Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

1. Designing the microfluidic platform for particle concentration

1. Design the pneumatic microfluidic platform consisting of one pneumatic valve for fluid flow in the 3D flow network and three pneumatic valves for sieve (Vs), fluid (Vf), and particle (Vp) valve operation (Figure 1).
   NOTE: Vs blocks concentrate particles from the liquid, and Vf and Vp allow fluid and particle release after concentration. Three pneumatic ports provide compressed air from the fluid/pneumatic supply layer (normally open) and the pneumatic valve light outlet to actuate the valve. The microfluidic channel network is designed with a CAD program^18,19.
2. Design the channel to be a pneumatic supply layer and a 3D channel network layer (Figure 2).
   NOTE: The fluid network is interconnected with the curved channels in the anterior part and the rectangular chamber in the posterior region. Vs block the inlet, and particles accumulate in the collection area of the curved fluid channel. Particle-free fluids (particle-free liquids) are exited through the Qf outlet and the concentrated particles through the Qp outlet (Figure 3).
3. According to the above conditions, prepare four types of SU-8 molds.
   NOTE: The four molds include a mold that allows the valve to be controlled via pneumatics, two molds that create fluid channels, and a clean mold without shape (Figure 4 and Table 1). The four types of molds mentioned are fabricated using standard photolithography processes. This mold making consists of a SU-8 mold on a silicon wafer as per previously published reports^18,19. Figure 5 depicts the device chip.

2. Fabrication of the microfluidic platform for particle concentration

NOTE: Figure 6 illustrates the fabrication of a microfluidic platform that concentrates particles.

1. Replicate the PDMS layer using a prepared pneumatic valve channel SU-8 mold (step 1.3) for pneumatically controlling the valve.
   1. Pour 10 mL of liquid PDMS and 1 mL of curing agent (see Table of Materials) into a prepared pneumatic valve channel mold (step 1.3) and heat-activate at 90 °C for 30 min.
   2. After the PDMS structures are cured, separate the SU-8 mold of step 2.1.1.
   3. Punch three 1.5 mm pneumatic ports (Vs, Vf, and Vp) into the pneumatic valve channel manufactured according to step 2.1.2 using a 1.5 mm puncture (see Table of Materials).
   4. Pour 10 mL of liquid PDMS and 1 mL of curing agent into a prepared clean SU-8 mold prepared in step 1.3 and spin-coat at 1,500 rpm for 15 s using a spin coater (see Table of Materials). Then heat-activate at 90 °C for 30 min.
   5. After the PDMS structures are cured, separate the SU-8 mold of step 2.1.4.
      NOTE: The valve diaphragm layer controls fluid flow according to the pneumatic pressure.
   6. Treat atmospheric plasma (see Table of Materials) to the PDMS structures prepared in steps 2.1.3 and 2.1.5 for 20 s.
   7. Align directly plasma-treated PDMS structures from step 2.1.6 according to the channel structure by checking with a microscope.
   8. Bond the aligned PDMS structures prepared in step 2.1.7 by heating at 90 °C for 30 min.
   9. Punch a 1.5 mm diameter hole in the fluid channel inlet (Qfp) and fluid channel outlets (Qf and Qp) within the pneumatic channel part to which the thin diaphragm layer is bonded, using a 1.5 mm puncture.
2. Replicate both sides of the PDMS layer using two SU-8 molds to make a microfluidic channel. Use a curved and rectangular microfluidic channel mold on the front and a microfluidic interconnection channel mold on the rear.
   1. Pour 10 mL of liquid PDMS and 1 mL of curing agent into the curved and rectangular microfluidic channel mold and spin-coat at 1,200 rpm for 15 s. Then create molds for the curved fluid chamber and fluid channels by thermal activation at 90 °C for 30 min (Figure 6A).
   2. Separate the PDMS layer on which the microfluidic channel is formed, then make a heat-activated mold covering the sealed vent wall by bonding to the glass wafer by treating atmospheric plasma for 20 s (Figure 6B).
   3. Pour 3 mL of liquid PDMS into the interconnection channel of the SU-8 mold (Figure 6C).
   4. Arrange the structure fabricated in step 2.2.2 with the interconnection channel mold in liquid PDMS on the microfluidic interconnect channel mold, and dry the superimposed structure at 130 °C for 30 min (Figure 6D).
      NOTE: While curing the rear structure, the PDMS mold fabricated in step 2.2.2 is inflated by the thermal pressure of the air layer, and the deformed PDMS layer is thermally activated (Figure 6E)¹⁶.
   5. After curing, remove the front SU-8 mold from the microfluidic channel network layer and carefully peel off the rear PDMS mold (Figure 6F).
      NOTE: The 3D fluidic network layer allows the creation of an anterior curved fluid chamber and microfluidic channels.
   6. Pour 10 mL of liquid PDMS and 1 mL of curing agent into a clean SU-8 mold. Then heat-activate at 90 °C for 30 min.
   7. After the PDMS structures are cured, separate the SU-8 mold.
      NOTE: This step creates the additional sealing layer.
   8. Treat the atmospheric plasma to PDMS structures prepared in steps 2.2.3 and 2.2.7 for 20 s.
   9. Align directly plasma-treated PDMS structures according to the channel structure by checking with a microscope.
   10. Bond the aligned PDMS structures by heating at 90 °C for 30 min.
3. Align the PDMS structures prepared in steps 2.1 and 2.2 according to the channel structure and bond them by treating atmospheric plasma for 20 s.

3. Setting up the device

NOTE: Figure 7 shows fabricating a microfluidic platform that concentrates particles.

1. Manually fill the microfluidic channel with bubble-free demineralized water using a 10 mL syringe.
2. To control the P_Qfp and the three pneumatic valves (P_Vs, P_Vf, and P_Vp) that control the microbead flow, insert a precision pressure controller with four or more output channels (see Table of Materials) for the working fluid (Qfp) into the microfluidic platform.
   NOTE: A precision pressure controller with four output channels can be replaced with multiple precision pressure controllers. In this experiment, the operating pressure of P_Qfp was 10 kPa, P_Vs was 15 kPa, and P_Vf and P_Vp were both 18 kPa (Figure 8 and Table 2). Figure 8 shows the working fluid flow rate over time as particles are concentrated by the microfluidic platform with P_Vs of 15 kPa, and Table 2 shows the actuation results according to the pneumatic valves.
3. Prepare carboxyl polystyrene test particles of various sizes in distilled water (see Table of Materials).
   NOTE: The particle sizes used in this experiment were 24.9, 8.49, and 4.16 µm; particles of various sizes can be used depending on the pressure of P_Vs.
4. To control the flow rate of the working fluid, fill a glass bottle half full with water (working fluid) and connect the glass bottle cap to the controller output channel and microvalve.
   NOTE: Connect one tube to the microvalve to receive compressed air from the controller and the other tube to inject water.
5. Observe platform operation through an inverted microscope for all platform operations and measure the operating flow rate over time at the outlet by a liquid flow meter (see Table of Materials).

4. Operation of the device

1. Inject the particle/fluid mixture under pressure at the inlet (Qfp) with Vp (Figure 9A).
   NOTE: The flow of particles and clean fluid from the outlet through the interconnected channels are controlled via Vp and Vf, respectively (Table 2).
2. Apply pressure to Vs at 15 kPa and Vp at 18 kPa to actuate the valve.
   NOTE: At this time, the diaphragm is deformed, the particles of the fluid Qfp are blocked in the contact space between the curved fluid channel and the curved fluid cantilever, and the unwanted Qfp fluid is released through the open Qf (Figure 9B,C).
3. When the particles are concentrated, apply pressure only to Vf.
   NOTE: At this time, when pressure is applied only to Vf, the clogged particles are released through Qp (Figure 9D).