m deep with walls spacing 150 m wide. The total length of the spiral channel differs for the two microfluidic devices, being 353 mm for Chip A and 1078 mm for Chip B. The spiral channel is connected with the exterior by two inlet/outlet holes.  
The fabrication of PDMS microfluidic devices started with the preparation of the SU-8 mold, as explained below. Firstly, silicon wafer support was coated by SU-8 (Microchemicals GmbH) using a spin coater (Spinner 150 WAFER SPINNER) by setting 10 s at 500 rpm and 60 s at 2000 rpm. Then a soft bake process in two thermal steps was performed: the first step at 65 C for 8 min and the second at 95 C for 80 min, both on the hot plate. The SU-8 was exposed for 30 s to standard UV photolithography by means of a double side mask aligner (Neutronix Quintel NXQ 4006) used in contact mode, ensuring the correct alignment between the SU-8 and the desired mask patterns. The post bake was characterized again by two thermal steps: the first at 65 C for 5 min and the second at 95 C for 25 min, on a hot plate. After that, the SU-8 was developed using Propylene glycol methyl ether acetate (PGMEA) for 20 s to obtain the finished mold. Finally, the released SU-8 structures were hard baked at 170 \({}^{\circ}\)C for 15 min. To guarantee the correct detachment of PDMS from the mold, a silanization step with trichloromethylsilane (CH\({}_{3}\)Cl\({}_{3}\)Si) and toluene in a 1:10 ratio was performed on mold surface. Before silanization, the SU-8 mold was subjected to an O\({}_{2}\) plasma treatment (300 W, O\({}_{2}\) 30%, 1 min). Next, mold surfaces were silanized through a wet phase for 4 h, then rinsed with 2-propanol and dehydrated on a hot plate at 170 \({}^{\circ}\)C for 10 min.  
To perform the mold casting of the reaction chamber, PDMS was prepared by hand mixing the pre-polymer and the curing agent in a 10:1 ratio, followed by a degassing step after which PDMS was poured on the mold. Curing reaction was performed at 120 \({}^{\circ}\)C for 15 min. The freshly cured spiral chambers were cleaned with acetone and 2-propanol, and dehydrated on a hotplate at 70 \({}^{\circ}\)C for 5 min. In the meantime, the silicon wafer acting as support was pre-cut, cleaned with sulphuric acid and hydrogen peroxide solution (3:1 v/v ratio) for 5 min, rinsed 3 times in water and dried with nitrogen. The silicon support was coated with a thin layer of PDMS by means of a spin coater (Spinner 150 WAFER SPINNER) with the following protocol: 5 s at 500 rpm and 60 s at 3000 rpm and cured at 120 \({}^{\circ}\)C on a hot plate for 5 min. The spiral chambers were bonded on the silicon support by means of a standard O\({}_{2}\) plasma treatment (300 W, O\({}_{2}\) 60%, 2 min). Once assembled, microfluidic devices were put at 90 \({}^{\circ}\)C for an hour in the oven. Figure 1 shows the PDMS microdevices and a scheme of experiment workflow.  
Figure 1: Computer aided design (CAD) representation of the channels featured in the two types of microdevices (Chip A in Panel (**a**) and Chip B in Panel (**b**)) used in this study. Panel (**c**): a wafer before dicing in single microdevices. Panel (**d**): experiment workflow. The microdevice is placed in a proper holder with inlet tube fed by a microsyringe pump and outlet tube to collect cDNA for RT-PCR analysis.