Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.
Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must be smaller than the incident wavelength of sound and the width of the fluidic channels are typically tens to hundreds of micrometers across, acoustofluidic devices typically use ultrasonic waves generated from a piezoelectric transducer pulsating at high frequencies (in the megahertz range). At characteristic frequencies that depend on the geometry of the device, it is possible to induce the formation of standing waves that can focus particles along desired fluidic streamlines within a bulk flow. Here, we describe a method for the fabrication of acoustophoretic devices from common materials and clean room equipment. We show representative results for the focusing of particles with positive or negative acoustic contrast factors, which move towards the pressure nodes or antinodes of the standing waves, respectively. These devices offer enormous practical utility for precisely positioning large numbers of microscopic entities (e.g., cells) in stationary or flowing fluids for applications ranging from cytometry to assembly.
Acoustofluidic devices are used to exert directional forces on microscopic entities (e.g., particles or cells) for their concentration, alignment, assembly, confinement or separation within quiescent fluids or laminar flowstreams.1 Within this broad class of devices, forces can be generated from bulk acoustic standing waves, surface acoustic standing waves (SSAWs)2 or acoustic travelling waves.3 While we focus on the fabrication and operation of devices supporting bulk acoustic standing waves, devices supporting SSAWs have received much attention recently due to their ability to precisely manipulate cells along surfaces4 and rapidly sort cells in continuous flow channels.5 Devices supporting bulk acoustic standing waves, however, rearrange particles based on the mechanical vibrations of the walls of the device generated by a piezoelectric transducer, which excites the standing waves in microfluidic cavities at geometrically defined resonant frequencies. This enables the potential for generating higher pressure amplitudes compared to SSAW devices, and thus, faster acoustophoretic transport of microscopic entities.6
These standing waves consist of a spatially periodic set of pressure nodes and antinodes, which are fixed in position as the pressure oscillates in time. Particles respond to the standing waves by migrating to the pressure nodes or antinodes, depending on the mechanical properties of the particles relative to the fluid, and which are described by the acoustic contrast factor:
where the variables ρ and β represent density and compressibility and the subscripts p and ƒ represent the suspended object (e.g., particle or cell) and the fluid, respectively.7 Entities that possess a positive acoustic contrast factor (i.e., ɸ >0) migrate to the pressure node(s); whereas, entities that possess a negative acoustic contrast factor (i.e., ɸ <0) migrate to the pressure antinodes.7 While the majority of synthetic materials (e.g., polystyrene beads) and cells exhibit positive acoustic contrast, elastomeric particles made from silicone-based materials,8 fatty molecules9 or other highly elastic constituents exhibit negative acoustic contrast in water. Elastomeric particles in acoustofluidic devices can be used to isolate small molecules10 and as means to confine synthetic particles11 or cells12 for the purposes of discriminate sorting.13
Acoustofluidic devices are usually manufactured from standard materials (e.g., silicon and glass) that have sufficient rigidity to support an acoustic standing wave. In many acoustofluidic devices (including the device shown herein), the mechanical waves are designed to resonate at the lowest harmonic mode, which consists of a half-wavelength standing wave spanning the width of the microchannel. This configuration has a pressure node at the center of the channel and pressure antinodes along the peripheries of the channel. It has been shown previously that these systems can be used for chip-based cytometry applications14-16 and applications ranging from the trapping of cells to the concentration of cells.17,18
We describe the process of fabrication, methods for use and representative performance capabilities of an acoustofluidic device that supports bulk acoustic standing waves. This device requires one photolithography step, one etching step and one fusing step to permanently bond a glass "lid" to the etched silicon substrate. We note that other acoustofluidic devices that support bulk acoustic standing waves can be fabricated from glass or quartz capillaries bound to piezoelectric transducers, which is described elsewhere.19,20 Silicon-based devices offer the advantages of robustness and control over the flow channel geometry, which together allow for numerous types of processing for samples containing suspensions of particles and cells. The devices are reusable provided they are properly cleaned between uses (i.e., by flushing the device with buffers and detergents).
1. Photolithography
2. Deep Reactive Ion Etching
3. Piranha Cleaning
4. Prepare the Borosilicate Glass Lid
5. Anodic Bonding
6. Finalizing the Acoustofluidic Device
7. Operating the Acoustofluidic Device
We designed the acoustofluidic device to contain a trifurcating inlet, a main channel with a width of 300 µm and a trifurcating outlet (Figure 1A–B). We note that we only used one inlet for all experiments in this study (i.e., to achieve sheathless focusing of particles via acoustic radiation forces) by blocking the other inlets with removable plugs. Following the procedures described above, we constructed a chip possessing a channel width of 313 µm, with an error of ∼4% due to imperfections during the microfabrication process (Figure 1C–D). We operated the device at a driving frequency of 2.366 MHz to induce a half-wavelength harmonic standing wave.
We used a signal generator connected to a power amplifier to generate the high frequency sinusoidal waveform to actuate the PZT transducer. We used an oscilloscope to measure the peak-to-peak output voltage (Vpp) generated from the power amplifier to verify the fidelity of the signal shape and amplitude. Using a syringe pump, we first injected a suspension of green fluorescent polystyrene beads at a rate of 100 µl/min without actuation of the PZT transducer as a negative control (Figure 2A). Next, we actuated the device at 2.366 MHz to form a half-wavelength standing wave across the width of the microchannel (Vpp = 40 V; Figure 2B). We found that these particles, which have a positive acoustic contrast factor, focused along the pressure node as expected.6 We also injected red fluorescent particles with a negative acoustic contrast factor (i.e., ɸ ≈ −0.88, synthesized from a process described previously)8 to verify that our device could induce their concentration along the pressure antinodes (Figure 2C).
Finally, we explored the extent of focusing of particles with a positive acoustic contrast factor at a range of flow rates (i.e., 0 to 1,000 µl/min as regulated by a syringe pump) and voltages (i.e., 0 to 50 Vpp). Videos comprised of 15 frames were collected for each condition. ImageJ software was used to sample five of the fluorescence intensity profile across the width of the microchannel. A numerical computing program was used to average the intensity profiles for each condition and to smooth the averaged data using an inline filtering program. As expected, the extent of particle focusing (i.e., as defined by the width of the fluorescence peak, corresponding to the width of the stream of particles) decreased with increasing flow rates (Figure 3A). We also found that the extent of particle focusing increased with increasing applied voltages (Figure 3B).
Figure 1: Acoustofluidic device supporting bulk acoustic standing waves. Schematic views of the top (A) and bottom (B) of a device comprised of an etched silicon substrate fused to a borosilicate glass "lid", polydimethylsiloxane (PDMS) blocks connected to silicone tubing and a piezoelectric transducer soldered to wires glued to the bottom of the device. Photographs of the top (C) and bottom (D) of the device are also shown. Please click here to view a larger version of this figure.
Figure 2: Acoustic focusing of particles with positive and negative acoustic contrast factors. (A) Prior to actuation of the lead zirconate titanate (PZT) transducer, particles with a positive acoustic contrast factor (10 µm, yellow-green polystyrene beads) flowing at 100 µl/min occupied the width of the microchannel. (B) After the PZT transducer is actuated (Vpp = 40 V and ƒ = 2.366 MHz), the particles in (A) are shown to focus along the pressure node of the standing wave. (C) Particles with a negative acoustic contrast factor focused along the pressure antinodes of the standing wave in the absence of applied flow (Vpp = 40 V and ƒ = 2.366 MHz). Please click here to view a larger version of this figure.
Figure 3: Focusing performance of an acoustofluidic device. Fluorescence intensity plots of polystyrene beads (shown in Figure 2A–B) are shown for (A) various flow rates (ranging from 0 to 1,000 μl/min) with a constant peak-to-peak voltage of 40 V and (B) various applied voltages (ranging from 0 to 50 Vpp) with a constant flow rate of 100 µl/min. Please click here to view a larger version of this figure.
Acoustophoresis offers a simple and rapid approach to precisely arrange microscopic entities within fluidic microchannels without the need of sheath fluids used in hydrodynamic focusing approaches.24 These devices provide several advantages over other methods of particle or cell manipulation (e.g., magnetophoresis,25,26 dielectrophoresis27 or inertial forcing28) due to their ability to process entities without high magnetic susceptibilities, electric polarizabilities or a narrow size dispersity. Furthermore, the focusing nodes of an acoustic standing wave can be positioned far from the source of excitation, which is something that is not possible by static magnetic or electric fields as per Earnshaw's theorem.29 An additional advantage is that acoustic devices can focus particles across a wide range of applied flow rates and independent of the flow direction, which is not possible in devices that rely on inertial forces for focusing,28 providing the means to efficiently transport particles or cells for enhanced particle inspection for applications such as flow cytometry and particle sizing.30,31 The ease of device fabrication and operation can directly allow for the implementation of similar devices for focusing, concentrating, fractionating and sorting objects suspended in fluids.32
We have shown that the primary radiation forces, which are the strongest forces produced by acoustic standing waves,1 can focus microparticles flowing through a microfluidic channel at flow rates exceeding 10 ml/hr for a single orifice design. For a fixed flow rate of 100 µl/min, we show that our device can focus particles into a narrow streamline (i.e., 50 µm across) without any sheath fluids at voltages as low as 20 V peak-to-peak, enabling a low-power method for the batchwise focusing of 10 million particles/min when processing densely concentrated solutions (e.g., 6 x 108 particles/ml), as an example. Furthermore, this throughput can be dramatically increased by fabricating multi-orifice acoustofluidic chips or channels that are actuated with higher harmonics to produce sets of parallel nodes.33
While the device shown herein only requires materials and methods used in conventional microfabrication, we emphasize that there are a handful of other techniques that can be used for constructing similar devices.19,34,35 The advantages of this approach include its simplicity as well as the durability of the final device.
The critical steps to the fabrication of these devices include photolithography to define the geometry of the microchannel, reactive ion etching to form the channel in the silicon and anodic bonding to fuse the silicon to a transparent "lid" for observation by fluorescence microscopy. All of these steps require clean room facilities to avoid the collection of dust or debris within the device. Once these steps are complete, however, bonding a PZT transducer and fluidic ports are relatively straightforward and can be performed outside of a clean room.
However, proper treatment of the device is essential for its longevity. This includes (1) incubating the device with passivating reagents (e.g., poly(ethylene glycol) silane) prior to each experiment to protect the channel from residue buildup and (2) flushing the device with detergents after each experiment. Buildup of debris may compromise the fidelity of the acoustic standing wave and may reduce the ability to efficiently focus particles or cells within the device. We also note that these devices are not well-suited for highly polydisperse samples or samples containing entities approaching half of the size of the standing wave.
Acoustofluidic devices provide enormous utility for a variety of applications spanning from colloidal assembly to cell separation and flow cytometry. The ability to process biological samples with precision at high flow rates can allow for the ability of increased throughputs by these microfluidic devices, while reducing costs from superfluous reagents, large sample volumes or bulky equipment for dispensing sheath fluids. The fabrication methods required to make acoustofluidic devices are straightforward and the procedures required for their operation are user-friendly. We hope these procedures will encourage the widespread development of similar devices to catalyze new areas of research for applications across materials science, biotechnology and medicine.
The authors have nothing to disclose.
This work was supported by the National Science Foundation (through grants DMR-1121107, CMMI-1363483 and Graduate Research Fellowships (GRF-1106401) to C.W.S., D.F.C. and K.A.O.) and the National Institutes of Health (R21GM111584). The authors have no conflicts of interest.
Silicon wafers | Addison Engineering, Inc. | 3P1 | 6” mechanical grade silicon wafer <111> |
AZ® 9260 photoresist | MicroChemicals GmbH | AZ9260-Q | Positive photoresist |
AZ® 400K developer | MicroChemicals GmbH | AZ400K CONC-CS | Dilute 1 part AZ 400k in 4 parts deionized H2O |
H2O2 | Sigma Aldrich, Co. | 216763 | 30 wt.% in H2O |
H2SO4 | Sigma Aldrich, Co. | 320501 | ACS reagent, 95.0-98.0% |
1165 Photoresist Remover | Dow Chemical, Co. | DEM-10018073 | 1-methyl-2-pyrrolidinone based |
Acetone | Sigma Aldrich, Co. | 320110 | ACS reagent, ≥99.5% |
Isopropyl alcohol | Sigma Aldrich, Co. | W292907 | ≥99.7%, FCC, FG |
Methanol | Sigma Aldrich, Co. | 322415 | Anhydrous, 99.8% |
Borosilicate glass (Nexterion glass B) | Schott AG | 2098576 | Size: 120×60 ±0.1 mm, Thickness: 1 ±0.005 mm |
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Thickness: 1 | |||
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Drill bit for glass and ceramic | McMaster-Carr, Inc. | 2954A1 | Drill bit size: 1/8” Overall length: 2 3/16” Shank diameter: 7/64” |
Overall length: 2 3/16” | |||
Shank diameter: 7/64” | |||
Polydimethylsiloxane (PDMS) kit | Sigma Aldrich, Co. | 761036 | Dow Corning, Co.; Sylgard 184®; 10 g clip-pack |
Biopsy punch | Ted Pella, Inc. | 15078 | Harris uni-core Tip ID: 3.0 mm Tip OD: 3.40 mm |
Tip ID: 3.0 mm | |||
Tip OD: 3.40 mm | |||
Lead zirconate titanate (PZT) transducer | APC International, Ltd. | Custom order, (841 WFB) | Length: 30.0 mm, Width: 5.0mm, Freq.: 2.46 MHz, 2.0 mm end wrap for leads |
(841 WFB) | Width: 5.0mm | ||
Freq.: 2.46 MHz | |||
2.0 mm end wrap for leads | |||
Silicone tubing | Cole Parmer Instrument, Co. | 07625-22 | 0.6 mm I.D. |
Polystyrene beads | Thermo Fischer Scientific, Inc. | F-8836 | 10 µm yellow-green fluorescence |