The overall goal of this fabrication approach is to create a versatile and robust acoustofluidic device to manipulate micron-sized colloidal particles in a contact-free manner using bulk acoustic standing waves. Our goal is to demonstrate a simple approach by showing how to fabricate acoustofluidic tools supporting bulk acoustic standing waves using standard equipment and procedures in hope of making this useful technology more accessible. A major advantage of acoustofluidics is that it offers a simple way to rapidly focus or separate microscopic entities which has broad implications in on-chip cytometry and cell sorting.

This technology offers the ability to arrange particles in a variety of flow rates in a gentle and discriminate fashion, all in a convenient miniaturized platform. In a clean room facility place a clean six-inch single side polished silicon wafer onto a spin coater polished side up. Deposit a positive photoresist directly onto the center of the wafer by carefully pouring until the photoresist covers most of the wafer.

Then, spin the sample to produce an even layer of photoresist. When finished, release the vacuum on the chuck and use wafer tweezers to retrieve the wafer. Next, place the wafer onto a hot plate and soft bake for the amount of time specified by the photoresist supplier.

While the photoresist bakes, load a photo mask, like the one shown here, into the holder of a mask aligner. Then, load the wafer and expose it with UV light to an energy dosage specified by the photoresist supplier. Next, remove the photopatterned wafer from the holder and place it in a solution of its corresponding developer.

Once finished, remove the wafer from the developer, wash it with a steady stream of deionized water, and dry it with nitrogen. Load the photopatterned wafer into the chamber of a deep reactive ion etching instrument and etch the fluidic channels into the wafer to the desired depth following standard etching procedures. When the etching process is complete, unload the sample from the chamber and place it into a large beaker containing a photoresist remover solution.

Ensure that the wafer is submerged in the solution and let it soak for one hour at 65 degrees Celsius. Remove the wafer from the beaker and rinse it with alternating streams of acetone and isopropyl alcohol. Then, dry the wafer with nitrogen.

In a well ventilated hood that is approved for acid use, prepare a Piranha solution in a large clean beaker by adding 30%hydrogen peroxide to sulfuric acid in a one to three ratio. Submerge the ion etched wafer in the Piranha solution with the etched features facing up and leave it for five minutes. Then remove the wafer and rinse it well with deionized water.

Resubmerge the wafer in the Piranha solution for an additional two minutes and then rinse it again with copious amounts of deionized water. In a separate well-ventilated hood that is dedicated to solvent use, wash the wafer with a steady stream of acetone followed by a steady stream of methanol and then dry the wafer with nitrogen gas. Using a scribe tool, etch straight lines into the wafer around the perimeter of the microfluidic chip so that it is smaller than the dimensions of the rectangular glass segment with predrilled holes.

Carefully snap the wafer along the etched lines. Rinse the silicon segment with a steady stream of acetone followed by a steady stream of methanol. Then, place the wafer on a hot plate at 95 degrees Celsius for two minutes for it to dry.

Next, carefully add the clean glass on top of the silicone segment with the etched features facing up. Make sure the holes are properly aligned. Then, carefully flip the segments while ensuring the holes are kept aligned.

Secure the two segments with double-sided conductive tape where half of the tape secures the vertical edges of the silicone segment and the other half of the tape secures the overhanging glass. Then, flip the segments again such that the glass segment is on top. Place the segments on top of a steel slab on a hot plate at 450 degrees Celsius.

Then, carefully add a second steel slab of at least five kilograms directly to the top of the assembled glass and silicon segments. This slab should not be in contact with the silicon segment or the conductive tape. Using a high-voltage power supply, connect the live lead to the steel slab on top of the assembled glass and silicon segments and the ground to the bottom steel slab.

Turn the voltage on the underlying hot plate to 1, 000 volts. Check the applied voltage using a multimeter by pressing one probe against the bottom plate and the other probe against the top plate. Return after two hours to turn off the hot plate and the DC power supply and remove the device from the metallic slabs.

Scrape the surface of the glass with a razor to remove any grime produced by the anodic bonding and then clean the surface of the glass with acetone. Next, prepare a sheet of polydimethylsiloxane that is approximately five millimeters thick by cutting it into several small square slabs approximately 10 millimeters by 10 millimeters. Use a 3-mm biopsy punch to cut one hole in the center of each slab.

Then, glue the slabs directly on top of the holes on the glass substrate using epoxy. Solder two wires to the two conductive areas on the transducer. Carefully glue the lead zirconate titanate transducer to the silicon segment on the back side of the device centered underneath the microchannel.

Finally, insert the silicone tubing through the holes in the slabs of polydimethylsiloxane and add additional glue around the slabs and the tubing to secure them in place. Securely mount the device onto a microscope stage with the microchannel directly underneath the objective. Take care so that the transducer does not make contact with the stage.

Next, connect the silicone tubes from the outlets of the device to syringes secured on syringe pumps. Place the silicone tube leading to the inlet of the device in a vial containing a suspension of either polystyrene beads or cells of interest. Then, place the vial containing the sample on a stir plate to continuously mix it in order to ensure that a constant concentration is maintained throughout the course of the experiment.

Connect the transducer to the output from a power amplifier in series with a function generator. Program the settings on the function generator and monitor the output on an oscilloscope. Then, turn on the function generator and power amplifier to begin actuating the transducer.

Next, turn on the microscope and ensure the microfluidic channel is clearly in focus. Also, turn on the syringe pump to introduce the sample into the device. Monitor the entities flowing through the device with a fluorescence microscope throughout the experiment.

Here, a syringe pump was used to infuse the microfluidic chamber with a suspension of green fluorescent polystyrene beads at a rate of 100 microliters per minute. Once the lead zirconate titanate transducer is activated and tuned to a frequency of 2.366 megahertz, a half wavelength standing wave forms across the width of this microchannel, which is 313 micrometers across. This focuses the stream of beads along the pressure node.

When the red fluorescent silicone particles, which have a negative acoustic contrast factor, were injected into the device they concentrated along the pressure antinodes. The ability of this system to focus particles is dependent on both the flow rate and the applied voltages. As the flow increases the distribution of particles across the microchannel spreads out.

Also increasing the applied voltages increases the extent of particle focusing. Once operational, this device can be used to manipulate particles and cells for a variety of microfluidic-based bioassays and experiments requiring fine spacial or temporal control. It is important to remember to take your time and be careful with each step as haste at any step can introduce imperfections in the final device.

Once the device is finished it can be used many times as long as proper care is given to clean the device between uses with proper detergents and wash buffers. After watching this video you should have a good understanding of how to fabricate an acoustofluidic device comprised of silicon and glass supporting bulk acoustic standing waves. Please remember that you are working with strong chemicals, such as the Piranha mixture, that can be extremely hazardous if handled poorly.

Please take proper care when handling these liquids to ensure safe chemical practice for all of your fabrication work.

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