阅读说明：本技术 声边缘效应 (Acoustic edge effect ) 是由 B·利普肯斯 B·罗斯-约翰斯鲁德 K·C·奇塔莱 K·N·库马尔 J·萨罗伊奥 于 2020-05-15 设计创作，主要内容包括：可以提供具有大量多向梯度的声场的声波用于形成相对于声波的界面区域的边缘效应。界面区域可以阻挡具有与界面区域的特性相关的某些特性的材料。受界面区域的声特性影响较小的其他材料可以通过声波。该技术允许使用边缘效应和界面区域分离材料。(An acoustic wave having a sound field with a large number of multidirectional gradients can be provided for creating an edge effect relative to the interface region of the acoustic wave. The interface region may block material having certain properties related to the properties of the interface region. Other materials that are less affected by the acoustic properties of the interface region may pass acoustic waves. This technique allows the materials to be separated using edge effects and interfacial regions.)

1. A method for separating a material from a fluid mixture, the method comprising:

flowing a fluid mixture containing the material into a chamber;

generating sound waves with an acoustic transducer at one end of the chamber;

establishing an interface region in the vicinity of the acoustic wave; and

using an acoustic field generated at the interface region to prevent the material from flowing with the fluid.

2. The method of claim 1, wherein the material is captured by a multidirectional acoustic standing wave.

3. The method of claim 1, wherein a pressure rise and acoustic radiation force on the material are generated at the interface region.

4. The method of claim, further comprising recirculating fluid passing through the acoustic wave back to the chamber.

5. The method of claim 1, wherein the multidirectional acoustic standing wave produces an acoustic radiation force having axial and lateral force components of the same order of magnitude.

6. An acoustic separation device, comprising:

an ultrasonic transducer coupled to an end of a chamber adapted to contain a fluid flow;

a controller coupled to the ultrasonic transducer and configured for causing the ultrasonic transducer to generate sound waves at an end of the chamber such that the sound waves have a sound field with gradients in a plurality of directions;

a fluid pump configured for generating a fluid flow in the chamber;

the material in the fluid flow interacts with the gradient to form an acoustic interface region that blocks the material from passing through the acoustic wave.

7. The device of claim 6, further comprising a reflector for reflecting the acoustic wave to create an acoustic standing wave.

8. The device of claim 7, wherein the reflector further comprises facets.

9. The device of claim 7, wherein the acoustic standing wave is a multidirectional acoustic standing wave.

10. The apparatus of claim 6, wherein the acoustic transducer is configured to be excited in a higher order mode.

Background

The separation of biological materials has been applied in various situations. For example, separation techniques for separating proteins from other biological materials are used in many analytical processes.

Acoustophoresis is a technique that uses acoustic waves (such as ultrasound) to separate particles and/or secondary fluids from primary or primary fluids. When there is a difference in density and/or compressibility, the acoustic wave may exert a force on the particles in the fluid, which is referred to as an acoustic contrast factor. The pressure distribution in the acoustic wave contains regions of local minimum pressure amplitude at the nodes and local maximum pressure amplitude at the antinodes. Depending on their density and compressibility, the particles may be driven to nodes or antinodes of the acoustic wave and trapped. Generally, the higher the frequency of the standing wave, the smaller the particles that can be captured.

Disclosure of Invention

This disclosure describes technologies relating to methods, systems, and apparatus for acoustic separation of materials. The material to be separated may be a biological material. Sound waves are generated in the fluid, creating pressure differentials at different locations. In a flowing fluid, a pressure rise may occur at the upstream interface region where the acoustic waves interact with the flowing fluid. The acoustic waves generate acoustic radiation forces that act on materials suspended in the fluid. The pressure rise and acoustic radiation force form a tunable barrier or filter at the interface region. This phenomenon is interchangeably referred to herein as interfacial effect or edge effect. The barrier or filter characteristic of the interface effect is located upstream of the fluid flow. The characteristics of the acoustic wave can be modified to change or control the characteristics of the interface region and the interface effects. For example, the frequency of the acoustic wave may be controlled such that a material having a specific acoustic contrast factor (acoustic contrast factor) is blocked by or passes through the acoustic wave. As another example, the characteristics of an acoustic wave may be modified to block or pass through a given range of sizes of material while blocking or passing through another different range of sizes of material.

In some embodiments, materials that are blocked or retained by interfacial effects are particles that can be designed to work with interfacial effects to achieve a certain result. As used herein, the term "particle" refers to any type of material that is different from the fluid in which the material is suspended. The particles can be used as a carrier structure for other compounds or biological materials. The particles may be beads, which may comprise a rigid component, such as glass, a polymer or a paramagnetic material, or may comprise a flexible component, such as a liquid or a gas, comprising an oil or a lipid. The functionalized material may be applied to a support structure having an affinity for one or more materials to be separated. The support structure may be mixed in a fluid containing the material. The fluid mixture may be provided to the interface region formed with the acoustic wave, for example, by flowing through a fluid chamber. The interface region formed by the acoustic wave may be distinguished from other materials in the fluid, such as by obstruction or by the support structure.

In some embodiments, the material adhered to the support structure with the functionalized material remains in the pillars, while other free materials in the fluid can pass through the acoustic waves to provide separation of the materials. The support structure may be implemented to have a particular acoustic contrast factor based on its density, compressibility, dimensions, or other properties, which allows the support structure to react more strongly to acoustic standing waves than other materials in the fluid mixture.

An acoustic transducer may be used to generate sound waves that may generate pressure in one or more directions. The acoustic standing wave force may be of the same order of magnitude in multiple directions. For example, the force in the direction of wave propagation may be of the same order of magnitude as the force generated in a different direction. An interface region may be created near the boundary or edge of the acoustic wave, which helps prevent the support structure from passing. Multiple transducers may be used, some for generating sound waves in one or more modes, and/or others for generating sound waves in a different mode. For example, the acoustic wave may be a standing wave that may create pressure in one dimension or direction or in multiple dimensions or directions. The acoustic wave may be generated in a manner that creates an interface region to prevent certain materials from passing while allowing other materials to pass. The acoustic wave may be generated in a mode that captures and clusters a particular material that increases in size until the gravitational or buoyant force on the clusters exceeds other forces on the clusters, such as fluid or acoustic forces, so that the clusters fall or rise from the acoustic wave.

The particles may comprise biological material, such as cells, and may comprise a support structure/biological material composite. Acoustic transducers comprising piezoelectric material for vibrating at ultrasonic frequencies can be used to generate acoustic waves. The acoustic transducer may be operated in one or more modes to achieve a desired effect and/or result. For example, the acoustic transducer may operate in a mode that preferentially traps or blocks particles having a certain density, size, compressibility, and/or other characteristics. The captured or blocked particles may be collected using an acoustic transducer operating in a collection mode, wherein the particles rise or settle out of the acoustic wave due to clustering and size increase to enhance buoyancy or gravity forces acting on the clustered particles such that the enhanced forces exceed acoustic and/or fluid drag forces. The rising or settling of the particles can be advantageously used to collect the separated particles and remove them from the fluid chamber. The mode of trapping particles for separation by rising or settling of the acoustic wave may be accompanied by a mode of obstructing or passing the particles in the fluid path. The modes prevented or allowed to pass through may be achieved with acoustic waves having an interface region that passes through the fluid path.

An example apparatus may include a fluid chamber configured to receive a fluid containing a functionalized material. The fluid chamber may be in the form of a column. The acoustic transducer is arranged relative to the fluid chamber, e.g. acoustically coupled to the fluid chamber, to provide acoustic waves or signals into the fluid chamber upon excitation. Excitation of the transducer in certain modes may produce a multi-directional acoustic field in the fluid chamber that encompasses multiple spatial locations with different acoustic pressure amplitudes. For example, some spatial locations may have relatively high sound pressure amplitudes compared to other spatial locations where relatively low sound pressure amplitudes are obtained.

In some exemplary modes, the particles may be driven to and held at some spatial location of the multi-directional acoustic field. In some exemplary modes, particles may be prevented from entering or passing through the acoustic field created by the acoustic wave by interfacial effects. In some example modes, the particles may pass through an acoustic field generated by sound waves due to fluid drag, buoyancy, and/or gravity. The fluid chamber may be configured for operation in different orientations. For example, where buoyancy or gravity is used in fluid chamber operation, the fluid chambers may be directional. The fluid chamber may be arranged at an angle to the vertical. Such an angled arrangement may provide advantages for fluid dynamic management or deployment of interface effects. The vertical flow may be upward or downward. An acoustic transducer may be coupled to one end of the fluid chamber to allow a fluidized or expanded bed to form in the fluid chamber. In such an arrangement, the particulate fluid mixture may flow into the fluid chamber, and the acoustic waves generated by the acoustic transducer may prevent the target particles from leaving the fluid chamber with the fluid flow, thereby forming an expanding or fluidized bed in the fluid chamber.

In some embodiments, the ultrasonic transducer is coupled to a flow path disposed in the fluid chamber. The ultrasonic transducer is energized to generate acoustic waves in the flow path. In terms of filtration, fluids and materials that pass through and out of the acoustic wave are referred to as filtrate or permeate, while materials that are impeded or retained by the acoustic wave are referred to as concentrate or retentate. The acoustic wave may be a standing or traveling wave, may be planar or multi-directional, or a combination of these waves. The acoustic transducer may be in a higher order mode to produce a multi-directional acoustic wave, or may operate in a "piston" mode to produce a unidirectional or linear acoustic wave. When operating in the higher order mode, a waveform is induced on the surface of the active element of the acoustic transducer, thereby emitting acoustic waves in multiple directions. In piston mode, the surface of the active element moves in a uniform back and forth motion, emitting sound waves in a single direction. In some embodiments, the ultrasonic transducer is configured to generate a multi-directional acoustic wave having an acoustic radiation force with an axial force component and a lateral force component of the same order of magnitude.

In the case of an acoustic standing wave, the acoustic energy reflector may be located opposite the acoustic transducer. The reflector may be disposed across the fluid chamber or flow path from the acoustic transducer. The reflector may be planar and reflect acoustic energy at the same angle of reflection as the angle of incidence. The reflector may be comprised of a plurality of elements arranged at different angles or extending different distances away from the reflector. Such a compound reflector may reflect acoustic energy at different angles of reflection depending on the elements of the reflector upon which the incident acoustic wave is incident. Complex reflectors can be designed and implemented to achieve a particular response, such as forming a particular shape or positioning of the interface region, or enhancing or reducing interface effects. Additionally, or alternatively, a plurality of ultrasonic transducers may be used to generate acoustic waves in the fluid chamber or flow path. For example, an ultrasonic transducer may be positioned opposite another ultrasonic transducer to facilitate the generation of an acoustic standing wave therebetween. Such a second ultrasonic transducer may be passive to reflect acoustic energy, or may be active to assist in the generation and/or control of the acoustic standing wave. Additionally, or alternatively, multiple ultrasonic transducers may be positioned along the fluid chamber or flow path to allow multiple acoustic waves to be generated at different locations.

Methods for forming the interfacial region and the interfacial effect are also disclosed herein. For example, the interface region may be formed by operating the ultrasonic transducer in a collection mode to capture and retain particles in the fluid stream against fluid drag forces. The collected particles can be used to form a pressure differential acoustic transducer, a method of using acoustic radiation forces to create a pressure rise upstream of the interface region.

In one exemplary implementation, separation devices are provided that are adapted to (i) receive a mixture comprising a primary fluid and an acoustically sensitive material; and (ii) using the acoustic standing wave to block the acoustically sensitive material when the mixture is presented to the acoustic standing wave, thereby changing the concentration of the material in the fluid. The concentration variation creates a pressure rise on the upstream interface region of the acoustic standing wave to assist in forming the interface region. The acoustic radiation force acting on the incoming suspension material further contributes to the formation of the interface region. The interfacial effect forming the interfacial region acts as a barrier to the suspended material.

Various operating parameters may be used to modulate and control the position of the interface region. For example, fluid dynamics of the mixture, control of fluid velocity, and/or control of properties of the fluid mixture may be controlled by various column design techniques. Additionally, or alternatively, the implementation of the acoustic wave may be controlled to affect various characteristics of the interface region. For example, control may be effected such that the interface region is formed upstream of the position of the ultrasonic transducer, or is formed to overlap with the position of the ultrasonic transducer. The frequency of the acoustic wave may be modified so that different contrast factor materials may be trapped or allowed to pass through the acoustic wave, or so that particles of one given size range are retained and particles of a second given range are allowed to flow through the acoustic wave. The acoustic waves that create the interface effect to form the interface region may also be modulated to pass the selective material at different times or at different speeds during operation.

Example implementations discussed herein include a separation device having at least one inlet for receiving a mixture of a fluid, a target material, and a non-target material, an ultrasonic transducer that generates ultrasonic waves and uses a pressure rise and an acoustic radiation force to generate an interface region to separate the target material from the mixture. An exemplary separation device includes an outlet port for outputting non-target material during separation and for outputting target material during a harvest stage. The fluid mixture may include particles such as mammalian cells, bacteria, cell debris, powders, proteins, exosomes, vesicles, viruses, plant cells, and insect cells.

In some exemplary implementations, the separation device includes a recirculation path to allow additional residence time of the target material in the fluid chamber or flow path. The separation device may be operated in several modes, including an initial loading mode to load a mixture of fluid and particles, a sample loading mode to introduce biological material into the separation device, a recirculation mode to enhance capture of target material, and an elution mode to harvest captured target material.

The details of one or more implementations of the subject matter described in this specification are set forth in the accompanying drawings and the description below.

Drawings

The drawings are provided to illustrate embodiments disclosed herein and not to limit the embodiments disclosed herein.

Fig. 1 is a diagrammatic view of a material separation device that implements the acoustic edge effect.

FIG. 2 is a schematic representation of a material separation device using perfluorohexane suspension to induce acoustic edge effects.

Fig. 3 is two side-by-side images of an implementation of the sound pressure distribution, with the image of the physical implementation on the left and the image of the analog implementation on the right.

Fig. 4 is an image of a simulated acoustic pressure gradient that contributes to the acoustic edge effect.

Fig. 5 is a graphical representation of the sound pressure and radiation force used to create the acoustic edge effect.

Fig. 6 is an analysis diagram of an acoustic interface.

FIG. 7 is a simulation model of the acoustic lateral force of a positive acoustic contrast particle.

FIG. 8 is a simulation model of the acoustic transverse force of a negative acoustic contrast particle.

Fig. 9 is a graph illustrating acoustic radiation force applied at an acoustic interface region.

FIG. 10 is a force diagram illustration of an object in an acoustic field.

FIG. 11 is a graph of interfacial force versus volume fraction for perfluorohexane suspensions.

FIG. 12 is a graph of interfacial force versus volume fraction for polystyrene suspensions.

Fig. 13 is a graph of the penetration of different sized particles.

Fig. 14 is a graph of penetration curves for different particle concentrations.

Fig. 15 is a graph of penetration curves for different particle concentrations and different sized particles.

FIG. 16 is a graph of the permeation curve of agarose beads.

Fig. 17 is a partial front view of an operating acoustic separation device illustrating acoustic edge effects.

Figure 18 is an isometric view of a multifaceted reflector.

Fig. 19, 20, 21 and 22 are partial front views of an operating acoustic separation device showing different acoustic interface region locations.

FIG. 23 is an illustration of an acoustic separation device that achieves an acoustic edge effect for particles having a density greater than water.

FIG. 24 is an illustration of an acoustic separation device that achieves an acoustic edge effect for particles having a density less than water.

FIG. 25 is an illustration of an acoustic separation device that achieves an acoustic edge effect in the absence of fluid flow.

FIG. 26 is a diagrammatic view of an acoustic separation device utilizing fluid flow to achieve an acoustic edge effect.

FIG. 27 is an illustration of an acoustic separation device having different geometries and fluid dynamics.

FIG. 28 is a diagram of an acoustic separation device illustrating vortices at an acoustic interface region.

FIG. 29 is a partial front view of an acoustic separation device having multiple interface regions.

FIG. 30 is a flow chart of a process for initiating an acoustic edge effect.

FIG. 31 is an illustration of an acoustic separation device configured for affinity separation of cellular material.

FIG. 32 is a diagrammatic view of an acoustic separation device and method for affinity separation of cellular material with perfluorohexane droplets.

Figure 33 is a graphical representation of purity and recovery of TCR cellular material.

Detailed Description

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

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

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

The term "comprising" is used herein to require the presence of the named component and to allow the presence of other components. The term "comprising" should be interpreted as including the term "consisting of … …" which allows for the presence of only the specified component as well as any impurities that may result from the manufacture of the specified component.

Numerical values are 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 value by less than the experimental error of conventional measurement techniques of the type described in the present application to determine 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 encompass 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 is also to be construed 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". The term "about" may refer to plus or minus 10% of the number indicated. For example, "about 10%" may mean a range of 9% to 11%, and "about 1" may mean 0.9 to 1.1.

It should be noted that many of the terms used herein are 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, but these terms may vary if the device is turned over. The terms "inlet" and "outlet" are with respect to a fluid flowing through them for a given structure, e.g., a fluid flowing through an inlet into a structure and flowing through an outlet out of a structure. 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, a first component may be described as being upstream and downstream of a second component.

The terms "horizontal" and "vertical" are used to denote 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 the surface of the top that is always higher than the bottom/base relative to absolute reference, i.e., the earth's surface. The terms "upward" and "downward" are also relative to an absolute reference; upward is always against the earth's gravity.

The present application relates to "same order of magnitude". Two digits are of the same order of magnitude if the quotient of the larger digit divided by the smaller digit is a value of at least 1 and less than 10.

Briefly stated, the present disclosure is directed to an acoustic separation device capable of producing an acoustic edge effect from one or more piezoelectric transducers. In some embodiments, the transducer is electrically excited into multiple modes of displacement mode of vibration to produce a multidirectional acoustic wave. Alternatively or additionally, the acoustic transducer may be excited in a piston mode to produce a planar or unidirectional acoustic wave. Combinations of sound waves may be generated, such as combinations of planar and multi-directional sound waves. Acoustic waves may be used to create interface effects or edge effects, and these terms are used interchangeably herein. The interface effect results in the formation of an interface region having an acoustic force field that prevents the material from passing through the acoustic wave and accompanying acoustic field. The interfacial effect may be configured to target certain types of materials, such as by blocking materials having density, compressibility, and/or dimensional characteristics within a particular range. The interface effect may be configured to pass through certain types of materials, such as by being configured to affect such materials to a much lesser degree than other particular materials to be blocked. The material to be passed is affected to a much lesser extent by sound waves and acoustic fields, so that other forces, such as gravity, buoyancy or fluid drag, prevail.

The acoustic edge effect results in an acoustic radiation force that can overcome the combined effects of fluid drag and buoyancy or gravity at certain flow velocities. As a result, the radiation force acts as a filter to prevent target particles (e.g., biological cells or acoustically responsive beads) from passing through the standing wave. Several forces generated by the acoustic wave can have an effect on the acoustic edge effect and the resulting interface area. For example, a multi-directional acoustic wave may generate a lateral force that acts on a material in a fluid stream presented to the acoustic wave. When the sound field generated by the sound waves impinges on the material, the scattering effect of the sound field away from the particles in the material generates a three-dimensional acoustic radiation force. The acoustic radiation force produces a net force effect on the particles. A non-zero net force may cause the particle to move in the acoustic field, while a near-zero net force may cause the particle to be trapped in the acoustic field. When the particles are small relative to the wavelength, the acoustic radiation force is proportional to the particle volume (e.g., the cube of the radius). The acoustic radiation force is proportional to the frequency and acoustic contrast factor of the material. The acoustic radiation force is proportional to the acoustic energy (e.g., the square of the acoustic pressure amplitude). For harmonic excitation, the sinusoidal spatial variation of force is the force that drives the particles to a stable position within the acoustic wave, particularly an acoustic standing wave. Particle capture can occur when the acoustic radiation force exerted on the particles is greater than the combined effect of the fluid drag force and the buoyancy/gravity force on the particles. In some modes of operation, lateral and axial acoustic forces generated by the acoustic waves act on the captured particles, resulting in the formation of tightly packed clusters through concentration, clustering, agglomeration, coagulation and/or coalescence of the particles, which continuously settle by enhanced gravity for particles heavier than the main fluid when a critical dimension is reached, or rise by enhanced buoyancy for particles lighter than the main fluid. In addition, secondary interparticle forces, such as the Bjerkness force, contribute to particle agglomeration. Clustering of the particles can aid in the formation of interfacial regions and enhance acoustic edge effects.

In the device of the present disclosure, during start-up, the fluid acoustically bound by the acoustic standing wave is clarified by the process of capturing clumped particles of increasing size until gravity or buoyancy dominates the sound and/or fluid drag force. At this point, the particle cluster is driven out of the acoustic wave. An acoustic standing wave is a three-dimensional acoustic field, which, in the case of excitation by a rectangular transducer, can be described as a fluid occupying a volume that is a substantially rectangular prism or cuboid. Typically, two opposing faces are the transducer and reflector, an adjacent pair of opposing faces are the walls of the device, and the last pair of opposing faces are the upstream and downstream faces of the cube through which the fluid flow is provided. The interface region is typically located more upstream relative to the acoustic standing wave field. This location is also referred to as the upstream interface region. The purging fluid is downstream of the interface region by the acoustic wave. Various combinations of operating parameters, such as flow rate, electrical power applied to the transducer, fluid concentration, or configuration of the acoustic wave, to name a few, may affect the location and characteristics of the interface region.

The transducer design can affect the performance of the system. A typical transducer is a layered structure having a ceramic piezoelectric element bonded to a backing layer and a wear plate. Because the transducer is loaded with the high mechanical impedance presented by the standing wave, conventional design criteria for wear plates, such as half-wavelength thickness for standing wave applications or quarter-wavelength thickness for radiation applications, and manufacturing methods may be inadequate. In contrast, in some exemplary embodiments of the present disclosure, the transducer lacks a wear plate or backing, allowing the piezoelectric element to vibrate in one of its eigenmodes, or in a combination of several eigenmodes, with a high Q factor. The vibrating ceramic piezoelectric element/disc may be directly exposed to the fluid flowing through the separation device.

Removing the backing (e.g., making the piezoelectric element an air backing) also allows the ceramic piezoelectric element to vibrate in higher order vibration modes (e.g., higher order modal displacements) with little damping. In a transducer having a backed piezoelectric element, the piezoelectric element vibrates like a piston with a more uniform displacement. Removing the backing allows the piezoelectric element to vibrate in a non-uniform displacement mode.

The size, shape and thickness of the piezoelectric material determine the displacement of the transducer at different excitation frequencies. In some exemplary implementations, the transducer operates at a frequency near the thickness resonance frequency (half wavelength). Transducers operating in higher order modes can produce a greater number of acoustic force gradients. Higher-order modal displacements produce sound waves with strong gradients in the sound field in multiple directions, thereby producing acoustic radiation forces in multiple directions.

In contrast to the vibrational modes in which piezoelectric material (e.g., a piezoelectric crystal) effectively moves as a piston with uniform displacement, the lateral force of the acoustic radiation force generated by the transducer can be increased by driving the transducer in a higher order mode shape. The sound pressure is proportional to the drive voltage of the transducer. The electrical power is proportional to the square of the voltage. The transducers are typically thin piezoelectric plates with the electric field in the z-axis and the main displacement in the z-axis. The transducers are typically coupled by air (i.e., an air gap within the transducer) on one side and by a fluid mixture on the other side. The type of wave generated in the plate is called a composite wave. A subset of the composite waves in the piezoelectric plate resemble leaky symmetric (also called compressive or tensile) Lamb waves. The piezoelectric properties of the plate typically result in the excitation of a symmetric Lamb wave. These waves are leaky because they radiate into the fluid layer, which results in the generation of acoustic waves in the fluid layer. Lamb waves exist in an infinite range of thin sheets with stress-free conditions on their surfaces. Because the transducers of this embodiment are inherently limited, the actual modal displacements are more complex.

In-plane displacement (x-displacement) and out-of-plane displacement (y-displacement) across the thickness of the plate, the in-plane displacement across the thickness of the plate being an even function and the out-of-plane displacement being an odd function. Due to the finite size of the plate, the displacement component varies across the width and length of the plate. In general, the (m, n) mode is a displacement mode of the transducer in which there are m fluctuations in the transducer displacement in the width direction and n fluctuations in the length direction. The maximum number of m and n is a function of the size of the piezoelectric material (e.g., piezoelectric crystal) and the excitation frequency. There are additional three-dimensional modes that are not of the (m, n) form.

The transducer is driven so that the piezoelectric element vibrates in a higher order mode of the general formula (m, n), where m and n are independently 1 or greater. Higher order modes generate acoustic waves in multiple directions, resulting in a greater number of nodes and antinode locations, characterized by strong gradients in the acoustic field.

In some example implementations, the ultrasound transducer is driven by an electrical signal that may be controlled based on voltage, current, phase angle, power, frequency, or any other electrical signal characteristic. In particular, the drive signal for the transducer may be based on a voltage, a current, a magnetic, an electromagnetic, a capacitive or any other type of signal to which the transducer is responsive. In embodiments, the signal driving the transducer may have a sinusoidal, square, sawtooth, pulsed or triangular waveform; and has a frequency of 500kHz to 10 MHz.

Referring to fig. 1, a diagram of an acoustic separation apparatus implementing an acoustic edge effect is illustrated. Acoustic edge effects: the acoustic radiation force forms an interface between the mixture of particles and fluid on one side and the cleaning fluid on the other side. The radiation force in the flow direction is generated by the transverse standing wave field component of the multi-dimensional field; the diffracted waves of the transducer and the scattered waves passing through the non-planar reflector of the interface result in additional radiation forces on the particles. The acoustic radiation force exerts a downward force on the particles that is greater than the fluid drag force on the particles, possibly assisted by gravity, thereby maintaining a stable interface. The acoustic forces block the particles from moving into the acoustic field. This effect can be established using a relatively low concentration of particles (e.g., -1-2%). One or more transducers and one or more reflectors may be used. The sound field may be angled. With the acoustic device turned on, the flow is turned on, and the acoustic edge holds the particles back and allows clear fluid to pass through, similar to filtration. The acoustic edge may also be below the transducer or in the acoustic field.

Some parameters that affect the formation and stability of the interface region include the concentration of particles, acoustic pressure/power, flow rate, acoustic contrast factor of the material, size of particles in the material, transducer frequency, and properties of the medium.

Figure 2 illustrates the operation of the acoustic separation device to form an interfacial zone with a Perfluorohexane (PFH) suspension. In the left hand illustration, the acoustic transducer T is not energized and fluid flows through the device unobstructed. In the right-hand illustration, the acoustic transducer is excited to form an interface region with acoustic edge effects. The interface region generates an acoustic force that is highly responsive to PFH particles. The obstruction of the PFH particles in the interface region helps to increase the acoustic edge effect to stabilize the interface region.

Fig. 3 illustrates the consistency of experimental and simulated data for generating edge effects. Experimental and simulation data confirm that a significant gradient is formed in the perpendicular or transverse axial direction of the acoustic wave. The pressure in the acoustic resonator used for the experiment is described by the following equation.

Fig. 4 illustrates a simulation showing a predicted acoustic pressure gradient, incorporating lateral radiation forces on the particles to help form the interface region. The transverse pressure gradient and transverse wave strength are much larger in the standing wave. Axial standing wave forces can affect and increase the transverse wave strength. The intensity of the transverse wave emanating from the acoustic standing wave decreases. Thus, the interface region may be formed with an edge at the lower edge of the standing wave due to the reduction of the lateral wave force.

Fig. 5 illustrates the acoustic forces that create a gradient in the acoustic separation device to form the interface region. Due to transducer mounting limitations, the gradient is weaker below the bottom cluster. The moving transverse wave pushes the particle edge towards the lower axial acoustic edge.

Fig. 6 shows a diagram of an analytical model of acoustic edge effects. The force generated at the interface is determined by the following equation.

ρ and c depend on the volume fraction of PFH at the interface

The contribution of this force is related to the difference in properties between the two fluids and is independent of the size of the particles. Through experimentation, it has been shown that particle size has a significant effect on edge effects. For some mixtures (e.g. polystyrene particle-mixtures) this force will add to the lateral radiation force, for other mixtures (e.g. PFH droplets) this force will be subtracted, which means that this force is opposite to the lateral radiation force. Together with the standing wave, there is a traveling wave across the interface. This phenomenon generates forces on the interface resulting from the radiation pressure at the interface between the cleaning fluid and the mixture.

The pressure in the standing wave of a typical rectangular transducer is given by the following equation,

P＝A cos(k_xx)cos(k_yy)cos(k_zz)e^jwt

in the above equation, dx is the length in the X direction, dy is the length in the Y direction, L is the path length of the standing wave, and k is_x、k_y、k_zAre the wave numbers in those directions, n, m, L are the number of modes taking values of 0, 1, 2, 3, for example, for a 3 × 3 mode, n ═ m ═ 3.

The lateral radiation forces block the particles from moving into the acoustic field. These particles stay below the acoustic edge forming the interface, as only the cleaning fluid is allowed to pass. As more particles are introduced by the flow from the bottom, the particles form a "self-cleaning channel" and the particles at the top fall by gravity, resulting in mixing. These particles do not clog or stagnate at the acoustic edges and are continuously removed. The acoustic device and flow can be periodically shut off to allow particles to settle to "force" the acoustic edge to clear. The transducer may also be non-rectangular, e.g. circular, hexagonal, octagonal, fractal geometry, it may be any shape, and similar calculations may be made to determine the radiation force in all directions.

Fig. 7 shows a graphical representation of the transverse force field component of a positive acoustic contrast particle. Fig. 8 shows a graphical representation of the transverse force field component of a negative acoustic contrast particle. Thus, the position where the particles find a stable position is different for positive and negative contrast particles in the transverse plane.

Fig. 9 is a graph of radiation force exerted on a particle at an interface region as a function of frequency. Assuming single particle approximation and integration along the interface, the force on the interface is calculated. This force is negative in the x-direction, meaning that it opposes the fluid drag force and blocks particles from entering the acoustic field, confirming the presence of the interface effect. An exemplary simulation implemented to determine the force of the interface region at a frequency of 2.16MHz verifies the assumption of a pressure release boundary condition at the edge of the sound field.

Fig. 10 is a graphical representation of the forces acting on a particle in an acoustic field. The standing wave field generates acoustic radiation forces on the particles in axial and transverse directions. The lateral radiation force in the flow direction is responsible for the acoustic trapping of particles in the acoustic field. The lateral radiation force comes from the vibration of the PZT/crystal/transducer in the lateral direction. Any combination of transducers, reflector assemblies that enhance lateral radiation forces may be suitable for acoustic edge effects, including the inclusion of scatterers, non-planar reflectors, focusing transducers, and focusing reflectors in the field. The following equation describes the forces that contribute to the acoustic edge effect.

Ger' kov formula¹

Fig. 11 is a graph illustrating the force of PFH beads suspended in water at an interface when the volume fraction of the PFH beads is changed. PFH beads are less dense than water and have a negative acoustic contrast factor.

Fig. 12 is a graph illustrating the force of polystyrene beads suspended in water at an interface when the volume fraction of the polystyrene beads is changed. Polystyrene beads are less dense than water and have a positive acoustic contrast factor.

FIG. 13 is a view showing the sameGraphical representation of the breakthrough curves for different bead sizes at concentration. As the particle size increases, the acoustic interface region is stable at lower power for the same flow rate. This result is as follows: the acoustic force on the particles is proportional to the particle size (R)²)。

Fig. 14 is a graph showing the penetration curves of beads with a size of 8.5 μm but at different concentrations. The acoustic interface region is stable at lower power for the same flow rate as the drop concentration is reduced. The increase in velocity results in stability due to the greater resistance of the fluid flow by the higher concentration of particles. The rate of flux of particles into the interfacial region increases with increasing particle concentration, which means greater acoustic force field strength to maintain the acoustic edge effect.

Fig. 15 is a graph showing combinations of penetration curves with different bead sizes and different concentrations. The combination of lower concentration and larger particle size means that the power for creating and maintaining the acoustic edge effect is smaller. As shown in fig. 15, the ratio of power to flow rate can be used as a guide set operating parameter for the acoustic separation device based on the bead size and bead concentration used in the fluid mixture.

Fig. 16 is a diagram showing the permeation curve of agarose beads. Agarose beads are less dense than water and have a positive acoustic contrast factor. Agarose beads are bioerodible, which may provide advantages for cell and gene therapy, where biological material collected using agarose beads is introduced into a patient as a therapeutic agent.

Fig. 17 is a partial front view of an operating acoustic separation device illustrating acoustic edge effects. Viewing is performed through a transparent reflector.

Figure 18 is an isometric view of a multifaceted reflector. The facetted reflector reflects acoustic waves from the transducer to create an acoustic standing wave in the fluid chamber or flow path. The facetted reflectors may help to increase the gradients in the acoustic field produced by the acoustic standing wave. Flat or faceted reflectors may be used to create the acoustic edge effect. The facetted reflector may maintain multiple parallel standing waves, increasing diffraction of the waves, which increases the gradient in the acoustic field, thereby increasing the radiation force, particularly in the lateral direction. The pressure distribution from the simulation shows the scattering pattern of the wavefield as a function of frequency. Pressure scattering causes a larger pressure gradient, resulting in higher radiation forces, especially in the lateral direction.

In an exemplary experiment, the column was loaded with 15% 5 μm droplets and an edge effect was established. Jurkat cells flowed in and the flush fraction was measured to determine their washout. No binding was performed in this experiment. The multi-faceted reflector shows more efficient cell clearance compared to a planar reflector at the same low power to flow rate ratio. With the facetted reflector, the cells were captured in the acoustic field, while for the same power with the planar reflector there was no visible evidence of the cells being captured in the acoustic field, indicating that the cells were more efficiently washed through the acoustic field. For structures with facetted reflectors, 5 Cylinder Volumes (CVs) are required to obtain an asymptotic value for the unit wash, and for a flat reflector, at least 13 CVs are required to achieve the same wash. The facetted reflectors show the ability to operate at lower power to flow rate ratios than planar reflectors.

Fig. 19, 20, 21 and 22 are partial front views of operating the acoustic separation apparatus, showing different acoustic interface region locations obtained using different example experiments. Experiments were performed with a planar reflector and a 1.5 "x 1.5" transducer. At lower power, the interface is formed at or slightly above the acoustic edge, as shown in fig. 19. When the power increases, the interface is pushed down and formed under the sound field, as shown in fig. 20. As the power increases, the distance from the acoustic edge forming the interface increases, as further shown in fig. 21. At lower power to flow ratios and higher flow velocities, interfaces form inside the rising sound field near the mid-section of the sound, as shown in fig. 22. In fig. 19 to 22, the transducer is on the left side and the reflector is on the right side. The interface may be formed at an angle from the transducer to the reflector, as best seen in fig. 22.

FIG. 23 is an illustration of an acoustic separation device that achieves an acoustic edge effect for particles having a density greater than water. FIG. 24 is an illustration of an acoustic separation device that achieves an acoustic edge effect for particles having a density less than water. A number of exemplary experiments were conducted using the configurations of fig. 23 and 24. The pillar geometry is important for the acoustic flow control behavior of the pillar. A diffuser at the top of the column allows the particles to settle more easily, contributing to a "self-cleaning" mechanism so that the acoustic edges are not saturated. Parameters such as diffuser height, diffuser angle, column diameter, etc. play a role in column operation. The column may also run in a straight section without a diffuser. In the interface region, there is a net zero balance of particle flow; as new particles flow through the flow field into the interface region, there is an equal and opposite flux of particles. There are a number of mechanisms for this opposite particle flow. For example, a diffuser at the top of the fluidizing column allows for a recirculating flow with a downward flow. The acoustic flow may establish a counter-rotating vortex with a downward flow. Additional inclined surfaces within the fluidized bed produce enhanced gravity settling as particles can slide down these surfaces.

For example, the particles may be solids, liquid droplets or gas bubbles. If the particles are heavier or denser than the fluid, the system operates in a vertically upward configuration. If the particles are lighter or less dense than the fluid, the system operates in a vertically downward configuration. In both cases, gravity on the particles contributes to the formation of the interfacial region. Examples of solid particles include microcarriers, PROMEGA beads and polymer particles. Examples of liquid particles include PFH droplets and PFP droplets. Examples of bubbles include microbubbles, Akadeum beads, hollow glass beads. As used herein, particles refer to all types of materials used to form beads, including solids, liquids, hollow materials, materials that are less dense than water and less dense than water, and any type of material that can be formed into discrete portions that can be entrained in a fluid stream.

The behavior of the acoustic edge is different for particles with different properties. The four quadrant classification is used to identify different attributes and acoustic edge effect behavior. In the first quadrant, X>0、Representing beads having a density greater than water and a positive acoustic contrast factor. These beads comprise Promega beads and microcarriers. The second quadrant comprises a first quadrant having X>0、Wherein the bead has a negative contrast factor and a density less than water. The third quadrant comprises a first quadrant having X<0、A particle of a character, wherein the bead has a negative acoustic contrast factor and a density less than water. These beads comprise PFH droplets. The fourth quadrant has X<0、A particle of a character wherein the bead is less dense than water and has a positive acoustic contrast factor. These beads include Akadeum beads and hollow glass spheres.

In each case in all four quadrants, an acoustic edge interface may be formed. The flow direction is opposite to the direction of gravity/buoyancy. The position of the acoustic edge may be different for different contrast factor particles.

FIG. 25 is an illustration of an acoustic separation device that achieves an acoustic edge effect in the absence of fluid flow. FIG. 26 is a diagrammatic view of an acoustic separation device utilizing fluid flow to achieve an acoustic edge effect. A multi-directional sound field is realized at one end of the column. A lateral standing wave force field and wave propagation for a given frequency of operation of the transducer is illustrated. A fixed number of particles in suspension in a fluid column is shown. The particles may be solid, liquid or gas. It is assumed herein that the particles are heavier than the fluid. Gravity acts vertically downwards. The acoustic edge effect is established as follows. As flow begins up through the particle mixture, the acoustic radiation force forms an interface between the particle mixture and the fluid on one side and the cleaning fluid on the other side. The radiation force in the flow direction is generated by the lateral standing wave field component of the multidirectional field. The acoustic radiation force exerts a downward force on the particles that is greater than the fluid drag force on the particles, possibly assisted by gravity, thereby maintaining a stable interface. The acoustic edge may be formed at the lower edge of the acoustic standing wave and may also be in the acoustic field. As shown in fig. 26, the dense concentration of particles near the acoustic edge is used to filter any flowing fluid or flowing fluid mixture.

The high columnar particle density near the acoustic edge acts as a filter. Particle size, density, system acoustic power and flow rate can be varied to produce extreme filtration. Unsteady acoustic reflections produce particle motion and self-cleaning. The filter particles in the column can be varied using a contrast factor, size or density factor. Smaller columnar particles may filter smaller target particles, and multi-sized columnar particles may further enhance filtering. Depending on the nature of the particles, the column may have a concentration gradient in the direction of flow, but is not necessary for the operation of the acoustic edge. Multi-sized column particles with appropriate contrast factors and densities can be used to perform extreme filtration. There are a number of control variables to filter particles of almost any size. Such extreme filtration could potentially be used to filter exosomes and viruses. For example, smaller particles may be selected to have a lower acoustic contrast factor so that they are more readily retained at the interface region than larger particles or particles having a larger acoustic contrast factor. By keeping the particles small at the interface region, the gaps between the particles can be reduced to provide very small pore extreme physical filtration.

As described above, the location of the interface region may be modified according to various influential parameters. For example, an angled interface region may be implemented similar to that shown in fig. 29. In this implementation, the lateral radiation force may be enhanced because the component of the primary force acts perpendicular to the interface. For most frequencies, the lateral force on the interface is considered negative, which means that the interface will be blocked and the particles will be blocked from moving in the flow direction (which is in the positive direction).

FIG. 27 is an illustration of an acoustic separation device having different geometries and fluid dynamics. The flow velocity at the acoustic edge remains constant in all three configurations. The column area convergence increases the flow velocity and increases the particle vertical gradient. This pooling may improve filtration. As the column area converges increases, particle circulation occurs near the acoustic edge. Particle circulation may improve "self-cleaning" and aid in particle mixing.

FIG. 28 is a diagram of an acoustic separation device illustrating vortices at an acoustic interface region. The stirring vortex created by the transverse waves reflected off the particle interface and/or the acoustic radiation force variations on the particles results in a self-cleaning acoustic filter interface. Acoustic flow is a steady flow in a fluid driven by reynolds stress and acoustic wave absorption. The high power may cause wave attenuation as the acoustic wave moves in a fluid that is absorbed in the propagation medium. This results in physical forces on the fluid driving fluid movement within the chamber. The more wave attenuation, the stronger the flow. If the particles are small enough, they will move with the bulk of the flowing fluid. This behavior has been observed in cells, especially when the particle mixture rises in an acoustic field with a small path length. The acoustic flow may also help create an acoustic edge by the action of counter-rotating vortices that keep the interface from rising. The acoustic flow is stronger in the traveling wave setting and the edges are unstable as the particles rise in the acoustic field. It may also provide additional mixing and self-cleaning mechanisms for the particles as long as the acoustic flow is stable and controllable.

FIG. 29 is a partial front view of an acoustic separation device having multiple interface regions. Multiple edge effects can be established in the same system. Figure 29 demonstrates the edge effect on a mixture of 6% VF leukocytes and 15% VF microdroplets. Since the droplets have a strong acoustic response, the edge is placed at the bottom of the acoustic field and all droplets remain below it. Compared to droplets, droplets like RBC have a weaker acoustic response and they rise in the acoustic field. Since the transducer typically provides a higher pressure in the center, the edge with the cell can be built up halfway in the acoustic field. In this way, multiple edge effects can be created in the same system for particles of different characteristics. This may be a way of using the edge effect to separate particles with different acoustic responses, and has been shown to reduce platelets in leukocytes by 70%, with a WBC recovery of 70%.

Edge effects can be established for only one particle type. In this way, particles with a lower acoustic response may flow or be flushed out of the column. As the concentration of particles forming the acoustic edge increases, the particles begin to act like a hydrodynamic filter. In this way, larger particles can be retained, while smaller particles such as viruses or platelets can pass.

Experiments were performed to separate the undiluted leukocyte fraction (5ml volume) without any affinity particles, run in a fluidized bed. Different power and flow rate conditions were operated in the experiment. The results indicate that the borders of RBCs and WBCs are established. Smaller platelets (3 μm) pass through to the outlet. Six to eight column washes were performed and 80% recovery of WBCs and 68% platelet reduction were observed. Some acoustic retention of platelets was observed. The number may be adjusted by adjusting the power and flow rate to achieve the desired recovery and/or consumption. Similar considerations apply to other particles such as exosomes, viruses, cell debris, etc.

FIG. 30 is a flow chart of a process for initiating an acoustic edge effect. The sound field was initially filled with a mixture of particles. The experiment was run with a frequency sweep and selecting an anti-resonance point near zero reactance or maximum resistance. Stopping the flow, turning on the acoustic device, and operating in a multi-dimensional standing wave field. By periodically turning the acoustic device on and off, particles in the acoustic field are trapped, clustered, and deposited under the acoustic edge. Once most of the particles leave the acoustic field, the flow is slowly turned on and ramped up to steady state. Once the interface is established, and as long as the proper power to flow rate ratio is maintained, the system can operate in a stable manner.

FIG. 31 is an illustration of an acoustic separation device configured for affinity separation of cellular material. The beads are held by an acoustic field generated at the interface region.

FIG. 32 is a diagrammatic view of an acoustic separation device and method for affinity separation of cellular material with perfluorohexane droplets.

Figure 33 is a graphical representation of purity and recovery of TCR cellular material. TCR depletion can be achieved using the acoustic separation techniques described herein. In one experiment, the system was loaded with 15% volume of functionalized droplets and TCR antibody was used to capture CD3+ cells. Diluted and undiluted leukocytes were flowed in and wash fractions of different cell types were analyzed, where the non-target WBC was CD 3-. This system was very effective (> 90% flush) in flushing RBCs and platelets. The system shows some retention of non-target WBCs such as monocytes, granulocytes, B cells or NK cells. Some of these cells, such as monocytes, are larger than the target T cells and can be acoustically captured. Some larger pores may also be trapped within the fluidized bed. With dilution, a higher percentage of non-target cells are washed away. This may be because a wider flow path is available due to the diluted feed. Experimental data indicate that the acoustic fluidized bed can retain particles/cells of interest but wash away particles/cells not of interest as long as there is sufficient acoustic difference between the different components.

The methods, systems, and apparatus discussed above are examples. Various configurations may omit, substitute, or add various procedures or components as appropriate. For example, in alternative configurations, the methods may be performed in an order different than described, and various steps may be added, omitted, or combined. In addition, features described with respect to certain configurations may be combined in various other configurations. Different aspects and elements of the configurations may be combined in a similar manner. Additionally, technology evolves and, thus, many of the elements are examples and do not limit the scope of the disclosure or claims.

Specific details are given in the description to provide a thorough understanding of the exemplary configurations (including implementations). However, configurations may be practiced without these specific details. For example, well-known processes, structures and techniques have been shown without unnecessary detail in order to avoid obscuring the configurations. This description provides exemplary configurations only, and does not limit the scope, applicability, or configuration of the claims. Rather, the foregoing description of the configurations provides a description for implementing the techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure.

Additionally, a configuration may be described as a process that is depicted as a flowchart or a block diagram. Although each may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be rearranged. The process may have additional stages or functions not included in the figure.

Having described several example configurations, various modifications, alternative configurations, and equivalents may be used without departing from the scope of the invention. For example, the elements described above may be components of a larger system, where other structures or processes may take precedence over or otherwise modify the application of the invention. Also, various operations may be performed before, during, or after the elements described above are considered. Accordingly, the above description does not limit the scope of the claims.

An assertion that a value exceeds (or is greater than) a first threshold is equivalent to an assertion that the value meets or exceeds a second threshold that is slightly greater than the first threshold, e.g., the second threshold is higher than the first threshold by a value in a resolution of the associated system. An expression that a value is less than (or within) a first threshold value is equivalent to an expression that the value is less than or equal to a second threshold value that is slightly lower than the first threshold value, e.g., the second threshold value is lower than the first threshold value by a value in the resolution of the associated system.