Microfluidic system and methods using acoustic waves

Abstract

The present document relates to systems and methods that implement acoustofluidics to manipulate particles within a microfluidic system.

Description

MICROFLUIDIC SYSTEM AND METHODS USING ACOUSTIC WAVESCROSS-REFERENCE TO RELATED PATENTS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 535.019, filed on August 28, 2023. The contents of that application are incorporated herein by reference in its entirety.BACKGROUND

[0002] Sample processing and preparation can be inefficient and ineffective, which can lead to sample loss and recovery. Even so, such methodologies can be difficult to implement in microfluidic systems.SUMMARY

[0003] The present document relates to systems and methods that implement acoustofluidics to manipulate particles within a microfluidic system. In particular embodiments, acoustic radiation with acoustic streaming actuation can be implemented to provide on-chip separation within a fluid sample.

[0004] In a first aspect, the present document encompasses a device including: a substrate (e.g., a piezoelectric substrate) comprising a top surface disposed on a piezoelectric portion; a first microchannel disposed on the top surface of the substrate; a first inlet configured to deliver a sample through the first microchannel; and a first active region. In some embodiments, the first active region includes a first electrode region and a second electrode region disposed on the top surface of the piezoelectric portion, wherein the first and second electrode regions are configured to launch an acoustic wave (e.g., a surface acoustic wave, a standing acoustic wave, and the like) through at least a portion the first microchannel. In some embodiments, the acoustic wave is configured to generate displacement of a particle or to modify a velocity of a fluid, thereby providing a non-oscillating velocity component to a liquid or a particle traveling through or within the first microchannel by way of acoustic radiation force and acoustic streaming.

[0005] In some configurations, the device further include one or more additional surfaces (e.g., formed of any useful material, such as silicon, glass, a polymer, and the like). In some embodiments, the surface is configured to support or surround the piezoelectric portion and / or the substrate. In some embodiments, the surface is in contact with the piezoelectric portion and / or the substrate. In some embodiments, a microchannel (e.g., the first microchannel) can be fully or partially disposed on a top surface of the piezoelectric portionand / or the substrate. In some embodiments, a microchannel can be fully or partially disposed on a top surface of a substrate, which in turn includes a piezoelectric portion (e.g., a piezoelectric substrate) and a non-piezoelectric portion (e.g., a semiconductor substrate, such as a silicon substrate, a quartz substrate, a glass substrate, and the like).

[0006] In some embodiments, the acoustic wave is configured to provide an asymmetrical or symmetrical amplitude profile of the acoustic wave in the active region. In some embodiments, a propagation direction of the acoustic wave is not parallel to a direction of flow within the first microchannel. In some embodiments, the propagation direction of the acoustic wave is perpendicular to the direction of flow within the first microchannel.

[0007] In some embodiments, a propagation direction of the acoustic wave is provided at an angle a to a direction of flow within the first microchannel. In some embodiments, a is not 0° and not 180° (e.g., 0° < a < 180° or 180° < a < 360°). In some embodiments, a is an oblique angle (e.g., greater than 0° and less than 90°; or greater than 90° and less than 180°).

[0008] In some embodiments, the first active region further comprises one or more further electrode regions. In some embodiments, the first, second, or further electrode regions comprises one or more electrodes. In some embodiments, the one or more electrodes are selected from the group consisting of planar electrodes, interdigitated transducers (IDTs), and the like.

[0009] In some embodiments, the device further comprises one or more reflectors, guide layers, or functionalized surfaces disposed on the top surface of the piezoelectric portion.

[0010] In some embodiments, the first microchannel comprises a functionalized surface configured to minimize adsorption of targets within a fluid sample; configured to sense, label, or capture targets within a fluid sample; and / or configured to exclude certain undesired components within a fluid sample.

[0011] In some embodiments, the device further includes a second inlet in fluidic communication with the first microchannel.

[0012] In some embodiments, the device further includes: a first inlet channel in fluidic communication with the first inlet; and a second inlet channel in fluidic communication with the second inlet. In some embodiments, the first inlet channel is configured to deliver a first fluid (e.g., comprising a first sample) through the first microchannel. In some embodiments, the second inlet channel is configured to deliver a second fluid (e.g., comprising a second sample or a first sheath fluid surrounding a flow of the first sample) through the first microchannel.

[0013] In some embodiments, the device further includes: a third inlet channel in fluidic communication with the second inlet or a third inlet. In some embodiments, the third inlet channel is configured to deliver a fluid (e.g., comprising a third sample or a second sheath fluid surrounding a flow of the first sample or the second sample) through the first microchannel.

[0014] In some embodiments, the second fluid and / or the third fluid comprises a sheath fluid configured to dilute a fluid sample within the first microchannel or configured to focus a flow of a fluid sample within the first microchannel.

[0015] In some embodiments, the device further includes one or more inlets or inlet channels in fluidic communication with the first microchannel. In some embodiments, the device further includes one or more outlets in fluidic communication with the first microchannel.

[0016] In some embodiments, the device further includes a plurality of microchannels, wherein each second channel is in fluidic communication with the first microchannel and a respective outlet.

[0017] In some embodiments, the device is configured to provide flow having an average fluid velocity of about 5 to 500 μm / s.

[0018] In another aspect, the present document encompasses a system comprising one or more devices (e.g., any device described herein); an electronics module configured to provide one or more electrical connections to at least one of the one or more devices; a fluidics module configured to provide one or more fluids to at least one of the one or more devices; and a temperature control module configured to regulate a temperature of the one or more devices.

[0019] In some embodiments, the system includes a plurality of devices (e.g., any described herein), wherein each device can be same or different.

[0020] In some embodiments, at least two of the plurality of devices are fluidically connected in series. In some embodiments, the plurality of devices comprises: a first device comprising a first inlet, a first outlet, and a first microchannel in fluidic communication with the first inlet and the first outlet; and a second device comprising a second inlet, a second outlet, and a second microchannel in fluidic communication with the second inlet and the second outlet. In some embodiments, the system further comprises: a fluidic interconnect disposed between the first outlet and the second inlet, thereby providing fluidic communication between the first outlet of the first microchannel and the second inlet of the second microchannel.

[0021] In some embodiments, at least two of the plurality of devices are fluidically connected in parallel. In some embodiments, the plurality of devices comprises: a first device comprising a first inlet, a first outlet, and a first microchannel in fluidic communication with the first inlet and the first outlet; and a second device comprising a second inlet, a second outlet, and a second microchannel in fluidic communication with the second inlet and the second outlet. In some embodiments, the system further comprises: a fluidic interconnect disposed between a fluid source, the first inlet, and the second inlet, thereby providing fluidic communication between the fluid source and the first outlet of the first microchannel and between the fluid source and the second inlet of the second microchannel.

[0022] In some embodiments, the electronics module comprises: a signal generator configured to generate a radiofrequency (RF) signal or an electrical signal; and a circuit configured to transmit the RF signal or electrical signal to the first and second electrode regions.

[0023] In some embodiments, the fluidics module comprises: a fluidic controller configured to deliver fluids to the first microchannel.

[0024] In some embodiments, the temperature control module comprises: a heat exchanger, a heat sink, an insulator, or a combination of any of these.

[0025] In another aspect, the present document encompasses a method of separating entities in a sample (e.g.. based on a characteristic, such as size), the method comprising: delivering the sample through a first microchannel; applying an acoustic wave through at least a portion of the first microchannel, thereby generating displacement of at least a portion of a first population and a second population traveling through the first microchannel by way of acoustic radiation force and acoustic streaming; and separating the sample into a first separated population and a second separated population.

[0026] In some embodiments, the sample comprises a first population of entities having a first characteristic and a second population of entities having a second characteristic that is different than the first characteristic. In some embodiments, the first separated population comprises a maj ority of entities having the first characteristic, and the second separated population comprises a majority of entities having the second characteristic.

[0027] In some embodiments, the first characteristic and the second characteristic are characterized by a difference in size, shape, hydrodynamic radius, composition, density, compressibility, shape, or a combination of any of these.

[0028] In some embodiments, the entities comprise particles (e.g., nanoparticles, microparticles, or mixtures thereof), proteins, polymers, lipids, nucleic acids, vesicles (e.g..exosomes, extracellular vesicles, and the like), cells (e.g., red blood cells, platelets, cancer cells, and the like), organisms (e.g., bacteria), contaminants, or a combination of any of these.

[0029] In some embodiments, said separating comprises using asymmetry in an amplitude of the acoustic wave traveling through the first microchannel to generate asymmetry in streaming or in a radiation field in the first microchannel.

[0030] In some embodiments, the majority’ of particles in the first separated population comprises a population in which more than about 50% (e.g., more than about 55%. 60%.65%, 70%, 75%, 80%, 85%, or more) of the particles are characterized as having the first characteristic.

[0031] In some embodiments, the majority' of particles in the second separated population comprises a population in which more than about 50% (e.g., more than about 55%. 60%.65%, 70%, 75%, 80%, 85%, or more) of the particles are characterized as having the second characteristic.

[0032] In some embodiments, said separating comprises flowing the first separated population into a first outlet channel and flowing the second separated population into a second outlet channel. In some embodiments, each of the first and second outlet channels are in fluidic communication with the first microchannel.

[0033] In some embodiments, the sample comprises a volume fraction of about 0.001% 1% occupied by the first population of entities and the second population of entities.

[0034] In some embodiments, the sample comprises a concentration of about 0.001% to 1 % (v / v) of the first population of entities and the second population of entities.

[0035] In some embodiments, the method further comprises: diluting the sample prior to said applying, wherein said diluting can occur before, during, or after said delivering the sample through a first microchannel. In some embodiments, said diluting is performed within the first microchannel.

[0036] In some embodiments, the method further comprises: delivering a sheath fluid to the first microchannel, thereby diluting the sample with the sheath fluid.

[0037] In some embodiments, the method further comprises: modifying (e.g., increasing or decreasing) a viscosity of the sample prior to said applying, wherein said modifying can occur before, during, or after said delivering the sample through a first microchannel. In some embodiments, said modify ing is performed within the first microchannel.

[0038] In some embodiments, the method further comprises: delivering a sheath fluid to the first microchannel, thereby modifying the viscosity of the sample with the sheath fluid.

[0039] In some embodiments, the method further comprises: collecting, separately, the first separated population and the second separated population.

[0040] In some embodiments, the method further comprises: analyzing the first separated population and the second separated population.

[0041] In yet another aspect, the present document encompasses a method of characterizing a sample, the method comprising: delivering the sample through a first microchannel; applying an acoustic wave through at least a portion of the first microchannel, thereby generating displacement of at least a portion of a first population and a second population traveling through the first microchannel by way of acoustic radiation force and acoustic streaming; separating the sample into a first separated population and a second separated population; collecting, separately, the first separated population and the second separated population; and analyzing the first separated population and the second separated population.

[0042] In some embodiments, the sample comprises a first population of particles having a first characteristic and a second population of particles having a second characteristic that is different than the first characteristic.

[0043] In some embodiments, the first separated population comprises a majority of particles having the first characteristic, and the second separated population comprises a majority of particles having the second characteristic.

[0044] In any embodiment herein, the acoustic wave is a surface acoustic wave and / or a standing acoustic wave.

[0045] In any embodiment herein, the acoustic wave is characterized as a cosine waveform, a sine waveform, a pulsed waveform, a sawtooth waveform, a non-continuous waveform, a non-standing waveform, or a combination of any of these.

[0046] In any embodiment herein, the acoustic wave is generated by applying least 1 watt (e.g., at least 1.1, 1.2, 1.3, 1.4, or 1.5 watts).

[0047] In any embodiment herein, the sample comprises plasma, serum, transdermal fluid, interstitial fluid, sweat, intraocular fluid, vitreous humor, cerebrospinal fluid, extracellular fluid, lacrimal fluid, tear fluid, sputum, saliva, mucus, a microorganism, a virus, a bacterium, a fungus, a parasite, a helminth, a protozoon, a cell, tissue, a fluid, a swab, a biological sample (e.g., blood, serum, plasma, saliva, etc.), an environmental sample (e.g., a water sample, a soil sample, etc.), or an agricultural sample.

[0048] In any embodiment herein, the sample comprises a plurality of particles. In some embodiments, the plurality' of particles comprises nanoparticles, microparticles, proteins.polymers, lipids, nucleic acids, vesicles (e.g., exosomes, extracellular vesicles, and the like), cells (e.g., red blood cells, platelets, cancer cells, and the like), organisms (e.g.. bacteria), contaminants, or a combination of any of these.Definitions

[0049] As used herein, the term “about” means + / - 10% of any recited value. As used herein, this term modifies any recited value, range of values, or endpoints of one or more ranges.

[0050] By “microfluidic” or “micro” is meant having at least one dimension that is less than 1 mm. For instance, a microfluidic structure (e.g., any structure described herein) can have a length, width, height, cross-sectional dimension, circumference, radius (e.g., external or internal radius), or diameter that is less than 1 mm.

[0051] By “nanofluidic” or “nano” is meant having at least one dimension that is less than 1 μm. For instance, a nanoparticle can have a length, width, height, cross-sectional dimension, circumference, radius (e.g., external or internal radius), or diameter that is less than 1 μm.

[0052] By “electrical connection,” as used herein, refers to any conductive or semi- conductive structure through which an electrical signal may pass. The electrical signal can be any useful change in electrical field, electric potential, current, or voltage. Non-limiting structures include lines, contact pads, busses, pins, connectors, bond lines, bond pads, etc., formed from any useful material (e.g., an ohmic material, a metal, etc.).

[0053] By “fluidic communication,” as used herein, refers to any duct, channel, tube, pipe, chamber, or pathway through which a substance, such as a liquid, gas, or solid may pass substantially unrestricted when the pathway is open. When the pathway is closed, the substance is substantially restricted from passing through. Typically, limited diffusion of a substance through the material of a plate, base, and / or a substrate, which may or may not occur depending on the compositions of the substance and materials, does not constitute fluidic communication.

[0054] As used herein, the terms “top,” “bottom,” “upper,” “lower,” “above,” and “below” are used to provide a relative relationship between structures. The use of these terms does not indicate or require that a particular structure must be located at a particular location in the apparatus.

[0055] Other features and advantages of the invention will be apparent from the following description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGs. 1A-B provides (Fig. 1A) a schematic of a non-limiting device 100 and (Fig. IB) a schematic of a non-limiting channel 1500.

[0057] FIGs. 2A-C provides schematics of a non-limiting integrated acoustofluidic device for nanoparticle purification and non-limiting working mechanisms. Fig. 2A. Schematic of the acoustofluidic device. A microfluidic channel was bonded to the LiNbOs substrate in between Interdigitated transducers. Fig. 2B. A standing surface acoustic wave (SSAW) was applied in a square channel of width = 200 μm and depth = 40 μm, coated with PEG (1% w / v). Fig. 2C. As the nanoparticles travel through the acoustofluidic device, they are subjected to four physical forces: drag forces induced by the applied Poiseuille flow, diffusion forces induced by gradients of chemical potential and Brownian motion, acoustic radiation forces induced by the SSAWs as well as secondary drag forces induced by the acoustic streaming.

[0058] FIGs. 3A-C shows differential separation of nanoparticles using SSAWs and acoustic streaming. Fig. 3A. Schematic representation of a non-limiting experiment. A sample including a mixture of fluorescent 1 μm particles. 200 nm particles and albumin in DI water was subjected to high-intensity SSAW at different flow rates of the Poiseuille flow. Fig. 3B. Fluorescent microscopy images showing the xy cross section (top view) of the microfluidic channel. Display of the effect of SSAWs on the mixture of fluorescent nanoparticles when the average flow velocity is v = 350 μm / s (Re = 4.2xl0‘4). Fig. 3C. Fluorescent microscopy images displaying the effect of SSAWs on the mixture of fluorescent nanoparticles when the average flow velocity is v = 10 μm / s (Re = 1.2xl0'5).

[0059] FIGs. 4A-D shows analysis of the spatial distribution of nanoparticles under SAWs at low flow velocities. Fig. 4A. Fluorescent microscopy image showing the top view of the microfluidic channel. The 200 nm particles exhibit different spatial distribution in the channel as a function of the applied input power to the IDTs, which increases the acoustic wave intensity. Low Intensity (P = 0.5 W) present no visible changes, medium intensity (P = 1 W) shows disruption of the particle distribution, and high intensity (P = 1.5 W) show particle focusing. Fig. 4B. Concentration of 200 nm particles along the channel width for different values of applied input power. Fig. 4C. Fluorescent microscopy images showing the spatial distribution of different nanoparticle sizes at low flow velocity (v = 10 μm / s) and high acoustic intensity (P = 1.5W). Fig. 4D. Concentration of nanoparticles collected at the right outlet of the microfluidic device after applying SSAWs with low flow velocity (v = 10 μm / s) and high acoustic intensity (P = 1.5W).

[0060] FIGs. 5A-H shows differential purification of nanoparticles using SSAWs in a microfluidic channel as a function of Pe. Fig. 5A. Schematic representation of a non-limiting experiment. A focused stream of a sample including a mixture of fluorescent 1 μm particles, 200 nm particles and albumin was flowed into the Acoustofluidic device along with a sheath flow of DI water. The beam was subjected to high-intensity SSAW at different flow rates of the Poiseuille flow. Fig. 5B. After applying SSAWs (P = 1.5W), change in concentration of fluorescent particles collected in the right outlet as a function of the Poiseuille flow average velocity in the microfluidic channel. Figs. 5C-E. Analysis of the effects of the Peclet number on the fractionation of nanoparticles using high intensity SSAWs. Plots show the change of total particle count collected on the right outlet of albumin (Fig. 5C), 200 nm particles (Fig. 5D), and 1 μm particles (Fig. 5E). Figs. 5F-H. Effect of the Peclet number on particle concentration using high intensity SSAWs. Plots show the change of concentration of albumin (Fig. 5F), 200 nm particles (G), and 1 μm particles (Fig. 5H) collected on the right outlet. Diamonds represent experimental results without SSAWs, and circles represent values when SAWs are applied. Peclet number (Pe) was determined as follows: Pe = v x LcI D, where v is the velocity of the fluid; Lc is the characteristic length of the channel, which was defined Lc= (2 x w x h) / (w + h), where w is the width and h is the height of the square channel; and D is the diffusivity of the particle in question.

[0061] FIGs. 6A-F shows purification of extracellular vesicles using high intensity SSAWs in microfluidic channels. A sample of fluorescently labeled extracellular vesicles (CD9+mcherry) is analyzed after acoustofluidic purification. Fig. 6A. Nano-tracking analysis (NT A) showing the size distribution and concentration of vesicles before and after differential separation using SSAWs. Fig. 6B. Particle recovery and Fig. 6C. Purity ratio of the samples collected. Applied power P = 1.5W, average flow velocity v = 10 μm / s. Figs. 6D-F. Size distribution and peaks of concentration identified by NTA for the samples at the channel inlet (Fig. 6D), channel right outlet (Fig. 6E), and channel center outlet (Fig. 6F). The concentration of particles at the channel left outlet was negligible.

[0062] FIGs. 7A-F shows non-limiting simulations predicting the differential separation of 150 nm nanoparticles based on the interplay between acoustic streaming and the acoustic radiation force with dampened SSAWs. Finite element simulations of a square microchannel subject to undampened and dampened standing acoustic waves. Figs. 7A-C. Acoustic streaming flow field (Fig. 7A), acoustic radiation force field (Fig. 7B), and histogram (Fig. 7C) of particle distribution after particle-tracing in a system with undampened standing waves. Figs. 7D-F. Corresponding acoustic streaming flow field (Fig. 7D), acoustic radiationforce field (Fig. 7E), and histogram (Fig. 7F) of particle distribution after particle-tracing in a system with dampened standing waves, where the acoustic intensity decays along the channel width.DETAILED DESCRIPTION

[0063] The present documents relates to a microfluidic device that uses acoustic waves to separate and purify particles (e.g., nanoparticles) from a mixture that contains other entities microparticles, soluble molecules, and the like in a liquid). This technology creates a wave that propagates though a piezoelectric material, in which the waves interact with the fluid solution as it flows through a microchannel placed in between a set of transducers. The flow rate and intensity of the wave can be controlled to displace particles within the microfluidic channel to collect different fractions of the particles into different outlets of the device. This displacement can be achieved by an interplay between the acoustic radiation forces and the acoustic streaming induced by the ultrasound field exerted.

[0064] FIG. 1A provides a non-limiting schematic of a device 100 including a substrate 101 and a channel 150 disposed on the top surface 101A of the substrate 101. The channel, in turn, can include an inlet configured to deliver a sample having a flow 155 that travels through the channel 150. The substrate can include a piezoelectric material (e.g., as in a piezoelectric substrate). In some embodiments, a substrate can include a piezoelectric portion (e.g.. a piezoelectric substrate disposed between the first and second electrodes) and a nonpiezoelectric portion (e.g., a semiconductor substrate, such as a silicon substrate). In some embodiments, a substrate can include a piezoelectric substrate.

[0065] The channel can be disposed in proximity to an active region. The active region can include a first electrode 110. a second electrode 120, and the area disposed between the first and second electrodes 1 10, 120. The electrodes can be disposed on the top surface 101A of the substrate 101. Furthermore, the first and second electrodes can be configured to launch an acoustic wave 130 (e.g., a surface acoustic wave, a standing acoustic wave, etc.) through at least a portion the channel 150. In some embodiments, the active region is disposed on the top surface of the piezoelectric portion.

[0066] In use, the acoustic w ave is characterized by a propagation direction 135 along the y-axis in FIG. 1A. Here, flow 155 within the channel 150 travels in a direction along the x- axis. In some embodiments, a propagation direction of the acoustic wave is not parallel to a direction of flow w ithin the channel. In other embodiments, the propagation direction of the acoustic wave is perpendicular to the direction of flow within the channel.

[0067] In some embodiments, the propagation direction of the wave coming out of the transducer will propagate in direction 135 (as depicted in FIG. 1A). while the reflected wave generated by the receiving transducer propagates in the opposite direction, thereby establishing a standing wave. In addition to standing waves, the displacement phenomena (e.g., described or observed herein) can be established by one traveling surface acoustic wave in propagating direction 135.

[0068] As seen in FIG. 1 A, a propagation direction 135 of the acoustic wave can be provided at an angle a to a direction of flow 155 within the channel 150. In some embodiments, a is not 0° and not 180°. In some embodiments, 0° < a < 180° or 180° < a < 360°. In some embodiments, a is an oblique angle.

[0069] The acoustic wave can be implemented to provide acoustic radiation force and acoustic streaming within the channel. In some embodiments, the acoustic wave is configured to generate displacement of a particle or modify a velocity of a fluid within the channel. As seen in FIG. IB, the acoustic wave can impart acoustofluidic effects 1600 within the first channel 1500. In some embodiments, the acoustic wave can be configured to induce particle displacement and modify the fluid-flow velocity within the channel by way of acoustic radiation forces and acoustic streaming. In other embodiments, the acoustic wave can be configured to provide a non-oscillating velocity component to a liquid or a particle traveling through the first microchannel by way of acoustic radiation force and acoustic streaming. In yet other embodiments, the acoustic wave is configured to provide an asymmetrical or symmetrical amplitude profile of the acoustic wave in the active region. In some embodiments, such a configuration can change, control, or modulate the acoustic radiation force and acoustic streaming along the channel cross-section.

[0070] The acoustic wave can be characterized as a cosine waveform, a sine waveform, a pulsed waveform, a sawtooth waveform, a non-continuous waveform, a non-standing waveform, or a combination of any of these. In some embodiments, the acoustic wave is generated by applying at least 1 watt (e.g., at least 1.1, 1.2, 1.3, 1.4, or 1.5 watts). For example, an intensify of the acoustic wave can be proportional to the input power applied to the RF signal generator that provides the electric signal.

[0071] Such acoustic waves can be generated and maintained within the active area. Furthermore, one or more active regions can be provided. For example, a plurality of active regions can be provided with a plurality of electrode pairs. In use, each electrode pair can be associated with an active region disposed between each electrode of the pair. The active region(s) with the electrodes and, optionally, reflectors can be disposed on a top surface ofthe piezoelectric portion and / or the substrate. One or more electronic circuits for the system can be provided to be in electric connection with the electrodes, the piezoelectric portion, the substrate, or other substrates that may be present.

[0072] To facilitate fluid transport, the device can include one or more channels. The channel can have any useful configuration to provide the sample to the active area. In some embodiments, the channel can be in fluidic communication with other channel(s) to deliver fluids (e.g.. samples, sheath fluids, and the like). In some embodiments, the device can include a first channel having a plurality of inlets (e.g., a first inlet and a second inlet). In turn, one or more inlet channels can provide fluid through the inlet(s). For example, as seen in FIG. IB. the device can include a first inlet channel 1510 in fluidic communication with the first inlet 1501 of a first channel 1500 and a second inlet channel 1520 in fluidic communication with the second inlet 1502 of a first channel 1500. In some embodiments, the first inlet channel can be configured to deliver a first fluid (e.g., including a first sample) through the first microchannel, and the second inlet channel can be configured to deliver a second fluid (e g., comprising a second sample or a first sheath fluid surrounding a flow of the first sample) through the first microchannel.

[0073] Additional inlet channels can be present. For example, as seen in FIG. IB, the device can include a third inlet channel 1530 in fluidic communication with a third inlet 1503 of a first channel 1500. In some embodiments, the third inlet channel is configured to deliver a fluid (e.g., including a third sample or a second sheath fluid surrounding a flow of the first sample or the second sample) through the first microchannel.

[0074] A plurality of inlet channels can be in fluidic communication, thereby forming any useful fluidic network. For example, the device can include a third inlet channel in fluidic communication with the second inlet, wherein the third inlet channel is configured to deliver a fluid (e.g., including a third sample or a second sheath fluid surrounding a flow of the first sample or the second sample) through second inlet and into the first microchannel.Optionally, a fourth inlet channel can be in fluidic communication with the second and third inlets of the first channel (e.g.. the second and third inlets 1502. 1503 of the first channel 1500) so that the same fluid from a same source can be delivered through the second and third inlets and into the first channel. Such a configuration may be useful, e.g., when the same sheath fluid is employed to surround a sample being delivered through a first inlet (e.g., first inlet 1501 in FIG. IB), in which the sheath fluid is delivered through both the second and third inlets (e.g.. the second inlet 1502 and the third inlet 1503 in FIG. IB).

[0075] The device can include one or more outlets in fluidic communication with the channel. As seen in FIG. IB, the device can include a first outlet 1511 in fluidic communication with the first channel 1500. Other outlets can be present. For example, the device can include a second outlet 1512 and a third outlet 1523. In some embodiments, the device includes the same number of inlets and outlets, and each inlet includes a respective outlet. For example, a first inlet 1501 can be associated with a respective first outlet 1511 based on the laminar flow that is present within the first channel 1500.

[0076] One or more outlet channels can be present. In some embodiments, the outlet channel can provide a fluidic communication to an outlet, which in turn can be used to collect separated samples. In some embodiments, the device further includes a first outlet channel 1515 in fluidic communication with a first outlet 1511, a second outlet channel 1525 in fluidic communication with a second outlet 1512, and a third outlet channel 1535 in fluidic communication with a third outlet 1513.

[0077] The channel can have any useful dimension, such as length, width, height, or cross-section (e.g., rectangular, circular, ellipsoid, triangular, etc.). In addition, any of these dimensions can be uniform (e.g., straight, curved, serpentine, etc.) or variable (e.g., tapered, widened, branched, etc.) along its length. In particular, the device can include an array of channels in any useful format (e.g., a format including a parallel array of channels having a main channel branching into a plurality of channels or an array having a plurality of channels converging into a main channel). Each channel, in turn, can include an inlet configured to receive fluid into the channel and an outlet configured to deliver fluid out of the channel. The fluid can have any useful flow velocity (e.g., an average fluid velocity of about 5 to 20 μm / s through a channel).

[0078] The channel can include a functionalized surface configured to minimize adsorption of targets within a fluid sample; to sense, label, or capture targets within a fluid sample; or to exclude certain undesired components within a fluid sample. The surface can include, e.g., one or more modifications, such to include a polymer (e.g., a poly(ethylene glycol) or a charged polymer), a protein (e.g., serum proteins), and the like, as well as combinations thereof.Electrodes

[0079] As described herein, the device can include at least a first electrode and a second electrode to provide an acoustic wave. Electrodes can have any useful configuration. The positions and configuration of the electrode regions can be optimized. For example, the electrode region can include one or more transducers, which in turn are arranged to providean acoustic wave that propagates along a first direction. In some embodiments, the electrodes include interdigitated transducers (IDT), in which comb electrodes are arranged to interdigitate the fingers of the electrodes. One of these electrodes can be an active electrode, and the other a grounded electrode. Each electrode can have any useful configuration and geometry. Other useful configurations and geometries for the electrodes can be employed. For instance, one, two. or more pairs of transducers can be employed. The design of the IDT can be selected from single finger electrodes, double split finger electrodes, pruned double split finger electrodes, or unidirectional electrodes.

[0080] Yet other exemplary electrodes include a planar electrode, a grating electrode, a thin film electrode, and / or a floating electrode having any useful thickness, period, material, or geometric arrangement and formed by any useful process, such as sputtering, vacuum deposition, or ion-plating. Non-limiting materials for electrodes include aluminum (Al), titanium (Ti), gold (Au), copper (Cu), tungsten (W), molybdenum (Mo), platinum (Pt), ruthenium (Ru), tantalum (Ta), chromium (Cr), osmium (Os), rhenium (Re), iridium (Ir). as well as alloys, doped forms, and multilayers thereof (e.g., TiW / AICu or TiW / Cu layers). Arrays of n electrodes or n pairs of electrodes (e.g.. n is 2, 4, 5. 10. 15, 20, 24, etc.) can also be incorporated with the devices and systems herein.

[0081] The device can further include one or more reflector regions disposed on a surface of the piezoelectric portion and / or the substrate. The reflector region(s) are arranged to provide an active area that confines the acoustic wave within a region of the piezoelectric portion. The reflectors can be formed of any useful material, including, but not limited to, aluminum, gold, chromium, silver, platinum, with an optional adhesion layer (e.g., including titanium), as well as alloys thereof. In one non-limiting example, the reflectors are composed of the same material as the electrodes, e.g., to simplify fabrication of the device.

[0082] The substrate can include any useful piezoelectric material. Exemplary piezoelectric materials include lithium tantalate (LiTaO2). lithium niobate (LiNbOs), potassium niobate (KNbCh), quartz (SiO2, such as an a-SiO2), langatate (La3Ga5.5Tao.5O14), langasite (La3Ga5SiOu). langanite (La3Ga5.5Nbo.5O14), lead zirconate titanate (Pb[ZrxTii-x]O3, where 0<x<l, such as PbZro.52Tio.4sO3), cadmium sulfide (CdS), berlinite (AIPO4). gallium phosphate (GaPO4), lithium iodate (LilOs), lithium tetraborate (Li2B4O7), bismuth germanium oxide (Bi12Ge020), zinc oxide (ZnO), aluminum nitride (AIN), etc., provided in any useful orientation, e.g., 36° YX LiTaOs, Y+36° cut LiTaOs, 0° X-cut LiTaOs, 128° XY LiNbOs, 41° YX LiNbOs, 64° YX LiNbOs, rotated Y-cut quartz, or 36° Y quartz. In someembodiments, the substrate include a piezoelectric material (e.g., any described herein) and a non-piezoelectric material (e.g., a semiconductor material, such as silicon).

[0083] A guide layer can overlie a top surface of the substrate, or a portion of this top surface. Such a guide layer can be used to propagate an acoustic wave confined to the guide layer. The guide layer can be formed of any useful material, such as a polymer (e.g., a polystyrene, a polyimide, a polynorbomene. a perfluoropolymer, a poly(xylylene) (e.g., parylene C or poly(chloro-p-xylylene)), poly(dimethylsiloxane), or a polymethylmethacrylate (PMMA)), an oxide (e g., ZnO), or a dielectric (e.g., a silicon oxide, such as SiO2; a silicon oxynitride, e.g., SiON; or a silicon nitride, such as SisN4, which can optionally including one or more dopants).Systems

[0084] One or more devices can be implemented with one or more modules within a system. In some embodiments, the system includes a plurality of devices (e.g., any herein), in which each device can be same or different. In some embodiments, at least two of the plurality of devices are fluidically connected in series or in parallel.

[0085] The inlets and outlets of devices can be configured to provide flow between devices. In some embodiments, the system includes: a first device comprising a first inlet, a first outlet, and a first microchannel in fluidic communication w ith the first inlet and the first outlet; and a second device comprising a second inlet, a second outlet, and a second microchannel in fluidic communication with the second inlet and the second outlet. Fluidic communication can be provided by way of a fluidic interconnect (e.g., a tubing, a manifold, etc.), which in turn can be disposed between the first outlet and the second inlet. In this way, fluidic communication can be established between the first outlet of the first microchannel and the second inlet of the second microchannel.

[0086] Configurations within the system can provide at least two of the plurality of devices are fluidically connected in parallel. For example, each device can be in direct connection with a fluid source. In some embodiments, the system includes: a first device comprising a first inlet, a first outlet, and a first microchannel in fluidic communication with the first inlet and the first outlet; and a second device comprising a second inlet, a second outlet, and a second microchannel in fluidic communication with the second inlet and the second outlet. Fluidic communication can be provided by way of a fluidic interconnect disposed between a fluid source, the first inlet, and the second inlet. In this way. fluidic communication can be established between the fluid source and the first outlet of the first microchannel and between the fluid source and the second inlet of the second microchannel.

[0087] The system can include an electronics module to provide electrical signals to the one or more devices. For example, the electronics module can include any number of electrical components configured to deliver an electrical signal to the piezoelectric portion and / or the substrate, in which that electrical signal is transduced to provide an acoustic wave. The electrical components can be configured to have an electrical connection with one or more electrodes configured in any useful manner. The electrodes can form a delay line, which can be optionally shorted. In addition, such lines can be unidirectional or bidirectional. The electrodes can be of any useful configuration (e.g., an interdigitated configuration, an arrayed configuration, a gate configuration, a one-port configuration, a two-port configuration, a delay line configuration, a unidirectional configuration, a bidirectional configuration, etc.), geometry (e.g.. bar electrodes, single finger electrodes, double finger electrodes, split finger electrodes, pruned double split finger electrodes, etc.), orientation (e.g., having a major axis that is orthogonal to a first direction that is the propagate direction of the acoustic wave and / or configured to provide an acoustic wave along a crystal cut or axis that supports acoustic waves), or electrical connection (e.g., shorted, grounded, open, closed, arrayed, etc.).

[0088] The electronics module can include any useful circuit components, such as an oscillator circuit (e.g., a Pierce circuit, a Colpitts circuit, or a Clapp circuit including an amplifier or a transistor, such as a bipolar junction transistor); one or more signal generators (e.g., configured to generate a radiofrequency (RF) signal or an electrical signal); one or more relay circuits (e.g.. including one or more circuits configured to transmit the RF signal or electrical signal to the first and second electrode regions); one or more attenuation networks (e.g., including one or more circuit components to reduce the amplitude of a signal, such as by use of one or more resistors); one or more filters (e.g., a frequency selective, a high pass filter, a low pass filter, or a phase shifting filter); one or more amplifiers (e.g., transistors); one or more impedance matching networks (e.g., including one or more circuit components configured to match the impedance of the device to another electronic component, in which exemplary7impedance matching networks can include an inductor with an optional resistor in series with the non-grounded electrode(s) of the device); and / or one or more coupling networks (e.g.. configured to provide an output, such as a measured frequency shift).

[0089] The fluidics module can include any useful port, via, chamber, and / or channel to deliver a sample to the active area of the device. The fluidics module can include a fluidic controller configured to deliver fluids to the channel(s) of the device. The fluidics module may be configured to be a disposable manifold that aligns with the device and the electronics module. Alternatively, the fluidics module may be reusable for repeat use. The fluidicsmodule can optionally include any useful microfluidic structure having a length, width, height, cross-sectional dimension, circumference, radius (e.g., external or internal radius), or diameter that is less than 1 mm.

[0090] A fluidics module can include a fluidics layer configured to be in fluidic communication with an active area of a device, in which the fluidics layer can include a channel designed to overlie the active area. In some embodiments, the fluidics layer can include a bulk substrate having one or more channels disposed thereof. The bulk substrate can include any useful material, including but not limited to glass, quartz, silicon, polymers, metals, or combinations thereof.

[0091] The temperature control module can include any components to regulate temperature of the device (e.g., the substrate, the channel, and the like). Such components can include one or more heat exchangers (e.g., surface plates with temperature control, a heat bath (e.g., a device in contact with a fluid to exchange heat, use of convective dissipation by flowing liquid or air in sealed devices, and others), coolers (e.g., Peltier coolers), heaters, heat sinks, insulations, or combinations of any of these.Methods

[0092] The present document also relates to methods of separating entities in a sample by employing any device or system herein.

[0093] The method can include separating entities in a sample (e.g., based on a characteristic, such as size). In some embodiments, the method includes: delivering the sample through a first microchannel, wherein the sample comprises a first population of entities having a first characteristic and a second population of entities having a second characteristic that is different than the first characteristic; and applying an acoustic wave through at least a portion of the first microchannel. In turn, at least a portion of the first and second populations traveling through the first microchannel can be displaced by way of acoustic radiation force and acoustic streaming. In some embodiments, said applying provides a non-oscillating velocity component to the first and second populations.

[0094] The method can further include separating the sample into a first separated population and a second separated population. Separating can include introducing asymmetry' in the amplitude of the acoustic wave traveling through the first microchannel or by introducing any other useful variation to such an acoustic wave that alters symmetry of the acoustic streaming and acoustic radiation force fields within the channel.

[0095] Separating can also include flowing the separated populations into different channels. For instance, a first separated population can be delivered into a first outletchannel, and a second separated population can be delivered into a second outlet channel. Each of the first and second outlet channels are in fluidic communication with the first microchannel. If more than two separated populations are present, then the device can include a plurality of channels configured to deliver such separated populations in any useful manner (e.g., to a collection output).

[0096] Optionally, separated populations can be collected. In some embodiments, the method can further include: collecting, separately, the first separated population and the second separated population. Separated populations can be stored, analyzed, or otherwise further processes.

[0097] In some embodiments, the first separated population includes a majonty of entities having the first characteristic, and the second separated population includes a majority of entities having the second characteristic. Any useful characteristic can be used to distinguish the first and second populations. In some embodiments, the first characteristic and the second characteristic are characterized by a difference in size, shape, hydrodynamic radius, composition, density, compressibility, shape, or a combination of any of these.

[0098] The separated population can include a majority of particles having that certain characteristics. In some embodiments, the majority of particles in the first separated population can include a population in which more than about 50% (e.g., more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more) of the particles are characterized as having the first characteristic. In some embodiments, the majority of particles in the second separated population can include a population in which more than about 50% (e g., more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more) of the particles are characterized as having the second characteristic.

[0099] Any useful entities can be separated. Non-limiting entities can include particles (e.g., nanoparticles, microparticles, or mixtures thereof), proteins, polymers, lipids, nucleic acids, vesicles (e.g., exosomes, extracellular vesicles, and the like), cells (e.g., red blood cells, platelets, cancer cells, and the like), organisms (e.g., bacteria), contaminants, or a combination of any of these.

[0100] Such entities can be present in any useful amount in any useful sample. Nonlimiting amounts include a volume fraction of about 0.001% to 10% occupied by the entities (e.g., 0.001% to 7%, 0.001% to 5%, 0.001% to 3%, 0.001% to 1%, or 0.001% to 0.1%); or a concentration of about 0.001% to 10% (v / v) of the entities (e.g., 0.001% to 7%, 0.001% to 5%, 0.001% to 3%, 0.001% to 1%, or 0.001% to 0.1% (v / v)). Non-limiting examples of samples include plasma, serum, transdermal fluid, interstitial fluid, sweat, intraocular fluid.vitreous humor, cerebrospinal fluid, extracellular fluid, lacrimal fluid, tear fluid, sputum, saliva, mucus, a microorganism, a virus, a bacterium, a fungus, a parasite, a helminth, a protozoon, a cell, tissue, a fluid, a swab, a biological sample (e.g., blood, serum, plasma, saliva, etc.), an environmental sample (e.g., a water sample, a soil sample, etc.), or an agricultural sample.

[0101] The methods herein can include one or more optional operations. For instance, a sample can be diluted. In some embodiments, the method can further include: diluting the sample (e.g., prior to applying an acoustic wave). Dilution can occur before, during, or after delivering the sample through a channel. In another instance, a sheath fluid can be provided to the channel. In some embodiments, the sheath fluid can be used to dilute the sample within the channel.

[0102] In another instance, a viscosity of the sample can be modified. In some embodiments, the method can further include: modifying (e.g., increasing or decreasing) a viscosity of the sample (e.g., prior to applying an acoustic wave). Modifications can occur before, during, or after said delivering the sample through a channel. In another instance, a sheath fluid can be provided to the channel to modify the viscosity of the sample. The sheath fluid can be characterized by a viscosity that is higher than, lower than, or same as a viscosity of the sample.

[0103] Samples can be characterized. In some embodiments, the method of characterizing a sample includes: delivering a sample through a first microchannel, wherein the sample comprises a first population of particles having a first characteristic and a second population of particles having a second characteristic that is different than the first characteristic; applying an acoustic wave through at least a portion of the first microchannel, thereby generating displacement of at least a portion of the first and second populations traveling through the first microchannel by way of acoustic radiation force and acoustic streaming; separating the sample into a first separated population and a second separated population, wherein the first separated population comprises a majority of particles having the first characteristic and wherein the second separated population comprises a majority of particles having the second characteristic; collecting, separately, the first separated population and the second separated population; and analyzing the first separated population and the second separated population.Uses

[0104] This acoustofluidic technology can be applied for various uses, including displacing, focusing, and / or filtering particles (e.g., nanoparticles), or removing both latermicroparticles and / or smaller molecules in a solution. This technology can be used for nanoparticle filtration in microfluidics. The use of acoustic fields can provide a contactless filter, which removes the risk of fouling or clogging of filtration membranes while maintaining a high concentration and / or high recovery (e.g., as compared to ultracentrifugation and / or size exclusion methodologies). This technology can be applied as a continuous and / or high-throughput nanofiltration system in various applications (e.g., treatment or filtration of various types of samples in various industries, such as water treatment, manufacturing, food and beverage, oil and gas, mining, and mineral processing, cosmetics, pharmaceuticals, diagnostics, and nanomedicine).

[0105] The devices, systems, and methods herein can be employed with any useful test sample, such as a fluid, a biological sample (e.g., blood, serum, plasma, saliva, etc.), an environmental sample, an agricultural sample, and the like. The sample can be obtained from any useful source, such as a subject (e.g., a human or non-human animal), a plant (e.g., an exudate or plant tissue, for any useful testing, such as for genomic and / or pathogen testing), an environment (e.g., a soil, air, and / or water sample), a chemical material, a biological material, or a manufactured product (e.g., such as a food or drug product).EXAMPLESExample 1: Differential purification of nanoparticles combining acoustic radiation forces and acoustic streaming in microfluidic channels

[0106] The separation of nanoparticles from heterogeneous mixtures can be useful for applications in photonics, metamaterials, photovoltaics, cosmetics, water treatment, drug delivery, medical diagnostics, and therapeutics. Conventional techniques commonly applied for nanoparticle separation include ultracentrifugation, electrophoresis, chromatography, filtration, size-selective precipitation, and solvent addition. Despite having high separation efficiency and good reproducibility', these techniques have significant limitations: multi-step preparation, performance in batch mode, low particle recovery, and need for a minimum sample volume. To overcome these limitations, technologies aim to achieve fast separation of nanoparticle samples with high recovery and minimal loses. Recently, scientists and engineers have shifted their attention towards microfluidic technologies for their remarkable ability to process multiple samples in parallel, with high throughput, scalability, and automation. In addition, microfluidics offer low cost, low sample volume, and minimal sample handling. Consequently, microfluidic methods can provide promising techniques to manipulate small volumes of samples, especially for biological and medical applications. Thesmaller dimensions, reduced costs, potential disposability, and lower sample volume requirements of these miniaturized systems add to the attractiveness of these methods for implementation into point-of-care diagnostic platforms.

[0107] Acoustofluidics is an emerging microfluidic technique for the precise manipulation of particles using the interaction of sound waves with solids, liquids, and gases. The combination of acoustic and microfluidic methods can address challenges in mixing, atomizing, droplet manipulation, nanofluidics, and biomedical research. In particular, there has been an emphasis on actuation of micro-objects, including bacteria, red blood cells, cancer cells, exosomes, and extracellular vesicles. Acoustofluidic separation offers a label- free filtration approach designed to displace particles with different sizes, as well as particles with different physical or mechanical properties. The ability to generate acoustic waves with frequencies in the kilohertz-to-megahertz range can allow' acoustofluidic techniques to directly manipulate particles across a length scale spanning more than five orders of magnitude ( 10 -7to 102- m). The acoustic pow er and frequencies employed have been safely- used in diagnostic applications, exhibiting remarkable biocompatibility. The versatility and biocompatibility of acoustofluidics highlight the technique’s potential to address challenges in biology and biomedicine for the isolation and detection of circulating biomarkers.

[0108] Of the different unit operations based on acoustofluidics, concentration of particles in suspension can be achieved using acoustophoresis. For examples, particles can be focused into a pressure node thereby depleting the surrounding medium. The flow can then be split into fractions containing concentrated particles or particle-free medium. Relevant applications of acoustic focusing include, e.g., : i) acoustofluidics on cylindrical capillaries, proposed as an alternative to hydrodynamic focusing on fluorescence activated cell sorting (FACS); ii) develoμment of a plasmapheresis chip capable of handling undiluted whole blood, removing the red blood cells and delivering clarified blood plasma; and iii) bead-based immunoassays performed in microtiter well plates, for the aggregation of antibody-activated microbeads to enhance sensitivity-. In addition, acoustic focusing can be used for free flow acoustophoresis (FFA), a field flow fractionation technique that exploits net acoustic forces driving particles towards a pressure node as they travel with the flow (e.g., as in a dual outlet FFA system for the enrichment of tumor cells (TCs) spiked into blood samples, which could be used to concentrate circulating tumor cells; separation of cultured cancer cells from white blood cells; and the separation of rare circulating tumor cells).

[0109] While most acoustic techniques relying on the acoustic radiation force as mechanism for concentration have been successful for applications w ith microparticles.recent efforts have shown moderate success on the purification of nanoparticles. Extracellular vesicles (EVs) — cell-secreted nanoparticles of 50-150 nm in diameter — represent an exciting set of vesicles with unique molecular cargoes that act as molecular fingerprints of the parent cell, and can be used to identify disease biomarkers from complex samples such as blood or lymph. Like other soft nanoparticles, the current technologies to isolate and purify EVs require expensive equiμment, are time consuming, produce high losses of valuable samples and exhibit low throughputs.

[0110] Recently, an acoustofluidic device using orthogonal standing surface acoustic waves (SAWs) was shown to achieve high purification and recovery of EVs by focusing and depleting cells and other microparticles from solution. Similarly, a tilted angle standing SAW was used to deplete larger particles from whole blood samples, yielding EVs with high recovery. These results show how acoustofluidics can be used effectively to purify vesicles, liposomes, viruses, and biologicals maintaining their viability’ and integrity. Despite these advancements, current acoustofluidic methods relying on acoustic radiation forces are unable to separate EVs or other nanoparticles from the soluble molecular compartment.Furthermore, these methods generally cannot concentrate and focus nanoparticles, preferring to actively deplete larger particles as the primary purification mechanism. Without wishing to be limited by theory' or mechanism, failure to focus nanoparticles has been attributed to molecular diffusion, thermal effects, and Rayleigh streaming, placing a practical lower limit on the particle size that can be manipulated by the radiation force. Although acoustic streaming has been considered detrimental for focusing and separation of particles in microfluidics, recent studies show that one can use highly focused, high-frequency SAWs to generate flow- pattern useful for particle actuation and particle displacement. These results show effective particle actuation based on acoustic streaming with promising results on submicron particles.[OHl] In contrast to previous acoustofluidic techniques where the acoustic radiation force and the acoustic streaming conflict, here we demonstrate a non-limiting acoustofluidic platform that couple particle focusing using acoustic radiation with acoustic streaming actuation. This collaboration of physical phenomena allows the differential purification of small nanoparticles from both larger micro-particles as well as soluble molecular contaminants. We provide a supporting numerical analysis based on governing equations pointing towards an interplay betw een asymmetrical acoustic radiation and acoustic streaming fields that arise as the wave amplitude diminishes traveling along the channel width. This acoustofluidic unit operation provides a single-step, on-chip nanoparticleseparation technique that overcomes traditional limitations of acoustofluidic purification systems.

[0112] From the interaction of waves with fluids emerge two distinct mechanisms that are commonly at odds when designing separation techniques: the acoustic radiation force and the acoustic streaming. Described herein is a microfluidic platform that combines both mechanisms, driving the separation of 150-300 nm particles from microparticles and small molecules in solution. Using this strategy, a differential separation of particles yielding 83% particle recovery and 75% reduction in concentration of solutes was obtained. The acoustofluidic focusing and separation was successfully used to isolate polystyrene particles and extracellular vesicles from cell culture. Simulations based on fundamental equations indicate that the interplay between the acoustic effects is driven by anisotropies in the force fields acting on the particles, arising from changes in wave amplitude. Taken together, the findings presented here offer promise for develoμment of high-throughput, biocompatible point-of-care purification systems, as well as other systems that would benefit from the processes described herein.Example 2: Non-limiting experimental methods

[0113] Device fabrication and operation: The acoustofluidic device included a microfluidic channel and a piezoelectric substrate generating standing-surface-acoustic-wave (SSAW). The microfluidic channels had a rectangular cross section, with depth h = 40 μm, width w = 200 μm, and length L = 12 mm. The channels were fabricated out of poly dimethylsiloxane (1: 10 curing agent to PDMS base; RTV 615, Momentive Performance Materials, Inc.. Niskayuna, NY) and bonded to the piezoelectric substrate after components were exposed to air plasma (Plasma Cleaner Model PDC-001, Harrick Plasma. Inc., Ithaca, NY). The channel walls were chemically treated with a 1% (w / v) aqueous solution of polyethylene glycol (PEG 8000, Sigma- Aldrich, Burlington, MA) to reduce surface adhesion. To minimize acoustic dissipation, the PDMS wall w as limited to 250 μm thickness in the acoustic region.

[0114] Interdigitated transducers (IDTs) were patterned into a 128° Y-cut of LiNbCE piezoelectric substrate (SAW grade, DSP, 0.5 mm, University' Wafer, Inc., South Boston, MA) using standard lithography techniques. The IDTs metal layer was added using vapor deposition: 10 nm platinum adhesion layer followed by 80 nm gold layer. The single electrode transducer pitch was set to 50 μm to achieve a wavelength of 200 μm. With N = 20, the IDT length was 9.7 mm, and the distance between transducers was set as 1.25 mm. Both IDTs were accompanied by acoustic reflectors on the opposite side to the channel. Highvoltage RF signals were generated using a GHz generator (Model E4431B, Hewlett Packard, Palo Alto, CA) subsequently amplified using a power amplifier (Model ZHL-1-2W-N+, Minicircuits, Brooklyn, NY). Filling of microfluidic channels and fluid control was achieved using a microfluidic flow controller (OBI MK3+, ELVESYS, Paris, France), while the flow rates were monitored using a microfluidic sensor (MFS-D-1, ELVESYS). The temperature was controlled using a temperature controller able to fit under the microscope (PE 120, Linkam Scientific Instruments Ltd., Surrey. United Kingdom).

[0115] Fluorescent particles and extracellular vesicles: Monodisperse, fluorescent polystyrene nanoparticles (FSDG, FSFR, FSSY, Bang Laboratories Inc., Fishers, IN) were used to generate both homogeneous and heterogeneous dilute, aqueous solutions of nanoparticles with concentrations between 0.1-5% (v / v). Some samples analyzed also contained 2% v / v fluorescent albumin from bovine serum (555 BSA, A34786, Invitrogen, Waltham, MA). Samples containing extracellular vesicles were collected and isolated from cell culture. Human melanoma cells transduced to express CD9+mcherry (Me260LN-CD9+) where maintained at 37°C in 5% CO2 in RPMI (Invitrogen) supplemented with 10% FBS. Once cell culture reached confluence, the culture medium was collected and centrifuged at 300 x g for 10 min to remove cell debris and apoptotic bodies. The supernatant was collected and concentrated using 100 kDa Amicon centrifugal filter after centrifugation at 2000 x g for 10 min. The remaining concentrated sample were detached from the filter and resuspended using phosphate-buffered saline (lx PBS; Thermo Fisher Scientific Inc., Waltham, MA) and used for analysis. Solutions contained EVs where prepared by dilution 5 pl of sample into 1 ml of lx PBS.

[0116] Particle characterization and fluorescent microscopy: The size and concentration of EVs / exosomes purified from cell culture supernatants were determined using Nano-tracking analysis (NanoSight NS300, Malvern Instruments Inc., Houston, TX), which is equipped with fast video capture and particle-tracking software. Concentration of the samples collected at the outlet was verified using fluorescence signals detected by Microplate reader (BioTek Synergy Neo2, Agilent Technologies. Inc., Santa Clara, CA). The protein content of cell culture samples validated using MicroBCA protein assay (Thermo Fisher Scientific Inc.). The interaction of samples with SSAWs within the microfluidic device was monitored using a motorized z-focus, motorized and coded 7x nosepiece, fully automated transmitted light axis, and fully automated 5x or 8x fluorescence axis research microscope (DM6000 B. Leica Microsystems GmbH. Wetzlar, Germany).

[0117] Numerical methods: We used COMSOL Multiphysics to perform 2D finite- element simulations of nanoparticle dynamics in acoustofluidics. For this purpose, we used the thermos-viscous acoustics module in the frequency domain to solve for the first-order terms of the expansion. We used the Laminar flow module to compute the second-order terms including the acoustic streaming velocity field. We defined then acoustic radiation force using the existing fields. The Particle tracing for fluid flow module was used to track the evolution of particles under the defined force fields. The Domain was defined as a rectangle of 200 μm width and 40 μm height, decomposed into a free triangular mesh with a boundary layer with 16562 elements. The particle radius was set as 0.075 μm with particle properties set as: pp= 1130 kg / m3, Cp=1500 J kg’1K’1, Kp= 0.19 W m^K’1, ap= 2.9 K’1, β?p=3.93xlO’10Pa’1. Fluid properties where set as those of water.Example 3: Differential separation of nanoparticle mixture using SSAWs

[0118] An acoustofluidic device was fabricated bonding a PDMS microfluidic channel between two interdigitated transducers (IDTs) deposited in the surface of a LiNbCh piezoelectric substrate (FIG. 2A). The microfluidic channel was manufactured using soft lithography techniques, having a square cross-section of height h = 40 μm, a width w = 200 μm, and being coated with a thin layer of PEG (1% (w / v)) to reduce particle adsorption to the channel walls (FIG. IB). The IDTs and accompanying reflectors were patterned with a pitch of 25 μm to obtain a wavelength of X = 200 μm, corresponding to 1 : 1 w / λ ratio. After applying an AC signal into the IDT, the transducers generate a standing SAWs (SSAWs) that propagates through the substrate and interacts with the solution of nanoparticles flowing within the microfluidic channel (FIG. 2A).

[0119] Without wishing to be limited by mechanism or theory, four mechanisms can be used to drive the dynamics of nanoparticles for such acoustofluidic device (FIG. 2C): I) Drag forces originating from the Poiseuille flow along the microfluidic channel; II) Diffusive forces caused by Brownian motion and gradients of chemical potential; III) Acoustic radiation forces originated by scattering from the SSAWs; and IV) Secondary drag forces that arise from acoustic streaming flows. Here, both the acoustic radiation and the streaming arise when the acoustic waves or radiation propagating in liquids impart a slow-, non-oscillating velocity component to the liquid or to small particles suspended in the liquid. The origin of these effects can be traced back to the non-linearity of the Navier-Stokes equation and the small but non-zero compressibility of ordinary liquids. The acoustic streaming is a phenomenon that emerges when the velocity field of the entire liquid acquires an additional,slowly varying component induced by the acoustic waves. On the other hand, the radiation force appears when small particles suspended in a liquid are moved by the momentum transfer from sound waves propagating in the liquid.

[0120] To derive the expressions describing both phenomena, we start by defining the variables and governing equations of this system. The motion of a fluid influenced by an acoustic oscillation is described by the scalar fields pressure p, temperature T, density p, and entropy 5, accompanied by the velocity vector field v. Changes in p and 5 can be given by the thermodynamic relations:which contains the specific heat capacity CPat constant pressure, the specific heat capacity ratio y, the isentropic compressibility K, and the isobaric thermal expansion coefficient a.Therefore, the system can be determined by specifying the values of T, p and v. Performing a perturbation expansion of these variables allows a decomposition of the physical problem into different order contributions. Assuming negligible higher order contributions, the perturbation series is written as follows:withat the wall. For this system, the continuity equation and the Navier-Stokes equations can be defined as:which dictate the evolution of the densify and the velocity field for Newtonian fluids. Neglecting products of first order terms and taking the time derivative of the continuity equation, we obtain a single equation for the spatiotemporal evolution of the density as follows:where Cais the speed of sound. Assuming a harmonic time dependence expressed by the complex phase we obtain the Helmholtz equation for a damped wave with angularfrequency co and w avenumber k: (9)

[0121] In the case of p = 0, this equation reduces to the familiar wave equation when y « 1, as is the case for most aqueous systems. Similarly, the thermodynamic first order equation for 7\ becomes as follows:

[0122] In general, the non-linearity of the Navier-Stokes equation indicates that a solution of the first-order fields cannot be exact. Therefore, a more accurate description of the system behavior can be obtained including the second order terms of the expansion. Assuming harmonic time dependence as before, the time average of the second-order continuity’ equation and Navier-Stokes takes the form as follows:

[0123] Generally, the velocity (v2) is non-zero, with the product of the first order terms acting as source terms. This velocity' indicates acoustic streaming, where the absorption of energy and momentum from the acoustic wave generates flows in the bulk.

[0124] The solutions for the first and second order acoustic fields can be used to determine the acoustic radiation force: time-averaged acoustic forces on a single suspended particle. These radiation forces are a consequence of scattering of the acoustic waves on the particles. On a single small particle of radius a, density pp, and compressibility’the acoustic radiation force is given by as follows:where is the compressibility of the fluid. The pre-factors frand f2are givenby:

[0125] In addition to the acoustic radiation force and the acoustic streaming, velocity fields generated by automated flow controllers in microfluidics can induce drag forces relevant to our analysis. When the fluid is driven through a long, straight, and rigid channel by imposing a pressure difference between the two ends of the channel, we develop a Poiseuille flow. Approximating the microfluidic channel as an infinite parallel-plate configuration, valid for systems with high aspect ratios, a constant pressure difference Ap maintained over a segment of length L of the channel generates a parabolic velocity field vxof the form:

[0126] The velocity profile for generalized rectangular cross-sections assumes a parabolic velocity profile, only disrupted near the comers of the channel. This velocity field is defined by the solution:where / / is the fluid kinematic viscosity, h is the channel height, and w the channel width. This velocity profile is directly proportional to the pressure difference and exerts drag forces that influence particle dynamics in solution.

[0127] Our first experiments explored the ability of our acoustofluidic strategy to separate nanoparticles in heterogeneous aqueous mixtures. Using SSAWs generated by applying 1.5 W of input power, we tested the differential fractionation of a sample containing 1 μm and 200 nm fluorescent particles, as well as fluorescent albumin (FIG. 3A). A strong displacement of 1 μm particles towards the pressure nodes was observed, with an average flow speed of 350 μm / s (FIG. 3B). The 1 μm particles concentrate in the in the side walls of the channel as well. For average flow speed of 350 μm / s, no significant change was observed in the concentration of both 200 nm particles and fluorescent albumin (FIG. 3B). The behavior of the 200 nm particles changes by reducing the flow rate in the channel (FIG. 3C). Applying the same 1.5 W input power to generate SSAW, we observed a focusing of the 200 nm particles towards one of the channel side w alls at an average flow speed of 10 μm / s.

[0128] Under these conditions, the 1 μm particles continue to migrate preferentially towards the pressure nodes and the walls as observed in FIG. 3B, while no changes in the concentration of albumin was noticeable.

[0129] To investigate the unusual stream of focused 200 nm particles observed in FIG. 3C, we performed experiments increasing the intensity of the SSAW while maintaining an average velocity of 10 μm / s in the microfluidic channel (FIG. 4A). While an applied input power up to 0.5 W had no observable effect on the distribution of the 200 nm particles, increasing the applied input power up to 1.0 W displayed the formation of strong, streaming flow patterns originating from the walls. These streaming-induced rolls disrupt visibly the homogenous concentration profile of the sample.

[0130] Further increasing the input power led to the formation of a highly concentrated stream of 200 nm particles towards one of the channel walls, followed by a significant depletion of particles in the center channel. Similarly, 100 nm particles were focused towards one of the channel walls (FIG. 4C), with no observable change on the concentration of albumin. These observations suggest that the operation of an acoustofluidic device using an average flow speed of 10 μm / s and input power of 1.5 W leads to a differential fractionation based on two different mechanisms: one dictating the behavior of 1 μm particles ( dominated by the acoustic radiation force), and another controlling the motion of 100-200 nm particles (Influenced by the acoustic streaming). Furthermore, the Poiseuille flow appears to compete with these mechanisms at lower velocities while dominating at sufficiently high flow rates. A quantification of the total number of particles in the right outlet of the device (see FIG. 3 A) showed that 87% of the 200 nm particles and 68% of the 100 nm particles were collected, while only 25% of 1 μm particles and 30% of 500 nm particles were collected. This indicates that this acoustofluidic technique can be used for differential separation of nanoparticles of 100-300 nm in diameter from larger particles. For particles smaller than 50 nm, the concentration profile was unchanged and thus, the total number of particles collected follows the outlet / inlet flow rate ratio (FIG. 4D).Example 4: Purification nanoparticle mixture using SSAWs and diluting sheath flows:

[0131] We tested the efficacy of this separation strategy when the particle mixture is forced into a narrow, straight beam by introducing two sheath flows without particles through two adjacent inlets (FIG. 5A). Sheath flows can be used to introduce a controlled stream of diluting medium able to significantly reduce the concentration of proteins and other small molecular contaminants. To test the potential of this technique for particle purification, we studied the fractionation of nanoparticles under the effect of: i) diffusion gradients induced bythe sheath flows; ii) and acoustic-induced forces resulting from the applied SSAWs (1.5 W input power); and iii) and drag forces consequence of the Poiseuille flow. The concentration of protein was dictated only by diffusion effects, exhibiting rapid mixing and a homogeneous concentration profile at sufficiently low values of Pe_ (FIG. 5C). Thus, the introduction of sheaths flows produced a 75% reduction in protein concentration in the channel right outlet. Furthermore, we observed particle focusing of 200 nm particles after applying the acoustic for low Pe number. This result strongly deviates from the diffusion-only case and is in agreement with the results in FIG. 4C. Quantification of particle concentration in the right outlet of the device after applying SSAWs at low Pe yielded an 83% recovery, with a corresponding 16% of particles collected in the center outlet (FIG. 5D). Under the same operating conditions, the particle recovery of 1 μm particles collected in the right outlet was shown to be -15% of the initial concentration, with most particles (-70% of the initial concentration) collected in the central outlet (FIG. 5E).

[0132] To test the effectiveness of the acoustofluidic separation method on other nanoparticle systems, we performed experiments aimed to purify extracellular vesicles (EVs) collected from cell culture media. Here, the sample is composed of a heterogeneous mixture of proteins, lipids, sugars, and ions with vesicles having high poly dispersity. For these experiments, we prepared a solution containing fluorescent vesicles derived from a genetically -modified human cell line expressing fluorescent CD9 — a surface protein enriched in EVs. The solution containing EVs is focused with sheath flows and separated from the cell culture medium using SSAWs at low Pe (1 .5 W, 10 μm / s). Nano-tracking Analysis (NT A) shows that the sample contained a distribution of particles ranging from 50 to 350 nm, with two peaks at 88 nm and 122 nm (FIG. 5A and FIG. 5D). NTA of the collected samples from the right outlet show a narrower size distribution with a peak at 115 nm (FIG. 5A and FIG. 5E). In contrast, the collected samples from the center outlet show a similar size distribution as the inlet with a notable decrease in concentration of EVs (FIG. 5 A and FIG. 5F). Quantification of the total number of EVs shows a recovery EVs of about 81%, with an increase in the sample purity ratio (FIG. B-C). These observations are consistent with the experimental results in FIGs. 4A-D. indicating the reproducibility of the acoustofluidic differential fractionation, which can be used for both rigid and soft nanoparticles.Example 5: Numerical study of nanoparticles acoustophoresis driven by acoustic radiation forces and acoustic streaming.

[0133] To understand the mechanisms driving the differential separation of nanoparticles observed in our experimental results, we implemented an algorithm performing numerical calculations that allow nanoparticle tracing in a thermotropic fluid after applying SSAWs. For this purpose, we adopted an idealization of the channel using the cross-section as the system's domain. Assuming hard walls and anti-symmetric bottom wall actuation oscillatory boundary conditions, we established the nanoparticles’ initial conditions to be those of a monodisperse suspension of non-interacting spherical particles. Next, we allowed the system of particles to evolve under the influence of streaming-induced drag and the acoustic radiation force. To perform this computation, we implemented a finite-elements simulation using COMSOL Multiphysics, following the methodology proposed by Muller et al.. A numerical study of microparticle acoustophoresis driven by acoustic radiation forces and streaming-induced drag forces, Lab on a Chip 2012;12(22):4617-4627. Here, we began by solving the first order acoustic field equations subject to the oscillating boundary conditions. This solution provides functional profiles for the first-order temperature 7^, density pr. and pressure p1;as well as the first-order velocity vector field. The results can then be used to compute the acoustic radiation force based on equation. In addition, we can use the Laminar flow physics interface to solve for the time-averaged second-order velocity (v2) obtaining a solution for the secondary acoustically induced streaming flows. Lastly, we used the particle tracing module considering both streaming and acoustic radiation forces to track the position of nanoparticles within the domain. Altogether, this algorithm allowed us to compute the dynamic evolution of nanoparticles in our model system.

[0134] First, we studied the dynamic evolution of nanoparticles after applying a standing acoustic wave with constant amplitude. The solution of the second-order expansion provided a Rayleigh streaming profile with eight streaming rolls with maximum at the top and bottom walls of the channel; local maxima were observed at z — h / 2 (FIG. 7A). The solution for the acoustic radiation force field showed maxima of force at a location corresponding to the inflection points of the sinusoidal acoustic wave (FIG. 7B). In similar fashion, the acoustic radiation force is lowest at the pressure wave nodes and anti-nodes. Utilizing these fields, we performed tracing experiments to follow the evolution of nanoparticles from initial conditions and computed the histogram of particle distribution along the channel width (FIG. 7C). Particles > 500 nm focus rapidly at the wave pressure nodes, driven by the strong acoustic radiation force that dominate over drag effects from the bulk. For smaller particles (150 nm),the simulation predicts pockets of increase concentration of nanoparticles at the side walls and in the vicinity of the pressure nodes. Particles circulate the space without further focusing but are unable to escape neighboring concentration pockets. The results observed in FIG. 7C are in disagreement with the experimental results in FIGs. 4B-C, suggesting that additional phenomenology might be missing from this initial theoretical computation. Motivated by this observation, we explored different configurations of parameters in our model that could lead to a particle distribution histogram where nanoparticles concentrate preferentially towards one of the channel side walls.

[0135] Among the different scenarios tested, we altered the particle distribution by introducing asymmetries in the standing waves. An additional set of simulations was performed applying a "‘dampened standing wave” where the amplitude is allowed to decrease as a function of y (FIG. 7D-F). As a result, the solution to the second-order expansion yielded an asymmetric streaming velocity profile. Despite exhibiting eight streaming rolls as before, the magnitude of the velocity obtained was significantly higher for the circulatory flows at y > w / 2. Similarly, the acoustic radiation force was strongest near the wall at y = w, while maintaining its periodicity and local maxima at the inflection points of the standing wave. In this scenario, particles larger than -500 nm are strongly attracted towards the wave pressure nodes as well, driven by the acoustic radiation force.

[0136] However, introducing these asymmetric force fields can change the dynamic behavior of 150 nm particles. Here, both the acoustic streaming and the acoustic radiation force are of the same order of magnitude, but the anisotropy of both fields promote the migration of the nanoparticles towards the side with faster flows. Once particles reach the faster streaming rolls and stronger radiation force areas within the channel, it becomes exceedingly difficult for particles to migrate towards other areas in the channel. Consequently, the particle distribution histogram shows a significant concentration of nanoparticles towards the channel wall at y = w (FIG. 7F). Altogether, this numerical study suggests that the displacement of small nanoparticles observed experimentally can be explained because of anisotropies in both the acoustic radiation force field and acoustic streaming flow originating from changes in the wave’s amplitude along the channel.

[0137] Taken together, we have demonstrated an acoustofluidic platform capable of differential separation of nanoparticles. The device allows us to separate particles 150-300 nm in diameter from (i) cells and microparticles > 1 μm in diameter; and (ii) molecular components in solution, including proteins, sugars, hormones, nucleic acids, and ions. Compared with other assays for nanoparticle isolation, our strategy provides continuous,label-free, contact-free, high-throughput separation with high reproducibility and potential for automation.

[0138] The power intensity and frequencies used to manipulate soft particles like EVs are know n to preserve morphology, content, and functions of the vesicles, highlighting the potential of these techniques for safe, on chip, point-of-care applications. While the EVs recovery of ~81% obtained is lower than other acoustofluidic systems that achieved > 90% separation yields, previous separation methods do not remove molecular contaminants from EV or other nanoparticle samples. The acoustofluidic method presented here exploits the fast diffusion of small molecules, reducing concentration of contaminants in the outlets enriched with nanoparticles while focusing and concentrating nanoparticles into specific outlet streams. In addition, this differential purification method can be combined with traditional orthogonal or tilted angle acoustofluidics techniques for precise and versatile filtration of complex samples in the form of multiple acoustic regions within the microfluidic device.

[0139] Our technology utilizes both acoustic radiation forces and acoustic streaming as mechanisms for the particle manipulation. In contrast, acoustofluidic focusing of nanoparticles traditionally consider streaming effects as a detrimental side effect setting a lower limit on particle size separation. Similarly, high-frequency nanoparticle actuation based on streaming circumvents radiation effect by limiting the size of the acoustic area. The combined use of diffusion, acoustic radiation, and acoustic streaming opens the door for novel acoustofluidic platforms that use a sophisticated yet controllable interplay of mechanisms for applications. As a tradeoff, stable focusing of nanoparticles using the acoustofluidic technique presented sets an upper limit on the flow' rate. Operation at high Pe disrupt the balance of forces, allowing drag forces from the Poiseuille flow to overcome the Rayleigh flows. Further work in this area can bypass this limitation to improve device through-put and process times.

[0140] The simulations performed suggest that the nanoparticle focusing through streaming arise as a consequence of a reduction of the acoustic wave amplitude along the channel width. Without wishing to be limited by mechanism and theory’, we hypothesize that the reduction of the acoustic w ave amplitude can be driven by thermos-viscous dissipation that transforms the energy of the acoustic field into heat. This is supported by the dramatic increase in temperature within the channel observed w ithout temperature control. As conventional acoustofluidic methods implement impedance matching networks and efficient IDT design to improve the energy transfer, better performance is expected. Thus, a detailedanalysis of the energy transfer and dissipation is important to optimize the performance of these devices and is poorly understood.

[0141] In some embodiments, the present documents provides methodologies that can include: i) employing temperature control systems to avoid overheating, cavitation, and device failure; ii) understanding an upper limit on flow rate for proper operation; iii) applying operating principles that can employ dilute samples; iv) isolating non-exosomal particles and protein aggregates in the nanoparticle compartment; and iv) controlling adsorption and accumulation of lipids and proteins in channel walls to minimize contamination of samples. In particular, the methodologies herein can be applied to understand the effect of high particle concentration on particle dynamics induced by streaming, the scattering disruption in high concentration, and other hydrodynamic effects, as well as the combined effects of these forces in droplets, droplet shells, nanocrystals, and non-spherical particles. Further adaptations to the devices, systems, and methods herein can include using different transducer designs, circuit optimization, cleaning protocols in continuum, integration of sensors, and additional external fields, among others.Other embodiments

[0142] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0143] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0144] Other embodiments are within the claims.Conclusion

[0145] Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. It should be noted that there are many alternative ways of implementing the processes, systems, and apparatus of the present embodiments. Accordingly, the present embodiments are to be considered asillustrative and not restrictive, and the embodiments are not to be limited to the details given herein.

Claims

CLAIMS1. A device comprising: a substrate comprising a top surface disposed on a piezoelectric portion; a first microchannel disposed on the top surface of the substrate; a first inlet configured to deliver a sample through the first microchannel; and a first active region comprising a first electrode region and a second electrode region disposed on the top surface of the piezoelectric portion, wherein the first and second electrode regions are configured to launch an acoustic wave (e.g., a surface acoustic wave, a standing acoustic wave) through at least a portion the first microchannel, wherein the acoustic wave is configured to generate displacement of a particle or modify a velocity of a fluid within the first microchannel by way of acoustic radiation force and acoustic streaming.

2. The device of claim 1, wherein the acoustic wave is configured to provide an asymmetrical or symmetrical amplitude profile of the acoustic wave in the active region.

3. The device of claim 1, wherein a propagation direction of the acoustic wave is not parallel to a direction of flow within the first microchannel.

4. The device of claim 3, wherein the propagation direction of the acoustic wave is perpendicular to the direction of flow within the first microchannel.

5. The device of claim 1, wherein a propagation direction of the acoustic wave is provided at an angle a to a direction of flow within the first microchannel, and wherein a is not 0° and not 180° (e.g., 0° < a < 180° or 180° < a < 360°).

6. The device of claim 5, wherein a is an oblique angle.

7. The device of any one of claims 1-6, wherein the acoustic wave is a surface acoustic wave and / or a standing acoustic wave.

8. The device of any one of claims 1-7, wherein the acoustic wave is characterized as a cosine waveform, a sine waveform, a pulsed waveform, a sawtooth waveform, a non-continuous waveform, a non-standing waveform, or a combination of any of these.

9. The device of any one of claims 1-8, wherein the acoustic wave is generated by applying at least 1 watt (e.g., at least 1.

1. 1.2, 1.3, 1.4, or 1.5 watts).

10. The device of any claim herein, wherein the first active region further comprises one or more further electrode regions.

11. The device of any claim herein, wherein the first, second, or further electrode regions comprises one or more electrodes.

12. The device of claim 11, wherein the one or more electrodes are selected from the group consisting of planar electrodes, interdigitated transducers (IDTs). and the like.

13. The device of any claim herein, further comprising one or more reflectors, guide layers, or functionalized surfaces disposed on the top surface of the piezoelectric portion.

14. The device of any claim herein, wherein the first microchannel comprises a functionalized surface configured to minimize adsorption of targets within a fluid sample, configured to sense, label, or capture targets within a fluid sample, or configured to exclude certain undesired components within a fluid sample.

15. The device of any claim herein, further comprising a second inlet in fluidic communication with the first microchannel.

16. The device of claim 15, further comprising a first inlet channel in fluidic communication with the first inlet, a second inlet channel in fluidic communication with the second inlet, wherein the first inlet channel is configured to deliver a first fluid (e.g., comprising a first sample) through the first microchannel, and wherein the second inletchannel is configured to deliver a second fluid (e.g.. comprising a second sample or a first sheath fluid surrounding a flow of the first sample) through the first microchannel.

17. The device of claim 16, further comprising a third inlet channel in fluidic communication with the second inlet or a third inlet, wherein the third inlet channel is configured to deliver a fluid (e.g., comprising a third sample or a second sheath fluid surrounding a flow of the first sample or the second sample) through the first microchannel.

18. The device of claim 16 or 17, wherein the second fluid and / or the third fluid comprises a sheath fluid configured to dilute a fluid sample within the first microchannel or configured to focus a flow of a fluid sample within the first microchannel.

19. The device of any claim herein, further comprising one or more inlets or inlet channels in fluidic communication with the first microchannel.

20. The device of any claim herein, further comprising one or more outlets in fluidic communication with the first microchannel.

21. The device of claim 20, wherein the device comprises a plurality of microchannels, and wherein each second channel is in fluidic communication with the first microchannel and a respective outlet.

22. The device of any claim herein, wherein the device is configured to provide flow having an average fluid velocity of about 5 to 500 μm / s.

23. A system comprising: one or more devices of any one of claims 1-22; an electronics module configured to provide one or more electrical connections to at least one of the one or more devices; a fluidics module configured to provide one or more fluids to at least one of the one or more devices; and a temperature control module configured to regulate a temperature of the one or more devices.

24. The system of claim 23, further comprising a plurality of devices of any one of claims 1-22, wherein each device can be same or different.

25. The system of claim 24, wherein at least two of the plurality of devices are fluidically connected in series.

26. The system of claim 25, wherein the plurality of devices comprises: a first device comprising a first inlet, a first outlet, and a first microchannel in fluidic communication with the first inlet and the first outlet; a second device comprising a second inlet, a second outlet, and a second microchannel in fluidic communication with the second inlet and the second outlet; and wherein the system further comprises a fluidic interconnect disposed between the first outlet and the second inlet, thereby providing fluidic communication between the first outlet of the first microchannel and the second inlet of the second microchannel.

27. The system of claim 24, wherein at least two of the plurality' of devices are fluidically connected in parallel.

28. The system of claim 27, wherein the plurality of devices comprises: a first device comprising a first inlet, a first outlet, and a first microchannel in fluidic communication with the first inlet and the first outlet; a second device comprising a second inlet, a second outlet, and a second microchannel in fluidic communication with the second inlet and the second outlet; and wherein the system further comprises a fluidic interconnect disposed between a fluid source, the first inlet, and the second inlet, thereby providing fluidic communication between the fluid source and the first outlet of the first microchannel and between the fluid source and the second inlet of the second microchannel.

29. The system of any one of claims 23-28, wherein the electronics module comprises:a signal generator configured to generate a radiofrequency (RF) signal or an electrical signal; and a circuit configured to transmit the RF signal or electrical signal to the first and second electrode regions.

30. The system of any one of claims 23-29, wherein the fluidics module comprises: a fluidic controller configured to deliver fluids to the first microchannel.

31. The system of any one of claims 23-30, wherein the temperature control module comprises: a heat exchanger, a heat sink, an insulator, or a combination of any of these.

32. A method of separating entities in a sample (e.g., based on a characteristic, such as size), the method comprising: delivering the sample through a first microchannel, wherein the sample comprises a first population of entities having a first characteristic and a second population of entities having a second characteristic that is different than the first characteristic; applying an acoustic wave through at least a portion of the first microchannel, thereby generating displacement of at least a portion of the first and second populations traveling through the first microchannel by way of acoustic radiation force and acoustic streaming; and separating the sample into a first separated population and a second separated population, wherein the first separated population comprises a majority of entities having the first characteristic and wherein the second separated population comprises a majority of entities having the second characteristic.

33. The method of claim 32, wherein the first characteristic and the second characteristic are characterized by a difference in size, shape, hydrodynamic radius, composition, density, compressibility, shape, or a combination of any of these.

34. The method of claim 32 or 33, wherein the entities comprise particles (e.g., nanoparticles, microparticles, or mixtures thereof), proteins, polymers, lipids, nucleic acids,vesicles (e.g., exosomes, extracellular vesicles, and the like), cells (e.g., red blood cells, platelets, cancer cells, and the like), organisms (e.g., bacteria), contaminants, or a combination of any of these.

35. The method of claim 32, wherein said separating comprises using asymmetryin an amplitude of the acoustic wave traveling through the first microchannel to generate asymmetry in streaming or in a radiation field in the first microchannel.

36. The method of any claim herein, wherein the majority of particles in the first separated population comprises a population in which more than about 50% (e.g., more than about 55%. 60%, 65%, 70%, 75%, 80%, 85%, or more) of the particles are characterized as having the first characteristic.

37. The method of any claim herein, wherein the majority of particles in the second separated population comprises a population in which more than about 50% (e.g., more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more) of the particles are characterized as having the second characteristic.

38. The method of any claim herein, wherein said separating comprises flowing the first separated population into a first outlet channel and flowing the second separated population into a second outlet channel, wherein each of the first and second outlet channels are in fluidic communication with the first microchannel.

39. The method of any claim herein, wherein the sample comprises a volume fraction of about 0.001% to 1% occupied by the first population of entities and the second population of entities.

40. The method of any claim herein, wherein the sample comprises a concentration of about 0.001% to 1% (v / v) of the first population of entities and the second population of entities.

41. The method of any claim herein, further comprising: diluting the sample prior to said applying, wherein said diluting can occur before, during, or after said delivering the sample through a first microchannel.

42. The method of claim 41, wherein said diluting is performed within the first microchannel.

43. The method of claim 42, further comprising: delivering a sheath fluid to the first microchannel, thereby diluting the sample with the sheath fluid.

44. The method of any claim herein, further comprising: modifying (e.g., increasing or decreasing) a viscosity' of the sample prior to said applying, wherein said modifying can occur before, during, or after said delivering the sample through a first microchannel.

45. The method of claim 44, wherein said modifying is performed within the first microchannel.

46. The method of claim 45, further comprising: delivering a sheath fluid to the first microchannel, thereby modifying the viscosity of the sample with the sheath fluid.

47. The method of any claim herein, further comprising: collecting, separately, the first separated population and the second separated population.

48. The method of claim 47, further comprising: analyzing the first separated population and the second separated population.

49. The method of any claim herein, wherein the sample comprises plasma, serum, transdermal fluid, interstitial fluid, sweat, intraocular fluid, vitreous humor, cerebrospinal fluid, extracellular fluid, lacrimal fluid, tear fluid, sputum, saliva, mucus, a microorganism, a virus, a bacterium, a fungus, a parasite, a helminth, a protozoon, a cell.tissue, a fluid, a swab, a biological sample (e.g., blood, serum, plasma, saliva, etc.), an environmental sample (e.g., a water sample, a soil sample, etc.), or an agricultural sample.

50. A method of characterizing a sample, the method comprising: delivering the sample through a first microchannel, wherein the sample comprises a first population of particles having a first characteristic and a second population of particles having a second characteristic that is different than the first characteristic; applying an acoustic wave through at least a portion of the first microchannel, thereby generating displacement of at least a portion of the first and second populations traveling through the first microchannel by way of acoustic radiation force and acoustic streaming; separating the sample into a first separated population and a second separated population, wherein the first separated population comprises a maj ority of particles having the first characteristic and wherein the second separated population comprises a majority of particles having the second characteristic; collecting, separately, the first separated population and the second separated population; and analyzing the first separated population and the second separated population.51 . The method of claim 50, wherein the particles comprise nanoparticles, microparticles, proteins, polymers, lipids, nucleic acids, vesicles (e.g., exosomes, extracellular vesicles, and the like), cells (e.g.. red blood cells, platelets, cancer cells, and the like), organisms (e.g., bacteria), contaminants, or a combination of any of these.

Images