C. Selection of aptamer using multilayered microfluidics device

Figure 3 shows a schematic illustration of the multilayered microfluidic chip and the mechanism of the air valve. The liquid flow layer contains four inlets and two outlets, where inlets 1, 2, 3, and 4 are used for the pre-incubation solution, wetting, MgCl2 buffer, and GOMNP solution, respectively. The air flow layer contains six inlets for air flow to operate the valves of each liquid channel. As shown in Fig. 3(a), these layers and slide glass were bonded together via an appropriate alignment to fabricate the multilayered microfluidic chip. Before performing the selection process, a pre-incubation solution was prepared, i.e., a mixture comprising 0.3 μM random ssDNA library and 1 μM adenosine; subsequently, it was incubated for 12 h. The pre-incubation conditions were optimized when multiple cycles of the SELEX were performed to identify a specific aptamer with high affinity. The waste and supernatant solutions flowed through outlets 1 and 2, respectively. The volume of the selection chamber was approximately 5 μl, and the height and width of the liquid and air channels were 50 and 80 μm and 250 and 400 μm, respectively [Fig. S5(a) in the supplementary material]. The solutions prepared for the selection of ssDNAs and adenosine were injected into the microchip using a syringe pump (Standard Infuse/Withdraw PHD ULTRA 4400, Harvard Apparatus, USA), and the GOMNP solution was introduced using a pressure pump (MFCS-EZ, Fluigent, Germany). The GOMNP solutions were stored in an ultrasonicator or vortex mixer (VM-10, Daihan-brand, Republic of Korea) to maintain a uniform dispersion during the injection. The electromagnet, controlled by the power supply, was mounted under the chip using a 3D-printed holder to contain the GOMNPs in the chamber. As shown in the top view [Fig. 3(b)], each liquid channel was perpendicular to the air flow channel of the control layer, and the air flow channel was connected to the pneumatic solenoid valves (MH1 miniature, Festo, USA). The cross sections of all fluid channels were designed to be rounded to operate the valve [Fig. 3(c)]. When pressurized air was applied to the upper channel, the thin PDMS layer deformed and obstructed the liquid flow channel [Figs. 3(c) and 3(d)], which was controlled to be operated independently using LabVIEW. We confirmed the performance of the valve under the same dimensions and conditions [Figs. S5(b) and S5(c) in the supplementary material].A single cycle for selecting the desired ssDNA from a random ssDNA pool using a microchip was performed, as shown in Fig. 4. For a clear understanding, the illustration of the cross-sectional view of the chamber is presented in Fig. S6 in the supplementary material. A MgCl2 solution was introduced through inlet 1 after ethanol was injected through inlet 2 to wet the entire channel and chamber [60 μl/min, Figs. 4(a) and S6(a) in the supplementary material]. GOMNP solutions were introduced into the chamber through inlet 4 until approximately 50% of the chamber was filled with GOMNPs as the electromagnet was turned on, whereas the valves for inlets 1, 2, and 3 and outlet 2 were closed [150 mbar, Figs. 4(b) and S6(b) in the supplementary material]. After filling the GOMNP, closing the valve for inlet 4, and opening the valve for inlet 3, a pre-incubated mixture comprising a random ssDNA pool and adenosine was introduced [20 μl/min, 35 μl, Figs. 4(c) and S6(c) in the supplementary material]. After 1 h of incubation with all valves closed [Figs. 4(d) and S6(d) in the supplementary material], the supernatant was obtained by injecting the MgCl2 solution through inlet 1 since the valves for inlets 3 and 1 were closed, and outlet 2 was opened [30 μl/min, 100 μl, Figs. 4(e) and S6(e) in the supplementary material]. Subsequently, the GOMNPs were washed out using Tris-EDTA buffer (TE) solution with bubbles through inlet 2 after the electromagnet was turned off [Figs. 4(f) and S6(f) in the supplementary material]. The supernatant obtained was amplified via an asymmetric PCR, and repeated steps of wetting after pre-incubating with the adenosine and a new randomized ssDNA pool were performed. The single-cycle selection on the chip was completed within 2 h, including the incubation time.