WO2006032048A2

WO2006032048A2 - Separation of particle types using a non-uniform acoustic field

Info

Publication number: WO2006032048A2
Application number: PCT/US2005/033362
Authority: WO
Inventors: King-Ho Holden Li; Thomas W. Kenny
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2004-09-15
Filing date: 2005-09-15
Publication date: 2006-03-23
Also published as: WO2006032048A3

Abstract

A method and device for separating (Fig.1) comprising a fluid flow channel with acoustic transducers (104, 106) providing an acoustic field with at least two steady-state regions having distinct acoustic field patterns with corresponding particle distributions (112, 114, 116), wherein particles are separated in a transition region (118) of the acoustic field.

Description

SEPARATION OF PARTICLE TYPES USING

A NON-UNIFORM ACOUSTIC FIELD

FIELD OF THE INVENTION

The present invention relates generally to separation of different particle types. More particularly, the present invention relates to separation of different particle types using a non-uniform acoustic field.

BACKGROUND

When an acoustic standing wave field is present in a fluid containing particles, the

particles tend to accumulate at the nodes if they are more dense than the fluid. Particles tend to accumulate at the antinodes if they are less dense than the fluid. This phenomenon has been exploited in various methods for separating or concentrating particles from the

fluid and/or from each other. For example, placement of a collection channel at a node (or antinode) with enhanced particle concentration provides separation of the particles from the fluid.

Variations on this theme have been considered in the art. For example, in WO 02/072235, Laurell et al. consider separation of blood cells from each other by adjusting the density of the fluid such that one type of blood cell is more dense than the fluid and another type of blood cell is less dense than the fluid. Thus one cell type will accumulate at the nodes and the other cell type will accumulate at the antinodes, thereby providing cell type separation. However, this method can be difficult or impossible to apply to particles having sufficiently similar densities. For example, blood cell types (which can differ in density by about 1%) can be difficult to separate with such a method.

Particle type separation by balancing acoustic forces with other forces has also been considered. For example, in US 4,763,361, Schram considers provision of an acoustic field having moving nodes for particle separation. A force balance between acoustic forces and viscous drag enables particle type separation. Similarly, Barmatz et al. (US 4,523,682) and Yasuda et al. (US 5,902,489) consider a balance between acoustic forces and non-acoustic forces (e.g., a gravitational force or an electric force) in order to separate particle types. However, these prior art particle separation methods tend to be quite complicated in practice. For example, the approach of Schram requires careful adjustment of the motion of the nodes (or antinodes) in order to provide efficient separation.

Accordingly, it would be an advance in the art to provide acoustic particle type separation in a simplified manner. It would be a further advance in the art to provide such simplified acoustic separation in a miniaturized configuration (e.g., having a microfluidic flow channel) suitable for incorporation into a handheld diagnostic device.

SUMMARY OF THE INVENTION

The present invention provides separation devices and methods for separating different particle types using a non-uniform acoustic field. The separation devices include a flow channel for fluid with one or more particle types as well as one or more acoustic transducers. The acoustic transducers provide an acoustic field within the flow channel that has several different regions. Two or more of the regions, called steady-state regions, have distinct steady-state acoustic field patterns and corresponding steady-state particle distributions. In contrast, one or more of the regions, called transition regions, has a dynamic acoustic field region and dynamic particle distributions. It is in the transition region that different particle types may be separated based, e.g., on size, density, and/or compressibility. The separated particle types can then be collected by one or more output channels. In one embodiment, two acoustic transducers are used to create the different acoustic field regions. In an alternative embodiment, one transducer is used to create the different acoustic field regions. Separation devices according to the invention may be used to separate any number of different particle types in a fluid, such as red and white blood cells in blood.

BRIEF DESCRIPTION OF THE FIGURES The present invention together with its objectives and advantages will be understood by reading the following description in conjunction with the drawings, in which: FIG. 1 shows an example of a separation device according to the present

invention;

FIGS. 2-4 show examples of other embodiments of separation devices according to the present invention;

FIG. 5 shows relevant dimensions of a separation device according to the present

invention;

FIG. 6 shows an example of micro-channel layout on a silicon wafer according to the

present invention; FIG. 7 shows an example of fabrication of a separation device according to the present invention; FIG. 8 shows an example of particles accumulated at acoustic field nodes according to the present invention;

FIG. 9 shows an example of particle image velocimetry analysis according to the present invention; ^

FIG. 10 shows a plot of velocity versus distance from the pressure node for different sized particles according to the present invention;

FIG. 11 shows a plot of velocity versus distance from the pressure node for different applied transducer voltages according to the present invention;

FIG. 12 shows a plot of velocity versus distance from the pressure node for different flow rates according to the present invention;

FIG. 13 shows image regions for video graphic analysis of particle separation according to the present invention; FIG. 14 shows the percentage of particles passing through a particular region that were green, in a separation device according to the present invention; FIG. 15 shows the effect of region width on efficiency of separation in a separation device according to the present invention; and

FIG. 13 shows the efficiency of a zig-zag separation device according to the present invention with varying slant angles of the slanted region.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows particle type separation in a separation device according to an embodiment of the invention. A flow channel 102 in substrate 103 contains a fluid with two or more particle types flowing in a direction shown by arrow 120. In this example, the fluid includes large particles 108 and small particles 110. First and second acoustic transducers 104 and 106, respectively, provide an acoustic field within flow channel 102. This acoustic field generally has at least two steady-state regions having distinct steady state field patterns. In the example of FIG. 1, the steady-state region provided by transducer 104 has standing wave nodes 112 and 114, and the steady-state region provided by transducer 106 has a standing wave node 116. There are various methods for establishing such acoustic field regions having different field patterns. For example, the frequency of the acoustic waves provided by transducers 104 and 106 can differ. Alternatively to or in combination with such a frequency difference, the position of acoustic field nodes can be varied by altering the position of transducers and/or acoustic reflectors (not shown) relative to flow channel 102.

Typically, an acoustic standing wave is obtained by superposition of oppositely directed acoustic traveling waves. Two oppositely directed acoustic transducers can be used to provide these two acoustic traveling waves. Alternatively, an acoustic reflection (e.g., from an interface between flow cell 102 and an air ambient) of an incident acoustic wave

can provide a back-reflected acoustic wave, which superposes with the incident wave to create an acoustic standing wave. A solid-air interface is typically a good acoustic reflector. The use of a reflection to generate the standing wave is preferable in cases where reduced power consumption and/or maximum simplicity is desired. The use of two transducers to generate the standing wave can be desirable to increase the acoustic energy density within the fluid. Sinewave excitations applied to the two transducers may be from

the same source and at the same relative phase, or there may be a phase difference between the two, which would laterally shift the position of the standing wave in the channel. Variations in the phase difference can be used to dynamically adjust the position of the bands during operation to maximize efficiency or to introduce a useful time variation in the operation of the system.

In the example of FIG. 1, it is assumed that particles 108 and 110 are more dense than the fluid in flow channel 102, so these particles will tend to accumulate at the acoustic field nodes as shown. If particles 108 and 110 are assumed to be less dense than the fluid, then particle accumulation as shown will occur if 112, 114 and 116 are antinodes instead of nodes.

Thus the different acoustic field steady-state regions each have corresponding different steady-state particle distributions. Particles in the steady-state region corresponding to transducer 104 accumulate on nodes 112 and 114. Particles in the steady-state region corresponding to transducer 106 accumulate on node 116. As the particles flow from one steady-state region to the other, there is a transition region 118 between the steady-state regions, where the particle distribution does not correspond to either of the steady-state particle distributions.

This transition region 118 is a key feature of the invention, since the transition particle

distribution can provide particle type separation. For example, large particles have been found to move to acoustic field nodes (or antinodes) more rapidly than smaller particles. (See Examples below). Thus, as shown in FIG. 1, transition region 118 can provide separation between large particles 108 and small particles 110. An output channel 122 can be disposed (as shown) to collect fluid and particles from a part of transition region 118 with an enhanced concentration of small particles 110. Alternatively, or in addition, an output channel can be disposed to collect fluid and particles from node 116 within transition region 118 having an enhanced concentration of large particles 108 (not shown).

The embodiment shown in FIG. 1 can be regarded as an example where a dynamic acoustic property (e.g., an acoustic force) is used to provide a spatial separation of particle types by flow between two regions each having different steady-state acoustic field patterns. It is particularly noteworthy in FIG. 1 that particle type separation is provided in transition region 118, and is not possible in regions where all particles have accumulated at the nodes (or antinodes).

Since the particle type separation provided by this embodiment of the invention may have a sensitive dependence on fluid flow rate, a feedback arrangement for controlling the flow rate can be employed. For example, the concentration (or purity) of a desired species in output channel 122 can be measured to provide a control signal. The flow rate through the flow cell can then be adjusted within a control loop designed to maximize this control signal. In this manner, the flow rate can be controlled to provide maximum concentration (or purity) of the desired species.

FIGS. 2 through 4 show alternative embodiments of the invention where a single acoustic transducer provides two acoustic field regions having different field distributions. Such single transducer embodiments are preferred to minimize device size and complexity. FIG. 2 shows a separation device having flow channel sections 202 and 204 in substrate 205. By making channel section 204 fractionally narrower than channel section 202, one acoustic transducer 206, generating one acoustic frequency, can be used to establish two steady-state acoustic field regions having distinct steady state field patterns. In this example, a first steady-state region, formed in channel section 202, has standing wave nodes 208 and 210, and a second steady-state region, formed in channel section 204, has standing wave node 212. If a fluid with large particles 214 and small particles 216, with densities less than that of the fluid, is advanced in the direction shown by arrow 218 through this flow channel, the particles will first accumulate at nodes 208 and 210 in the first steady-state region, and will then accumulate at node 212 in the second steady-state region. As the particles flow from the first region to the second region, they will flow through transition region 220, where the particle distribution does not correspond to either of the steady state particle distributions. Large particles 214 and small particles 216 can therefore be separated in transition region 220. Output channels 222 can then be disposed to collect fluid and particles from a part of transition region 220 with an enhanced concentration of small particles 216. Alternatively, or in addition, an output channel can be disposed to collect fluid and particles from node 212 from within transitional region 220 having an enhanced concentration of large particles 214 (not shown).

When an acoustic wave passes through an interface between two materials at an oblique angle, it produces both reflected and refracted (or transmitted) waves. This refracted wave will either bend towards or away from the incident normal depending on the difference

between the velocities of acoustic waves within the two materials. These velocities, and hence the refraction angle, are determined by material properties such as elastic modulus and density. One can take advantage of this property of acoustic waves to design a flow channel for which one acoustic transducer, with a fixed frequency, will generate two or more acoustic field regions.

FIG. 3 shows a zig-zag flow channel 302, with direction of fluid flow X, preferably formed in single-crystal silicon 304. An acoustic transducer 306 provides an acoustic field region designated by arrows 308 in a straight, first section 310 of flow channel 302 and an acoustic field region designated by arrows 312 in a slanted, second section 314 of flow channel 302. Regions 308 and 312 will generally have different acoustic field patterns due to acoustic anisotropy and the nonzero tilt angle θ. In the example of FIG. 3, it is

assumed that θ is less than the total reflection angle for longitudinal waves from flow

channel 302 into the fluid flowing through it, so that region 312 includes acoustic waves in the fluid. Preferably, a third section 316, parallel to first section 310, and having a similar

acoustic field pattern, designated by arrows 318, to second section 314 is used to minimize the geometry and provide greater acoustic field region control of the separation device.

FIG. 4 shows a zig-zag flow channel 402, preferably formed in single-crystal silicon 404,

with direction of fluid flow X. Flow channel 402 has a first, straight, section 406, a second, slanted, section 408, and a third, straight, section 410 that is parallel to first straight section 406. An acoustic transducer 412 provides an acoustic field region designated by arrows 414 in first section 406 and an acoustic field region 416 in third section 410. Regions 414 and 416 will generally have different acoustic field patterns due to differing distances from channel 402 to transducer 412. In the example of FIG. 4, it is assumed that θl is greater than the total reflection angle for longitudinal waves from flow channel 402 into the fluid flowing through it. Thus, in this example, no acoustic excitation would be present in second section 408 of channel 402. In an alternative aspect of this embodiment, acoustic excitation can be eliminated in second section 408 by varying both the angle between first section 406 and second section 408 (θl), and the angle between

second section 408 and third section 410 (Θ2). Another parameter that can be varied in

this embodiment is the distance M between first section 406 and third section 410 (see below).

FABRICATION OF A SEPARATION DEVICE In a preferred embodiment of the invention, the flow channel is a micro-channel and the acoustic transducer is a piezoelectric element. Examples of piezoelectric elements are macroscopic ceramic transducers made of PZT, quartz, or other commonly available piezoelectric materials. To minimize attenuation of force transmission from the transducer to the flow channel material, the bonding of the transducer to the flow channel material

should have adequate stiffness to ensure that the maximum generated force is transferred to the micro-channels. Many bonding materials may be used for this purpose, such as epoxy resins. Alternatively, a thin film PZT deposition technique could be used to deposit

piezoelectric films on an outer surface of the flow channel. This technique would eliminate the requirement for a bonding material. Thus, reduction in force from the bonding material, as well as non-uniformities caused by differences in bonding layer thickness could be avoided.

The mechanism for generating an acoustic wave in the flow channel is an important consideration when fabricating a separation device according to the invention. PZT acoustic transducers can be made with natural frequencies up to above 10MHz. Preferably, the acoustic transducer generates a frequency above about 1.5 MHz. Operation at these frequencies generally eliminates a cativation effect associated with operations below 1 MHz.

In order to produce standing waves in a micro-channel, the selection and placing of the acoustic transducer is important. FIG. 5 shows the dimensions that can be varied in a separation device such as that shown in FIG. 1. The separation device shown in FIG. 5 has two transducers, 502 and 504, which are attached to silicon material 506 with bonding material 508. Alternatively, the transducers may be clamped into force contact with the channel structure (not shown). Flow channel 510 is formed in silicon material 506. Dimensions a and c, the widths of the silicon material 506, are not critical. For ease of imaging in experimentation, a and c are preferably between about 15mm and 30mm. By modeling the attenuations of the acoustic waves generated by acoustic transducers 502 and 504, it is possible to calculate a micro-channel width b that gives one steady-state acoustic field region with two pressure nodes and another steady-state acoustic field region with one pressure node, as shown in FIG. 1. For example, if transducer 502 generates a frequency of 5 MHz and transducer 504 generates a frequency of 3 MHz, micro-channel

width b should be about 400 μm. Depending on the volume of fluid flowing through the

separation device, the operating frequency of the acoustic transducer can be minimized by choosing an appropriate micro-channel width. Typically, this width would be in the range of hundreds of microns. Small, several-micron changes in width b, such as those caused by typical fabrication errors, are not significant given the wavelengths used. The upper flow channel length, m and n, should be set such that there is enough time for both large particles and small particles to accumulate at the pressure nodes in the steady- state acoustic field region generated by transducer 502. For a volumetric flow rate of about 10 μl/min and a linear flow rate of about 2 mm/s, typical values for m and n are in

the range of about 10mm to about 20mm. The upper flow channel length is not critical, as changing it would not have a drastic effect on the separation mechanism.

The length p of the transitional region can be calculated based on the speed of congregation of large particles versus small particles at the node in the steady-state acoustic field region generated by transducer 504. For example, if it takes about 0.2 sec for a large particle to reach the new node, and a much longer time for a smaller particle to reach the new node, dimension p should be about 400 μm given the above flow rates.

Dimension p is the most critical dimension for the success of the separation device.

In a preferred embodiment, the micro-channels are constructed from a silicon wafer, preferably a single-crystal silicon wafer. FIG. 6 shows an example of micro-channels 602 laid out in a wafer 604. Preferably, the fabrication process is based on a simple one-mask

layout. The mask can be, for example, a conventional chrome mask or a transparency mask. One of the most important controls in the process is the ability to ensure that the patterns of the micro-channels are printed either parallel or orthogonal to the major flat of the wafer as shown in FIG. 6. The reason for doing so is to ensure that acoustic propagation is always in the same axis across the silicon wafer, thereby enabling consistency of experimental results. This can be done, e.g, by drawing a series of parallel

lines with different thicknesses over the mask. One of the lines can then be lined up with the wafer flat, e.g. with the help of a microscope setup in a manual lithography machine using precision micro stage control.

FIG. 7 shows an example of a fabrication process flow according to the invention. First, patterns of micro-channels are defined by photolithography. FIG. 7 A illustrates the results of this step, with a pattern of photoresist 702 on silicon wafer 704. Next, as shown in FIG. 7B, photoresist 702 is etched to give micro-channel 706 using, e.g., Deep Reactive Ion Etching (DRIE). Finally, as shown in FIG. 7C, silicon wafer 704 and micro-channel 706 are sealed by anodic binding of glass wafer 708. An important fact to note is that the DRIE machine can give a different etch rate across a silicon wafer during long periods of etching. Though the depth of the micro-channels is not an absolutely critical dimension in the design, it is logical to check on the depth of each device after the manufacturing process. The etching step is based on time control.

The micro-channels may also be made of plastic. For example, plastic can be injection molded to form the micro-channels. Alternatively, thermoplastics may be hot embossed to stamp the micro-channels. Acrylic is a preferred plastic for this invention, as it has a very low background fluorescence compared to other plastics.

Once the micro-channels are molded from the plastic, they may go through post processing steps in which channel surfaces are treated to be either hydrophilic or hydrophobic. In many cases, hydrophilic surfaces are preferred so that surface tension can be used as a means of driving the fluid sample into the micro-channels without any consideration of precise volume control. In other cases where volume control is necessary, a hydrophobic surface coupled with a precision pumping mechanism would ensure a certain level of volume control. This pumping mechanism could be, e.g., a mechanical pump or an in-line micro-pump.

There are many methods and sets of materials that have been used for fabrication of microfluidic channels for many applications in recent years. The requirements for this invention are simple enough that most if not all prior art examples for this fabrication would be acceptable. For this application, the materials and fabrication method should be chosen on the basis of cost, convenience, and compatibility between materials and fluid. The important design issues unique to this application relate to the width of the channel and the placement of the transducers relative to the acoustic properties of the materials, and these design considerations must always be taken into consideration once the materials are selected.

SEPARATION OF BLOOD

Separation devices according to the present invention are suitable for separating white blood cells (WBCs) and red blood cells (RBCs). At any fixed pressure, the displacements of WBC and RBC are dependent on their radii, compressibility, and previous locations. Due to the larger size of WBCs, they are able to reach the new nodes more quickly than RBCs, thus allowing separation in the transition zone.

Whole blood is more than four times as viscous as water, making it difficult to separate without dilution. Blood could be diluted in several ways. For example, the whole blood could be diluted with phosphate buffered saline. For precision volume mixing, a cutoff mechanism could be used such that a fixed amount of blood is mixed with a predefined quantity of diluting fluid.

Another issue with whole blood is coagulation of blood and clumping of blood cells. Thus, preferably, an anticoagulant is used and the whole blood is diluted with EDTA.

A third difficulty in using whole blood is the low number density of WBCs compared to RBCs in the blood. To circumvent this problem, the whole blood may be centrifuged and the "buffy coat", a mixture of WBCs and platelets sandwiched between plasma and RBCs, may be extracted to increase the overall number density of WBCs.

EXAMPLES

Concentration of particles along pressure nodes

Acoustic separation in the microscale using a standing wave was tested in a fairly large channel of width 1 mm. A stereomicroscope, Stemi SV6 (Carl Zeiss, Thornwood, NY) was used as it provided a large working distance between the lens and the sample.

Polystyrene beads of 10 μm in diameter, supplied by Duke Scientific (Palo Alto, CA),

were used. An external pump, Model 11 (Harvard Apparatus, Holliston, MA) provided continuous flow for the micro-channel specimen. It allows precision volumetric control of the fluid dispensed in the system and is useful in standardizing experiments. Alternating current was fed into a PZT transducer by a power supply (Fluke Instruments, Everett,

WA). FIG. 8 illustrates concentration of lOμm particles 802 along pressure nodes 804

using this system. Investigation of parameters for concentration of particles along pressure nodes A non-intrusive, widely used method to measure the velocities of particles in fluids is known as particle image velocimetry (PIV). This method uses cross-correlations of successive images, taken by a camera system at up to 30 frames per second, to track particle movement and to predict the velocity of particles. For micro-fluidics applications, a very comprehensive study and protocol in using micro-particle image velocimetry (μ-

PIV) has been developed in Professor Juan Santiago's Stanford Microfluidics Laboratory (Devasenathipathy, S. et al., "Particle Tracking Techniques for Electrokinetic MicroChannel Flows," Analytical Chemistry, 2002, Vol. 74, No. 15, pp. 3704-3713).

Fluorescent microspheres, supplied by Duke Scientific (Palo Alto, CA), were used for these experiments. As these microspheres are not naturally buoyant, the salinity of the fluid was adjusted to produce a fluid having the same density as the microspheres. The experimental setup included an epi-fleorescense microscope (Axioskop 2, Carl Zeiss), camera system (COHU, San Diego, CA), image capturing DAQ card (National Instrument, Austin, TX), Lab View program and Matlab software for numerical calculations. To ensure consistency in experiments, micro-channels were used at least three times with different sizes of particles.

Three sizes of particles were used in the μ-PIV experiments, 3μm, 7μm and lOμm. The

particles were introduced into the same piece of micro-channel system separately to ensure consistency in the acoustic field. Experiments were formulated with the focus of investigating the influence of particle size, acoustic pressure and lateral flow rate in the micro-channels. FIG. 9 shows an example of the type of data that can be obtained from a μ-PIV experiment. FIG. 9A and FIG. 9B show digitally processed images taken of

microspheres at time 0 and 3 sec, respectively. In FIG. 9 A, microspheres 902 are randomly distributed throughout the micro-channel. In contrast, in FIG. 9B, microspheres 902 are concentrated along pressure node 904, indicated by a dashed line. A particle velocity field derived from a series of images taken between time 0 and time 3 sec is shown in FIG. 9C, with pressure node 904 indicated by a dashed line. For this and the remaining figures, the x-direction is the direction of fluid flow, and the y-direction is the lateral movement of the particle as it moves in the x-direction down a micro-channel.

For the first experiment, the different sized microspheres were each suspended in fluid and introduced into a micro-channel with a bulk flow rate of 1 μl/min, with a PZT operating at

1.5

MHz and an applied voltage of 30 MHz. FIG. 10 shows plots of velocity in the y-direction versus distance across the micro-channel away from the pressure node for 3-μm (solid

line), 7-μm (dotted line), and 10-μm (dashed line) microspheres. As can be seen from this

figure, larger particles have a higher velocity towards pressure nodes as compared to smaller particles.

For the next experiment, the different sized microspheres were each suspended in fluid and introduced into a micro-channel with a bulk flow rate of 1 μl/min, with a PZT

operating at 1.5 MHz with applied voltages ranging from 18V to 48V. The result for a 3 μm microsphere is shown in FIG. 11. As can be seen from this figure, a higher applied

voltage results in a higher particle velocity towards pressure nodes. A similar trend was seen with 7μm and lOμm microspheres. Finally, 7μm microspheres were suspended in fluid and introduced into a micro-channel

with a PZT operating at 1.5MHz with a constant applied voltage of 30V. Bulk flow rates were varied from 0.5 μl/min to 2.5 μl/min. As can be seen from FIG. 12, flow rate did not

have a significant effect on particle velocity towards pressure nodes.

Based on the results of these experiments, it is possible to estimate the actual acoustic force acting on the microspheres. For a typical 3 micron microsphere in an acoustic field

provided by a PZT operating with an applied voltage of 30V, the largest force the microsphere could experience is about 2 x 10^'13 N, which translates to the equivalent of less than 14g force. For the maximum operating voltage achieved by PZT, this force could go up to about the equivalent of 4Og. This magnitude and scale of acoustic forces is much less than what a typical cell is subjected to in a centrifuge of up to 10,000g and above. Thus, there is little basis for concerns in the medical community as to the "harmfulness" of introducing such technology in a μTAS (Thrombolytic Assessment

System) application.

Separation of two different particle types using a separation device according to one embodiment of the invention The micro-channels used in this experiment had a channel width of 600 μm and two

different PZTs, operating at 3MHz and 1.5MHz. The widths of the outlets were designed such that a center outlet, where bigger particles are collected, was similar in diameter to the two original pressure nodes. The reason for this design was to cater to the use of the device with blood, in which WBCs can vary in size from 8 to 20 μm. Because of this size variance, larger WBCs will move more quickly to the new pressure node while smaller WBCs will remain relatively close to the original two pressure nodes. By having the center outlet a similar diameter to the original pressure nodes, all WBCs will thus be collected.

The sample used in this experiment contained two groups of fluorescent microspheres having diameters of 3μm and 7μm, respectively. The smaller microspheres emitted a red

fluorescence, while the larger ones emitted green fluorescence under an epi-fluorescence microscope. The number densities were set at 20: 1 between the red and green microspheres. This was to emulate the situation of red blood cells RBCs being more population dense than WBCs in actual whole blood, although the real case is somewhat on the order of 700:1.

In order to quantify the effectiveness and purity of this separation mechanism, video

graphic analysis was used to count the number of particles collected at each downstream outlet. Video files were first converted from an mpeg file into avi file format. Then, the files were broken down into individual frames of pictures by a simple Matlab function code. These images were fed into National Instruments' Vision Assistant program, which

then counted the number of green particles that were in proximity to where each outlet would be. (As red particles were at a much higher number density than green particles, the focus was purely on setting the search criterion for green particles using the color recognition feature of the program). Images of micro-channels were divided into three regions, as diagrammed in FIG. 13. FIG. 13 shows a transitional region in a micro- channel 1302, with fluid containing lOμm (green) particles 1304 and 7μm (red) particles 1306. Image Regions 1, 2, and 3 are positioned where outlets would be located in a separation device according to the invention.

FIG. 14 shows results of the percentage of particles that passed through image Region 2 that were green. Results are shown for four separate experiments, as indicated by the four bars. No green particles were seen in Regions 1 and 3 in any of the four experiments. An average of 76% purity for the bigger, green, particles was observed in Region 2 of the micro-channel based on four different sets of video clips. From an analysis of the video clips, it was estimated that more than 78% purity was possible using a separation device according to the invention.

Region 2 in this analysis was set at l lOμm. A further analysis was conducted to

determine the optimum size of this Region, and hence the outlet, for optimum separation. FIG. 15 shows the percentage of particles in Region 2 that were green for varying widths of Region 2. The results from four video clips are shown, indicated by the asterisks. As can be seen from this figure, as the width of Region 2 gets narrower, the percentage of particles that flow through this region that are large green particles increases.

Separation of two different particles types using a separation device according to another embodiment of the invention

A series of zig-zag separation devices, as shown in FIGS. 3 and 4, were fabricated with varying θl and Θ2, and varying lengths of M. The micro-channels were more than 3 cm

long near the outlet to provide ample room for the objective lens to move along the micro- channels to investigate separation efficiency. Micro-channels were built with a fixed width of 400μm. Polystyrene microspheres of size 3 and 7 μm, as described above, were used to

test the efficiency of separation of the device.

FIG. 16 shows the overall efficiency of a zig-zag separation system, such as that shown in

FIG. 3. In this experiment, M = 600 μm or 1 mm at varying θ (θl and Θ2 are set to be

equal in this case). The separation device includes a transducer operating at 3MHz with

an applied voltage of 40V, and fluid flows through the device at a flow rate of 1 μl/min.

Generally, one would expect an increase in the overall efficiency of separation as θ is

increased, but this trend is only demonstrated in the case where M = 600 μm. This is

partly due to the fact that as the slanted region becomes longer, it introduces more

secondary effects such as the influence of reflected shear waves in the silicon material that

re-introduce back as longitudinal waves in the fluid in the micro channels.

Table 1 shows the results of varying θl and Θ2 such that (Θ2 - θl) is always larger than the

critical angle. In this experiment, the flow rate was 1 μl/min and the transducer operated at

3MHz with an applied voltage of 40V. At certain variations, such as setting θl and θ2 to

be 30° and 45° respectively, with M = 600 μm, there is no effective separation at all. This

is due to the small difference in distance between the new pressure nodes and the original

ones. Either a stronger acoustic field or a slower flow rate could improve the separation.

Table 1

Thus, it is possible to have a separation system at different angles of incidence provided that there is sufficient acoustic energy being transmitted into the micro channels. Another important consideration factor is the liquid bulk flow rate. Although a slow flow rate reduces the overall throughput, it is very important in the case of a weak oblique angle incidence wave propagation as it enables particles to spend more time in each acoustic field region.

CONCLUSION

In conclusion, this invention provides methods and devices for separating different particle types using a flow channel and an acoustic field with two or more steady-state regions. As

one of ordinary skill in the art will appreciate, the present invention is not limited to or defined by what is shown or described herein. One of ordinary skill in the art will appreciate that various changes, substitutions, and alterations could be made or otherwise implemented without departing from the principles of the present invention. Accordingly, the scope of the present invention should be determined by the following claims and their

legal equivalents.

Claims

CLAIMSWhat is claimed is:

1. A separation device, comprising:

a. a flow channel for a fluid comprising two or more particle types;

b. one or more acoustic transducers providing an acoustic field within said flow

channel, wherein said acoustic field has at least two steady-state regions having

distinct steady-state acoustic field patterns and corresponding distinct steady-

state particle distributions, wherein said acoustic field has at least one transition

region having a particle distribution other than any of said steady-state particle

distributions, and wherein said two or more particle types are separated in said

transition region; and

c. one or more output channels disposed to collect one or more of said particle

types from said transition region.

2. The separation device as set forth in claim 1, wherein one of said steady-state

regions is in a first location, wherein another of said steady state regions is in a

second location, and wherein said transition region is located in an intermediate

location between said first and said second locations.

3. The separation device as set forth in claim 2, wherein said fluid flows at a

steady rate sequentially through said first location, said intermediate

location, and said second location.

4. The separation device as set forth in claim 1, wherein said flow channel

comprises a first channel section and a second channel section.

5. The separation device as set forth in claim 4, wherein said second channel

section is tilted at a nonzero angle with respect to said first channel

section.

6. The separation device as set forth in claim 4, wherein said second

channel section has a narrower width than said first channel section.

7. The separation device as set forth in claim 4, further comprising a third

channel section parallel to said first channel section.

8. The separation device as set forth in claim 7, wherein a single

acoustic transducer provides said electric field, wherein one of said

steady-state regions is located in said first channel section, wherein

another of said steady-state regions is located in said third channel

section, and wherein said second channel section has substantially

no acoustic excitation.

9. The separation device as set forth in claim 1, wherein said one or more acoustic

transducers comprise piezoelectric elements.

10. The separation device as set forth in claim 9, wherein said piezoelectric

elements comprise macroscopic ceramic transducers or films deposited

on an outer surface of said flow channel.

11. The separation device as set forth in claim 1, wherein said two or more particle

types are distinguished on the basis of differences in size, density or

compressibility.

12. The separation device as set forth in claim 1, wherein said two or more particle

types are red and white blood cells.

13. A method of separating two or more particle types in a fluid, comprising: a. establishing an acoustic field in a flow channel using one or more acoustic transducers, wherein said acoustic field has at least two steady-state regions having distinct steady-state acoustic field patterns, and wherein said acoustic field has at least one transition region having a dynamic acoustic field pattern; b. advancing said fluid with said two or more particle types through said at least two steady state regions and said at least one transition region, wherein said two or more particle types have a first steady-state distribution in a first steady- state region, a second steady-state distribution in a second steady-state region, and a dynamic distribution in said at least one transition region; c. separating said two or more particle types in said transition region; and d. collecting one or more of said separated particle types.

14. The method as set forth in claim 13, wherein said establishing of said acoustic field comprises placing at least two acoustic transducers at different locations along said flow channel to generate said at least two steady-state regions and said at least one transition region.

15. The method as set forth in claim 13, wherein said establishing of said acoustic field comprises using one acoustic transducer to generate said at least two

steady-state regions and said at least one transition region.

16. The method as set forth in claim 15, wherein said establishing further comprises dividing said flow channel into two or more sections of different widths.

17. The method as set forth in claim 15, wherein said establishing further comprises dividing said flow channel into two sections, wherein said sections are titled at a nonzero angle with respect to each other.

18. The method as set forth in claim 15, wherein said establishing further comprises dividing said flow channel into three sections, wherein a first section and a third section are parallel to one another, and a second section is tilted at a nonzero angle with respect to said first and third sections.

19. The method as set forth in claim 13, wherein said separating of said particle types is based on differences in particle type size, density or compressibility.

20. The method as set forth in claim 13, wherein said collecting comprises providing an output channel from said flow channel in said transition region.