Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

Podsumowanie

The present protocol describes a pneumatic microfluidic platform that can be used for efficient microparticle concentration.

Streszczenie

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. This platform has a three-dimensional (3D) network with curved fluid channels and three pneumatic valves, which create networks, channels, and spaces through duplex replication with polydimethylsiloxane (PDMS). The device operates based on the transient response of a fluid flow rate controlled by a pneumatic valve in the following order: (1) sample loading, (2) sample blocking, (3) sample concentration, and (4) sample release. The particles are blocked by thin diaphragm layer deformation of the sieve valve (Vs) plate and accumulate in the curved microfluidic channel. The working fluid is discharged by the actuation of two on/off valves. As a result of the operation, all particles of various magnifications were successfully intercepted and disengaged. When this technology is applied, the operating pressure, the time required for concentration, and the concentration rate may vary depending on the device dimensions and particle size magnification.

Wprowadzenie

Due to the importance of biological analysis, microfluidic and biomedical microelectromechanical systems (BioMEMS) technologies^1,2 are used to develop and study devices for the purification and collection of micromaterials^2,3,4. Particle capture is categorized as active or passive. Active traps have been used for external dielectric⁵, magnetophoretic⁶, auditory⁷, visual⁸, or thermal⁹ forces acting on independent particles, enabling precise control of their movements. However, an interaction between the particle and external force is required; thus, the throughput is low. In microfluidic systems, controlling the flow rate is very important because the external forces are transmitted to the target particles.

In general, passive microfluidic devices have micropillars in microchannels^10,11. Particles are filtered through interaction with a flowing fluid, and these devices are easy to design and inexpensive to manufacture. However, they cause particle clogging in micro-pillars, so more complex devices have been developed to prevent particle clogging¹². Microfluidic devices with complex structures are generally suitable for managing a limited number of particles^{13,14,15,16,17,18}.

This article describes a method to fabricate and operate a pneumatically driven microfluidic platform for large particle concentrations that overcomes the shortcomings¹⁸ as mentioned above. This platform can block and concentrate particles by deformation and actuation of the thin diaphragm layer of the sieve valve (Vs) plate that accumulates in curved microfluidic channels. Particles accumulate in curved microfluidic channels, and the concentrated particles can separate by discharging the working fluid via the actuation of two PDMS seals on/off valves¹⁸. This method makes it possible to process a limited number of particles or concentrate a large number of small particles. Operating conditions such as the magnitude of flow rate and compressed air pressure can prevent unwanted cell damage and increase cell trapping efficiency.

Protokół

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).

Wyniki

Figure 8 shows the flow rate of the fluid rates for a four-stage platform operation, as mentioned in Table 2. The first stage is the loading state (a state). The platform was supplied with fluid with all valves open, and the working fluid (Qf) and particles (Qp) are almost identical as the microfluidic channel network exhibits structural symmetry. In the second stage (b state), compressed air was transported to Vs to block the particles, and as the Vs diaphragm deformed, the...

Dyskusje

This platform provides a simple way to purify and concentrate particles of various sizes. Particles are accumulated and released through pneumatic valve control, and no clogging is observed because there is no passive structure. Using this device, the concentration of particles of three sizes is presented. However, the operating pressure, the time required for concentration, and the rate may vary depending on the device dimensions, particle size magnification, and the pressure at Vs^18,<...

Materiały

Name	Company	Catalog Number	Comments
1.5 mm puncture	Self procduction	Self procduction	This puncture was made by requesting a mold maker based on the Miltex® Biopsy Punch with Plunger (15110-15) product.
4 inch Silicon Wafer/SU-8 mold	4science	29-03573-01	4 inch (100) Ptype silicon wafer/SU-8 mold
Carboxyl Polystyrene Crosslinked Particle(24.9 μm)	Spherotech	CPX-200-10	Concentrated bead sample1
Flow meter	Sensirion	SLI-1000	Flow measurement
High-speed camera	Photron	FASTCAM Mini	Observation of concentration
Hot plate	As one	HI-1000	heating plate for curing of liquid PDMS
KOVAX-SYRINGE 10 mL/Syringe	Koreavaccine	22G-10ML	Fill the microfluidic channel with bubble-free demineralized water.
Laboratory Conona treater/Atmospheric plasma	Electro-Technic	BD-20AC	Chip bonding/atmospheric plasma
Liquid polydimethylsiloxane, PDMS	Dow Corning Inc.	Sylgard 184	Components of chip
Microscope	Olympus	IX-81	Observation of concentration
PEEK Tubes	SAINT-GOBAIN PPL CORP.	AAD04103	Inject or collect particles
Polystyrene Particle(4.16 μm)	Spherotech	PP-40-10	Concentrated bead sample3
Polystyrene Particle(8.49 μm)	Spherotech	PP-100-10	Concentrated bead sample2
Pressure controller/μflucon	AMED	μflucon	Control of air pressure
Spin coater	iNexus	ACE-200	spread the liquid PDMS on SU-8 mold