Abstract:
The disclosure describes a prism containing a microfluidic channel. By coupling bulk acoustic wave generators to opposing sides of the prism, a standing bulk acoustic wave field can be excited in the prism and in the microfluidic channel. Because the microfluidic channel is titled with respect to the nodes of the bulk acoustic wave field, the prism microfluidic channel device can be used to separate microparticles and biological cells by size, compressibility, density, shape, or mass distribution. This technology enables high throughput cell sorting for biotechnology applications such as cancer cell detection.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit under 35 U.S.C. §119 of Provisional Application Ser. No. 62/283,999, filed Sep. 17, 2015, and Provisional Application Ser. No. 62/390,207, filed Mar. 22, 2016, each of which is incorporated herein by reference. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
       [0002]    This invention was made with government support under Grant No. CMMI-1235145 awarded by the U.S. National Science Foundation. The government has certain rights in the invention. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    This invention relates generally to the separation of particles. More specifically, the invention relates to the separation of particles suspended in a fluid through the use of acoustic forces. 
         [0004]    Separating and sorting particles is an important activity in many applications including biological research, cellular engineering, biological and chemical analyses, and lab-on-a-chip technologies. Several methods exist for particle separation, but each has its own drawbacks. For example, magnetic, electrokinetic, and dielectrophoretic separation methods rely on the particles having an inherent charge, polarization, or magnetization. When the particles are biological cells, modifying the cells to obtain these properties can adversely impact the cell physiology and viability, rendering the techniques unusable for cellular engineering applications. Hydrodynamic separation mechanisms, including flow cytometry, can be continuous and passive, but these techniques are limited in volumetric flow rate or require contact between the particle and channel structure. 
         [0005]    Alternatively, using acoustic forces in microfluidic particle and cell separation devices provides a promising solution to the limitations of many other methods. Acoustic particle separation is label-free and can separate particles based on physiological properties including shape, density, size, and compressibility. The use of the acoustic force is contactless and does not require any specific particle property, labeling, or tagging. Moreover, the acoustic force on biological cells does not appear to have a significant deleterious effect on the particle viability, making acoustic microfluidics an ideal method for cell separation, especially when downstream processing and analyses requires the cell physiology to remain unmodified. 
         [0006]    According to one known acoustic method, particles are separated using standing surface acoustic waves (SAW). These types of devices, which are typically molded with soft polymers (such as in a polydimethylsiloxane), are not robust enough for use outside of a lab and are expensive to fabricate. Further, the flowrate of fluid in the device is limited because the useful standing waves are confined to a distance from the bottom surface of the channel, typically resulting in a shallow channel. It would therefore be advantageous to develop an improved device for sorting particles using acoustic forces, and a method of manufacturing the same, that are more robust and offer greater throughput. 
       BRIEF SUMMARY 
       [0007]    According to one embodiment of the present invention is a device employing standing bulk acoustic waves to separate particles flowing in a channel, where the nodes of the standing waves are at an angle to the direction of flow of a fluid through the channel. Within the microfluidic channel, the particles experience an acoustic force towards the acoustic nodes, which is proportional to the particle volume. The acoustic force is opposed by the drag force on the particle from the fluid, which is proportional to particle radius. Consequently, particles with a larger volume move faster towards acoustic nodes than smaller volume particles. By applying the acoustic forces at an angle to the direction of flow, it is possible to separate particles by size or other characteristic in a continuous flow device. 
         [0008]    In one embodiment, the device comprises a prism containing a microfluidic channel and bulk acoustic wave transducers positioned on opposing sides of the prism. Using the transducers, bulk acoustic wave energy can be excited through the prism to create acoustic pressure fields in the microfluidic channel. Specifically, nodes (and antinodes) form in parallel lines within the fluid passing through the microfluidic channel. Notable, the standing wave is present throughout the depth of the channel and is not limited to a surface of the channel. In one embodiment, the node line spacing is on the order of 100 μm for particles of interest in the range of 1-20 μm. 
         [0009]    The present invention can be used to separate many different types of microparticles, such as bacteria or cells. One example application includes cancer cell detection in blood. Particle separation using the acoustic force can be done with live cells or bacteria and is not destructive so it can be integrated into other lab-on-a-chip type devices to examine particles after separation. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0010]      FIG. 1  is a diagram illustrating the effect of drag and acoustic forces on different particles. 
           [0011]      FIG. 2  is cross section views of the particle separation device according to one embodiment. 
           [0012]      FIG. 3  is an alternative view of the particle separation device. 
           [0013]      FIG. 4  path lines for particles ranging in size from 3 μm to 9 μm in a tilted angle acoustic field with microfluidic flow. 
           [0014]      FIG. 5  is an image showing the path of two different sized particles in a separation device according to one embodiment. 
           [0015]      FIG. 6  is an illustration of another example embodiment. 
           [0016]      FIG. 7  is a graph depicting various regions defining a particle path. 
       
    
    
     DETAILED DESCRIPTION 
       [0017]    According to one embodiment, the separation device comprises a prism  100 , a microfluidic channel  110  within the prism  100 , and a pair of bulk acoustic wave generators  120  attached to the prism. As shown in  FIG. 2 , the prism  100  is generally rectangular in shape and is constructed from poly (methyl methacrylate) (PMMA) in one embodiment, although other rigid polymers or materials can be used. The acoustic wave generators  120  are coupled to opposing external sides  101  of the prism  100  and are used to create standing or traveling waves within the microchannel  110 . However, separation along node lines is more effective in standing wave environments. To create standing waves, the acoustic wave generators  120  are positioned parallel to each other on opposing sides of the channel  110 . In one embodiment, the acoustic wave generators  120  are lead zirconate titanate (PZT) transducers; however, other types of bulk acoustic wave generators can be used.  FIGS. 2-3  depict alternative embodiments of the separation device. 
         [0018]    The channel  110  is fabricated at an angle with respect to the transducers  120 . This configuration is identified as the “tilted angle” geometry. Because the standing waves are substantially parallel to the transducers  120 , the standing waves are angled to the direction of flow in the channel  110 . In one embodiment, the angle of the channel  110  relative to the standing waves is between  10  and  45  degrees. However, as will be discussed, the angle of the channel  110  can vary depending on the particles to be separated and other design parameters.  FIG. 1  is an illustration of the titled angle geometry where the node lines are shown at an angle to the direction of flow. 
         [0019]    Applying the acoustic force at an angle with respect to the flow is advantageous because the separation distances are limited only by the size of the channel  110 . In contrast, when parallel configurations are used the separation distance is limited to a fraction of the acoustic wavelength. The title angle geometry enables a device with higher throughput and separation of particles closer in size. 
         [0020]    To introduce the fluid to the prism and collect the effluent, inlet  102  and outlet  103  are provided on the prism  100 . The inlet  102  provides an entry point for the particles into the microchannel  110 . The particles are suspended in a fluid such as water, traveling through the channel  110  in a laminar or streamline flow. In one embodiment, the inlet  102  and outlet  103  are coplanar with the channel  110 . 
         [0021]    In an alternative embodiment, additional inlets  104  and  105  are provided for sheath flow, as seen in  FIG. 6 , around the inlet flow. More specifically, the additional inlets  104  and  105  provide a flow of fluid that does not contain particles around the particle stream, concentrating the particles to the center of the channel  110 . In the embodiment with sheath flow, the particles are concentrated at the center of the channel  110  before separation begins by controlling the ratio of the suspended particle volumetric flow rate to the sheath fluid volumetric flow rate. Without sheath flow, the particles start distributed across the channel  110  and the separation will be incomplete since some small particles are positioned at the part of the channel where the larger particles are ending. In addition to focusing particles within the width of the cannel  110 , the particles can be focused towards the mid-height of the channel  110  by making the inlet  103  shallower than the additional inlets  104  and  105 . This is particularly advantageous in bulk acoustic devices since the acoustic wave is present through the depth of the channel  110 . 
         [0022]    For example, in one embodiment, the node lines in the channel are oriented vertically along the height of the channel and spaced evenly along the width of the channel. The channel must be wide enough to include multiple node lines (more node lines can allow for greater separation), but narrow enough for both acoustic uniformity and a sufficient flow velocity (given the volumetric flow rate) for separation. 
         [0023]    When the channel is tilted with respect to the transducers  120  (and thus tilted to the node lines of the standing waves), the drag and acoustic forces have components transverse to the channel. Because of the difference in the relationship between the acoustic force and the drag force on particles of different sizes in the same acoustic field, larger particles more closely follow the node lines of the acoustic field, taking an undulating path from node line to node line. Conversely, the smaller particles more closely follow the streamlines of the flow. Thus, the difference in forces enables particle separation. Acoustic forces can also affect particles differently based on their density, compressibility, shape, and mass distribution.  FIG. 1  depicts the differences in magnitude between drag and acoustic forces for two different sized particles. 
         [0024]    With the appropriate choice of device and operational parameters, the balance between the acoustic force and the drag force on particles enables separations. However, depending on particle type, flow rate, acoustic energy, and channel size, particles can take different paths within the channel  110 . While it is beneficial to have the particles to be separated follow one of two different paths, several different trajectories are possible depending on the parameters of device construction and operation. In the separation device, a particle can follow one of three distinct trajectories: (1) the particle follows the fluid streamline with no “deflection” by acoustic effects; (2) the particle follows an undulating path; (3) the particle follows a path to a node line and remains in the node line.  FIG. 4  is an example of paths followed by different particle sizes ranging from 3 μm to 9 μm. Notice the smallest particle follows a fairly straight path, whereas the largest particle undulates from node to node. 
         [0025]    Plotting acoustic pressure against water velocity, four parabolas are produced that divide the design space into five regions. The plot is shown in  FIG. 7 . The condition of four distinct regions (labeled I through IV) are described physically as follows. 
         [0026]    In region I, the acoustic force on a particle is sufficiently large, relative to the fluid force, that 15 μm and 2 μm particles all deflect to a node line, and separation does not occur. 
         [0027]    In region II, 15 μm particles deflect to a node line while 2 μm particles follow an undulating path, because the trajectory of a large particle is more sensitive to acoustic pressure than that of a small particle. Separation is possible, in principle, but is not maximally effective. 
         [0028]    In region III, 15 μm particles deflect to a node line while 2 μm particles do not deflect. This achieves maximal, robust separation. 
         [0029]    In region IV, 15 μm particles follow an undulating path 2 μm particles do not deflect. Separation is possible, in principle, but not maximally effective. 
         [0030]    (Region V, not labeled, is the area on the abscissa of low acoustic pressure in which neither particle deflects.) 
         [0031]    Given that PMMA prisms  101  are rapidly prototyped, different sizes can be created if the velocity and pressure parameters cannot be varied sufficiently to achieve the desired separation. 
         [0032]    To fabricate the prism  100 , micromachining technology is utilized. For example, the microfluidic channel  110  can be milled at a sub-mm scale on a Mini-Mill desktop CNC system from Minitech Machinery Corp. In alternative embodiment, the each part of the prism is printed using additive manufacturing techniques (or 3D printing). However the device can also be fabricated using embossing or casting techniques. 
         [0033]    In one example fabrication method using micromachining, the prism  100  is formed from two half-pieces (a first part  151  and a second part  152 ) joined together. In this example, the first part  151  and the second part  152  are manufactured from PMMA stock 3.175-mm thick. As shown in  FIG. 2 , the first part  151  is milled is milled with a 0.05 inch end mill to create a groove  153  300 microns deep on its surface. The second part  152  has a flat surface. Both half-pieces  151 , 152  are milled to form inlet/outlet segments, alignment mechanism  106 , and transducer through-slots. The alignment mechanism  106  (shown in  FIG. 2 ), such as an alignment key, allow ‘snap-together’ assembly with sufficient precision to create complex channels  110  and to present a planar face on the exterior of the prism  100  for uniform acoustic coupling. For example, the transducers  120  are contained within slots and coupled acoustically to the prism  100  with water, way oil, or acoustic coupling gel. To complete assembly, the two half-pieces  151 ,  152  are bonded by cleaning first with soap and water, ashing the faces to be bonded in a Harrick Plasma Cleaner (in air) for five minutes, and then clamping the two pieces between glass plates and baking in an oven at 305 degrees F. for 60 minutes. This fabrication technique allows for very rapid prototyping of different designs. 
         [0034]    Given the relative ease of micromachining a custom channel  120  profile, in one embodiment the method of fabricating a device further comprises simulating the separation dynamics to determine the optimal device configuration. For example, according to one embodiment, COMSOL 4.3b Multiphysics Software is used to model particle motion in a channel 1270 μm wide and 150 μm deep tilted 20° with respect to the acoustic field. However, any simulation software can be used. The fluidic medium is water with a velocity of 10 mm/s. Using the particle tracing with mass postanalysis function in COMSOL, 3 μm to 9 μm diameter particles are simulated including the drag force from the fluid and the force due to the acoustic field. An analytic expression was used for the acoustic force given by: 
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         [0035]    where {circumflex over (x)} is the particle coordinate along the channel, ŷ is the particle coordinate across the channel, p 0  is the acoustic pressure which controls the amplitude of the force, r is the particle radius, ρ is the density of the particles and the medium, water, p and m respectively, β is the compressibility of the particles and the medium, λ is the wavelength, 200 μm, and θ is the tilt angle of the channel. 
         [0036]    This equation represents the force from the acoustic field created by acoustic waves incident from both sides of the channel. Using this simulation, parameters can be varied to achieve separation for a given particle size. 
         [0037]    Because bulk acoustic waves enter the microfluidic channel from the sides (unlike a SAW device where the waves enter from the bottom), the nodal separation is dependent only on the acoustic frequency and acoustic velocity in the fluid. Thus, node lines with 100 micron spacing require a frequency of about 7.41 MHz. In one example device, separation of 2 μm and 15 μm polymer spheres suspended in water was accomplished by driving the transducers  120  with 33V amplitude at 6.8 MHz, which is a resonant frequency of some PZT transducers  120 , and gives a node spacing of about 113 microns. This example is shown in  FIG. 5 . 
         [0038]    In one example embodiment, the channel has a width of 1.27 mm, a height of 300 μm, a length of about 4-5 mm, and a tilt angle of 15° or 30° with respect to the exterior walls  101  of the prism  100 . In this particular embodiment, the prism  100  has a height of 5.08 mm and a width of 4.34 mm. However, a wide range of dimensions, shapes, and other design parameters may work. 
         [0039]    While the disclosure has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modification can be made therein without departing from the spirit and scope of the embodiments. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.