A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

1. Acoustophoretic Device Design and Photomask Layout

NOTE: General considerations and guidance for microfabrication process design and mask layout can be found in texts on microfabrication and tutorials of photomask design.^19–21

1. Lay out Mask 1, the Fluidic Layer (front side), using appropriate CAD software. Choose a geometry that permits sample injection and separation appropriate for the desired application.
   1. For acoustic focusing, set the fluidic channel width w to provide a resonant frequency f_n greater than 1 MHz according to the equation f_n = nc/2w, where c is the speed of sound in the relevant fluid and n is the number of standing-wave nodes (e.g., for a 900-µm wide channel, the two-node resonance is expected at f₂ = 1.65 MHz).
      NOTE: Particles occupying distinct lateral positions near the end of the separation channel should exit from distinct outlets. In this protocol, particles are separated by size, so the outlets are designated SPO and LPO, for small-particle and large-particle outlet, respectively, as shown in Figure 1.
   2. Set the fluid channel length to control the length of time particles are exposed to the separation field at a specific flow rate. Longer on-chip residence time for particles to migrate due to separation forces must be traded off against the larger required chip footprint.
      NOTE: In our acoustic focusing devices, the flow channel makes three passes down the chip for increased residence time (Figure 2a). At a typical total flow rate of 200 µl/min, particles flowing through the 300 x 200 µm cross-section, 117-mm long separation channel spend on average 2.1 sec in the acoustic field.
2. Include fluidic ports in the mask layout for connections to standard tubing arranged on a standardized grid (5-mm pitch). Include suitable fiducial marks for alignment of masks to each other during fabrication and dicing of individual devices.
3. Lay out Mask 2, the Via Layer (back side), which only includes fluidic ports. Include fiducial marks for alignment to Mask 1.

Figure 1. Acoustofluidic Device. Schematic sketches of acoustophoretic device architecture. (a) Top view, showing the overall H-filter configuration (not to scale). (b) Schematic of the channel cross-section at the location marked by the black dashed line in (a), showing the pressure field (blue dotted lines), and the sense of the acoustic primary radiation forces (PRF) that drive particles toward nodal planes (red arrows). The channel cross-section is 900×200 µm with a wall approximately 10 µm thick separating the main (300 µm wide) and bypass channels. (c) 3D representation of particle separation. Please click here to view a larger version of this figure.

2. Cleanroom Fabrication of Microfluidic Chips for Acoustophoresis

1. Pattern the back-side fluid ports using Mask 2 on a double-side polished 0.5-mm thick, 100-mm diameter <100> silicon wafer by standard positive-resist photolithography.  Etch this geometry by deep reactive ion etching (DRIE) to a depth of 350–400 µm.
2. Turn the wafer over, and pattern the front-side fluid channel geometry using Mask 1 by standard positive-resist photolithography onto the other side of the silicon. Then, mount the device wafer to a second (blank silicon) carrier wafer using photoresist.
3. Etch the channels, also by DRIE, to a depth of 200 µm, through-etching the silicon in the port locations (the carrier wafer protects the DRIE tool surface). De-mount the device wafer from the blank Si wafer by soaking in resist-stripping solution.
4. Clean the device wafer and a featureless 0.5 mm-thick borosilicate glass wafer using Piranha solution (sulfuric acid and hydrogen peroxide in a 3:1 ratio).
5. Seal the fluid channels by anodically bonding the glass and silicon wafers using the following parameters: chamber pressure at 3 mTorr, piston pressure at 1,000 N, temperature at 350 °C, and apply 750 V until the current drops below 0.2 mA.
6. Cut the individual chips apart with a diamond blade on a dicing saw.

3. Final Device Assembly

1. Piezo Transducer Attachment
   NOTE: Ultrasound is generated in the microfluidic chip by a piezoceramic transducer attached to the silicon side.
   1. From a two-component low-viscosity epoxy kit, weigh out the recommended ratio of both components and mix them thoroughly.
   2. Dispense the epoxy mixture with a pipette and spread evenly across a lead zirconate titanate (PZT) piezoceramic to create a thin, even layer (approximately 10 µl of epoxy mixture for a piezo with dimensions of 37.5 × 10 × 0.5 mm).
   3. Using a suitable jig or fixture, align the epoxy side of the piezoceramic with the microfluidic chip, providing an overhanging area on one side for subsequent wire attachment (see Figure 2a), and bring the two components into contact. Clamp the assembly in a vice, taking care not to crack any of the components, and cure at the temperature and duration recommended by the epoxy manufacturer.
   4. After the epoxy has cured, attach fine-gauge wire leads to each side of the piezoceramic, by soldering with a fine-tip soldering iron, making the briefest possible contact with the piezo to avoid thermally de-polarizing it. Alternatively, use a conductive adhesive to glue wires to the piezo.

Figure 2. Fluidic Breadboard, Chip Mounting, and World-to-Chip Interface. Photos of (a) the acoustic microfluidic chip (external dimensions of 70×9×1 mm) with attached piezo transducer wire leads, showing three passes of the separation channel down the chip, (b) custom fluidic screw fittings and machined tubing components for the chip-to-world interface, (c) the chip mounted to the underside of the fluidic breadboard using clamping fixtures, spanning an opening in the breadboard to allow fan cooling, (d) a top view of the breadboard with attached tubing connections and cooling fan, and (e) a cross-sectional schematic of the screw fittings that interface the tubing with the mounted microfluidic chip. Please click here to view a larger version of this figure.

2. Device Mounting and World-To-Chip Interfacing
   1. Attach the chip to the “fluidic breadboard” (a plate with a grid of regularly-spaced threaded through-holes) using clamping fixtures. Attach a cooling fan to the breadboard to regulate temperature during acoustic experiments (see Figure 2).
      NOTE: Operating without the cooling fan at typical drive voltages raises the device temperature to 70–80 °C. This significantly shifts the resonant frequency due to altered sound velocity in the fluid, and may negatively impact the viability of any biological particles that are being processed.
   2. Screw in chip-to-world connectors, previously described elsewhere²² and shown in Figure 2. Join these interface tubes to additional tubing at the inlets and outlets using standard ¼”-28 unions for 1/16” tubing.

4. Characterization of Acoustic Focusing Performance

NOTE: System components required for acoustic focusing characterization are grouped together in the Materials List. Steps 4.1 and 4.2 below apply to any Core Device used with this platform, whereas subsequent steps describe operations specific to the acoustofluidic device discussed here.

1. System Configuration
   1. Assemble the microfluidic chip using world-to-chip connections on the fluidic breadboard, as described in Section 3. Make connections using tubing (such as 1/16” outer diameter fluoropolymer tubing) to a fluid pump and collection vials. Mount the breadboard assembly on the stage of a microscope capable of fluorescence imaging equipped with a CCD camera.
   2. Connect lengths of small inner diameter (ID) tubing (0.006” is recommended) immediately after the chip (see Figure 4) to serve as flow restrictors, which stabilize the system and control the flow splitting between the chip outlets. Use larger ID tubes, such as 0.01” or 0.03”, for all other connections.
      1. Estimate the hydrodynamic flow resistance R_h of each piece of tubing with length L and inner diameter D using the equation R_h = 128 μL/πD⁴, where μ is the fluid’s dynamic viscosity. The pressure drop ΔP due to each length of tubing at a given flow rate Q is given by ΔP = QR_h.
      2. Choose a ratio of restrictor lengths to split the flow between the outlets as appropriate for the separation method being used. Optimal separation with the acoustic chips in this protocol requires an SPO:LPO flow ratio of approximately 65:35%.
      3. Choose the length of the outlet flow restrictors such that their fluidic resistance is at least 3–4 times larger than the total resistance in the rest of the system (appropriately summed in series or parallel). For the acoustophoresis device used in this work, tubing lengths of 35 and 65 cm for the LPO and SPO are suitable.
         NOTE: Careful consideration must be given to R_h of any microfluidic Core Device. For our acoustic focusing chip in this work, R_h is low due to its relatively large channel dimensions, so the connected tubing resistances easily exceed it. For devices with smaller channel dimensions, the chip’s resistance may dominate the rest of the system tubing, in which case design and control of on-chip channel R_h is an additional consideration during Step 1 of this protocol. Detailed design principles and guidelines are available in the literature.^23,24
2. System Verification
   1. Check for leaks to verify that the microfluidic device has no defects and all tubing connections are sealed. Dispense water through the inlet tubes as needed with a syringe and monitor for any fluid leaking in the main channel.
   2. Dispense a known volume through the chip, and measure the volumes collected from the outlets to ensure that the flow ratio is as expected. Deviations from the expected volume ratio may indicate blockages in one of the outlets or leaking connections.
      1. To maintain system cleanliness and prevent clogging flush the entire system (fluidic tubing and chip) with appropriate cleaning solutions (e.g., bleach, ethanol, water) before and after running any experiments.
      2. In the event of clogs or blockages in one of the outlets, flush the system while plugging the clear outlet to clear the blockage. If this is unsuccessful, reverse the flow direction during flushing, optionally applying rapid pulses of reverse flow (using a manually-actuated syringe). Finally, if the blockage still cannot be removed, replace the tubing or chip, as necessary.
3. Frequency Scan Setup for Acoustic Focusing
   1. Fill the bypass channel (see Figure 1) with fluid such as deionized water, ethanol, or phosphate buffered saline (PBS) by manual injection with a syringe. Note that this fluid does not come into contact with the sample being processed. The details of adjusting node position using different bypass fluids are described elsewhere^16,17. In brief, if the node needs to be closer to the dividing wall, use a lower-density bypass fluid, such as ethanol; for node placement closer to the input sample stream, choose a denser fluid, such as a glycerol solution.
   2. Make a bead solution of approximately 0.01% (w/v) 5–8 µm fluorescent polymer beads suspended in a buffer such as PBS with 0.05% Tween-20 and fill the sample inlet syringe with the bead solution. Fill the buffer inlet syringe with the same buffer (this does not need to be the same fluid as in the bypass channel). Note that in general, the buffer is chosen to suit the application (see Step 5.1).
   3. With the fluidic breadboard on the microscope stage, focus approximately halfway into the depth of the channel at a straight channel region just before the outlet with both channels (separation and bypass) in the field of view. An additional oblique-angle external light source may be required to make the channel walls visible, for successful post-processing of image data.
4. Automated Frequency Scan and Image Capture
   1. Make electrical connections using shielded cables (e.g., RG-58 equipped with BNC connectors) to a function generator with radio frequency (RF) amplifier to deliver the excitation signal to the piezo transducer. Optionally, connect an oscilloscope to the output of the function generator for monitoring the actual voltage applied to the transducer.
   2. Turn on the cooling fan, and set the function generator such that the output of the RF amplifier to the piezo transducer is in the range of 12–25 volts peak-to-peak (V_pp).
   3. Set both syringes to the same flow rate between 50 and 200 µl/min. Using a single syringe pump to drive both syringes with the same motor is recommended to minimize perturbations to the flow.
   4. Perform a frequency scan procedure by specifying the start and end frequencies, the step size between frequency values, and the piezo drive voltage using a laboratory automation toolkit such as National Instruments LabVIEW.
   5. At each frequency step, apply the voltage for 15 sec to allow the system to equilibrate, then capture 10 images of beads flowing through the chip for subsequent analysis (exposure times between 10 and 100 msec are recommended).
   6. Between each applied frequency step, turn off the voltage for approximately 20 sec to let the beads redistribute uniformly across the channel and remove bias in focusing from the previously applied frequency step.
5. Image Analysis to Determine Resonant Frequency and Focusing Position
   1. Run an image analysis script (such as the AF_freqScanPlotter.m MATLAB script provided with this Protocol) and enter the required information at the prompts: select the list of image files generated in Step 4.4, enter the scan start and stop frequencies and step size, the full channel width, the wall location, and finally select the image region to analyze, including both the separation and bypass channels.
   2. Observe the analysis script average the set of images captured at each frequency step, and average the intensity values along the flow direction. This results in a cross-section line-scan of fluorescence intensity.
      NOTE: The frequency corresponding to the highest intensity is defined as the resonant frequency (Figure 3, middle row), and the position in the channel where the highest intensity occurs is the focusing position (Figure 3, bottom row).

Figure 3. Representative Frequency Scan. Example of frequency scan data for well coupled (A) and poorly coupled (B) piezo and chip. Top row: fluorescent intensity of beads (red represents high, and blue represents low intensity). Middle row: maximum intensity at each frequency.  Bottom row: location of maximum intensity, where the red dashed line indicates predicted focusing position, and red diamonds indicate resonant frequency as determined by the maximum intensity. Please click here to view a larger version of this figure.

5. Automated Separation

NOTE: The automated separation experiment is run to separate large particles from small particles due to the size-dependent acoustic focusing forces applied in the microfluidic chip. The required system components are grouped together in the Materials List.

1. System Configuration and Sample Preparation
   1. Connect the microfluidic chip to the syringe pump, computer-controlled multi-port selection valves, PC-interfaced flow meters, and tubing, as shown in Figure 4. This configuration enables automated processing of samples through the microfluidic separation chip, as well as automated cleaning steps between experiments to remove cross-contamination and sample carry-over.
   2. Use a sample buffer suitable for the cells or particles to be separated, or as required by the analysis assay to be used after separation. As in Step 4.3.2, the recovery buffer (but not necessarily the bypass fluid) must match the sample fluid.
      NOTE: Any aqueous buffers typically used with biological samples (e.g., PBS) have acoustic properties similar to water and will not appreciably change the performance of the acoustic device. The use of sample fluids with density and viscosity significantly different from water is possible, but only recommended for operators with significant acoustophoresis experience.

Figure 4. Acoustic System Configuration for Automated Separation Experiments. The blue lines trace the main flow path through the system. All green and black lines are 0.03” inner diameter (ID) tubing, and all blue and gray-colored lines are 0.01” ID tubing, with the exception of the holding coils, which are 0.03” ID, and the flow restrictors, which are 0.006” ID. The syringes are filled with buffer, and the holding coils have sufficient volume (550 µl) to prevent uptake of any samples or cleaning reagents into the syringes. Please click here to view a larger version of this figure.

2. Separation Procedure
   1. Pre-run state. Prior to running the separation, ensure that the cleaning reagent reservoirs (bleach, ethanol, buffer) have sufficient fluid, the waste reservoirs are not full, and the fluid lines are primed (i.e., filled with solution). If the latter condition is uncertain, or if the system is being run for the first time after idling (e.g., the first time on a given day), run an automated cleaning procedure (see Step 5.3 below). Set Valves 3 and 4 at the chip outlets to flow to waste reservoirs initially.
   2. Turn on the cooling fan, and set the function generator at the resonant frequency for the acoustic chip being used, as determined in step 4.5. Adjust the voltage set point on the function generator so that the RF amplifier outputs between 12 and 25 V_pp, as appropriate for the desired separation.
   3. Connect the Sample Input Vial, Buffer Input Vial, and appropriate collection vials to the system. Just before attaching the Sample Input Vial to its pickup tubing, vortex the vial briefly to re-suspend any particles that may have settled. Then, attach the vial and initiate the separation routine without delay.
      CAUTION: If the sample to be processed contains potentially infectious agents, the vials should be of a screw-top type, to maintain a sealed system and prevent sample aerosolization. In addition, when working with biohazardous materials, handle all vials and tubing while wearing the necessary protective equipment and using the required hazard controls and procedures for the Biological Risk Group and Biosafety Level appropriate to the material. Consult institutional policies and protocols in case of any uncertainty.
   4. Use a program in a laboratory automation toolkit to control the valves, flow sensors and pump to carry out the fully-automatic separation routine.
      NOTE: The routine switches the valves, actuates syringe pump withdrawal and infusion, and monitors flow sensor data for correctly timing the collection of output sample fractions. The major steps are summarized below in 5.2.4.1 through 5.2.4.3, as if carried out manually.
      1. Prime the pickup tubing. Withdraw approximately 15 µl from the Sample Input Vial to completely fill the tube connecting it to Valve 1. Simultaneously, prime the tube connecting the Buffer Input Vial to Valve 2. In a similar fashion, prime the air inlet of Valve 1 to ensure that it contains no fluid. Finally, switch Valves 1 and 2 to expel to waste any excess fluid or air that has entered the loading coil.
      2. Load the sample coil, as shown in Figure 5a. A typical loading sequence for processing a 250-µl sample is to withdraw 25 µl of air at 50 µl/min, then 250 µl of sample at 200 µl/min, followed by 35 µl of leading buffer at 200 µl/min, and finally another 25 µl of air at 50 µl/min.
         NOTE: All volumes and flow rates are user-selectable. Note that this loading sequence is in reverse order of how the fluid plugs will flow through the separation device.
      3. Begin sample infusion. Set the syringe pumps to infuse the loaded fluid plugs at the desired rate (typically 100 µl/min). Monitor the flow rates at the SPO and LPO with flow sensors to ensure that flow is steady and at the ratio determined in Step 4.1, and that clogging has not occurred.
      4. Collect separated fractions. When the flow sensors detect a spike in flow rate, indicating passage of the first air gap, switch the corresponding output valve from waste (where it started in Step 5.2.1) to a sample collection vial.
      5. After passage of sample from the chip, observe the flow sensor detect the second air gap. At this point, switch the output valves back to waste. After the full volume loaded from Step 5.2.4.2 is dispensed, stop the syringe pump infusion and terminate the automation routine when the flow rate reaches zero.
   5. After the separation experiment is complete, disconnect the SPO and LPO sample collection vials and store them appropriately for subsequent analysis.
3. Automated Cleaning and Decontamination
   NOTE: Before processing each sample, flush and decontaminate the whole fluidic system using the following automated routine.
   1. Secure the SPO and LPO collection vial tubes, as well as the sample pickup tube in empty vials to collect excess cleaning solutions that will be flushed through the tubes.
   2. Start the automated system cleaning routine. As with the Automated Separation procedure in Step 5.2, the program should control the valves and syringe pump to sequentially load the holding coil with cleaning reagents, and flush them through the system.
   3. Perform the following cleaning routine: flush with 10% bleach, then with 70% ethanol, and end with water or an appropriate saline buffer (e.g., 1x PBS or buffer used for sample processing). Use the following flush volumes: 450 µl for bleach and ethanol, and 1,000 µl for water/buffer.
   4. During flushing steps, flush each reagent into the SPO while the LPO outlet valve is set to a port that is blocked, then vice versa, to remove any potential clogs in the outlet flow restrictors. Maintain withdrawal and infusion flow rates of 300–500 µl/min to minimize bubble generation in the tubing and backpressure buildup when either the SPO or the LPO is blocked.
   5. Discard the excess flushing solutions and the vials in which they collected by following appropriate procedures for handling biological or chemical waste.