CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62/316,933, filed on April 1 , 2016; and to U.S. Provisional Patent Application Serial No. 62/154,690, filed on April 29, 2015, the disclosures of which are hereby fully incorporated by reference in their entireties.

BACKGROUND

[0002] In the medical field, it often is desirable to separate low concentration cells from a fluid mixture with no harm to the cells, wash cells, concentrate cells in a fluid mixture, differentiate cells based on key parameters, or even fractionate many different types of cells. Such processes are key in the development of possible cures to many common diseases. It may also be desirable to separate particles or cells different in size, density and or acoustic contrast factor through the use of an acoustic field where the particles may be separated from each other as well. Examples include the separation of live from dead cells, and the separation of differentiated from undifferentiated cells. The methods described herein provide for such a separation or fractionation method that is label-free.

[0003] In the food and beverage industry, filter cartridges and filter membranes have conventionally been used to filter particles from liquids. Such filters are expensive and become clogged and non-functional as material is processed. In contrast, acoustophoresis provides, among other possible advantages, a solid-state, low-cost alternative to filter cartridges and filter membranes that is capable of processing large quantities of a host medium, for example water or beer, that is laden with yeast or other suspended particles.

[0004] In the food and beverage industry, host fluid is flowed through filters at flow rates up to ten times greater than those through conventional acoustophoresis devices. At these higher flow rates, trapping of the particles in the host fluid is decreased, thereby leading to decreased separation efficiency. It would therefore be desirable to provide systems and methods capable of separating a second fluid or a particulate from a host fluid at much higher flowrates, or at much lower concentrations, than conventional macro-scale acoustic separators.

[0005] In the oil and water industry, efficiently and economically separating oil and other contaminants from water has become an important process. The rise of fracking techniques has led to many settling ponds and large costs for transportation of contaminated water. These settling ponds are a challenge to the environment and better means are needed to more effectively clarify fracking water. Acoustophoresis provides, among other possible advantages, a solid-state, effective means of clarifying fracking, but the flow rates associated with such macro-scale acoustophoresis devices is still too low to be feasible. It would therefore be desirable to provide systems and methods capable of separating a second fluid, cell, or particulate from a host fluid at much higher flowrates.

BRIEF DESCRIPTION

[0006] The present disclosure relates, in various embodiments, to mini to macro- scale systems, devices, and methods for acoustophoresis to separate, fractionate, isolate, concentrate, wash, detect, or even differentiate cells or particles in fluid suspension. The devices and methods include a flow chamber containing an ultrasonic transducer and reflector that set up an angled acoustic standing wave oriented at an acute angle relative to the direction of mean flow through the flow chamber, which includes the particle path through the angled acoustic standing wave. At higher flow rates, acoustic standing waves may be used to deflect the particles in a desired direction, without causing the particles to become trapped in the standing wave. By applying the acoustic standing wave to the host fluid at an angle thereto, desired deflection of the particles can be achieved.

[0007] Disclosed herein is an acoustophoresis device comprising: a flow chamber through which is flowed an initial mixture of a host fluid and at least one of a second fluid, a cell, or a particulate, the flow chamber defining a direction of mean flow; at least one ultrasonic transducer located on a wall of the flow chamber, the transducer including a piezoelectric material driven by a voltage signal to create an angled acoustic standing wave in the flow chamber oriented at an acute angle relative to the direction of mean flow through the flow chamber; and a reflector located on a wall on an opposite side of the flow chamber from the at least one ultrasonic transducer, and the reflector is designed and positioned to create a standing wave along the acute angle direction. As examples, the transducer may be in direct contact with the fluid in the chamber, it may be adhesively attached to a polymer film, or it may be used to excite a second material to generate acoustic standing waves. Further, the transducer may utilize a piezoelectric material that is ceramic, such as a PZT - 8, or polymer such as polyvinylidene fluoride (PVDF).

[0008] In particular embodiments of the device, the angled acoustic standing wave is oriented at an angle of about 20° to about 70° relative to the direction of mean flow through the flow chamber. The multi-dimensional acoustic standing wave can be a three-dimensional acoustic standing wave. The angled acoustic standing wave may also be a planar acoustic standing wave, or a combination of planar acoustic standing waves and multi-dimensional acoustic standing waves.

[0009] In certain embodiments of the device, the acoustophoresis device further comprises an inlet at a first end of the flow chamber and a clarified fluid outlet at a second end of the flow chamber opposite the first end. The acoustophoresis device may further comprise a concentrate outlet at the second end of the flow chamber. The at least one concentrate outlet at the second end of the flow chamber may lead to a further process such as cell washing, cell concentration or cell fractionation where the cells are biological cells such as T cells, B cells and NK cells. In certain embodiments, the cells separated are Chinese hamster ovary (CHO) cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, or human cells. The use of mammalian cell cultures including the aforementioned cell types has proven to be a very efficacious way of producing/expressing the recombinant proteins and monoclonal antibodies required of today's pharmaceuticals.

[0010] In certain embodiments of the device, the acoustophoresis device further comprises a deflection wall below the clarified fluid outlet, the deflection wall extending substantially perpendicular to the direction of mean flow through the flow chamber. The acoustophoresis device can include a concentrate outlet at a lower end of the deflection wall. The angled acoustic standing wave can be a multi-dimensional acoustic standing wave that results in an acoustic radiation force having an axial force component that deflects the second fluid, cell, or particulate into the deflection wall. The angled acoustic standing wave can be a three-dimensional acoustic standing wave.

[0011] In certain embodiments of the device, the acoustophoresis device can include a plurality of ultrasonic transducers arranged in series, each transducer including a piezoelectric material driven by a voltage signal to create an angled acoustic standing wave in the flow chamber oriented at an angle of about 20° to about 70° relative to the direction of mean flow through the flow chamber. Each transducer of the plurality of transducers can be oriented at the same angle relative to the direction of mean flow through the flow chamber.

[0012] In particular embodiments, the acoustophoresis device can further comprise an upper inlet duct through which the initial mixture of the host fluid and at least one of the second fluid, cell, or particulate flows into the acoustophoresis device; a lower inlet duct through which a cell wash flows into the acoustophoresis device; an upper duct exit through which the host fluid of the initial mixture flows out of the acoustophoresis device; a middle duct exit through which the wash fluid flows out of the acoustophoresis device; and a lower duct exit where the second fluid, cell, or particulate concentrates after passing from the flow of the initial mixture through the upper inlet duct through the cell wash flow.

[0013] The acoustic chamber or chambers may also incorporate a straight path, such as that generated by a glass tube that runs down the center line of the angled wave acoustic device. In this instance, the acoustic wave is transmitted through the wall of the glass tube and the main flow through the acoustic device is not disrupted by the angular portions of the transducers and reflectors at the edges of the acoustic device.

[0014] Also disclosed is a method of separating a second fluid, a cell, or a particulate from a host fluid. The method comprises: flowing an initial mixture of the host fluid and at least one of the second fluid, the cell, or particulate through an acoustophoresis device; sending a voltage signal to drive the at least one ultrasonic transducer to create the angled standing wave in the flow chamber to deflect the second fluid, cell, or particulate; and collecting the second fluid, cell, or particulate from the acoustophoresis device. The acoustophoresis device comprises a flow chamber through which is flowed the initial mixture of the host fluid and at least one of the second fluid, the cell, or the particulate, the flow chamber defining a direction of mean flow; at least one ultrasonic transducer located on a wall of the flow chamber, the transducer including a piezoelectric material driven by a voltage signal to create an angled acoustic standing wave in the flow chamber oriented at an acute angle relative to the direction of mean flow through the flow chamber; and a reflector located on a wall on an opposite side of the flow chamber from the at least one ultrasonic transducer. In particular embodiments of the method, the angled standing wave is oriented at an angle of about 20° to about 70° relative to the direction of mean flow through the flow chamber. The angled acoustic standing wave can be a multi-dimensional acoustic standing wave, such as a three-dimensional acoustic standing wave. The angled acoustic standing wave can be a three-dimensional acoustic standing wave. The flow chamber of the acoustophoresis device can further include an upper inlet duct through which the initial mixture of the host fluid and at least one of the second fluid, cell, or particulate flows into the acoustophoresis device; a lower inlet duct through which a cell wash flows into the acoustophoresis device; an upper duct exit through which the host fluid of the initial mixture flows out of the acoustophoresis device; a middle duct exit through which the wash fluid flows out of the acoustophoresis device; and a lower duct exit where the second fluid, cell, or particulate concentrates after passing from the flow of the initial mixture through the upper inlet duct through the cell wash flow.

[0015] In certain embodiments of the method, the acoustophoresis device further comprises an inlet at a first end of the flow chamber and a clarified fluid outlet at a second end of the flow chamber opposite the first end. The acoustophoresis device used in the disclosed method may further comprise a concentrate outlet at the second end of the flow chamber.

[0016] In yet another embodiment, there may be two parallel inlets, one containing a fluid and cell mixture, e.g., from a cell culture, and the second a washing fluid. The device also contains two outlets, one for the cell culture fluid, and the other for the washing fluid. The action of the angled acoustic standing wave is to move all suspended cells from the original cell culture fluid into the washing fluid, thereby accomplishing a washing process. [0017] In another embodiment, there is a single inlet to the device containing a fluid mixture containing microcarriers, e.g., cytodex beads, and cells in suspension, e.g., from an adherent cell culture after cells have been separated from the microcarriers through, e.g., a trypsinization process. The action of the angled acoustic standing wave results in the separation of the fluid into two streams, one a fluid stream containing all the cells, and the other a fluid stream containing all the microcarriers.

[0018] In certain embodiments of the method, the acoustophoresis device can include a plurality of parallel collection ducts designed to collect cells or particulates of different properties that were fractionated by the angled acoustic wave forces.

[0019] In certain embodiments of the method, the acoustophoresis device can include an operating mode coupled to at least two exit ducts used to collect cells or particles differentiated by the angled wave as a result of property differences.

[0020] In certain embodiments of the method, the acoustophoresis device further comprises a deflection wall below the clarified fluid outlet, the deflection wall extending substantially perpendicular to the direction of mean flow through the flow chamber. The acoustophoresis device used in the disclosed method can include a concentrate outlet at a lower end of the deflection wall. The angled acoustic standing wave can be a multidimensional acoustic standing wave that results in an acoustic radiation force having an axial force component that deflects the second fluid, cell, or particulate into the deflection wall. The second fluid, cell, or particulate can be collected from the acoustophoresis device via the concentrate outlet after deflection into the deflection wall.

[0021] In certain embodiments of the method, the acoustophoresis device can include a plurality of ultrasonic transducers arranged in series, each transducer including a piezoelectric material driven by a voltage signal to create an angled acoustic standing wave in the flow chamber oriented at an angle of about 20° to about 70° relative to the direction of mean flow through the flow chamber. Each transducer of the plurality of transducers can be oriented at the same angle relative to the direction of mean flow through the flow chamber.

[0022] The second fluid, cell, or particulate can be collected from the acoustophoresis device at a draw rate of about 200 to about 350 milliliters per minute. The mixture of the host fluid and at least one of the second fluid, cell, or particulate can be flowed through the acoustophoresis device at a flow rate of about 400 to about 700 milliliters per minute. The voltage signal sent to the at least one ultrasonic transducer can be from about 5 V to about 200 V, or, more preferably, from about 5 V to about 50 V. The ultrasonic transducer can be operated at a frequency of about 0.2 MHz to about 200 MHz, or, more preferably, from about 0.5 MHz to about 10 HHz.

[0023] In particular embodiments of the method, the angled acoustic standing wave results in an acoustic radiation force on the second fluid, cell, or particulate; the flow of the mixture of the host fluid and at least one of the second fluid, cell, or particulate through the acoustophoresis device results in a viscous drag force on the second fluid, cell, or particulate; and a ratio of the acoustic radiation force to the viscous drag force is about 0.1 to about 0.9. In some embodiments, the acoustophoresis device is operated such that the acoustic radiation force is large enough to retard the second fluid, cell, or particulate from passing through the angled acoustic standing wave. In other embodiments, the acoustophoresis device is operated such that the second fluid, cell, or particulate passes through the angled acoustic standing wave.

[0024] In some constructions, the at least one ultrasonic transducer includes a plurality of ultrasonic transducers arranged in series and rotated relative to each other at an angle such that their acoustic standing waves are not parallel to each other. For example, the transducers may be angled 90° from each other. Each transducer includes a piezoelectric material driven by a voltage signal to create an angled three- dimensional acoustic standing wave in the flow chamber oriented at an angle of about 20° to about 70° relative to the direction of mean flow through the flow chamber to benefit differentiation, separation, concentration or fractionization of the second fluid, cell, or particulate.

[0025] These and other non-limiting characteristics are more particularly described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

[0027] FIG. 1 schematically illustrates the flow velocity components of a particle as it approaches a left-running acoustic standing wave that deflects the particle in the direction of the velocity component V _T .

[0028] FIG. 2 schematically illustrates the flow velocity components of a particle as it approaches a right-running acoustic standing wave that deflects the particle in the direction of the velocity component V _T .

[0029] FIG. 3 schematically illustrates the particle deflection effect caused by increasing and decreasing the velocity component of a particle as it approaches an acoustic standing wave normal thereto and is deflected away from the standing wave axial direction (i.e., V _T ).

[0030] FIG. 4 is a graph illustrating the net particle deflection angles of a particle at different wave angles with a fixed acoustic radiation force ratio of 0.5.

[0031] FIG. 5 is a graph illustrating the net particle deflection angles of a particle at different fluid velocities with a fixed wave angle of 60°.

[0032] FIG. 6 represents universal operating characteristics of any angled wave acoustic chamber presenting particle or cell deflection versus wave angle for any operating parameter M.

[0033] FIG. 7 represents a plot from FIG. 6 giving both the deflection angle versus M for a fixed wave angle, as well as defining the operating regions of such a system.

[0034] FIG. 8 illustrates particle trajectory computational results showing predicted particle deflections versus particle size for a yeast particle in a device operating at a flow velocity of 18 cm/min, at a frequency of 2 MHz, and at an acoustic pressure amplitude of 1 MPa, with an acoustic standing wave oriented at an angle of 45° relative to the direction of mean flow. Three lines are shown: the uppermost line represents a particle size of 10pm, the middle line represents a particle size of 7pm, and the lowermost line represents a particle size of 5pm. The numbers on the right hand side represent particle axial acceleration in (m ² /s). [0035] FIG. 9 illustrates particle trajectory computational results showing predicted particle deflections versus flow velocities for a 7 micron yeast particle in a device operating at flow velocities of 6 cm/min, 12 cm/min, 18 cm/min, and 24 cm/min, at a frequency of 2 MHz, and at an acoustic pressure amplitude of 1 MPa, with an acoustic standing wave oriented at an angle of 45° relative to the direction of mean flow.

[0036] FIG. 10 illustrates particle trajectory computational results showing predicted particle deflections versus particle size for a Chinese hamster ovary (CHO) cell in a device operating at a flow velocity of 18 cm/min, at a frequency of 2 MHz, and at a pressure of 1 MPa, with an acoustic standing wave oriented at an angle of 45° relative to the direction of mean flow. Three lines are shown: the uppermost line represents a cell size of 20pm, the middle line represents a cell size of 18pm, and the lowermost line represents a cell size of 16pm. The numbers on the right hand side represent particle axial acceleration in (m ² /s).

[0037] FIG. 11 illustrates particle trajectory computational results showing predicted particle deflections versus flow velocities for Chinese hamster ovary (CHO) cells having different contrast factors in a device operating at a flow velocity of 18 cm/min, at a frequency of 2 MHz, and at a pressure of 1 MPa, with an acoustic standing wave oriented at an angle of 45° relative to the direction of mean flow.

[0038] FIG. 12 illustrates the initial, or development length region, for a particle entering an acoustic standing wave at the start of a negative force region.

[0039] FIG. 13 illustrates the initial, or development length region, for a particle entering an acoustic standing wave at the start of a positive force region.

[0040] FIG. 14 illustrates a free body diagram showing the forces experienced by a particle suspended in an acoustic standing wave that is oriented at an angle relative to a direction of mean flow through a flow chamber.

[0041] FIG. 15 illustrates an exemplary acoustophoresis device according to a first embodiment of the present disclosure.

[0042] FIG. 16 illustrates an exemplary acoustophoresis device according to a second embodiment of the present disclosure.

[0043] FIG. 17 illustrates an exemplary acoustophoresis device according to a third embodiment of the present disclosure. [0044] FIG. 18 illustrates an exemplary acoustophoresis device according to a fourth embodiment of the present disclosure.

[0045] FIG. 19 illustrates an exemplary acoustophoresis device according to a fifth embodiment of the present disclosure.

[0046] FIG. 20 illustrates a generated flow profile in which the flow rate is higher at the bottom of the angled acoustic standing wave than at the top thereof.

[0047] FIG. 21 is a cross-sectional diagram of a conventional ultrasonic transducer.

[0048] FIG. 22 is a cross-sectional diagram of an ultrasonic transducer of the present disclosure. An air gap is present within the transducer, and no backing layer or wear plate are present.

[0049] FIG. 23 is a cross-sectional diagram of an ultrasonic transducer of the present disclosure. An air gap is present within the transducer, and a backing layer and wear plate are present.

DETAILED DESCRIPTION

[0050] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0051] Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure of the embodiments selected for illustration in the drawings, and are not intended to define or limit the scope of the disclosure. In the drawings and the following description below, it is to be understood that like numeric designations refer to components of like function.

[0052] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0053] The term "comprising" is used herein as requiring the presence of the named component and allowing the presence of other components. The term "comprising" should be construed to include the term "consisting of, which allows the presence of only the named component, along with any impurities that might result from the manufacture of the named component. [0054] Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0055] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of "from 2 grams to 10 grams" is inclusive of the endpoints, 2 grams and 10 grams, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values.

[0056] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier "about" should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of "from about 2 to about 10" also discloses the range "from 2 to 10." The term "about" may refer to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 1 1 %, and "about 1 " may mean from 0.9-1.1.

[0057] It should be noted that some of the terms used herein may be relative terms. For example, the terms "upper" and "lower" are relative to each other in location, i.e. an upper component is located at a higher elevation than a lower component in a given orientation, but these terms can change if the device is flipped. The terms "inlet" and "outlet" are relative to a fluid flowing through them with respect to a given structure, e.g. a fluid flows through the inlet into the structure and flows through the outlet out of the structure. The terms "upstream" and "downstream" are relative to the direction in which a fluid flows through various components, i.e. the flow fluids through an upstream component prior to flowing through the downstream component. It should be noted that in a loop, a first component can be described as being both upstream of and downstream of a second component.

[0058] The terms "horizontal" and "vertical" are used to indicate direction relative to an absolute reference, i.e. ground level. However, these terms should not be construed to require structures to be absolutely parallel or absolutely perpendicular to each other. For example, a first vertical structure and a second vertical structure are not necessarily parallel to each other. The terms "top" and "bottom" or "base" are used to refer to surfaces where the top is always higher than the bottom/base relative to an absolute reference, i.e. the surface of the earth. The terms "upwards" and "downwards" are also relative to an absolute reference; upwards is always against the gravity of the earth. It is to be understood that gravity, or the effects of gravity, are negligible in the angled wave deflection process described herein, because the process works on individual particles, not much larger particle clusters as used in other systems.

[0059] The term "parallel" should be construed in its lay sense of two surfaces that maintain a generally constant distance between them, and not in the strict mathematical sense that such surfaces will never intersect when extended to infinity.

[0060] The present application may refer to "the same order of magnitude." Two numbers are of the same order of magnitude if the quotient of the larger number divided by the smaller number is a value of at least 1 and less than 10.

[0061] As explained previously, in conventional acoustophoresis devices, acoustic standing waves cause particles in a host fluid to collect, agglomerate, aggregate, clump, cluster, or coalesce at the nodes or anti-nodes of the acoustic standing wave, depending on the particles' acoustic contrast factor relative to the host fluid, forming clusters that eventually fall out of the standing wave due to enhanced gravity when the clusters have grown to a size large enough to overcome the holding force of the standing wave (e.g. by coalescence, clustering, or agglomeration). For fluids/particles that are more dense than the host fluid (e.g., cells), the clusters sink to the bottom of the device and can be collected separately from the clarified host fluid. For fluids/cells/particles that are less dense than the host fluid, the buoyant clusters float upwards to the top of the device and can be collected therefrom. In conventional acoustophoresis devices, the acoustic standing waves created therein generate acoustic radiation forces in the axial direction (i.e., in the direction of the standing wave) and in the lateral directions (i.e., perpendicular to the direction of the standing wave). In these devices, the axial force typically is perpendicular to the flow direction, and, as the mixture flows through the flow chamber, particles in suspension experience a strong axial force component in the direction of the standing wave. Since this acoustic force is perpendicular to the flow direction and the drag force, it quickly moves the particles to pressure nodal planes or anti-nodal planes, depending on the contrast factor of the particle. The clusters of particles form quickly as a result of lateral radiation forces and then drop out of the mixture due to enhanced gravity.

[0062] The present disclosure relates to acoustophoretic devices that employ multidimensional ultrasonic acoustic standing waves, planar acoustic standing waves or combinations of planar and multidimensional acoustic standing waves (collectively referred to herein as angled acoustic standing waves) oriented at an angle relative to the direction of mean flow through the device. The direction of mean flow through the chamber is to be understood to include the path traveled by a second fluid, cell, or particulate that is flowed through an angled acoustic standing wave generated in the device. These angled acoustic standing waves deflect particles in a host fluid stream, rather than trapping the particles for agglomeration. This is an important distinction from many current acoustophoresis devices. These devices disclosed herein can operate at high flowrates and can be used to replace costly and clog-prone filter cartridges and filter membranes in various industries. The devices and methods of the present disclosure rely primarily on the axial force component to deflect the particles out of the acoustic field, rather than relying on trapping, agglomeration, and gravitational and buoyancy forces. The devices and methods presented herein are capable of being operated independent of gravity (i.e., in any orientation), and do not rely on gravitational settling. In this way, the axial force of an angled acoustic standing wave oriented at an angle relative to the flow direction is capable of advantageously deflecting particles in fluid streams at high flow rates of up to about 400 mL/min, and more preferably up to about 600 mL/min or about 700 mL/min in devices with a cross section of 1 inch by 1 inch.

[0063] Thus, bulk acoustic standing waves angled relative to a direction of flow through a device can be used to deflect, collect, differentiate, or fractionate particles or cells from a fluid flowing through the device. The angled acoustic standing waves can be used to separate or fractionate particles in the fluid by size, density, speed of sound, or shape. The angled acoustic standing wave can be a three-dimensional acoustic standing wave. The acoustic standing wave may also be a planar wave where the piezoelectric material is excited in a piston fashion or the acoustic standing waves may be a combination of the planar acoustic standing waves and the multidimensional acoustic standing waves. For purposes of this disclosure, a standing wave where the lateral force is not the same order of magnitude as the axial force is considered a "planar acoustic standing wave." This can be used to separate live cells from dead cells, damaged cells from healthy cells, or differentiated from undifferentiated cells. The deflection of the particles by the standing wave can also be controlled or amplified by the strength of the acoustic field, the angle of the acoustic field, the properties of the fluid, the three dimensionality of the standing wave, the frequency of the standing wave, the acoustic chamber shape, and the mixture flow velocity.

[0064] When acoustic standing waves propagate in liquids, the fast oscillations may generate a non-oscillating force on particles suspended in the liquid or on an interface between liquids. This force is known as the acoustic radiation force. The force originates from the non-linearity of the propagating wave. As a result of the non- linearity, the wave is distorted as it propagates and the time-averages are nonzero. By serial expansion (according to perturbation theory), the first non-zero term will be the second-order term, which accounts for the acoustic radiation force. The acoustic radiation force on a particle, or a cell, in a fluid suspension is a function of the difference in radiation pressure on either side of the particle or cell. The physical description of the radiation force is a superposition of the incident wave and a scattered wave, in addition to the effect of the non-rigid particle oscillating with a different speed compared to the surrounding medium thereby radiating a wave. The following equation presents an analytical expression for the acoustic radiation force on a particle, or cell, in a fluid suspension in a standing wave. where p _m is the speed of sound in the fluid medium, p is density, φ is acoustic contrast factor, V _p is particle volume, λ is wavelength, k is 2π/λ, P ₀ is acoustic pressure, X is the axial distance along the standing wave (i.e., perpendicular to the wave front), and where p _p is the particle density, p _m is the fluid medium density, β _ρ is the compressibility of the particle, and p _m is the compressibility of the fluid medium.

[0065] For a multi-dimensional standing wave, the acoustic radiation force is a three- dimensional force field, and one method to calculate the forces is Gor'kov's method, where the primary acoustic radiation force F _R is defined as a function of a field potential U, F _v = -v (c/ ) , where the field potential U is defined as and and f ₂ are the monopole and dipole contributions defined by

f _ L_ _f _ 2(Λ - 1)

J l 2 J 2

Ασ ^ζ 2Λ + 1

where

P,

σ = Λ =

c

f Pf Pf ^c i where p is the acoustic pressure, u is the fluid particle velocity, Λ is the ratio of cell density p _p to fluid density p _f , σ is the ratio of cell sound speed c _p to fluid sound speed Cf, V ₀ is the volume of the cell, and < > indicates time averaging over the period of the wave.

[0066] Gork'ov's model is for a single particle in a standing wave and is limited to particle sizes that are small with respect to the wavelength of the sound fields in the fluid and the particle. It also does not take into account the effect of viscosity of the fluid and the particle on the radiation force. As a result, this model cannot be used for the macro-scale ultrasonic separators discussed herein since particle clusters can grow quite large. A more complex and complete model for acoustic radiation forces that is not limited by particle size was therefore used. The models that were implemented are based on the theoretical work of Yurii llinskii and Evgenia Zabolotskaya as described in AIP Conference Proceedings, Vol. 1474-1 , pp. 255-258 (2012). These models also include the effect of fluid and particle viscosity, and therefore are a more accurate calculation of the acoustic radiation force.

[0067] The acoustic radiation force on a particle is seen to be a symmetric function having a period that is one half the acoustic wavelength. This means a particle will be accelerated and decelerated exactly the same by the radiation force. FIG. 1 schematically shows a mixture flowing through a standing wave, with the standing wave oriented at an angle relative to the direction of mean flow. The angled acoustic standing wave can be a three-dimensional acoustic standing wave. The acoustic standing wave may also be a planar wave where the piezoelectric material is excited in a piston fashion or the acoustic standing waves may be a combination of the planar acoustic standing waves and the multidimensional acoustic standing waves.

[0068] In FIG. 1 , V is the velocity of an initial mixture of a host fluid and particles or particulates. The particles are deflected toward the wave front, or away from the wave axial direction as shown. FIG. 1 depicts a left running wave (i.e., the wave moves to the left when looking in the direction of the fluid flow). The fluid velocity can be decomposed into a velocity component V _T parallel to the left running wave, and a velocity component V _N normal to the wave, as shown in FIG. 1. In this case, a particle in suspension will be deflected in the V _T direction. The direction of mean flow through the chamber is to be understood to follow the path traveled by the bulk mixture that is flowed through an angled acoustic standing wave generated in the device. In this regard, it is noted that when V _T is in the upward direction (such as in FIG. 1 ), the acute angle λ is on the lower downstream portion of the acoustic standing wave.

[0069] The particles are deflected in the direction of the tangential velocity component. FIG. 2 depicts a right running wave (i.e., the wave moves to the right when looking in the direction of the fluid flow). In this case, any particle in suspension will again be deflected in the V _T direction. Again, the direction of mean flow through the chamber is to be understood to follow the path traveled by the bulk mixture that is flowed through an angled acoustic standing wave generated in the device. In this regard, it is noted that when V _T is in the downward direction (such as in FIG. 2), the acute angle λ remains on the lower downstream portion of the acoustic standing wave. In other words, the angle λ (i.e., the angle of the acoustic standing wave relative to the direction of mean flow) is always measured from the lower downstream portion of the acoustic standing wave in the V _T direction.

[0070] An angled acoustic standing wave, such as those shown in FIG. 1 and FIG. 2, can often be analyzed more simply by using a Galilean transformation. This transformation amounts to looking at the same problem while running along the wave at a velocity V _T (i.e., parallel to the wave). Thus, the velocity component V _T plus V (along the direction of mean flow) is equivalent to the velocity component V _N (normal to the wave, irrespective of the wave angle). In other words, the physics of the problem do not change with such a transformation, which resultantly amounts to solving the flow through a standing wave with the flow direction perpendicular to the wave, or in the axial direction of the wave. In this direction, the acoustic radiation force variation, as presented in Equation 1 , will result in a symmetrical series of velocity increases and decreases in the normal flow direction. Using v as the particle perturbation velocity resulting from the acoustic radiation forces on a particle as the mixture flows through a normal acoustic standing wave, the following governing equation can be generated (i.e., from Newton's second law, Equation 1 and Stokes drag):

(dv\ 3nP ₀ ² V _{P m r}

PpVp ^) + 6πμΓ _ρ ν = — φ(β, ρ) sin(2/cx) (2)

[0071] As such, v is actually AV _N , or the change in particle velocity normal to the standing wave resulting from the effects of the acoustic radiation forces on the particles as generated by the standing wave. The viscosity effects oppose the perturbation velocity, and act in a direction toward the mean velocity. As a result, the viscosity drives the particle perturbation velocity to fluctuate about the mean flow velocity with an amplitude of AV _N . This effect is further verified by assuming the inertial term in Equation 2 is small. This assumption infers that the particles in suspension are small enough to instantly react to the viscous and radiation forces. With this assumption, the first term on the left side drops out, and Equation 2 can be reduced to: v = C sin(2/cx)

where

3 μλ where C is a function of the acoustic pressure, r is the particle radius, φ is the particle contrast factor, μ is the fluid viscosity, and λ is the acoustic wavelength. With this assumption, the particle velocity instantly adjusts to the Stokes velocity generated by the radiation force.

[0072] Turning now to FIG. 3, the particle deflection effect caused by the decrease and increase of the velocity component normal to the acoustic standing wave is presented when the standing wave is at a 45-degree angle to the flow. As inferred by the Galilean transformation, the tangential velocity component has to remain constant as the velocity component normal to the acoustic standing wave varies symmetrically about the mean normal velocity. There are no forces tangent to the waves. Therefore, the tangential velocity component has to remain constant.

[0073] As seen by the flow triangles depicted in FIG. 3, this results in a visible difference in the particle deflection by the alternating normal velocity variation. The particle will have a net deflection angle away from the standing wave axial direction. Neglecting gravity effects, this will be true for both left and right running waves. Using this phenomena and the resulting geometry in FIG. 3, the following expression was generated for the change in particle flow angle ΔΘ (measured from the mixture flow velocity direction entering the standing wave) as a function of the change in normal velocity of the particle:

[0074] The expression in Equation 4 was used to generate both the maximum upward deflection (+ΔΘ = 1 1.3°) and the maximum downward deflection (-ΔΘ = 18.4°), as shown in FIG. 3. These two deflections can be combined to generate the net deflection (ΔΘ = 7.1 °), as presented in FIG. 3.

[0075] The same expression was used to generate the net particle deflection angles at different wave angles. These results are presented in the graph of FIG. 4. Rotating the standing wave angle up or down can generate this effect. FIG. 4 shows, for the condition analyzed, that maximum particle deflection occurs with a wave angle between about 50° and about 75°. At an angle of 90°, no deflection occurs because the tangential velocity component, V _T , is zero.

[0076] A similar study was conducted by fixing the wave angle and changing the fraction of normal velocity variation about the mean fluid velocity component. Either increasing the flow velocity with fixed acoustics or changing the acoustic radiation force on the particle with fixed flow velocity can generate this effect. FIG. 5 shows a graph generated with these parameters. As can be seen in the graph of FIG. 5, the particle deflection increases with increases in AV _N A/ _N and reaches a maximum when AV _N A/ _N = 1 .0.

[0077] When AV _N A/ _N = 1.0, the normal velocity reaches zero in the standing wave and the particle travels along the wave. For further clarification, with a constant velocity mixture approaching an acoustic standing wave oriented at an angle of 60° relative to the direction of mean flow of the mixture, any particle that is stopped in the normal direction by the standing wave radiation forces is deflected at an angle upwards of 60°, or travels parallel to the wave front. The particle that is only slowed down by the standing wave will be deflected at a constant angle away from the normal direction, or axial direction of the standing wave. [0078] A universal solution for particle or cell deflection by angled acoustic waves was generated using a newly developed, non-dimensional parameter M. It is defined as follows: π τ _ρ ² βΡ$φ

(5)

V 3 μλΥ

AV N,

(6)

V

Particle Radiation Force

M

Viscous Drag Force

where C is the maximum normal velocity perturbation (AV _N ) from Equation 3 and V is the fluid free stream velocity. This non-dimensional parameter, M, is extremely important because it represents the ratio of acoustic radiation force on a particle to the viscous drag force on the particle. M is the key parameter for particle deflection by an angled standing wave. Both acoustic power and particle size are squared in the expression. This means they are the most dominant factors for determining particle deflection. An accurate expression for any particle deflection in an angled wave, in terms of M, can be obtained by solving particle movement with the normal wave exactly, and then transforming the results to the angled wave flowfield.

[0079] Calculated particle deflection angles are presented in FIG. 7 versus wave angle and the non-dimensional deflection parameter M. All possible particle deflection angles are seen to fall on, or lie on curves below the 45-degree line as shown in FIG. 6. The forty-five degree line represents maximum particle deflections for any angled acoustic wave. The maximum deflections represent a particle deflection angle equal to the acoustic wave angle, and always fall on the forty-five degree line. Each M curve in FIG. 6 is seen to have a discontinuity near the maximum deflection value where the particle deflection jumps from the difference between the up and down deflection regions shown in FIG. 3, to the down deflection only. This steep gradient represents a change in the physical mode of the deflection process. This occurs when the radiation force in the downward deflection region reaches a value large enough to stop the particle motion through the wave, which is described in greater detail herein. The results show that particles flowing in a fluid suspension can be deflected down an acoustic standing wave of any strength, if the wave angle is small enough. The different M curves in FIG. 6 can represent the effects of power on particle deflection versus wave angle while particle size, fluid compressibility factor, acoustic wavelength, fluid viscosity, and fluid velocity are all held constant at the baseline condition. The baseline condition in FIG. 7 is at M=0.8 which represents: mixture free stream velocity, V = 7.75 x 10 ^"4 m/sec; acoustic standing wave wavelength, λ = 7.4 x 10 ^"4 m; mixture viscosity, μ = 1 .0 x 10 ^"3 Pa-sec; contrast factor, X = 0.12; mixture compressibility, β = 4.06 x 10 ^"10 m ² /N; particle radius, r = 3 x 10 ^"6 m; and acoustic pressure amplitude, Po = 1 .0 MPa.

[0080] The particle deflection curve presented in FIG. 6 for M=0.8 is for all wave angles. The wave angles are varied from zero to ninety degrees. The particle deflection, at any constant M value, becomes equal to the wave angle as the wave angle is increased. At this point, the particle is stopped from moving through the waves by the normal radiation forces, and moves along the wave front direction. The particle deflection reaches a maximum of 53.1 °, for M=0.8, at a wave angle of 53.1 °. At a wave angle of 55° with M=0.8, the particle deflection angle drops to 28°. At a wave angle of 60° with M=0.8, the particle deflection is 23°.

[0081] FIG. 7 presents the particle deflection variation with M that occurs through waves angled at 53.1 °. M is varied from 0 to 1 in FIG. 7. The discontinuity in the deflection curve near maximum deflection is evident in the curve. The magnitude of the discontinuity increases with increasing M. This discontinuity is extremely useful, since it can allow for the separation of particles due to slight property differences. The differences could represent live versus dead cells, tagged versus untagged cells, mutated versus original cells, or even healthy versus unhealthy cells. Region 1 shown in FIG. 7 is where the acoustic radiation force is large enough to stop the particles from moving through the waves. The particles are seen to move parallel to the wave front and ΔΘΜ = γ in Region 1 . Theoretically, in Region 1 , all the particles will be deflected down the wavefront in the first wave. In Region 2, the particles pass through all the waves, and get deflected down (for the right running wave shown) a constant angle, ΔΘΜ _, which is significantly lower than γ. The particle net deflection in Region 2 is the difference between the downward deflection (particle slowed down by the radiation forces) and upward deflection regions (particle sped up by the radiation forces). Region 1 and Region 2 represent two different modes of operation. This discontinuity is extremely useful, since it could allow the separation of particles with very small size, stiffness, or density differences.

[0082] FIGS. 8-11 present particle trajectory computational results for yeast particles and CHO cells showing the effect on the particle deflection experienced under different particle/cell sizes and varied flow rates. In FIGS. 8-11 , the shaded and lined area represents the angled acoustic standing wave. In this regard, the flow from right to left in FIGS. 8-11 , and all of the particles/cells enter the angled acoustic standing wave at the same point on the left side of the right-running wave. The lines then represent the deflection trajectories of the different sized cells/particles and/or the cells/particles flowed at varied flow rates.

[0083] FIG. 8 presents particle trajectory computational results to further verify the physics of angled standing waves and the predictions presented. These results were obtained by numerically solving Equation 2 in its entirety and include inertial effects. Viscosity modifies inertial effects to generate a symmetrical perturbation velocity about the mean normal velocity component, to obtain a constant deflection as shown in FIG. 8

[0084] FIG. 8 presents particle trajectory computational deflection results of different size yeast particles (i.e., CFD predicted particle deflection versus particle size). The smaller particles deflect less since they have lower magnitude radiation forces acting on them. In addition to varying the size of the particles, lower radiation forces can be generated in many different ways, as will be appreciated by those skilled in the art. As a result, angled standing waves can be used to separate or fractionate particles in suspension by size, density, speed of sound, and shape. This technique may allow live cells to be separated from dead cells, or even damaged cells from healthy cells. The deflection of the particle by the standing wave can also be controlled or amplified by the strength of the acoustic field, the angle of the acoustic field, the properties of the fluid, the three dimensionality of the standing wave, the frequency of the standing wave, the acoustic chamber shape, and the mixture flow velocity. As can be seen beginning from the left side of FIG. 8, particle deflection in the first few wavelengths can vary depending on the exact location where the particle enters the standing wave (referred to as a length effect). The viscosity damps this initial length effect out quickly. The CFD results verify the constant angle of deflection across a large number of waves, as presented above. Based on the theory presented, the deflection of the particle will be a function of the non-dimensional deflection parameter, M.

[0085] Turning now to FIG. 9, particle trajectory computational results are presented, which verify the effects of normal velocity variation on the particle deflection resulting from a mixture flowing into an acoustic standing wave at an angle of 45 degrees (i.e., predicted particle deflection versus flow velocity). As the flow velocity increases, AVN/VN decreases and particle deflection angles are shown to decrease. This effect provides another means to increase the ability to detect minor differences in particle properties based on this procedure. As the flow velocity was increased (from about 0 cm/min to about 24 cm/min in FIG. 9), AVn/Vn decreased and particle deflection angles were shown to decrease. This effect provides another means to increase the ability to detect minor differences in particle properties using the methods and devices according to the present disclosure.

[0086] The particle trajectory computational results verify that particles in a fluid flowing through an acoustic standing wave, with the acoustic standing wave oriented at an angle relative to the fluid flow with a constant velocity, will be deflected a constant angle from the fluid flow direction. It is expected that this angular deflection phenomena will have a short (few waves), initial development region where viscous dissipation will force any non-symmetrical perturbation velocity distribution generated by either inertia effects, or the location at which the particle enters the standing wave, to be symmetrical about the fluid mean flow. The flow angle deflection can vary in this initial region, but the region is so short that the result is insignificant to the overall particle deflection for a macro-scale system, such as those systems specifically disclosed herein.

[0087] The computational framework discussed above can easily be extended in three dimensions. For small cells or emulsions the drag force F _v can be expressed as:

where U _f and U _p are the fluid and cell velocity, R _p is the particle radius, _f and μ _ρ are the dynamic viscosity of the fluid and the cells, and μ = μ _ρ I μ _/ is the ratio of dynamic viscosities. The gravity/buoyancy force F _B is expressed as:

F _B =→R _p ³ (p _f - _Pp )g

[0088] For small particles, this force can typically be neglected as it is several orders of magnitude smaller than the other force components, making this disclosure essentially gravity independent. The primary acoustic radiation force FA has been defined before.

[0089] A general particle trajectory model then becomes: m _p = F _V + F _A + F _B

where m _p is the mass of the particle, F _v is the drag force of the fluid on the particle, F _R is the acoustic radiation force, and F _B is the gravity/buoyancy force which can typically be neglected. These equations can then be integrated to find the particle trajectories for a given fluid flow, particle, and initial conditions.

[0090] In another embodiment, the angled acoustic standing wave field can be oriented such that it has both a polar and azimuthal angle with respect to the fluid flow, which then would result in particle deflections to a corner of the fluid channel.

[0091] FIG. 12 and FIG. 13 schematically present the initial, or development length region, for a particle that enters a standing wave at different locations. The pluses and minuses represent acoustic radiation force directions, from Equation 1 , that slow or speed up a particle, respectively. A zero radiation force occurs at each dashed vertical line shown. The force varies as a sine wave between these dashed lines. FIG. 12 represents a particle entering the wave system at the start of a negative force region. The solid staggered lines represent residual perturbation velocities generated due to particle inertia. As seen in FIG. 12, the particle is still moving down due to inertia when the radiation force approaches zero at the second vertical dashed line. This effect is exaggerated to explain the physics present. In most cases, inertia effects can be neglected from a macro sense. A similar effect is shown in FIG. 13, but in the opposite direction. FIG. 13 represents a particle entering the wave system at the start of a positive force region. Again, the solid staggered lines represent residual perturbation velocities generated due to particle inertia. These inertial effects generate different force regions where the viscous and radiation forces add or subtract as shown by F _R +F _V and FR-F _v . In this manner, these schematics in FIG. 12 and FIG. 13 show how these different flow regions force the perturbation velocity to be repetitive and symmetric about the mean flow velocity. This symmetrical perturbation about the mean velocity results in a constant angle deflection of the particle. This effect distinguishes the macro devices and systems disclosed herein from previous MEMS work, which operated in the development region and which can have many different deflections of the same particle. Additionally, the processes and devices disclosed herein use bulk acoustic standing waves, not surface waves used previously in other work.

[0092] It is contemplated that a specific velocity profile can be used to enhance particle diffraction. For example, the flow velocity may decrease with height (i.e., the flow rate is progressively lower at the top of the flow chamber than at the bottom). The resulting particle deflections by the angled standing wave would thereby be amplified because the percent normal velocity variations would increase dramatically with height because of the incoming velocity profile. As will be appreciated by those skilled in the art, coupling the velocity profile to the angle of the standing wave can be tuned to any specific, desired fractionation output. In this regard, the velocity profiles of the incoming mixture can be generated by any suitable means, including screens, ducts, plenums, or diffusers.

[0093] As another means of explaining many of the same concepts presented above, FIG. 14 provides a free body diagram showing the forces experienced by a particle suspended in an acoustic standing wave that is oriented at an angle relative to a direction of mean flow through a flow chamber. In FIG. 14, FV represents the drag force in the direction of mean flow, FB represents the force due to buoyancy forces, which is generally negligible, and FR represents the acoustic force in the direction of the acoustic standing wave, which has force components in the x and y directions. It is to be understood that reliance on gravitational or buoyancy forces for settling or rising is not necessary for efficient separation using the devices and methods according to the present disclosure

[0094] Conventional macro-scale ultrasonic separators use acoustic standing waves to generate tightly packed clusters of suspended fluid or particulate, which continuously drops out of a flowing fluid mixture. Such conventional macro-scale separators generally operate with flow Reynolds numbers less than 50, particle concentrations up to 20%, ultrasonic standing wave field frequencies of 1 -3MHz, and acoustic pressure amplitudes of about 1 MPa. Although these systems are effective, their flow rates are limited by the strength of the lateral acoustic radiation forces. Consequently, such systems are undesirable for applications requiring high flow rates. For example, as explained above, applications in the food and beverage industry require flow rates up to ten times faster than these conventional separators can support.

[0095] The present disclosure relates to acoustophoretic devices that employ angled ultrasonic acoustic standing waves that are angled relative to the flow direction and, as a result, deflect cells, particulates, or a second fluid in a host fluid stream. The acoustophoresis devices and methods disclosed herein utilize the axial radiation forces of a multi-dimensional acoustic standing wave. The axial radiation forces in a standing wave can be significantly higher than the lateral forces, though they are within an order of magnitude. Thus, significant performance improvements can be generated by using axial, rather than lateral, radiation forces to collect particles or cells in a fluid suspension.

[0096] FIG. 15 presents a first exemplary embodiment of such an axial force, macro- scale acoustophoretic device designated generally as 100. The acoustophoretic device 100 generally operates so as to use the axial radiation forces from an angled acoustic standing wave oriented at an angle relative to the direction of mean flow through the device 100. The acoustophoretic device 100 depicted in FIG. 15 includes a flow chamber 110, an ultrasonic transducer 120, and a reflector 130.

[0097] The flow chamber 110 is the region of the device 100 through which is flowed an initial mixture of the host fluid and a second fluid or particulate and defines a direction of mean flow therethrough. The direction of mean flow is designated generally as 116 in FIG. 15. In particular embodiments, the initial mixture of the host fluid and at least one of the second fluid or particulate is flowed through the device 100 at a flow rate of about 400 mL/min to about 700 mL/min. The flow chamber is formed by a sidewall, and has a cross section of 1 inch by 1 inch..

[0098] In certain embodiments, the initial mixture of the host fluid and second fluid or particulate enters the flow chamber 110 through an inlet 140. As shown in FIG. 15, the inlet 140 is generally located at a first end 112 of the flow chamber 110 (i.e., upstream of the ultrasonic transducer 120 and reflector 130).

[0099] In particular embodiments, such as that shown in FIG. 13, the device 100 also includes a clarified fluid outlet 150, which is generally located at a second end 114 of the flow chamber 110. As seen in FIG. 15, the second end 114 of the flow chamber 110 is opposite the first end 112 of the flow chamber 110 (i.e., the second end 114 is downstream of the ultrasonic transducer 120 and reflector 130). In this way, the inlet 140 permits fluid ingress into the device 100, and the clarified fluid outlet 150 permits fluid egress from the device 100.

[0100] The device 100 further includes at least one ultrasonic transducer 120. The ultrasonic transducer 120 may generally be located within or on the sidewall of the flow chamber 110, and the sidewall is shaped to hold the transducer at an acute angle relative to the direction of mean flow 116. In FIG. 15, the device 100 includes four ultrasonic transducers 120. In this regard, it is to be understood that the device 100 includes at least one ultrasonic transducer, but may otherwise include as many or as few transducers as is desired for a particular application. Each ultrasonic transducer 120 is driven by a voltage signal to create an angled acoustic standing wave 122 in the flow chamber 110 between the ultrasonic transducer 120 and a reflector 130 located on the sidewall on an opposite side of the flow chamber 110 from the transducer 120. In particular embodiments, the voltage signal sent to the ultrasonic transducer 120 is from about 25 V to about 50 V. The ultrasonic transducer 120 is generally operated at a frequency of about 2 MHz to about 3 MHz. The angled acoustic standing wave 122 created by the ultrasonic transducer 120 results in an acoustic radiation force having an axial force component (i.e., in the direction of the standing wave, between the transducer and the reflector, angled relative to the flow direction). It is to be understood that the angled acoustic standing waves utilized herein can be generated between a transducer 120 and a reflector 130 such as is shown in the first, leftmost acoustic chamber depicted in FIG. 15, or can be generated between two transducers positioned opposite one another, such as between transducer 121 and transducer 123, as depicted in the last, rightmost acoustic chamber depicted in FIG. 15.

[0101] Due to the orientation of the ultrasonic transducer 110 relative to the flow chamber 110, the angled acoustic standing wave 122 created by the ultrasonic transducer 110 is oriented at an angle A relative to the direction of mean flow 116 through the flow chamber 110. As shown in FIG. 13, the angle A at which the angled acoustic standing wave 122 is oriented relative to the direction of mean flow 116 through the flow chamber 110 is generally an acute angle (i.e., less than 90°). In certain embodiments, the angle A at which the angled acoustic standing wave 122 is oriented relative to the direction of mean flow 116 through the flow chamber 110 is about 20° to about 70°. In particular, it has been found that maximum deflection of particulates entrained in the host fluid occurs at an angle of about 60° to about 70°.

[0102] As previously explained, in certain embodiments the device 100 includes a plurality of ultrasonic transducers. In the embodiment shown in FIG. 15, all four of the transducers 120 have the same angle relative to the direction of mean flow 116 through the flow chamber 110. It is also contemplated that when a plurality of transducers are provided, the transducers can create angled acoustic standing waves that are oriented at different angles relative to the direction of mean flow 116 through the flow chamber 110. For example, each transducer may create angled acoustic standing waves in the flow chamber 100 oriented at an angle of about 20° to about 70° relative to the direction of mean flow 116 through the flow chamber 110, which angle may be the same or different than the other transducers present within the device 100. Moreover, each transducer can be operated so as to create different standing waves (e.g., of different frequencies) in the flow chamber.

[0103] In particular embodiments, the acoustophoresis device further includes a concentrate outlet 160. The concentrate outlet 160 is also located at the second end 114 of the flow chamber 110, adjacent to but spaced apart from the clarified fluid outlet 150. The concentrate outlet 160 and the clarified fluid outlet 150 have flow paths that are angled apart from each other at a relatively shallow acute angle. In operation, the transducers 120 cause desired particles to be deflected into the concentrate outlet 160, permitting clarified fluid to flow out through the clarified fluid outlet 150. The clarified fluid has a relatively lower concentration of the particles compared to the fluid entering through inlet 140. Please note that although here the concentrate outlet 160 is shown above the clarified fluid outlet 150, their locations can be reversed if desired.

[0104] In the device of FIG. 13, the M operation point can be set for the device to operate in Region 1 described with respect to FIG. 7. As a result, the particle, cell, or second fluid is deflected down the wave angle as shown. Some particles may collide and/or pass through several waves, but eventually most particles will be deflected down toward the lower chamber wall. In this way, the concentrate outlet 160 will collect concentrated mixture, while the clarified fluid outlet 150 will collect clarified fluid. In this manner, the device depicted in FIG. 13 will provide high speed separation, clarification, or concentration of the mixture.

[0105] In certain other embodiments, such as that shown in FIG. 16, the device 100 includes a deflection wall 170 below the clarified fluid outlet 150. In such embodiments, the concentrate outlet 160 is generally located at a lower end 172 of the deflection wall 170. In the embodiment shown in FIG. 14, the deflection wall extends substantially perpendicular to the direction of mean flow 116 through the flow chamber 100. In other embodiments, the deflection wall 140 can be angled or tilted relative to the mean direction of fluid flow. In the embodiment of the device 100 depicted in FIG. 16, the deflection wall 170 further serves as the second end 114 of the flow chamber 110, opposite the first end 112 of the flow chamber 110, which is generally defined by the inlet 140.

[0106] As explained above, the angled acoustic standing wave 122 results in an acoustic radiation force having an axial force component (i.e., in the direction of the standing wave, between the transducer and the reflector, angled relative to the flow direction). The axial force component deflects the second fluid or particulate into the deflection wall, as explained in great detail herein. Upon being deflected, the second fluid or particulate can then be collected from the device 100. As will be appreciated by those skilled in the art, the second fluid or particulate may be collected from the device by any suitable means, such as via the concentrate outlet 160 after deflection into the deflection wall 170. In particular embodiments, the second fluid or particulate is collected from the device 100 via the concentrate outlet 160 at a draw rate of about 200 to about 300 mL/min. While the transducers 120 are depicted at the top end of the flow chamber 110 (i.e., above the reflectors 130), it is to be understood that their locations can be reversed, such that the reflector 130 is located above the transducer 120. It is specifically contemplated, for example, that the device of FIG. 16 could be inverted, such that the reflector 130 is located at an upper end of the device and the transducer 120 is located at a lower end of the device. The device could then be operated so as to deflect particles in a host fluid flowing therethrough upward in the direction of the angled acoustic standing wave (i.e., upward toward the reflector 130) and to the concentrate outlet 160, which can be located at the upper end of the device (e.g., where the clarified fluid outlet 150 is located in FIG. 16). Resultantly, the clarified fluid outlet 150 could be located at a lower end of the device (e.g., where the concentrate outlet 160 is located in FIG. 16), and the deflection wall 170 could be repositioned as necessary to achieve the desired deflection.

[0107] In certain other embodiments, such as that shown in FIG. 17, a device 1700 according to the present disclosure may include one or more inlet ducts and one or more outlet ducts from the flow chamber. As can be seen, device 1700 is substantially similar to device 100 of FIG. 15, except as otherwise explained herein. For example, the device 1700 in FIG. 17 includes two inlet ducts and three outlet ducts. An initial mixture of a host fluid and at least one of a second fluid, cell, or particulate flows into the angled waves through an upper inlet duct 1701. A cell wash flows into the device via a lower inlet duct 1702. The angled wave is designed to operate at an M that generates the Region 1 process described above with reference to FIG. 7. As a result, the particles/cells are deflected along the wave angle as shown. The cells/particles pass from the mixture flow, through the wash flow, and concentrate in a lower duct exit 1703. The host fluid of the mixture primarily leaves the chamber through an upper exit duct 1704. The wash fluid primarily exits the chamber through a middle duct exit 1705. In this manner, particles/cells in a mixture can be isolated, washed, and concentrated in a single process. It is further contemplated that any of these steps could also be done separately through a different angled wave process, where the M operation point is set for the system to operate in Region 1 described above with reference to FIG. 7.

[0108] In certain other embodiments, the initial mixture of a host fluid and at least one of a second fluid, cell, or particulate can be flowed through a device according to the present disclosure with M set to operate the device either in Region 2, or in the steep gradient region described above with reference to FIG. 7. If operating in Region 2, multiple angled transducer-reflector pairs arranged in series may be necessary, such as is shown in device 100 of FIG. 15, with multiple outlets. In this mode, the different particles are deflected at different angles and the device fractionates many particles based on property differences. The same device can be operated with at least two outlets and with an M such that it is in the steep gradient region between 1 and 2, as described above with reference to FIG. 7. In this operation mode, very small differences will cause the particle to enter different outlets and the device can be operated as a property differentiator, such as by differentiating between live cells versus dead cells.

[0109] As previously explained, the acoustophoresis devices according to the present disclosure can be used for various purposes, including cell washing, cell concentration or cell fractionation. FIG. 18 depicts a cross-sectional view of one such embodiment of a device according to the present disclosure including at least two inlets and at least two outlets. The device in FIG. 18 is depicted with two inlets 1801 , 1802 and four outlets 1803, 1804, 1805, 1806. The first inlet 1801 can be of any suitable size and shape and is generally used as the inlet through which a mixture of a host fluid and at least one of a second fluid, cell, or particulate is introduced to the device. The second inlet 1802 can likewise be of any suitable size and shape and may have a cross-sectional width that is greater than a cross-sectional width of the first inlet 1801 , such as is shown in FIG. 18. The first inlet 1801 is located above the second inlet 1802. The second inlet 1802 generally serves as the inlet through which a wash fluid can be introduced into the device. Alternatively, the second inlet 1802 can be used to carry another host fluid containing cells or particulates therein that can be the same or different than the first host fluid, with the cells or particulates having the same or different properties as one another.

[0110] The device further includes cavities 1810, 1820 in which transducers/reflectors can be located. As explained herein, the device can include one transducer and one reflector or two opposing transducers to create the angled acoustic standing wave. For example, cavity 1810 could hold a transducer and cavity 1820 could hold a reflector, cavity 1810 could hold a reflector and cavity 1820 could hold a transducer, or both cavity 1810 and 1820 could hold transducers. As can be further seen from FIG. 18, the cavities 1810, 1820 can be separated from the flow chamber by secondary chambers 1812 and 1822, respectively. In this way, secondary chamber 1812 separates cavity 1810 from the flow chamber 1850 and secondary chamber 1822 separates cavity 1820 from the flow chamber. The secondary chambers 1812, 1822 are generally filled with a fluid (e.g., water) or gel that is acoustically transparent, such that the transducer(s) and/or reflector located in cavities 1810 and 1820 are capable of generating an angled acoustic standing wave therebetween.

[0111] The device includes outlets 1803, 1804, 1805, and 1806. The uppermost outlet 1803 generally serves as a clarified fluid outlet through which the host fluid, which has been clarified of cells or particulates, flows out of the device. The middle outlets, outlets 1805 and 1806 can be used to recover a wash fluid that is used in the device. Alternatively, it is to be understood that the wash fluid could be removed via the same outlet as the host fluid, such as any of outlets 1803, 1805, and 1806. Finally, lowermost outlet 1804 can be used to remove the second fluid, cell, or particulate from the device after being deflected toward outlet 1804 by the angled acoustic standing wave. The uppermost outlet 1803 is above the middle outlets 1805, 1806, and the lowermost outlet 1804 is below the middle outlets 1805, 1806. As will be appreciated by those skilled in the art, any of the outlets can be uses to remove any fluid or material therefrom. For example, depending on the particular application and orientation of the device, an of the outlets can be used to remove a host fluid, a wash fluid, or a second fluid, cell, or particulate from the device. Put another way, any outlet can be used for any desired output. [0112] In one embodiment, such as the embodiment of the acoustophoresis device shown in FIG. 19, the angled wave field can be a combination of two or more angled wave fields designed to generate three-dimensional displacement of the particles with respect to the fluid direction. Such a field can be generated by arranging two transducers in series and tilting the transducers such that the angled acoustic standing waves generated by each individual transducer are not parallel to one another. This arrangement is depicted in FIG. 19, in which transducer T1 is arranged in series with transducer T2 and arranged such that they are angled 90° from one another. The system would operate at an M required for Region 2 operation. As such, all the larger particles would move more to the top of the duct in the side view as a result of the first angled wave system. All the particles would move to the right of the duct as a result of the second angled wave in series, and the net results of the two wave systems in series are shown in the view from the exit plane as the 3D particle collection on the right side of FIG. 19.

[0113] In certain other embodiments, such as that shown in FIG. 20, the angled wave field separation effect can be combined with a generated flow profile, at the acoustic chamber entrance, specifically designed to enhance particle separation, or fractionation. The decrease in velocity with height (i.e., the velocity is lower at the top of the angled acoustic standing wave than at the bottom thereof), as shown in FIG. 20, will increase M with height increasing the deflection variation of a particle with height. Such flow profiles can be obtained using duct wall contouring, screens, obstructions, valving or other flow manipulations.

[0114] Some further explanation of the ultrasonic transducers used in the devices, systems, and methods of the present disclosure may be helpful as well. In this regard, the transducers use a piezoelectric crystal, usually made of PZT-8 (lead zirconate titanate). Such crystals may have a 1 inch diameter and a nominal 2 MHz resonance frequency, and may also be of a larger size. Each ultrasonic transducer module can have only one crystal, or can have multiple crystals that each act as a separate ultrasonic transducer and are either controlled by one or multiple amplifiers. The crystals can be square, rectangular, irregular polygon, or generally of any arbitrary shape. The transducer(s) is/are used to create a pressure field in the standing wave direction (axial), namely axial forces that deflect particles in the host fluid out of the pressure field.

[0115] FIG. 21 is a cross-sectional diagram of a conventional ultrasonic transducer. This transducer has a wear plate 50 at a bottom end, epoxy layer 52, ceramic crystal 54 (made of, e.g. PZT), an epoxy layer 56, and a backing layer 58. On either side of the ceramic crystal, there is an electrode: a positive electrode 61 and a negative electrode 63. The epoxy layer 56 attaches backing layer 58 to the crystal 54. The entire assembly is contained in a housing 60 which may be made out of, for example, aluminum. An electrical adapter 62 provides connection for wires to pass through the housing and connect to leads (not shown) which attach to the crystal 54. Typically, backing layers are designed to add damping and to create a broadband transducer with uniform displacement across a wide range of frequency and are designed to suppress excitation at particular vibrational eigen-modes. Wear plates are usually designed as impedance transformers to better match the characteristic impedance of the medium into which the transducer radiates.

[0116] FIG. 22 is a cross-sectional view of an ultrasonic transducer 81 of the present disclosure. Transducer 81 is shaped as a disc or a plate, and has an aluminum housing 82. The piezoelectric crystal is a mass of perovskite ceramic crystals, each consisting of a small, tetravalent metal ion, usually titanium or zirconium, in a lattice of larger, divalent metal ions, usually lead or barium, and O2- ions. As an example, a PZT (lead zirconate titanate) crystal 86 defines the bottom end of the transducer, and is exposed from the exterior of the housing. The crystal is supported on its perimeter by a small elastic layer 98, e.g. silicone or similar material, located between the crystal and the housing. Put another way, no wear layer is present. In particular embodiments, the crystal is an irregular polygon, and in further embodiments is an asymmetrical irregular polygon.

[0117] Screws 88 attach an aluminum top plate 82a of the housing to the body 82b of the housing via threads. The top plate includes a connector 84 for powering the transducer. The top surface of the PZT crystal 86 is connected to a positive electrode 90 and a negative electrode 92, which are separated by an insulating material 94. The electrodes can be made from any conductive material, such as silver or nickel. Electrical power is provided to the PZT crystal 86 through the electrodes on the crystal. Note that the crystal 86 has no backing layer or epoxy layer. Put another way, there is an air gap 87 in the transducer between aluminum top plate 82a and the crystal 86 (i.e. the air gap is completely empty). A minimal backing 58 and/or wear plate 50 may be provided in some embodiments, as seen in FIG. 23.

[0118] The transducer design can affect performance of the system. A typical transducer is a layered structure with the ceramic crystal bonded to a backing layer and a wear plate. Because the transducer is loaded with the high mechanical impedance presented by the standing wave, the traditional design guidelines for wear plates, e.g., half wavelength thickness for standing wave applications or quarter wavelength thickness for radiation applications, and manufacturing methods may not be appropriate. Rather, in one embodiment of the present disclosure the transducers, there is no wear plate or backing, allowing the crystal to vibrate in one of its eigenmodes (i.e. near eigenfrequency) with a high Q-factor. The vibrating ceramic crystal/disk is directly exposed to the fluid flowing through the flow chamber.

[0119] Removing the backing (e.g. making the crystal air backed) also permits the ceramic crystal to vibrate at higher order modes of vibration with little damping (e.g. higher order modal displacement). In a transducer having a crystal with a backing, the crystal vibrates with a more uniform displacement, like a piston. Removing the backing allows the crystal to vibrate in a non-uniform displacement mode. The higher order the mode shape of the crystal, the more nodal lines the crystal has. The higher order modal displacement of the crystal creates more trapping lines, although the correlation of trapping line to node is not necessarily one to one, and driving the crystal at a higher frequency will not necessarily produce more trapping lines.

[0120] In some embodiments, the crystal may have a backing that minimally affects the Q-factor of the crystal (e.g. less than 5%). The backing may be made of a substantially acoustically transparent material such as balsa wood, foam, or cork which allows the crystal to vibrate in a higher order mode shape and maintains a high Q-factor while still providing some mechanical support for the crystal. The backing layer may be a solid, or may be a lattice having holes through the layer, such that the lattice follows the nodes of the vibrating crystal in a particular higher order vibration mode, providing support at node locations while allowing the rest of the crystal to vibrate freely. The goal of the lattice work or acoustically transparent material is to provide support without lowering the Q-factor of the crystal or interfering with the excitation of a particular mode shape.

[0121] Placing the crystal in direct contact with the fluid also contributes to the high Q-factor by avoiding the dampening and energy absorption effects of the epoxy layer and the wear plate. Other embodiments may have wear plates or a wear surface to prevent the PZT, which contains lead, contacting the host fluid. This may be desirable in, for example, biological applications such as separating blood, or the food and beverage industry, where contamination of the host fluid must be avoided. Such applications might use a wear layer such as chrome, electrolytic nickel, or electroless nickel. Chemical vapor deposition could also be used to apply a layer of poly(p- xylylene) (e.g. Parylene) or other polymers or polymer films. Organic and biocompatible coatings such as silicone or polyurethane are also usable as a wear surface.

[0122] One specific application for the acoustophoresis devices and methods disclosed herein is in the processing of a second fluid or particulates entrained in a beverage, such as yeast in beer. Through the use of acoustophoresis, the deflection, fractionation, and separation of the particulates is achievable in macro-scale systems requiring high flow rates. This is an improvement over current filtration processes (filter cartridges, depth filtration, and the like), which routinely become clogged or fouled at the required high flow rates. It is to be further understood that the acoustophoresis devices and processes disclosed herein, through the use of angled acoustic standing waves, may also be coupled with standard filtration process upstream or downstream, such as beverage sheets, filter cartridges, depth filtration, tangential flow filtration (TFF), or other physical or mechanical filtration processes.

[0123] Desirably, flow rates through the devices of the present disclosure can be a minimum of 400 mL/min per cm ² of cross-sectional area of the acoustic chamber. Even more desirably, the flow rate can range as high as 600 mL/min/cm ² to 700 mL/min/cm ² , or even higher.

[0124] The present disclosure has been described with reference to exemplary embodiments. Obviously, modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the present disclosure be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.