CN110998311A

CN110998311A - Separation using angled sound waves

Info

Publication number: CN110998311A
Application number: CN201880036435.8A
Authority: CN
Inventors: B·利普肯斯; J·迪翁; W·M·小普雷茨; K·C·奇塔莱; B·罗斯-约翰斯鲁德
Original assignee: Flodesign Sonics Inc
Current assignee: Flodesign Sonics Inc
Priority date: 2017-03-30
Filing date: 2018-03-30
Publication date: 2020-04-10
Also published as: WO2018183968A1; AU2018243774A1; EP3631436A4; SG11202003768SA; EP3631436A1; CA3071640A1; AU2022259774A1

Abstract

The present invention relates to methods and systems for separating material from a host fluid using an acoustophoretic device. These methods and systems can deflect materials (e.g., secondary fluids, cells, beads or other particles, exosomes, viruses, oil droplets) at high flow rates in the primary fluid stream.

Description

Separation using angled sound waves

Cross Reference to Related Applications

This application is a continuation-in-part application of 15/613,790, 15/613,790 is a divisional application of 15/143,481, 15/143,481 claiming priority from U.S. provisional patent application No. 62/316,933 filed 2016, 4/1 and U.S. provisional patent application No. 62/154,690 filed 2015, 4/29. Priority is also claimed for U.S. provisional patent application No. 62/479,309 filed on 30.3.2017 and U.S. provisional patent application No. 62/485,229 filed on 13.4.2017. All of these applications are incorporated by reference and as described herein.

Background

In the medical field, it is often desirable to separate low concentrations of cells from a fluid mixture without harming the cells in the fluid mixture, washing the cells, concentrating the cells, differentiating the cells based on key parameters, or even fractionating many different types of cells (fractionates). Such an approach is critical to the development of a possible cure for many common diseases. It is also desirable to use an acoustic field to separate particles or cells of different sizes, densities and/or acoustic contrast factors, where the particles may also be separated from each other. Examples include the separation of live cells from dead cells and the separation of differentiated cells from undifferentiated cells. The methods described herein provide such separation or fractionation methods without labeling.

In the food and beverage industry, filter cartridges (filter cartridges) and filter membranes are commonly used to filter particles from liquids. Such filters are expensive and become clogged and non-functional with the treatment material. In contrast, among other possible advantages, acoustophoresis provides a low-cost solid-state alternative to filter cartridges and membranes that can handle large quantities of host media (e.g., water or beer) loaded with yeast or other suspended particles.

In the food and beverage industry, the primary fluid is caused to flow through the filter at a flow rate that is up to ten times higher than the flow rate through conventional acoustophoresis devices. At these higher flow rates, particle entrapment in the host fluid is reduced, resulting in reduced separation efficiency. It is therefore desirable to provide systems and methods that are capable of separating a second fluid or particles from a host fluid at much higher flow rates or at lower concentrations than conventional macro-scale acoustic separators.

In the oil and water industry, efficient and economical separation of oil and other contaminants from water has become an important process. The creation of fracturing technologies has resulted in many settling ponds and high costs for transporting contaminated water. These settling ponds are environmentally challenging and require better means to more effectively clarify the frac water. Among other possible advantages, acoustophoresis provides an effective solid-state means of clarifying the frac water, but the flow rates associated with such macro-scale acoustophoresis devices are still too low to be practical. It is therefore desirable to provide systems and methods that are capable of separating a second fluid, cells or particles from a host fluid at a much higher flow rate.

Disclosure of Invention

The present disclosure describes various embodiments of micro-scale to macro-scale systems, devices, and methods for acoustophoretic separation, fractionation, separation, concentration, washing, detection, or even differentiation of cells or particles in a fluid suspension. The apparatus and method include a flow chamber and an ultrasonic transducer and reflector that provide an angled acoustic standing wave oriented at an acute angle relative to a mean flow direction through the flow chamber, the flow chamber including a particle path through the angled acoustic standing wave. At higher flow velocities, the acoustic standing wave can be used to deflect particles in a desired direction without the particles being trapped in the standing wave. By applying the acoustic standing wave at an angle to the main fluid, a desired deflection of the particles can be achieved.

These systems and methods may use a bulk ultrasonic standing wave oriented at an angle γ relative to the fluid velocity to separate, classify, and distinguish various particles. This method provides a sensitive separation capability related to the size of the particles and the acoustic contrast.

In one aspect, a system for separating material from a host fluid comprises: a flow chamber defining a mean flow direction; an ultrasonic transducer comprising a piezoelectric material configured to be excited to generate an angled bulk acoustic standing wave having a wavelength and an acoustic radiation force in the flow chamber and oriented at an acute angle relative to a mean flow direction through the flow chamber, wherein the flow chamber has a minimum internal dimension that is at least 10 times the wavelength of the angled acoustic standing wave; a reflector opposite the at least one ultrasonic transducer; a first inlet fluidly connected to the flow chamber; a second inlet fluidly connected to the flow chamber; a first outlet fluidly connected to the flow chamber; and a second outlet fluidly connected to the flow chamber. Embodiments of these systems may include one or more of the following features.

In some embodiments, the first inlet is at least 0.1 inch (e.g., 0.2 inch, 0.3 inch, 0.4 inch, 0.5 inch, or 1 inch) from the angled bulk acoustic standing wave.

In some embodiments, the system further comprises a first channel terminating at the first inlet, wherein the first channel has a substantially straight section extending at least 0.1 inch (e.g., 0.25, 0.5, 0.75, or 1 inch) from the first inlet.

In some embodiments, the space between the ultrasound transducer and the reflector includes a first portion inside the flow chamber and a second portion outside the flow chamber. In some cases, the system further includes an acoustically transparent material separating the first portion from the second portion. In some cases, the system further includes a cooling water system fluidly connected to the second portion. In some cases, the second portion is filled with a solid material having an acoustic impedance equal to an acoustic impedance of the primary fluid.

In some embodiments, the example system comprises a plurality of ultrasound transducers.

In some embodiments, the first inlet and the second inlet are coaxial. In some cases, the first outlet and the second outlet are coaxial. In some cases, the first inlet has a rectangular cross-section. In some cases, the rectangular cross-section of the first inlet has an area of at least 0.05 square inches (e.g., 0.1, 0.25, 0.5, 0.75, or 1 inch).

In some embodiments, the first inlet has an aspect ratio of at least 5 (e.g., 10, 15, 20, 25, or 50).

In some embodiments, the system further comprises a third outlet, wherein the second outlet is disposed between the first outlet and the third outlet, and the cross-sectional area of the third outlet is less than the cross-sectional area of the second outlet. In some cases, the second outlet has a rectangular cross-section and the third outlet has a rectangular cross-section. In some cases, the width of the second outlet is the same as the width of the third outlet. In some cases, the height of the second outlet is at least 2 times the height of the third outlet.

In some embodiments, the system further comprises a plurality of third outlets, each offset from the axis of the second outlet in the direction of deflection of the angled acoustic wave.

In some embodiments, the system further comprises a first channel terminating at a first inlet, wherein the first channel has a substantially straight section extending at least 0.1 inch (e.g., 0.25, 0.5, 0.75, or 1 inch) from the first inlet and at a first acute angle relative to a plane perpendicular to the angled acoustic standing wave. In some cases, the second channel terminates at a second inlet, wherein the second channel has a substantially straight segment that extends at least 0.1 inch (e.g., 0.25, 0.5, 0.75, or 1 inch) from the second inlet and forms a second acute angle with respect to a plane perpendicular to the angled acoustic standing wave. In some cases, the first acute angle and the second acute angle are equal. In some cases, the system further includes a third channel terminating at the first outlet, wherein the third channel has a substantially straight segment extending from the first outlet and forming a third acute angle with respect to a plane perpendicular to the angled acoustic standing wave. In some cases, the first acute angle and the third acute angle are equal. In some cases, the system further includes a fourth channel terminating at the second outlet, wherein the first outlet is located in a direction of deflection of the angled sound wave relative to the second outlet, wherein the fourth channel has a first cross-sectional area, the third channel has a first portion with the first cross-sectional area and a second portion with a second cross-sectional area smaller than the first cross-sectional area, and the second portion of the third channel is located between the first outlet and the first portion of the third channel. In some cases, the third passageway has a substantially straight segment extending from the first outlet at a third acute angle. In some cases, the first acute angle is 80 to 90 degrees.

In some embodiments, the wall of the flow chamber adjacent to the first outlet in the direction of deflection of the angled acoustic wave extends at an acute angle relative to a plane perpendicular to the angled acoustic standing wave. In some cases, the acute angle is 1 to 20 degrees (e.g., greater than 2 degrees, greater than 3 degrees, greater than 5 degrees, greater than 10 degrees, less than 15 degrees, less than 10 degrees, less than 7.5 degrees, less than 5 degrees).

In one aspect, a system for separating material from a host fluid comprises: a flow chamber extending between a first end and a second end; an inlet at the first end of the flow chamber; a first outlet located between a first end of the flow chamber and the second end of the flow chamber, the inlet and the first outlet defining an average flow direction through the flow chamber; an ultrasonic transducer comprising a piezoelectric material configured to be excited to produce an angled acoustic standing wave between the inlet and the first outlet, the angled acoustic standing wave having a wavelength and an acoustic radiation force in the flow chamber and being oriented at an acute angle relative to a mean flow direction through the flow chamber; and a reflector opposite the at least one ultrasonic transducer; wherein the first outlet is spaced apart from the second end of the flow chamber.

In some embodiments, the flow chamber has a minimum internal dimension that is at least 10 times the wavelength of the angled acoustic standing wave.

In some embodiments, the first outlet is at least 0.5 inches from the second end of the flow chamber.

In some embodiments, the flow chamber has a distance between the first end and the second end, and the first outlet is at least 30% of the distance from the second end. In some cases, the first outlet is at most 70% of the distance from the second end.

In some embodiments, the system further comprises a second outlet at the second end of the chamber.

In one aspect, a method of separating material from a host fluid comprises: flowing an initial mixture of the primary fluid and the material at a flow rate into an acoustophoresis device via an inlet, the acoustophoresis device comprising: an acoustic isolation chamber in communication with the inlet; an ultrasonic transducer coupled to the acoustic isolation chamber and arranged to be excited to produce acoustic waves at an angle to the mean flow direction of the initial mixture; controlling a ratio of an acoustic radiation force generated by the ultrasonic transducer to a viscous drag force of the initial mixture to deflect the material passing through a first subset of the acoustic waves at a different angle than a second subset of the material, thereby allowing separation of the first subset from the second subset. Embodiments of these methods may include one or more of the following features.

In some embodiments, the method further comprises controlling the ratio by controlling one or more of an angle, a flow rate, an excitation frequency of the ultrasound transducer, or a power supplied to the ultrasound transducer.

In some embodiments, the method further comprises controlling the ratio based on one or more characteristics of the subset. In some cases, the method further comprises controlling the ratio based on one or more of material size, density, compressibility, or acoustic contrast factor.

In some embodiments, the method further comprises controlling the ratio to deflect at least some of the material at an angle of the acoustic wave.

In some embodiments, the material further comprises a third subgroup different from the first subgroup and the second subgroup, and the controlling the ratio further comprises deflecting the third subgroup at a different angle than the first subgroup or the second subgroup.

In some embodiments, the method further comprises controlling the ratio to be within a range determined by the characteristics of the subset of materials in the mixture to be separated. In some cases, the range is determined by the relative sizes of the materials in the subgroups to be separated. In some cases, the range spans at least one order of magnitude.

In some embodiments, the method further comprises collecting the first or second subset in a collection conduit in communication with the sound isolation chamber.

In some embodiments, the material comprises at least two sub-sets of particles, cells, or fluids having different properties.

A "bulk acoustic standing wave" may indicate an acoustic wave that propagates through a volume of a medium, such as water, with little attenuation. In contrast, a "surface acoustic standing wave" is an acoustic wave that travels along the surface of a material that exhibits elasticity, with an amplitude that generally decays exponentially with depth into the substrate. The surface acoustic waves do not penetrate very far into the volume of the medium (e.g. water), e.g. from the substrate up to a few millimeters into the water volume.

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

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 techniques of the type described in the present application for determining the value.

All ranges disclosed herein are inclusive of the recited endpoints and independently combinable (e.g., a range of "2 grams to 10 grams" is inclusive of the endpoints, 2 grams and 10 grams, and all 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 close to these ranges and/or values.

The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. The modifier "about" when used in the context of a range should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, a range of "about 2 to about 10" also discloses a range of "2 to 10" and the term "about" can refer to the number referred to plus or minus 10%. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may represent 0.9-1.1.

It should be noted that some of the terms used herein may be relative terms. For example, the terms "upper" and "lower" are positionally opposite one another, i.e., in a given orientation, the upper component is at a higher elevation than the lower component in a given orientation, but these terms may vary if the device is turned over. The terms "inlet" and "outlet" are associated with a given structure and are relative to a fluid flowing through them, e.g., the fluid enters the structure through the inlet and exits the structure through the outlet. The terms "upstream" and "downstream" are relative to the direction of fluid flow through the various components, i.e., fluid flows through an upstream component before flowing through a downstream component. It should be noted that in a loop (loop), a first component may be described as being both upstream and downstream of a second component.

The terms "horizontal" and "vertical" are used to indicate directions relative to an absolute reference (i.e., the ground plane). However, these terms should not be construed as requiring structures to be absolutely parallel or absolutely perpendicular to each other. For example, the first and second vertical structures need not be parallel to each other. The terms "top" and "bottom" or "base" are used to refer to a surface that is always higher at the top than at the bottom/base relative to an absolute reference (i.e., the surface of the earth). The terms "upward" and "downward" are also relative to absolute references; upward is always opposite to the gravitational force of the earth. It will be appreciated that gravity or the effects of gravity are negligible during the deflection of the angled waves described herein, as this process affects individual particles, rather than much larger clusters of particles as used in other systems.

The term "parallel" should be interpreted in the straight-white sense (in its laysense) of maintaining a substantially constant distance between two surfaces, rather than in the strict mathematical sense of the surfaces not intersecting when they extend indefinitely.

If the quotient of the larger number divided by the smaller number is a value of at least 1 and less than 10, then the two numbers are of the same order of magnitude.

The details of one or more embodiments of the systems, apparatuses, and methods are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

Drawings

Fig. 1 is a schematic illustration of particle deflection by acoustic radiation force of an angled acoustic standing wave oriented at an angle γ relative to a flow velocity V.

Fig. 2A and 2B are schematic diagrams of normal and tangential velocity components of a left going acoustic standing wave (fig. 2A) and a right going acoustic standing wave (fig. 2B).

FIG. 3 is a schematic diagram of a Galileo transform that decomposes the angled acoustic standing wave system into a system of two equations, i.e., normal to the wavefront and tangential to the wavefront.

Fig. 4 is a schematic illustration of the net particle deflection caused by the tangential velocity component after propagating half a wavelength in the direction normal to the wavefront.

FIG. 5 is a plot of particle deflection angle Δ θ for values of M parameter from 0 to 1_MGraph against wave angle γ.

FIG. 6 is the particle deflection angle Δ θ for acoustic standing wave angles of 30 °, 45 °, and 60 °_MGraph against M parameter.

FIG. 7A shows the particle deflection angle Δ θ_MGraph against M-parameter curve, highlighting two possible regions of particle deflection. FIG. 7B is the particle deflection angle Δ θ at angles less than the wave angle γ_MFIG. 7C is a schematic view of a particle deflection angle Δ θ_MEqual to the wave angle gamma.

FIGS. 8A and 8B show the numerical particle deflection trajectories of CHO cells having (a) an M/sin lambda value less than 1 and (B) an M/sin lambda value greater than 1 for a frequency of 2MHz, an acoustic pressure amplitude of 1MPa, a particle size of 18 μ M, and an acoustic contrast factor of 0.03.

FIG. 9A shows the numerical particle deflection trajectories for CHO cells with diameters of 16, 18 and 20 μm and an acoustic contrast factor of 0.03. FIG. 9B shows the numerical particle deflection trajectories of CHO cells with 20 μm diameter and acoustic contrast factors of 0.03, 0.035, 0.04, 0.045 and 0.05. The frequency is 2MHz, the sound pressure amplitude is 1MPa, and the speed amplitude is 6 cm/min.

FIG. 10 shows the numerical particle trajectory of CHO cells as a function of the magnitude of the velocity of fluid through the channel.

Fig. 11A is a graph comparing general analytical predictions of particle deflection to numerical particle trajectories over a wide range of M values. Fig. 11B and 11C are graphs comparing general analytical predictions of particle deflection with numerical particle trajectories over a wide range of M values.

Fig. 12A-12G illustrate an Angled Wave Device (AWD) system having standing waves angled at 45 °. Fig. 12A is a photograph of an AWD system with multiple inflow ports on the right side and multiple outflow ports on the left side. FIG. 12B is a schematic diagram showing the placement of the transducer, reflector, and flow channel. Fig. 12C is a schematic diagram showing one possible mode of operation of the AWD, where the dashed lines indicate the nodal surface position of the standing wave. Fig. 12D is a schematic illustration of the flow distribution within the AWD. Fig. 12E is a cross section of AWD, and fig. 12F and 12G are cross sections of alternate duct arrangements of AWD.

FIG. 13 is the particle size distribution of the polystyrene beads used in the experiment.

Fig. 14A-14F are photographs of polystyrene bead deflection as a function of electrical power to a 1MHz transducer that establishes an acoustic standing wave at 45 °.

Fig. 15A and 15B illustrate an AWD system configured to concentrate particles or cells by lowering the mixture conduit and constricting the lower buffer stream.

Fig. 16 is a schematic diagram of an AWD system configured for particle fractionation.

17A, 17B, and 17C are schematic diagrams illustrating aspects of an Angled Fluid Device (AFD) system. Figure 17A shows the geometry and flow characteristics of the system. Fig. 17B shows particle transfer between fluids. Fig. 17C schematically depicts the fluid flow direction of the system.

Fig. 18A is a photograph of an AFD and fig. 18B is a schematic diagram showing the setting of fluid streamlines from a CFD prediction.

Fig. 19A and 19B are photographs of an acoustic chamber window of an AFD system showing movement of particles through the AFD system without the use of acoustics (fig. 19A) and with the use of acoustics (fig. 19B).

Fig. 20A and 20B are a cross-section and schematic diagram, respectively, of a system in which a flow configuration is used to increase the concentration of a mixture of particles separated using the system.

FIG. 21 is a schematic diagram of an AFD system designed for particle fractionation.

22A, 22B, and 22C are a schematic, a graph of modeled flow rates, and a cross-section, respectively, of an AFD system designed to collect particulates.

Fig. 23A and 23B are a cross-section and schematic diagram, respectively, of a low angle AFD system.

Fig. 24A-24C show the results of using the AWD system to fractionate T cells from 35um beads. Fig. 24A and 24B are schematic diagrams showing the expected isolation of T cells from beads. Fig. 24C is a graph of the results.

Fig. 25A-25C show the results of using the AWD system to fractionate a mixed population of beads. Fig. 25A is a schematic showing the expected separation of larger beads from smaller beads. Fig. 25B and 25C are graphs of the results.

Fig. 26A-26C show the results of using an AWD system to fractionate a population of PMMA beads. 26A, 26B and 26C show the distribution of beads between the central outlet and the buffer outlet when no acoustics is used, 1W of power is applied and 1.2W of power is applied.

Fig. 27 shows a 10 degree AWD system with a central channel and a buffer channel around it.

Fig. 28 shows an AWD system with one small inlet on one side, a buffer stream on top of it and 5 outlets where the different fractions from the mixture population will end up.

Fig. 29 shows an AWD system in which the flow of viewing is made possible by 2 glass windows.

Like reference symbols in the various drawings indicate like elements.

Detailed Description

The present disclosure relates to acoustophoretic devices that employ multi-dimensional ultrasonic standing waves, planar acoustic standing waves, or a combination of planar and multi-dimensional acoustic standing waves (collectively referred to herein as angled acoustic standing waves) oriented at an angle relative to a mean flow direction through the device. The mean flow direction through the chamber is understood to include the path followed by the second fluid, cell or particle flowing through the angled acoustic standing wave generated in the device. These angled acoustic standing waves deflect particles in the main fluid stream rather than trapping the particles for agglomeration. This is an important difference compared to many current acoustophoresis devices. These devices described can operate at high flow rates and can be used to replace expensive and easily clogged filter cartridges and membranes in a variety of industries. The apparatus and methods of the present disclosure rely primarily on axial force components to deflect particles out of the acoustic field, rather than on trapping, agglomeration, and gravity and buoyancy. The devices and methods presented herein are capable of operating independently of gravity (i.e., in any direction) and do not rely on gravity settling. In this manner, axial forces of the angled acoustic standing wave oriented at an angle relative to the direction of flow can advantageously deflect materials (e.g., second fluids, cells, beads or other particles, exosomes, viruses, oil droplets) at high flow rates of up to about 400mL/min, more preferably up to about 600mL/min or about 700mL/min in a device having a cross-section of 1 inch x 1 inch in the main fluid flow. Devices having a total flow path of 0.5 inch x 0.5 inch were also produced with a central inlet of 0.1 inch x 0.1 inch. For these devices, the volumetric flow rate is about 0 to 100ml/min, typical buffer flow rates are 20 to 100ml/min, and the central flow rate is 1 to 10 ml/min. This corresponds to a linear velocity of about 1 to 100mm/s, regardless of the size of the device.

Thus, a standing bulk acoustic wave that is angled relative to the direction of flow through the device can be used to deflect, collect, differentiate, or separate particles or cells flowing through the device. The angled acoustic standing wave may be used to separate or separate particles in a fluid by size, density, speed of sound, or shape. The angled acoustic standing wave may be a three-dimensional acoustic standing wave. The acoustic standing wave may also be a planar wave, in which the piezoelectric material is excited in a piston fashion, or a combination of a planar acoustic standing wave and a multi-dimensional acoustic standing wave. For the purposes of this disclosure, standing waves having lateral forces at least one order of magnitude less than the axial forces are considered "planar acoustic standing waves". However, standing waves that are not planar acoustic standing waves. May also be used with the methods described in this disclosure. Can be used to separate live cells from dead cells, damaged cells from healthy cells, or undifferentiated cells. The deflection of the particles by the standing wave can also be controlled or amplified by the intensity of the acoustic field, the angle of the acoustic field, the properties of the fluid, the three dimensions of the standing wave, the frequency of the standing wave, the shape of the acoustic chamber and the flow rate of the mixture.

When an acoustic standing wave propagates in a liquid, the rapid oscillations may produce non-vibratory forces on particles suspended in the liquid or interfaces between the liquids. This force is called the acoustic radiation force. The source is derived from the non-linearity of the propagating wave. Due to the non-linearity, the wave is distorted as it propagates and the time average is not zero. By series expansion (according to perturbation theory), the first non-zero term will be the second term, which will account for the acoustic radiation force. The acoustic radiation force on a particle or cell in a fluid suspension is a function of the difference in radiation pressure across the particle or cell. The physical description of the radiation force is the superposition of the incident and scattered waves, except for the effect of non-rigid particles oscillating at different velocities compared to the surrounding medium, thereby radiating out the waves. The following equation represents the acoustic radiation force F on a particle or cell in a fluid suspension in a standing wave_RThe analytical expression of (2).

Wherein, β_mIs the speed of sound in the fluid medium, ρ is the density, X is the acoustic contrast factor, V_PIs the volume of the particles, λ is the wavelength, k is 2 π/λ, P₀Is the acoustic pressure amplitude, x is the axial distance along the standing wave (i.e., perpendicular to the wavefront), and

where ρ is_ρIs the particle density, p_fIs the density of the fluid medium, β_ρIs the compressibility of the granules, β_fIs the compressibility of the fluid medium.

The acoustic radiation force on a particle is considered to be a symmetric function with a period of half the wavelength of the acoustic wave. This means that the radiation force distribution repeats every half wavelength. This also means that the particles will be accelerated and decelerated by the radiation force represented by equation (1).

Figure 1 schematically shows the deflection of particles, which force variation will occur when the mixture flows through the stationary wave at an angle y. V is the velocity of the mixture of fluid and particles. The sign in the figure indicates the direction of the radiation force. The positive sign indicates that the radiation force is in the direction of flow and increases the particle velocity, while the negative sign indicates that the radiation force slows the particle. As shown, the particles will always be deflected towards the wave front or away from the wave axis. Fig. 1 is a wave traveling to the left, or inclined to the left when viewed in the direction of fluid mixture flow.

Fig. 2A and 2B are schematic diagrams of normal and tangential velocity components of a left going acoustic standing wave (fig. 2A) and a right going acoustic standing wave (fig. 2B). As shown in FIGS. 2A and 2B, the fluid velocity (V) in FIG. 1 can be decomposed into a velocity component (V) perpendicular to the traveling wave_N) And a component (V) parallel to the wave_T). The particles are always deflected in the direction of the tangential velocity component. It is the tangential fluid motion that carries or drags the particles at a constant velocity as the normal velocity component is slowed or accelerated by the axial radiation force. In this case, any particles in the suspension will again be at V_TDeflecting in the direction.

By utilizing a Galileo transform, as shown in FIG. 3, the angled flow problem as shown in FIG. 2 can generally be analyzed more simply. This transformation is equivalent to a transformation at wave velocity V_TThe same problem is seen when travelling along a wave. Theoretically, the physics of the problem does not change with this transformation. As shown in fig. 3, this is equivalent to solving for standing waves with flow direction perpendicular to the wavefront or in the axial direction of the wave. On the one handUpward, changes in acoustic radiation force, as shown in equation (1), will result in a series of symmetrical increases and decreases in velocity in the normal flow direction. Using v as the velocity of particle perturbation caused by the acoustic radiation force on the particle when the mixture flows through normal acoustic standing waves, the following control equation can be generated to describe the particle trajectory (i.e., according to Newton's second law, equation (1), and Stokes' resistance), where r is_pIs the particle radius:

thus, V is actually Δ V_NOr a change in particle velocity perpendicular to the standing wave caused by the effect on the particles of acoustic radiation forces generated by the standing wave relative to the normal fluid flow velocity. The viscosity effect is always opposite to the disturbance velocity and acts in the direction towards the average velocity. Thus, viscosity always drives the particle perturbation velocity around the average flow velocity by the amplitude Δ V_NFluctuating. It is assumed that the particles in the suspension are small enough to react immediately to viscosity and radiation forces. With this assumption, the first term on the left disappears and equation 3 can be simplified as:

v＝C sin(2kx) (4)

wherein the content of the first and second substances,

c is the maximum perturbation velocity in the normal direction and is considered as a function of sound pressure amplitude, particle radius, acoustic contrast factor, fluid viscosity and sound wavelength. With this assumption, the particle velocity is immediately adjusted to the Stokes velocity generated by the radiation force.

Figure 4 schematically shows the effect of particle deflection caused by a decrease and increase in the velocity component perpendicular to the acoustic standing wave when the standing wave is at an angle gamma to the flow. As inferred by the galileo transform, the tangential velocity component must remain constant because the velocity component perpendicular to the acoustic standing wave varies symmetrically around the average normal velocity.

The fluid trajectory relative to the average trajectory of the particles is also shown in fig. 4. P₁P₂Is during a time period Δ t₀The fluid trajectory of (1). P₁P₃Is the average particle trajectory. V_NIs the component of velocity, V, perpendicular to the wave_TIs the tangential component of velocity along the wavefront, V is the velocity of the incoming mixture, t is the time, Δ θ_MIs the deflection of the particles relative to the direction of the fluid. P₁Is where the mixture enters half the wavelength of the standing wave. A planar standing wave is assumed. The radiation force does not deflect the fluid but is in the direction of the fluid velocity at P₁Horizontally aligned P₂Away from half a wavelength. On the other hand, as shown, the tangential component of the fluid velocity deflects the particle from the wavefront, down to P₃. The term "deflection direction of the angled sound wave" is used to refer to the direction of this deflection.

The problem of interest is to determine particle deflections with acoustic angles under different flow and acoustic conditions. Δ V_NIs the maximum normal velocity perturbation, C is associated with the sinusoidal acoustic radiation force acting on the particle, as shown in equation (4).

The deflection of a particle or cell can be expressed as Δ V_NV, which is a dimensionless parameter, defined as M in the following analytical equation solution:

can be unfolded into

Where C is the maximum normal velocity disturbance (Δ V) from equation (4)_N) And V is the fluid free flow velocity. This dimensionless parameter M is important because it represents the ratio of the acoustic radiation force on the particle to the viscosity resistance on the particle. M is a key parameter for deflecting particles by an angled standing wave. Both the sound pressure and the particle size are squared in the expression. This means that they are the most important factor in determining particle deflection. By solving for particle motion using normal waves, and then transforming the result intoThe angular wave flow field (i.e., using the Galileo transform shown in FIG. 3) can yield an accurate representation of the particle deflection in the angular wave, denoted by M. The galileo transformation has no effect on time. Thus, the propagation time between half wavelengths (repetitions) is the same in the normal wave plane as well as in the transformed angled wave plane.

Equation 7 represents Δ t_MIs the time it takes for the particles in suspension to pass one-half wavelength of the normal standing wave (i.e. each half wavelength of the process repeats) as they are accelerated and decelerated by the acoustic axial radiation force. Equation 8 is Δ t_oIs the time it takes for the fluid to pass through one half wavelength of the normal wave. These two time values are independent of the Galileo transform and, in conjunction with FIG. 4, can be used to obtain particle deflection from the direction of fluid flow.

The ratio of these times is defined as

Equations

10 and 11 use epsilon in conjunction with wave angle gamma to produce an expression of particle deflection in the angular wavefield.

Fig. 4 helps explain equations (10) and (11). The angled waves in FIG. 4 are represented by passing V_TThe result of transforming the normal wave is added to all velocities. P₁Is the point at which the flowing mixture enters the standing wave. Standing waveAt an angle gamma with respect to the flow direction. The dashed lines represent the regions in the standing wave where the radiation force on the particles is zero. When crossing the dotted line shown in fig. 4, the direction of the radiation force is reversed. P₂And P₃Is on the zero force line relative to P₁Is a point of lambda/2. The particles flowing in suspension through P₁When passing through P, the sound wave is deflected₃As shown in fig. 4. P₂Is the point that passes without acoustic radiation force, which represents the fluid flow direction. Connection P₁And P₃The dashed line of (a) indicates the average trajectory of the particle through one cycle of acoustic radiation force. Theta_MIs the total angle of the same line with the normal direction of the wave. Thus, Δ θ_MIs generated by sound waves with respect to the direction of flow (i.e. the connection P)₁And P₂Dashed line of (d) measured particle deflection angle. The particle travel time calculated from the normal wave analysis is used, together with the tangential velocity transformation, to derive the displacement of the particle in the direction of the wavefront. The particle wavefront distance produced by the transformation in the absence of radiation force is Δ t_oV_TThe particle wavefront distance resulting from both Galileo's transformation and the combined effect of acoustic radiation force on motion is Δ t_MV_T. Difference (Δ t)_oV_T-Δt_MV_T) Is the deflection of the particle in the direction of the wave front caused by the sinusoidal acoustic radiation force acting on the particle. For delta theta to be calculated at different wave angles and different deflection parameters M_MOr particle deflection angle, the integral expression of epsilon must be solved in equation (11).

With the substituted variables, an analytical solution for the particle deflection is obtained as a function of the wave angle and the dimensionless parameter M, which is defined by the ratio of the acoustic radiation force and the viscosity force on the mixture flowing through the acoustic standing wave. This analytical solution, which allows predicting the particle deflection angle for all values of M and γ, is shown in equation (12).

Fig. 5 shows the calculated deflection angle of the particles from equation (12) as a function of the wave angle γ and the dimensionless deflection parameter M. The different M curves in fig. 5 may represent the effect of power on particle deflection versus wave angle, while particle size, fluid compressibility, acoustic wavelength, fluid viscosity, and fluid velocity remain constant at baseline conditions. The wave angle varies from zero to ninety degrees. At any constant value of M, particle deflection starts at zero, with a wave angle of zero and moves up along a 45 ° line until a maximum value is reached. Increasing the wave angle increases the component of the radiation force, slowing the particles when M is fixed. Under some wave angle conditions, the particles are stopped from moving through the wave by normal radiation forces and are forced by the fluid to move in the direction of the wave front. At this point, the particle deflection reaches a maximum value for this value of M (i.e., for example, M ═ 0.667, wave angle 42 degrees).

The triangular solution area below the 45 line shown in fig. 5 represents all possible particle deflections with the mixture flowing at an angle to the standing bulk acoustic wave. It can be applied to any fluid, standing wave, particle or acoustic pressure. It represents the deflection of the particles at all angles of wave as a function of a dimensionless parameter M, which is the ratio of the acoustic radiation force to the viscous drag on the particles. As shown in fig. 5, the deflection angle is seen to fall or lie below the 45 ° line. 45 degree line represents deflection angle Δ θ_MAnd the sound wave angle γ is equal. This is the maximum particle deflection for any angled acoustic wave, which occurs when M/sin γ ≧ 1, i.e., the acoustic radiation force equals or exceeds the viscous drag. This analytical solution enables the design and control of an angled wave system to provide the M values needed to achieve the desired results, as discussed in more detail later in this disclosure.

It is seen that each M-curve in fig. 5 has a steep gradient around the maximum deflection value, where the particle deflection transitions from the difference between the upper and lower deflection regions shown in fig. 1 for the left traveling wave to only upward deflection. This steep gradient represents a change in the physical mode of the deflection process and is reflected in the experimental results presented later in this disclosure. This occurs when the radiation force in the upward deflection zone reaches a value large enough to prevent the particles from moving through the wave. The results show that particles flowing in a fluid suspension can be deflected along an acoustic standing wave of any intensity if the wave angle is sufficiently small. The different M-curves in fig. 5 may represent the effect of acoustic pressure on particle deflection versus wave angle, while particle size, fluid compressibility, acoustic wavelength, fluid viscosity, and fluid velocity remain constant at baseline conditions.

For example, the graph where M ═ 0.8 can represent many different applications. One exemplary application of M-0.8 has a fluid mixture velocity V-7.75 × 10^-4m/s, acoustic standing wave wavelength λ 7.4 × 10^-4m, viscosity of mixture is 1.0X 10%^- ³Pa.s, contrast factor X0.12, mixture compressibility β_f＝4.06×10^-10m²N, particle radius r_p＝3×10^-6m, sound pressure amplitude P₀1.0MPa, as a point of discussion. The particle deflection curves for the various M parameters shown in fig. 5 are for all wave angles. Viewing this curve as a wave angle, varying from zero to ninety degrees, helps explain physics. The particle deflection initially moves up the 45 line. Along this line, the particle stops between the waves and moves tangentially along the wave front. This effect continues with increasing wave angle until the axial radiation force no longer opposes the normal velocity component of the particle. At this point, the particle moves through the plurality of waves and is deflected by each wave through which it passes. For M-0.8, the particle deflection is at a maximum of 53 ° at a wave angle of 53 °. At a wave angle of 55 ° where M ═ 0.8, the particle deflection angle decreased to 38 °; at a wave angle of 60 ° where M is 0.8, the particle deflection is 26.5 °.

Fig. 6 shows the variation of particle deflection with M occurring through waves at angles of 30 °, 45 ° and 60 °. In fig. 6M varies from 0 to 1. Particle deflection angle Δ θ_MIncreasing with increasing M value. The rate of increase of the deflection angle of the particles also increases with the value of M. A steep gradient of the deflection curve is observed for all curves around the maximum deflection angle. The magnitude of the gradient is seen to increase with increasing wave angle gamma. This steep gradient provides a mechanism for separating particles with only minor differences in acoustic properties.

Fig. 7A, 7B and 7C show particle deflection curves relative to M for acoustic angles of only 45 °. In the region 1The particles pass through all waves and are deflected downward at a constant angle (for the right traveling wave shown), Δ θ_MLess than γ, as shown in fig. 7B. The net deflection of particles in zone 1 is the difference between downward deflection (particles slowed by the radiation force) and upward deflection (particles accelerated by the radiation force). The curve in fig. 7A shows the large gradient that occurs when zone 1 transitions to zone 2. In the vicinity of this transition, a small change in M produces a particle deflection angle Δ θ_MLarge variations of (c). Separation of particles with a slight size, stiffness or density difference can be achieved in this transition region. Region 2 represents the operating parameter space where the acoustic radiation force is large enough to prevent particles from moving through the wave. In region 2, the particle moves parallel to the wavefront, and Δ θ_Mγ. In theory, in zone 2, all particles will be deflected downward along the wavefront in the first wave, as shown in fig. 7C.

As shown in fig. 5, the results of the analytical model predict that particles in suspension can be deflected downward by an acoustic standing wave of any intensity if the wave angle is small enough. As the wave angle γ decreases, the fluid and particle velocities normal to the wave decrease. At some point the acoustic radiation force will overcome the oncoming normal velocity component of the particle and as a result the particle will stop moving through the wave and propagate along the wave front. This process will occur when the wave angle is low enough to cause the resulting particle velocity component normal to the wave to reach zero. The forty-five degree line in fig. 5 represents the locus of these points. Analysis predicts that for any value of M, the maximum deflection always falls on this 45 degree trajectory. Since the acoustic power parameter M is equal to C/V, where C represents the maximum particle normal velocity perturbation produced by the acoustic radiation force, it can also be interpreted as Δ V_NV, where V is the oncoming fluid and particle velocity. When Δ V_NAt V sin (γ), the acoustic perturbation velocity is equal to the fluid normal velocity component of the wave. Thus, at any power or acoustic pressure of an acoustic standing wave, the standing wave will have an angle at which the radiation force can stop the particle velocity perpendicular to the wave. This point is defined by the following equation, which represents the maximum particle deflection and where the deflection curve for a given value of M intersects the 45 degree line in fig. 5:

Δθ_max＝sin^-1(M)＝γ (13)

equation 13 defines the maximum deflection angle possible and the wave angle γ required for maximum particle deflection using an angled acoustic standing wave as a function of the dimensionless parameter M.

The M parameter may also be used to determine desired operating characteristics, for example, for deflecting very small particles in a suspension. The smaller the particle size, the lower the M factor. Assuming that the flow rate is reduced as low as possible for the feasibility of the system and the power is increased as large as possible, the M-operation curve dictates that the system should be operated at as low a wave angle as possible, since the particle deflection is maximized at lower wave angles for low values of M. This indicates that systems used with small particles or nanoparticles should operate at very small angles (e.g., <5 °, <4 °, <3 °, <2 °, <1 °).

The predictions presented above are based on an analytical procedure for ideal standing waves and flow velocity fields and are used as guidelines for more accurate numerical particle trajectory studies and experimental validation tests, indicating the use of acoustic standing waves to deflect, collect, differentiate, separate, purify, or fractionate a population of particles or cells from a mixture that may contain many different types of particles (i.e., differing in size and/or material properties such as density or compressibility).

Given some initial conditions of the particle, the particle trajectory can be solved by numerically integrating the equation of motion of the particle, equation (3). The equation is solved by a fourth-order Runge Kutta method with automatic time steps. In the following results, a uniform velocity distribution of the fluid using a one inch wide flow channel was used. Typical conditions used in the calculation are an acoustic standing wave with a frequency of 2MHz and a sound pressure amplitude of 1 MPa. The acoustic standing wave has a width of one inch and an angle of 45 °.

FIGS. 8A and 8B show deflection results for particles with properties similar to Chinese Hamster Ovary (CHO) cells. CHO cells are of interest because they are widely used for the production of recombinant proteins and monoclonal antibodies. A typical CHO cell diameter is 18 μm and the acoustic contrast factor is 0.03.

FIGS. 8A and 8B show the numerical particle deflection trajectories of CHO cells for M/sin γ values less than 1 and M/sin γ values greater than 1, respectively. The simulations used were: the frequency is 2MHz, the sound pressure amplitude is 1MPa, the diameter of the CHO cell is 18 μm, and the acoustic contrast factor of the CHO cell is 0.03. The results of the numerical particle trajectories further verify the physical properties of the angled standing waves and the analytical predictions for both the case of M/sin γ <1 and M/sin γ ≧ 1. These results include inertial effects. The viscosity changes the inertial effect to generate a symmetric perturbed velocity around the average normal velocity component, resulting in a net constant deflection as shown in fig. 8A and 8B. Thus, the particle deflection in the first half wavelength may vary depending on the exact position of the particle relative to the standing wave, as shown in fig. 8, where the initial particle positions of two particles differ by a quarter wavelength in the y-direction. Viscosity will quickly diminish the effect of this initial length. The results demonstrate a constant angle of deflection as the particle passes through each half wavelength of the standing wave. When M/sin γ ≧ 1 (i.e., the condition in FIG. 8B), the particle deflection angle is equal to the standing wave angle. After an initial transient of particle motion, the particles deflect along the wave angle.

FIG. 9A shows the numerical particle trajectories for CHO cells with diameters of 16 μm, 18 μm and 20 μm and an acoustic contrast factor of 0.03. FIG. 9B shows the numerical particle trajectories for CHO cells of 20 μm diameter and acoustic contrast factors of 0.03, 0.035, 0.04, 0.045 and 0.05. The frequency used in simulation is 2MHz, the sound pressure amplitude is 1MPa, and the speed amplitude is 6 cm/min.

FIG. 9A shows the deflection of CHO particles of three slightly different sizes (16 μm, 18 μm and 20 μm, representing a change in size of about + -10%). The minimum particle deflection is that of particles with an M/sin gamma value of less than 1. The 18 μ M particles deflect according to a M/sin γ value of less than 1 but greater than 16 μ M particles, resulting in a larger deflection. The 20 μ M particle deflection is a deflection of M/sin γ ≧ 1 type trajectory. These small size differences result in larger differences in particle trajectories. Fig. 9B shows similar results, but as a function of small changes in the acoustic contrast factor (i.e., values of 0.03, 0.035, 0.04, 0.045, and 0.05). These results indicate that angled standing waves can be used to separate or classify particles in suspension by size, acoustic contrast factor, i.e., density and compressibility, and shape. This technique allows live cells to be separated from dead cells and even damaged cells to be separated from healthy cells. For example, table 1 lists acoustic contrast factors for several cell types.

TABLE 1

Cell type	Density (g/cc)	Speed of sound (m/s)	Acoustic contrast factor
				Jurkat T cells	1.06	1615	0.079
Primary T cells	1.04	1560	0.049
				Yeast	1.1	1700	0.12
CHO	1.03	1550	0.03

FIG. 10 shows the numerical particle trajectories of CHO cells as a function of the velocity magnitude of the fluid through the channel. These particle trajectories demonstrate the effect of normal velocity variations on particle deflection due to the mixture flowing into the acoustic standing wave at a 45 ° angle. Δ V with increasing flow velocity_Nthe/V decreases and the particles deflect at an angle. This effect provides a means to improve the ability to detect small differences in particle properties by manipulating the fluid velocity. The deflection of the particles by the standing wave may also be controlled and/or amplified by the intensity of the acoustic field, the angle of the acoustic field, the properties of the fluid, the three dimensions of the standing wave, the frequency of the standing wave, the shape of the acoustic cavity, and the mixture flow rate.

Fig. 11A compares a general analytical prediction of particle deflection with a numerical particle trajectory over a wide range of M values. The different lines in the figure represent the analytical predictions of figure 5. Each symbol represents numerical data of CFD. Each line or symbol type in fig. 11 represents a different value of M. The agreement between the analytical predictions and the numerical results is good. The errors seen in the narrow region around the wave angles of 0 ° and 90 ° are believed to be the result of anomalies occurring in these two extreme cases. The results demonstrate the importance of the deflection parameter M, the location of the maximum deflection and the presence of a steep gradient region near the point of maximum deflection.

Fig. 11B and 11C show experiments using an angled wave device with two outer channels adjacent to a central channel and a wave angle of 45 degrees. The apparatus was operated under the following conditions: 2.1MHz, a flow rate of 2ml/min for the central channel and 40ml/min for the outer channels. The outer channel contains a clarifying or buffering fluid and the central channel is provided with a fluid containing beads of a given size. For each of the four different sets of beads, the power was varied and the deflection angle of the beads with respect to the power was measured and plotted in fig. 11B. In addition, for each of the four different sets of beads, the deflection angle of the beads relative to the M factor was measured as a function of power and plotted in fig. 11C. The bead size of each of the four different sets of beads falls within a different size range for each set of beads. The size of the groups represented by circles in the figure is between 10 and 20 microns. The size of the groups indicated by triangles in the figure is in the range of 27-32 microns. The size of the groups represented by the diamonds in the figure is in the range of 32-38 microns. The size of the groups represented by squares in the figure is in the range of 45-53 microns. As shown in fig. 11B, the deflection angle of the beads was varied with power. As shown in fig. 11C, the M-factor of all beads matched well with the analysis shown by the solid black squares.

The numerical particle trajectory model can be easily modified to account for more realistic acoustic and flow fields. Computational fluid dynamics simulations can be performed to determine fluid velocities in actual fluid channel geometries. Similarly, a numerical solver for the sound field produced by the piezoelectric transducer can be used to predict a more accurate solution for the sound field. The particle trajectory model can then use the numerically predicted acoustic and fluid velocity fields to obtain a more realistic prediction. Another extension is to include gravitational and buoyant forces acting on the particles.

Two large, ultrasonic, angled wave separator configurations were prepared and tested. Two different methods are used to generate the desired fluid/acoustic interaction. The first concept is that of an Angled Wave Device (AWD), in which an angled acoustic standing wave propagates through one or more parallel fluid streams flowing in a straight pipe. The second is an Angled Fluidic Device (AFD) in which narrow fluid streams are injected and controlled to flow through the acoustic standing wave cavity at an angle to the standing wave. These large ultrasonic separators have been shown to have the potential to operate effectively at significantly higher flow rates and/or significantly lower particle concentrations than conventional acoustic separators. For example, while earlier acoustic separators typically operated at linear velocities of less than 1mm/s, the systems described in this disclosure may operate at linear velocities of up to 100 mm/s. The test results validated the analytical predictions and demonstrated the potential for separating or classifying particles in suspension by size, density and speed of sound using angled acoustic standing waves.

Fig. 12A, 12B, and 12C illustrate an AWD system 100 with a 45 ° angled standing wave. Fig. 12A is a photograph of an AWD system 100 having a plurality of

inflow ports

110, 112 on the right side and a plurality of

outflow ports

114, 116 on the left side. FIG. 12B is a schematic diagram of the system 100 showing the location of the transducer 118, reflector 120, and fluid channels. Fig. 12C is a schematic diagram showing one possible mode of operation of the AWD system 100, where the dashed lines indicate the nodal surface position of the standing wave. Fig. 12D is a schematic diagram of the flow distribution within the AWD system 100. Fig. 12E is a cross section of the AWD system 100 and fig. 12F and 12G are cross sections of alternate piping arrangements for the AWD system.

The AWD system 100 can operate in both horizontal and vertical directions. The right side shows a plurality of

inlets

110, 112, while the left side shows a plurality of

outlets

114, 116. Inlet 100 and inlet 112 are coaxial rectangular tubes having an axis 107. In the orientation shown, fluid flows horizontally through the flow chamber 109 from right to left, in this case a rectangular tube. Typically, the AWD system includes a piezoelectric material configured to be excited to produce an angled acoustic standing wave having a wavelength and an acoustic radiation force within the flow chamber that is at an acute angle relative to a mean flow direction through the flow chamber, and a minimum internal dimension of the flow chamber is at least 10 times (e.g., at least 50 times, at least 100 times, or at least 1000 times) the wavelength of the angled acoustic standing wave. In the AWD system 100, standing waves are generated at an angle of 45 to the flow direction by PZT-8, 1MHz, 1 inch by 1 inch transducer, and a stainless steel reflector. Optionally, some systems include multiple transducer/reflector pairs. The minimum internal dimension of the flow chamber 109 of the system is the height 108 of the flow chamber, which is about 0.75 inches. In the tests described in more detail below, the AWD system 100 was run vertically, flowing downward, to eliminate the gravitational effects of particle deflection. The mixture of polystyrene beads and water was pumped down through a 0.2 inch central inlet channel at a rate of 155 cm/min. In the AWD system 100, the intermediate inlet passage (inlet 110) has a cross-sectional area of about 0.15 square inches. Typically, the mixture inlet of an AWD system has a cross-sectional area of between 0.01 and 2 square inches (e.g., 0.05, 0.1, 0.25, 0.5, 0.75, or 1 square inch).

The space between the ultrasonic transducer and the reflector has a first portion within the flow chamber and a second portion outside the flow chamber. In the acoustic chamber of the AWD system 100, a thin acoustically transparent membrane 122 is used to separate the mixture flow from the prismatic void area (i.e., the second portion outside the flow chamber) created by the angled transducer and reflector. Optionally, the system may include a cooling water system fluidly connected to the prismatic void region. For example, a pump may circulate water through these zones to maintain a constant fluid temperature. In some systems, these prismatic void regions are filled with a solid material having an acoustic impedance equivalent to the primary fluid. It has been found that this approach eliminates the flow problems associated with triangular regions while allowing angled waves to pass with minimal reflection.

As shown in fig. 12E, the straight rectangular duct comprises an inner duct (inlet 110) through which the mixture of particles and host fluid flows and an outer duct (inlet 112) through which the buffer stream flows (inlet 112). The buffer flow conduit (inlet 110) completely surrounds the mixture flow conduit (inlet 112). The mixture flow conduit stops before the acoustic standing wave passes through the acoustic region of the system at an angle to the direction of flow. Then, after the acoustic standing wave, the mixture flow conduit (outlet 114) continues in a rectangular piping system. As a result, the angled acoustic standing wave passes through both the mixture flow and the buffer flow, as shown in fig. 12C. The system has two inlet streams entering the acoustic standing wave and two outlet streams exiting the standing wave. The inlet and outlet conduits are aligned. The acoustic standing wave is at an angle to the direction of flow in the pipe.

The flow rate is set to produce laminar flow in the chamber and operates below 200 reynolds number based on equivalent conduit diameter. A low reynolds number results in shear-dominated flow, with no turbulence. The flow rates are set in three of the four streams. Two inlet flow rates are provided to push the flow, and either outlet flow conduit can be provided to pull the flow. This push and pull operation ensures that the flowing fluid remains laminar and straight, and also provides a method of modifying the flow profile for desired particle separation. The average buffer flow rate may be set higher or lower than the average flow rate of the mixture stream. As the mixture flow passes through the angled acoustic standing wave, the particles in suspension will be deflected downward along the wave front, as shown. The deflection of the particles from the horizontal direction may vary from zero to the wave direction. The deflection is a factor of the M factor. If the M factor is large enough to block flow through the wave, the particle will propagate along the wave angle. The particles will be carried by the fluid velocity component parallel to the wave. The primary fluid direction will be acoustically unaffected and will travel horizontally to the mixture outlet duct as shown.

A typical velocity profile through the acoustic portion of the AWD system 100 is shown in fig. 12D. The flow is at a very low reynolds number, which rapidly creates shear flow in the pipe. The fluid is to flow in multiple layers or laminar flow. This is why at low reynolds numbers the cylinder has a lower drag coefficient than the sphere. Three-dimensional regions in the shape of pipes should be avoided. The inner mixing flow conduit should have a high aspect ratio (e.g., at least 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 100:1) such that the mixture conduit provides for stable, approximately two-dimensional flow. In the AWD system 100, the aspect ratio is about 7: 1.

As shown in fig. 12E, the buffer flow around the pipe at the side edges is expected to limit the wall boundary flow effect. Due to viscous dissipation at low reynolds numbers, no eddy currents will be present. As a result, as shown, a fully developed two-dimensional laminar flow profile will develop rapidly in the pipe and into the vicinity of the acoustic region in both the mixture pipe and the buffer pipe. The buffer flow rate is set to provide rapid strengthening of the shear layer between the streams to provide a near constant velocity in the flow of the mixture through the angled standing wave. As shown in fig. 12D, the mixture flow conduit terminates before the acoustic region to eliminate shear layers between the flows. Typically, the distance d between each inlet and the space between the ultrasound transducer and the reflector 120 (where the angled waves are formed)₁Between 0.025 inches and 2 inches (e.g., 0.5, 0.05, 0.25, 0.5, 0.75, or 1 inch). In the AWD system 100, the distance d between each entrance and the space between the ultrasonic transducer 118 and the reflector 120 (where the angled waves are formed)₁About 0.5 inches.

Fig. 13 is a particle size distribution of polystyrene beads used to test the AWD system 100. The beads used had an average diameter of about 150 μm and a size as small as 20 μm and as large as 220 μm. The mixture contained two grams of beads per liter of water. This allows visual observation of the mixture flow. A water buffer stream was pumped around and parallel to the mixture at a rate of 23 cm/min. The electrical power of the transducer was varied from zero to 3.2 watts (W) and the particle deflection was recorded. A wide range of particle sizes results in greater variation of the M parameter. It is expected that for a certain power and fluid velocity, larger particles will deflect at a wave angle of 45 °, while smaller particles will not deflect at all or at a small angle.

Fig. 14A-14F are photographs of polystyrene beads 122 flowing through the AWD system 100 during testing to show bead deflection as a function of electrical power input to a 1MHz transducer establishing an acoustic standing wave at 45 degrees. These figures show that bead deflection increases with increasing power (0W in fig. 14A, 0.8W in fig. 14B, 1.5W in fig. 14C, 1.8W in fig. 14D, 2.4W in fig. 14E and 3.2W in fig. 14F). In these photographs, the mixture flow is from right to left, gravity is from right to left, and the acoustic standing wave axis is from top left to bottom right in the direction of the model window shown. Figure 14A shows the flow of the mixture without sound. In the absence of acoustic forces, the beads 122 flow horizontally with the fluid and no particle deflection is observed, all beads flow to the outlet region 123.

The M-factor and particle deflection increase directly with the power supplied to the transducer. For powers up to 1.5W, the flow of the mixture is seen to deflect downwardly at an angle less than the wave angle as it moves from right to left through the angled wave (see fig. 14B). At 1.5W, the larger beads start to deflect along the angled wavefront, while the smaller particles pass directly through the acoustic field, exhibiting a grading phenomenon (fig. 14C). When the power was increased above 1.5W, the medium and smaller sized beads were deflected at a wave angle of 45 ° (fig. 14D and 14E) until the power was 3.2W and all visible beads were deflected along the standing wave (fig. 14F). Furthermore, as the power increases, the exit areas 123 of the smaller beads that are not deflected along the standing wave begin to exhibit a gradually increasing deflection.

Using the M factor, bead diameter changes were calculated based on the power change measured from the first noted bead deflection along the wavefront to all bead deflections. Analytical calculations show that large particles of 200 μm are deflected along the wavefront at a power of 1.5W, whereas most of all particles larger than 130 μm are deflected along the wavefront at a power of 3.2W. Analysis predicts that the same value of the product of particle size and the square of the acoustic energy (which is proportional to power) produces the same particle deflection. The results are in good agreement with the bead size distribution recorded and the observed bead behaviour. These test results validate the analytical model and demonstrate the ability to select and differentiate dimensions or material properties using angled wave techniques.

Some AWD systems have a third outlet configured to concentrate the deflected material. For example, these systems may have a second outlet disposed between the first outlet and a third outlet, wherein the cross-sectional area of the third outlet is less than the cross-sectional area of the second outlet.

Fig. 15A and 15B illustrate an AWD system 200 having such a piping configuration. The AWD system 200 is configured to concentrate particles or cells by lowering the mixture conduit 110 and compressing the lower buffer stream. After flowing through the acoustic waves, an outlet mixture conduit 114 is attached to the sidewall to provide higher concentration particle collection. The attachment of this mixture conduit is done after the flow through the acoustic field so that the buffer flow surrounds the inlet mixture conduit 110 before the acoustic wave and thus provides a good flow distribution and particle concentration. The tubing flow rate may be varied by a push/pull mechanism as described above to help achieve the desired separation and concentration. The mixture conduit wall attachment isolates the outlet conduit 124 from the buffer flow conduit (outlet 116). D is the height of the outlet mixture conduit 114. h is the height of the buffer flow conduit 116 above the mixer conduit 114. d is much smaller and is the height of the third outlet duct 124. In the AWD system 200, the width of the catheter is the same. If the velocities in the two conduits are the same, the concentration ratio should ideally be D/D. D/D may vary accordingly. In AWD systems having this configuration, the height D of the outlet mixture conduit 114 is typically between 2 and 100 times (e.g., 3, 5, 10, 25, 50, 75 times) the height D of the third outlet wall buffer conduit 124. If the velocities in the two pipes are the same, the ratio of the flow velocity of the mixture to the lower buffer stream should ideally be D/D. In the AWD system 200, the height D of the mixture conduit 114 is about 3 times the height D of the third outlet 124. The height h of the buffer inlet 112 and buffer outlet 116 may be much smaller than shown in the figures and is chosen for the specific application with the CFD.

Some AWD systems have a plurality of third outlets, each of which is offset from the axis of the second outlet in the direction of deflection of the angled acoustic wave.

Fig. 16 is a schematic diagram of an AWD system 300 configured for particle classification. In the orientation of fig. 16, the direction of deflection of the angled sound waves is downward, and a plurality of collection conduits 124 a-124 e are provided below the mixing conduit (outlet 114) to collect particles of different sizes. The AWD system 300 has five collection conduits 124 a-124 e, but some AWD systems configured as a cascade have more collection conduits (e.g., 10 collection conduits, 15 collection conduits, or 20 collection conduits) or fewer collection conduits (e.g., 4 collection conduits, 3 collection conduits, or 2 collection conduits). The M factor is set in zone 1, which, together with the push/pull flow rate setting, provides for operation with different deflections with different particle sizes. One example configuration has an overall system height of one inch, with five collection channels, with an overall spacing of a typical distance of 0.4 inches. The system can be scaled up or down as needed to accommodate smaller or larger flow rates. Fractionation systems can be used, for example, to enrich cells from leukopack (e.g., to fractionate different cells, such as erythrocytes, monocytes, granulocytes, and lymphocytes); for ranking an initial population of T cells by size; for fractionating the affinity bead/cell complexes from unbound free cells; or for fractionating populations of free cells, affinity bead/cell complex a and affinity bead/cell complex B. The use of an M factor may facilitate the design and operation of the acoustic separation system.

In one example, it is desirable to separate a mixed population of two particles (one 5 micron in size and one 10 micron in size) of the same material in a 45 ° angled wave device. The operating parameters (e.g., flow rate, power, and frequency) are selected so that the M factor of the larger particles is M₁₀0.8. To pairFor a 45 ° angled wave device, this M factor results in a 45 degree particle deflection for a ten micron particle. Since M is proportional to the square of the particle radius, the M factor for smaller particles is M₅0.8/4-0.2. The deflection angle of the particles was about 2 degrees. Thus, a suitable angled wave setting with a wave angle of 45 ° can rank the two populations.

In a second example, the aim is to fractionate three different cells (i.e. lymphocytes, monocytes and neutrophils), which are cells present in the leukocyte population. A typical size of lymphocytes is 6 microns. Monocytes and neutrophils were about 10 microns. In addition, the acoustic contrast ratio of lymphocytes (acoustic contrast factor) is smaller than that of monocytes. The 45 ° angled wave device (45 ° shaped wave device) can be adjusted so that the monocyte M-factor (M-factor) is 0.75. Neutrophils of the same size and slightly smaller acoustic contrast coefficient have a slightly smaller M-factor of about 0.725. Smaller lymphocyte M factor as monocyte M factor (6/10)²Either 0.36 or 36% scaling, results in an M factor of 0.27. The deflection curve at a wave angle of 45 ° indicates that monocytes and neutrophils are deflected at 45 ° while lymphocytes are deflected at about 5 °. A system with appropriately designed outlets enables the independent harvesting of monocytes and neutrophils in one channel and the harvesting of lymphocytes in a separate outlet, thereby isolating and enriching lymphocytes.

In a third embodiment, the goal is to fractionate the output of the affinity cell selection process. 25 micron affinity beads were used for TCR + T cell negative cell selection treatment. The TCR + T cells are bound to the affinity beads and form a complex of the affinity beads and a plurality of TCR + T cells attached to the beads. TCR + T cells are not bound and thus remain in solution as free unbound cells. The tilted wave device was then used to fractionate the two populations, free unbound TCR cells from the affinity bead/TCR + cell complex. The radius of the T cells was about 6 microns. Thus, the ratio of the M factors is (25/6)²17. Selecting system parameters such that affinity is achievedThe sex/cell complex has an M factor of 1, which causes the complex to deflect at wave angles. Unbound free cells then have an M factor of 1/17 ═ 0.06, which means that the free cells are deflected at an angle of less than 1 °, completing the fractionation process of the affinity bead/cell complexes from the free cells.

In a fourth example, the goal was to fractionate mixed cell populations composed of cells of similar size but different acoustic contrast coefficients, where cell a had a contrast coefficient of 0.03 and cell B had a contrast coefficient of 0.06. Cell a was separated from cell B using a 45 ° inclined wave system. The system was adjusted so that the M factor of cell B was 0.75, thereby deflecting cell B at an angle of 45 °. Since the M factor is scaled by the contrast ratio, the M factor for cell a is 0.75/2 to 0.375, resulting in cell a deflection at about 5 °. A properly designed system should be able to separate cells a deflected at 5 ° from cells B deflected at 45 °.

17A, 17B, and 17C are schematic diagrams of aspects of an example AFD system 400. Fig. 17A shows the system geometry and flow characteristics. Fig. 17B shows particle transfer between fluids.

FIG. 17A shows an AFD system 400 having an acoustic chamber with an ultrasonic transducer 118 on one side, a reflector 120 on the opposite side of the chamber, and a plurality of

flow inlets

110, 112 and flow

outlets

114, 116. the ultrasonic transducer 118 and chamber are designed to produce a bulk ultrasonic standing wave that moves horizontally within the chamber, as shown in FIG. 17A. the vertical hatching shown in this figure represents the nodal plane location of the standing wave. two

inlets

110, 112 are shown at the top right of the chamber and two

outlets

114, 116 are shown at the bottom left of the chamber. a first channel 110 'terminates at the first inlet 110 and a second channel 112' terminates at the first inlet 112. the channels 110 ', 112' make an α angle of 60 ° with the plane perpendicular to the angled acoustic standing wave (in the horizontal direction). in the AFD system 400, the first channel 110 'and the second channel 112' each have a substantially straight portion that extends at least 0.5 inches from their

respective inlets

110, 112.

The two lower outlet ducts are at an β angle of 70 ° from horizontal, in some systems, the angles α and β are the same, in some systems, the four ducts each enter the acoustic chamber at different angles, however, these angles vary between 0 degrees and 90 degrees in some systems, the angle α is 30 ° to 88 ° (e.g., greater than 35 °, greater than 40 °, greater than 45 °, greater than 50 °, greater than 55 °, greater than 60 °, less than 80 °, less than 75 °, less than 70 °, less than 65 °, less than 60 °, less than 55 °, less than 50 °).in some systems, the angle α is 30 ° to 88 ° (e.g., greater than 35 °, greater than 40 °, greater than 45 °, greater than 50 °, greater than 55 °, greater than 60 °, less than 80 °, less than 75 °, less than 70 °, less than 65 °, less than 60 °, less than 55 °, less than 50 °).

Fluid enters the acoustic chamber through an inlet conduit and exits the chamber through an outlet conduit. Typical conduit dimensions are 0.5 to 1 inch channel depth and 0.1 to 0.4 inch channel width. The flow rate is set to produce laminar flow within the chamber and operates at a reynolds number of less than 200 based on the conduit diameter. A low reynolds number results in a shear dominated flow without turbulence. The flow velocity is set in three of the four conduits attached to the acoustic chamber in fig. 17A. The convection current is both pushing and pulling. Two inlet flow rates are set to push the flow and an outlet flow carrying particles is set to pull the flow. This push-pull operation ensures that the flow (streams) can flow to the desired place. The outlet mixture flow may be set above or below the flow rate into one of the inlet conduits. A typical flow profile is shown in the chamber of fig. 17A. A well developed laminar flow profile enters the acoustic chamber as shown. The wall shear layer between the chamber inlets is rapidly mixed. A fairly uniform flow develops for a while near the interface of the two injected flows as shown in fig. 17A. The flow shear forces on the fluid at the corners cause the flow to rotate and will generate large scale vortices as shown. This flow rotation will be a slow solid rotation because of its low reynolds number. The flow rates at both the inlet and outlet are controlled. In most operations, two inlet flow conduits are specified and one of the outlet flow conduits is specified. This type of operation is known as push/pull. The acoustic standing wave may be planar (planar) or three-dimensional. Planar standing waves are preferred. The wall of the flow chamber adjacent the first outlet in the direction of deflection of the inclined acoustic wave may extend at an acute angle relative to a plane perpendicular to the inclined acoustic standing wave. In the AFD system 400, the lower wall of the chamber is tilted downward by an angle γ as shown. The wall inclination angle is designed to help collect particles deflected by acoustic radiation forces. Some AFD systems have wall tilt angles of 1 to 20 ° (e.g., greater than 2 °, greater than 3 °, greater than 5 °, greater than 10 °, less than 15 °, less than 10 °, less than 7.5 °, less than 5 °).

Fig. 17B presents the AFD system 400 operating with a fluid mixture having suspended particles entering through the inlet 110 and clarified fluid entering the chamber in the inlet 112. The particles are considered to have a positive acoustic contrast coefficient, which means that they will deflect towards the nodal plane surface as shown. In this way, all particles are deflected in a downward direction. The inclined walls at the bottom of the chamber allow particles to fall from the acoustic field without being trapped in the wall shear layer or being retained by acoustic edge effects.

Fig. 17C schematically depicts the fluid flow direction of the AWD system 400. The fluid velocity is decomposed into a component orthogonal to the acoustic standing wave nodal plane and a component tangential to the acoustic standing wave nodal plane. The normal direction represents the axial direction of the standing wave. For plane waves, this is the direction of the radiation force acting on the particles in the mixture. Thus, the radiation force slows down and accelerates the normal velocity component of the particle relative to the fluid normal velocity. The tangential velocity component of the particles remains the same as the fluid. As a result of this effect, the particles are deflected at an angle towards the downward direction to the fluid. If the radiation force is large enough, the normal velocity of the particles approaches zero and the particles will move vertically downward as the fluid continues to flow through the chamber toward the outlet conduit. It is important to recognize that these particle deflections use fluid velocity, and more specifically the component of fluid velocity in the downward direction. The fluid carries the particles downward. This effect is completely decoupled from gravity. This process is gravity independent.

Fig. 18A is a photograph of a prototype of the AFD system 400 tested, and fig. 18B is a schematic diagram showing the placement of the fluid streamlines from the CFD predictions. In fig. 18B, red indicates the mixture flow and blue indicates the buffer flow. The CFD results show that the streams are regular and homogeneous without any mixing between the streams. There are two

inflow ports

110, 112 and two

outflow ports

114, 116 in the AFD system 400. The top inlet is at an angle of 60 deg. to the horizontal. The outlet is at 70 deg.. A pump is used to control the amount of flow entering the acoustic chamber through the inlet and the amount of flow exiting the outlet (push-pull control). The AFD device was tested using a 1MHz acoustic standing wave running at 1W. The two streams are angled at about 30 deg. with a standing wave in the acoustic chamber.

Fig. 19A and 19B are photographs of a sound chamber window of an AFD system showing particle motion through the AFD system without acoustic effects (fig. 19A) and particle motion through the AFD system with acoustic effects (fig. 19B). The test was performed with a flow of 200ml/min through all inlets and outlets. This produced a mixture flow velocity of about 160 cm/min. The flow of the mixture of polystyrene beads 122 and water is readily visible. The mixture was 2 grams of beads 122 per liter of water. The beads 122 are those described in connection with fig. 13.

In fig. 19A (acoustic effect off), the mixture flow is seen to flow from the upper left inlet 110 directly to the lower left outlet 116, as predicted by CFD. The second stream is water, which is not visible in the photograph. Fig. 19B (acoustic effect on) shows the effect of the tilted standing wave on the movement of the beads 122. The tested M/sin (gamma) parameter is greater than 1.0. The beads 122 are deflected along the sloping wave front (wave front) almost immediately as they enter the acoustic chamber. This causes all visible beads 122 to move vertically from the mixture stream to the buffer stream, down to the bottom of the chamber and into the lower right outlet 114. This result occurs at all buffer flow rates, illustrating the ability of this system to be used for particle washing, or for particle separation and/or collection at high flow rates when compared to conventional ultrasonic separation systems. The AFD system 400 is not limited to two streams and may be modified to include a number of different angle changes. The AFD system 400 has the potential to work with a variety of fluid/particle mixtures where the suspended matter can be beads, cells, exosomes (exosomes), viruses, oil droplets, or any material with a different density, compressibility, or contrast coefficient than the host fluid. The system can work with nanoparticles because the acoustic radiation force effect is amplified by the angle created by the acoustic wave for flow.

Some systems are configured to provide fractionation by providing constriction within an outlet channel positioned to receive deflected material. For example, the fourth channel terminating at the second outlet may have a first cross-sectional area. The third passageway terminating at the first outlet may have a first portion with a first cross-sectional area and a second portion with a second cross-sectional area, wherein the second cross-sectional area is smaller than the first cross-sectional area, the second portion of the third passageway being located between the first outlet and the first portion of the third passageway.

Fig. 20A and 20B illustrate this method of increasing the concentration of a mixture of particles extracted with an AFD system. In the AFD system 500, the lower outlet duct 114 is constricted proximate the acoustic chamber. This constriction is shown in the particle outlet, which is pulled to the desired flow rate, for example by a pump. Since the flow rate is set by the pull rate, any area shrinkage results in an increase in velocity. Exit conduit constriction D/D increases flow velocity by the D/D of the two three-dimensional conduits shown in fig. 20A and 20B. Fig. 20B presents an approximate flow profile with moderate constriction, where the pull flow velocity is such that the peak velocity in the acoustic chamber occurs in a region near the constriction region of the outlet conduit. The higher velocities present near the entrance of the

outlet conduits

114, 116 and near the separation streamlines separating the two streams will provide better separation for fewer particles moving back into the mixture stream. Higher velocity means a higher tangential velocity component downward carrying the particles (e.g., beads 122). For example, if the flow rates in all four conduits are set to be the same by the push-pull mode and are contracted to 90% of the conduit area, the velocity near the inlet of the Q4 outlet conduit increases by a factor of 10 when compared to the second outlet conduit flow rate Q3 or relative to the inlet flow rates of the two conduits (Q1 and Q2). This effect is the reflected flow distribution shown in fig. 20B. The length of the constriction region shown in the schematic of the velocity profile provides the velocity direction towards the constriction channel. This means that there is less chance of particles re-entering the original mixture stream and better separation efficiency. In the same configuration, and still employing 90% constriction, if the flow rate is set so that the Q4 outlet conduit has one tenth of the flow rate into the inlet conduit, the throttling velocity is nearly or exactly the same as the unthrottled outlet conduit velocity in Q3, thus still providing the downward velocity component required to separate particles, while allowing much less fluid to flow at the outlet conduit flow rate Q4. This arrangement results in a mixture concentration of perhaps 10 times achieved with each pass through the AFD separator.

FIG. 21 presents an AFD system 600 designed for particle fractionation. Four

inlet conduits

110, 111, 112, 113 and four

outlet conduits

114, 115, 116, 118 are shown. The different shading represents the CFD prediction, which shows the ability to maintain four oblique flows through the acoustic chamber. Again, the push/pull operation allows for unique confinement within the acoustic chamber. Some AWD systems contain many more streams. All four streams flow through the chamber at an angle to the acoustic standing wave. If the top or blue stream is a mixture of a fluid mixture with a plurality of particle size suspended particles, the particles can be fractionated into the lower three collection conduits shown in FIG. 21 using an oblique wave deflection method. The system may be operated with an M factor that allows different particles to be deflected for the collection configuration shown. The same AFD system shown in fig. 21 can be expanded to have five or more inlet conduits and five or more outlet conduits. The incoming mixture and buffer streams can then flow through a number of different pairs of adjacent conduits. Operating with push/pull technology, this causes particle separation to occur at a variety of different wave angles. In the same way, different conduit flows can be used to set different velocity profiles for different particle distribution requirements.

22A, 22B, and 22C are each a schematic, graph, and cross-section of a simulated flow velocity and AFD system 700 designed for particle collection. Fig. 22A shows flow through the system. The system contains one inlet conduit 110 and one outlet conduit 116 attached to the acoustic chamber. The collection region 130 is shown below the flow through the acoustic chamber at an angle to the standing wave as shown. This collection is enhanced by the large scale collection of vortices shown in figure 22A. This collection vortex is driven by the flow flowing through the chamber and provides a tangential velocity component parallel to the nodal plane which can carry particles from the mixture stream down into a collection region. The trapped vortex can be further enhanced by drawing fluid through a collection outlet 114 located at the bottom of the system. Switching the acoustic effect in a suitable manner to allow the particles to fall to the collection bottom may also improve performance.

Fig. 23A and 23B are a cross-section and schematic, respectively, of a low angle AFD system 700. The M parameter can be used to determine the operational characteristics required for deflecting very small particles (e.g., 100nm to 1000nm, or 10nm to 100nm, or 1 to 10nm nanoparticles; bacteria; viruses, such as lentiviruses (lentiviruses) or retroviruses (retroviruses), adeno-associated viruses, exosomes, microvesicles (microviscles), and other nano-sized particles) in suspension. The smaller the particle size, the lower the M factor. In systems where the flow velocity is reduced as low as possible for system feasibility, the power is increased as much as possible, and then the M operating curve specifies that the system should be operated at as low a wave angle as possible. Typical operating parameters for these systems are angles close to between 0 and 15 degrees, frequencies of 2 to 50MHz, sound pressure amplitudes of 1 to 20 MPa; and the line speed is about 10mm/s, 1mm/s or 0.1 mm/s. For low values of M, the deflection peak is at a lower wave angle. The AFD system 700 is configured for use with nanoparticles. Two

inlet flow conduits

110, 112 flow into the acoustic chamber and two

outlet flow conduits

114, 116 are used to exit the flow. Entrance angle

And exit angle

Are all about 5. In other ASWD systems, the entry angle

And exit angle

At different angles (e.g., 2 ° to 10 °, greater than 3 °, greater than 4 °, less than 9 °, less than 8 °, less than 7 °, less than 6 °). Again, push/pull techniques may be used to control the flow to produce the desired particle fractionation.

The systems and methods described in this disclosure may provide large ultrasonic separators that use a bulk acoustic standing wave angled to the direction of the fluid mixture flow field to create particle deflection that may be used to collect, differentiate, separate, purify, or fractionate one particle or population of cells from a mixture that may contain multiple different types of particles. The particle trajectory equations provide key physical properties. A generic prediction curve developed for particle deflection at all wave angles as a function of a dimensionless parameter M defined by the ratio of acoustic radiation force to viscous force acting on the particle can be used in system design and operation. The particle deflection, measured by the direction of fluid flow, varies continuously from zero to a maximum equal to the wave angle γ (which is the angle produced by the flow direction for the standing wave). The analysis result is quite consistent with the track numerical calculation and the model test result. The sound pressure amplitude, particle diameter and wave angle are shown to have the greatest effect on particle deflection.

The results also show that for any sound pressure amplitude of the standing wave, there is a wave angle of the standing wave at which the radiation force ceases to be orthogonal to the wave; thus, the particles start to move along the wavefront. This is defined by a dimensionless parameter M, the wave angle γ and the operation close to this point produce a large particle deflection with small controllable parameter (e.g. acoustic power or flow velocity) variations. This operating point is very useful because it allows the separation of particles having slight size, rigidity or density differences.

Some of these systems and methods use standing waves at an angle to the flow channel or narrow streams injected at an angle through a stationary acoustic chamber. These two systems are shown to effectively separate polystyrene beads from a high velocity flowing mixture when compared to conventional ultrasonic separators. It has also been shown that large scale ultrasonic separators operate effectively at much higher flow rates or at much lower particle concentrations than conventional ultrasonic separators. The model test results agree well with theory and confirm the developed prediction system. The tilted wave system may work with a fluid/substance mixture in which the suspended substance may be microcarrier beads, cells, exosomes, viruses, oils or any material with a different density, compressibility or contrast coefficient than the main stream. Analytical models predict that the system can work theoretically even with nanoparticles, since the acoustic radiation force effect is amplified by the angle produced by the flowing acoustic waves.

Fig. 24A-24C present the results of using an AWD system similar to the system shown in fig. 12A-12E to fractionate T cells from 35 μm beads. The system had a 30 ° wave angle and was operated at a frequency of 2.1MHz with a flow rate of 5ml/min of T cell/bead mixture through the central inlet and a flow rate of 30ml/min of buffer through the buffer inlet. Fig. 24A and 24B are schematic diagrams illustrating the expected separation of T cells from beads. Fig. 24C presents the results. When the system was operated without the use of acoustic effects, 98% of the T cells and 95% of the beads flowed through the central outlet. When a power of 2.3W was applied, 92% of the T cells flowed through the central outlet and 100% of the beads were deflected into the buffer outlet.

FIGS. 25A-25C present results of using the same AWD system to fractionate mixed populations of beads predominantly 10 μm-29 μm and 32 μm-42 μm in size. The system had a 30 ° wave angle and was run at a frequency of 2.1MHz with a flow rate of 2ml/min of bead mixture through the central inlet and a flow rate of 40ml/min of buffer through the buffer inlet. The resulting linear flow velocity was 48 cm/min. Fig. 25A is a schematic illustrating the expected separation of larger beads from smaller beads. Fig. 25B and 25C present the results. When the system is operated without the use of acoustic effects, a majority of the two sizes of beads flow through the central outlet. When a power of 1.5W is applied, most of the smaller beads still flow through the central outlet but most of the larger beads are deflected into the buffer outlet.

FIGS. 26A-26C present the results of using the same AWD system to fractionate populations of PMMA beads ranging in size from 5 μm to 20 μm. The system had a 30 ° wave angle and was run at a frequency of 2.1MHz with a flow rate of 2ml/min of bead mixture through the central inlet and a flow rate of 40ml/min of buffer through the buffer inlet. The resulting linear flow velocity was 48 cm/min. Fig. 26A, 26B and 26C show the distribution of beads between the central outlet and the buffer outlet in three cases: the case where the acoustic effect is not used, the case where the power of 1W is applied, and the case where the power of 1.2W is applied. When the system is operated without the use of acoustic effects, most of the beads flow through the central outlet. When 1W of power is applied, the larger beads start to be preferentially diverted into the buffer outlet. When a power of 1.2W was applied, most beads larger than 12 μm were deflected into the buffer outlet. These results illustrate the ability to selectively fractionate materials with minimal differences.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

For example, fig. 27 shows a 10 ° AWD system 900 with a central channel and a buffer channel in the vicinity thereof. The central passage may be circular or rectangular in cross-section. A typical cross-section of the system may be 0.1 '. times.0.1' to 1 '. times.1' channel with a central channel width of 1/2 to 1/10 of the channel cross-section. Applications include cell fractionation, bead-cell fractionation.

In another embodiment, fig. 28 shows an AWD system 930 with a small inlet on one side and a buffer flow on top of it, and with 5 outlets where different parts of the mixture population end. Typical dimensions for the channels may range from 0.25 "by 0.25" to 1 "by 1", with the opposite side entrance widths varying from 1/2 for the channel width to 1/10 for the channel width. Applications include, for example, leukopack fractionation, T cells.

In another embodiment, fig. 29 shows an AWD system 960, where the flow can be viewed through 2 glass windows. An attachment is included so that the system can be suspended vertically with the aid of a metal rod. The tilted wave system 960 is identical to the 10 deg. AWD system 900. The system may be configured at wave angles of 5 to 85 degrees.

Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A system for separating material from a host fluid, the system comprising:

a flow chamber defining a mean flow direction;

an ultrasonic transducer comprising a piezoelectric material configured to be excited to generate an angled bulk acoustic standing wave having a wavelength and an acoustic radiation force in the flow chamber and oriented at an acute angle relative to a mean flow direction through the flow chamber, wherein the flow chamber has a minimum internal dimension that is at least 10 times the wavelength of the angled acoustic standing wave;

a reflector opposite the at least one ultrasonic transducer;

a first inlet fluidly connected to the flow chamber;

a second inlet fluidly connected to the flow chamber;

a first outlet fluidly connected to the flow chamber; and

a second outlet fluidly connected to the flow chamber.

2. The system of claim 1, wherein the first inlet is at least 0.1 inches from the angled bulk acoustic standing wave.

3. The system of claim 1, further comprising a first channel terminating at the first inlet, wherein the first channel has a substantially straight section extending at least 0.1 inches from the first inlet.

4. The system of claim 1, wherein a space between the ultrasound transducer and the reflector comprises a first portion inside the flow chamber and a second portion outside the flow chamber.

5. The system of claim 4, further comprising an acoustically transparent material separating the first portion from the second portion.

6. The system of claim 4, further comprising a cooling water system fluidly connected to the second portion.

7. The system of claim 4, wherein the second portion is filled with a solid material having an acoustic impedance equal to an acoustic impedance of the host fluid.

8. The system of claim 1, wherein the system comprises a plurality of ultrasound transducers.

9. The system of claim 1, wherein the first inlet and the second inlet are coaxial.

10. The system of claim 9, wherein the first outlet and the second outlet are coaxial.

11. The system of claim 9, wherein the first inlet has a rectangular cross-section.

12. The system of claim 11, wherein the rectangular cross-section of the first inlet has an area of at least 0.01 square inches.

13. The system of claim 1, wherein the first inlet has an aspect ratio of at least 5.

14. The system of claim 1, further comprising a third outlet, wherein the second outlet is disposed between the first outlet and the third outlet, and the cross-sectional area of the third outlet is less than the cross-sectional area of the second outlet.

15. The system of claim 14, wherein the second outlet has a rectangular cross-section and the third outlet has a rectangular cross-section.

16. The system of claim 15, wherein the width of the second outlet is the same as the width of the third outlet.

17. The system of claim 16, wherein the height of the second outlet is at least 2 times the height of the third outlet.

18. The system of claim 1, further comprising a plurality of third outlets, each offset from an axis of the second outlet in a direction of deflection of the angled acoustic wave.

19. The system of claim 1, further comprising a first channel terminating at the first inlet, wherein the first channel has a substantially straight segment extending at least 0.1 inches from the first inlet and forming a first acute angle with respect to a plane perpendicular to the angled acoustic standing wave.

20. The system of claim 19, further comprising a second channel terminating at the second inlet, wherein the second channel has a substantially straight segment extending at least 0.1 inches from the second inlet and forming a second acute angle with respect to a plane perpendicular to the angled acoustic standing wave.

21. The system of claim 20, wherein the first acute angle and the second acute angle are equal.

22. The system of claim 19, further comprising a third channel terminating at the first outlet, wherein the third channel has a substantially straight segment extending from the first outlet and forming a third acute angle with respect to a plane perpendicular to the angled acoustic standing wave.

23. The system of claim 22, wherein the first acute angle and the third acute angle are equal.

24. The system of claim 22, further comprising a fourth channel terminating at the second outlet, wherein the first outlet is located in a direction of deflection of the angled sound wave relative to the second outlet, wherein the fourth channel has a first cross-sectional area, the third channel has a first portion having the first cross-sectional area and a second portion having a second cross-sectional area smaller than the first cross-sectional area, and the second portion of the third channel is located between the first outlet and the first portion of the third channel.

25. The system of claim 22, wherein the third passageway has a substantially straight segment extending from the first outlet at a third acute angle.

26. The system of claim 19, wherein the first acute angle is 80 to 90 degrees.

27. The system of claim 1, wherein a wall of the flow chamber adjacent to the first outlet in a deflection direction of the angled acoustic wave extends at an acute angle relative to a plane perpendicular to the angled acoustic standing wave.

28. The system of claim 27, wherein the acute angle is 1 to 20 degrees.

29. A system for separating material from a host fluid, the system comprising: a flow chamber extending between a first end and a second end;

an inlet at a first end of the flow chamber;

a first outlet located between a first end of the flow chamber and a second end of the flow chamber, the inlet and the first outlet defining an average flow direction through the flow chamber;

an ultrasonic transducer comprising a piezoelectric material configured to be excited to produce an angled acoustic standing wave between the inlet and the first outlet, the angled acoustic standing wave having a wavelength and an acoustic radiation force in the flow chamber and being oriented at an acute angle relative to a mean flow direction through the flow chamber; and

a reflector opposite the at least one ultrasonic transducer;

wherein the first outlet is spaced from the second end of the flow chamber.

30. The system of claim 29, wherein the flow chamber has a minimum internal dimension that is at least 10 times a wavelength of the angled acoustic standing wave.

31. The system of claim 29, wherein the first outlet is at least 0.5 inches from the second end of the flow chamber.

32. The system of claim 29, wherein the flow chamber has a distance between the first end and the second end, and the first outlet is at least 30% of the distance from the second end.

33. The system of claim 32, wherein the first outlet is at most 70% of the distance from the second end.

34. The system of claim 29, further comprising a second outlet at a second end of the chamber.

35. A method of separating material from a host fluid, comprising:

flowing an initial mixture of the primary fluid and the material at a flow rate into an acoustophoresis device via an inlet, the acoustophoresis device comprising:

an acoustic isolation chamber in communication with the inlet;

an ultrasonic transducer coupled to the acoustic isolation chamber and arranged to be excited to produce sound waves at an angle to the average flow direction of the initial mixture;

controlling a ratio of an acoustic radiation force generated by the ultrasonic transducer to a viscous drag force of the initial mixture to deflect the material passing through a first subset of the acoustic waves at a different angle than a second subset of the material, thereby allowing separation of the first subset from the second subset.

36. The method of claim 35, further comprising controlling the ratio by controlling one or more of the angle, the flow rate, an excitation frequency of the ultrasound transducer, or a power supplied to the ultrasound transducer.

37. The method of claim 35, further comprising controlling the ratio based on one or more subsets of characteristics.

38. The method of claim 37, further comprising controlling the ratio based on one or more of material size, density, compressibility, or acoustic contrast factor.

39. The method of claim 35, further comprising controlling the ratio to deflect at least some of the material at an angle of the acoustic wave.

40. The method of claim 35, wherein the material further comprises a third subset different from the first subset and the second subset, and the controlling the ratio further comprises deflecting the third subset at an angle different from the first subset or the second subset.

41. The method of claim 36, further comprising controlling the ratio to be within a range determined by characteristics of a subset of materials in the mixture to be separated.

42. The method of claim 41, wherein the range is determined by the relative sizes of the materials in the sub-groups to be separated.

43. The method of claim 42, wherein the range spans at least one order of magnitude.

44. The method of claim 35, further comprising collecting the first or second subset in a collection conduit in communication with the acoustic isolation chamber.

45. The method of claim 35, wherein the material comprises at least two subsets of particles, cells, or fluids having different properties.