CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/352,333, filed June 15, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Biological analytes may be stored and tested in large-scale nanowell arrays in microfluidic devices. Samples may be loaded onto wells from a sample source.

SUMMARY

[0003] An aspect of the present disclosure provides a system for redistributing one or more particles in a sample. In some embodiments, the system comprises a motor assembly configured to supply one or more cycles. In some embodiments, said motor assembly is attached to a rotor comprising one or more sides. In some embodiments, the system comprises an actuating arm coupled to said rotor at an attachment point such that said actuating arm is free to rotate about said attachment point when said actuating arm is in motion. In some embodiments, the system comprises a section of tubing. In some embodiments, said section of tubing is threaded through an end of said actuating arm that is distal to said rotor. In some embodiments, said section of tubing is configured to contain said sample comprising said one or more particles wherein, said motor assembly, when in operation, provides said one or more cycles that cause said actuating arm to come into motion and manipulate said section of tubing, thereby homogenizing said one or more particles in a sample.

[0004] In some embodiments, said rotor has one side. In some embodiments, said rotor is circular. In some embodiments, said motor assembly is attached to a base. In some embodiments, said actuating arm comprises a slot that captures a pivot pin mounted on said base. In some embodiments, a first end of said section of tubing is fluidically connected to one or more sample sources. In some embodiments, first end of said section of tubing draws fluids from two different sources. In some embodiments, said fluids from said two different sources are mixed in said section of tubing.

[0005] In some embodiments, said section of tubing is attached to said base. In some embodiments, said section of tubing has sufficient slack to follow the full travel of said actuating arm while said actuating arm is in motion. In some embodiments, the system further comprises a microfluidic device. In some embodiments, said microfluidic device comprises a channel in fluidic communication with one or more wells. In some embodiments, a second end of said section of tubing is fluidically connected to said microfluidic device. In some embodiments, said section of tubing is fluidically connected to said microfluidic device via a device input line.

[0006] In some embodiments, said device input line is configured to flow said sample into said microfluidic device via said channel. In some embodiments, said microfluidic device comprises one or more wells. In some embodiments, said one or more wells have a volume of less than about 2 nanoliters (nL). In some embodiments, said one or more wells have a volume of less than about 1 nL.

[0007] In some embodiments, said microfluidic device comprises at least about 5,000 of said wells. In some embodiments, said microfluidic device comprises at least about 50,000 of said wells. In some embodiments, said microfluidic device comprises at least about 500,000 of said wells. In some embodiments, said section of tubing is comprised of a polymeric material. In some embodiments, said section of tubing is comprised of PEEK (poly etheretherketone) or Teflon.

[0008] In some embodiments, said section of tubing has an inner diameter of at least about 1000 micrometers (pm) or less. In some embodiments, said section of tubing has an inner diameter of at least about 500 micrometers (pm) or less. In some embodiments, said section of tubing has an inner diameter of at least about 300 micrometers (pm) or less.

[0009] In some embodiments, the system further comprises a power supply electrically connected to said motor. In some embodiments, the system further comprises one or more computer processors individually or collectively programmed to operate said power supply. In some embodiments, the system further comprises a driver board electrically connected to said motor. In some embodiments, the system further comprises one or more computer processors individually or collectively programmed to operate said driver board. In some embodiments, said motor is operated in pulse width modulation mode using said power supply and said driver board.

[0010] In some embodiments, said pulse width is programmed to obtain a rotational velocity in the range of at least about 5 revolutions per minute (rpm) to at least about 20 rpm. In some embodiments, said actuating arm is caused to come into motion with respect to two directions normal to the axis of said section of tubing.

[0011] Another aspect of the present disclosure provides a method comprising: (a) providing a system for redistributing one or more particles in a sample, comprising: a motor assembly that supplies one or more cycles, wherein said motor assembly is attached to a rotor; an actuating arm attached to said rotor at an attachment point such that said actuating arm is free to rotate about said attachment point when said actuating arm is in motion; and a section of tubing, wherein a first end of said section of tubing is attached to the end of said actuating arm that is distal to said rotor, and wherein said section of tubing contains said sample comprising said one or more particles and (b) operating said motor assembly of to provide said one or more cycles to said actuating arm, there causing said actuating arm to come into motion and manipulate said section of tubing.

[0012] In some embodiments, said rotor has one side. In some embodiments, said rotor is circular. In some embodiments, the method further comprises homogenizing said one or more particles in said sample. In some embodiments, said motor assembly is attached to a base. In some embodiments, said actuating arm comprises a slot that captures a pivot pin mounted on said base plate. In some embodiments, a first end of said section of tubing is fluidically connected to one or more sample sources.

[0013] In some embodiments, said fluids from said two different sources are mixed in said section of tubing. In some embodiments, said section of tubing is attached to said base plate by one or more anchor points. In some embodiments, said section of tubing has sufficient slack to follow the full travel of said actuating arm while said actuating arm is in motion. In some embodiments, the method further comprises a microfluidic device. In some embodiments, said microfluidic device comprises a channel in fluidic communication with one or more wells.

[0014] In some embodiments, a second end of said section of tubing is fluidically connected to said microfluidic device. In some embodiments, said section of tubing is fluidically connected to said microfluidic device via a device input line. In some embodiments, said device input line is configured to flow said sample into said microfluidic device via said channel. In some embodiments, said one or more wells have a volume of less than about 2 nanoliters (nL). In some embodiments, said one or more wells have a volume of less than about 1 nL. In some embodiments, said one or more wells have a volume of less than about 0.5 nL.

[0015] In some embodiments, said microfluidic device comprises at least about 5,000 of said wells. In some embodiments, said microfluidic device comprises at least about 50,000 of said wells. In some embodiments, said microfluidic device comprises at least about 500,000 of said wells. In some embodiments, said section of tubing is comprised of a polymeric material. In some embodiments, said section of tubing is comprised of PEEK (poly etheretherketone) or Teflon.

[0016] In some embodiments, said section of tubing has an inner diameter of at least about 1000 micrometers (pm) or less. In some embodiments, said section of tubing has an inner diameter of at least about 500 micrometers (pm) or less. In some embodiments, said section of tubing has an inner diameter of at least about 300 micrometers (pm) or less. In some embodiments, the method further comprises a power supply electrically connected to said motor. In some embodiments, the method further comprises one or more computer processors individually or collectively programmed to operate said power supply. In some embodiments, the method further comprises a driver board electrically connected to said motor.

[0017] In some embodiments, the method further comprises one or more computer processors individually or collectively programmed to operate said driver board. In some embodiments, said motor is operated in pulse width modulation mode using said power supply and said driver board. In some embodiments, said pulse width is programmed to obtain a rotational velocity in the range of at least about 5 revolutions per minute (rpm) to at least about 20 rpm. In some embodiments, said actuating arm is caused to come into motion with respect to two directions normal to the axis of said section of tubing.

[0018] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0019] In some aspects, the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0020] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0021] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0023] FIG. l is a side view and a top view of a mechanical actuation device for randomizing (i.e., homogenizing or mixing) a particle suspension contained in a tubing.

[0024] FIG. 2 illustrates a microfluidic device loading system that includes the mechanical actuation device of FIG.1.

[0025] FIG. 3 is a flow diagram illustrating an example of a method of using the microfluidic device loading system to populate an array of wells in a microfluidic device.

[0026] FIG. 4 shows a schematic diagram of a comparison of loading a particle suspension into a well array of a microfluidic device using a conventional flow approach vs a mechanical actuation-assisted flow process.

[0027] FIG. 5 shows a computer system 501 that is programmed or otherwise configured to control acoustic retrieval processes as disclosed herein. The computer system 501 can regulate various aspects of flow homogenization processes of the present disclosure.

DETAILED DESCRIPTION

[0028] While various embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0029] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0030] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0031] “Target” means any particulate or molecular material of interest that can be manipulated by a system of the invention, e.g., targets may be one or more, cells, cell components (e.g., organelles), biomolecules (e.g., nucleic acids or proteins), particles (e.g., beads). [0032] “Sample” means any liquid that includes target material. A sample may also include other components, such as reagents, such as processing reagents and/or assay reagents. In some cases, the sample is an aqueous solution. In some cases, the sample is a biological liquid, such as blood, plasma, serum, urine, or cerebrospinal liquid, or a solution including a biological liquid.

[0033] “Particle suspension” means a preparation containing finely divided particulate or molecular material distributed uniformly throughout a fluid vehicle, e.g., a particle suspension may include cells, cell components (e.g., organelles), biomolecules (e.g., nucleic acids or proteins), beads (e.g., capture beads with a biomolecule of interest bound thereon) in a buffer solution.

[0034] The present disclosure provides acoustic retrieval systems, microfluidic devices, and methods for using an externally applied acoustic field for selective retrieval of contents from the microfluidic device in the case where the fluid is a single phase (e.g., aqueous).

[0035] The present disclosure may provide an acoustic retrieval system. The present disclosure provides a microfluidic device. The present disclosure may provide a method for generating an acoustic field that can be used to produce a localized fluid flow in the microfluidic device containing a single fluid phase. The acoustic field may be generated by an external acoustic transducer that is coupled to the microfluidic device using a coupling medium.

[0036] The present disclosure provides an acoustic retrieval system, microfluidic device, and method for using an acoustic field to produce a localized fluid flow in the microfluidic device that can be used to actuate on demand the contents of a single selected nanowell within a high- density array of nanowells in the device for downstream processing.

[0037] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of a single cell (i.e., single cell analysis (SCA)), its contents, and/or selected biomolecules (e.g., secreted proteins or nucleic acids).

[0038] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of a single cell in clinical applications such as screening for rare immune cells, for example B-cells and T-cells, in a sample.

[0039] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of a single cell, its contents, and/or selected biomolecules in a synthetic biology application (e.g., yeast).

[0040] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of a single cell, its contents, and/or selected biomolecules in a drug discovery application. System and Device

[0041] The present disclosure provides system for redistributing one or more particles in a sample. The system may comprise a motor assembly. The motor assembly may be configured to supply one or more cycles. The motor assembly may be attached to a rotor. The rotor may be attached to an actuating arm at an attachment point. The actuating arm may be free to rotate about the attachment point when the actuating arm is in motion. The system may further comprise a section of tubing, wherein the first end of the section of tubing is threaded through the end of the actuating arm that is distal to the rotor. The section of tubing may contain a sample. The same may comprise one or more particles. The motor assembly, when in operation, may provide one or more cycles that cause the actuating arm to come into motion and manipulate the section of tubing. The system may homogenize one or more particles in a sample. The system may be used to uniformly distribute a suspension of particles flowed in a tubing from a sample source and into a microfluidic device. The microfluidic device may include an array of wells for partitioning the suspension of particles in the sample into subsamples for downstream analysis. The mechanical actuation device may be used to distribute the particles efficiently and uniformly into individual wells of the well array. The system may further comprise a microfluidic device loading system and a method for randomizing (i.e., homogenizing) a particle suspension flowed from a sample source for injection (loading) into a microfluidic device. Uniformity in loading a particle suspension into a microfluidic device is essential for efficiently filling a large well array.

[0042] Other elements of the device, system and methods are possible, including the elements described in U.S. Patent 10,488,321, entitled “Devices and Methods for High-Throughput Single Cell and Biomolecule Analysis and Retrieval in a Microfluidic Chip”, issued on November 26, 2019, the entire disclosure of which is incorporated herein by reference.

[0043] Motor assembly

[0044] The present disclosure provides a system for redistributing one or more particles in a sample. The system may comprise a motor assembly. The motor assembly may be configured to supply one or more cycles. FIG. l is a side view and a top view of a mechanical actuation device 100 for randomizing (i.e., homogenizing or mixing) a particle suspension contained in a tubing. Mechanical actuation device 100 may include a motor assembly 110. Motor assembly 110 may be mounted on a base plate 115. Base plate 115 may, for example, be a 2 x 3-inch base plate. Motor assembly 110 may include a motor 120. Motor assembly 110 may include a rotor 122 attached to motor 120 on a motor axile 124. The rotor may have one or more sides. The rotor may have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more sides. The rotor may be circular. The motor assembly may include a wheel attached to the motor on a motor axile. Motor 120 may, for example, be a DC motor, such as a SparkFun M260 motor (SparkFun Electronics, Niwot, Colorado).

[0045] Attached to rotor 122 may be an actuating arm 130. Actuating arm 130 may be attached to rotor 122 at an attachment point 132 such that actuating arm 130 is free to rotate about attachment point 132. Actuating arm 130 may include a slot 134 that captures a pivot pin 136 mounted on base plate 115 by a pivot pin post 137. The diameter of pivot pin 136 may be less than the width of slot 134 in actuating arm 130, so that actuating arm 130 is free to slide along pivot pin 136.

[0046] The components of motor assembly 110 may be sized to fit the dimensions of base plate 115

[0047] Systems as disclosed herein may further comprise a power supply and a driver board. As depicted in FIG. 2, systems may further include a power supply 230 and a driver board 235. In one example, power supply 230 may be a 12V power supply. In one example, driver board 235 may be a TB6612FNG driver board. Driver board 235 may be controlled by a computer 240 (e.g., a Dell T3600 computer). Power supply 230 and driver board 235 controlled by computer 240 may be used to activate motor 120.

[0048] Tubing

[0049] The present disclosure provides a system for redistributing one or more particles in a sample. The system may comprise a section of tubing comprising a sample. The sample may comprise one or more particles. The section of tubing may be connected to the motor assembly. The section of tubing may be threaded through the end of the actuating arm that is distal to the rotor. The section of tubing may have a first end and a second end. The first end of said section of tubing may be fluidically connected to one or more sample sources. The first end of said section of tubing may be connected to two or more sample sources. The two or more sample sources may comprise fluids. The fluids from the two or more sample fluids may be mixed in the section of tubing when the actuating arm is in motion. The second end of the section of tubing may be fluidically connected to a microfluidic device. The section of tubing may contain a sample. The same may comprise one or more particles. The motor assembly, when in operation, may provide one or more cycles that cause the actuating arm to come into motion and manipulate the section of tubing. The system may homogenize one or more particles in a sample. The system may be used to uniformly distribute a suspension of particles flowed in a tubing from a sample source and into a microfluidic device.

[0050] As depicted in FIG. 1, the section of tubing 140 may be threaded through a tubing hole 138 at the end of actuating arm 130 that is distal to rotor 122. Tubing 140 may be made of a flexible polymer material, such as PEEK (polyetheretherketone) or Teflon®. The inner diameter of tubing 140 may, for example, be about 500 pm or less. In one example, tubing 140 may be an IDEX 1569 PEEK tubing with an 793.75 pm outer diameter and an 500 pm inner diameter. Tubing 140 may be attached at two anchor points 142 on base plate 115. Tubing 140 may be anchored on base plate 115 with sufficient slack to allow the tubing to follow the full travel of actuating arm 130 as rotor 122 is rotated. Anchor points 142 hold tubing 140 in place and provide strain relief so that the motion of the tubing does not propagate beyond base plate 115. This configuration of tubing 140 threaded through tubing hole 138 in actuating arm 130 and anchored by anchor points 142 is used to randomize the particle positions within the section of tubing using mechanical actuation (i.e., vigorous shaking motion of the tubing). Base plate 115, rotor 122, actuating arm 130, and anchor points 142 may be fabricated by stereolithographic 3D printing. In one example, the printing material may be a plastic material such as Accur ClearVue plastic (3D Systems, Inc.). Tubing 140 may be used to draw particle suspension liquids from two different sources for mixing in the tubing. Tubing 140 may be used to flow a particle suspension from a source container for loading into a microfluidic device. For example, one end of tubing 140 may be connected to a sample source container (not shown) and the other end of tubing 140 may be connected to a microfluidic device (not shown).

[0051] Microfluidic device

[0052] The present disclosure provides a system for redistributing one or more particles in a sample, the sample may be flowed via a section of tubing to a microfluidic device, wherein said microfluidic device may further process said sample.

[0053] FIG. 2 illustrates a microfluidic device loading system 200. Microfluidic device loading system 200 includes mechanical actuation device 100. Microfluidic device loading system 200 may include a sample source 210 and a microfluidic device 220. Mechanical actuation device 100 may be used to uniformly distribute a suspension of particles flowed from sample source 210 into microfluidic device 220. Sample source 210 may be configured to supply a suspension of particles. For example, sample source 210 may include a volume of a sample liquid 212 that includes a suspension of particles. In one embodiment, the particles may be cells in a buffer solution. In one embodiment, the particles may be beads (e.g., capture beads with a biomolecule of interest bound thereon) in a buffer solution. In one embodiment, the buffer solution may be an aqueous buffer. Sample source 210 may be coupled to tubing 140 by a sample input line 214.

[0054] A microfluidic device 220 may be configured to support automated high-throughput processes to isolate, screen, and/or retrieve single cells or biomolecules in a biological sample. Microfluidic device 220 may include an array of wells (not shown) for compartmentalizing the biological sample into a plurality of subsamples for downstream analysis. In some embodiments, a well is a nanowell. In some embodiments, microfluidic device 220 can include at least about 100 nanowells, or at least about 1,000 nanowells, or at least about 10,000 nanowells, or at least about 100,000 nanowells, or up to about 1,000,000 nanowells. The volumes of wells in the array of wells can be uniform or may vary. In some embodiments, a well is a nanowell. In some cases, the nanowell has a volume of about 2 nL or less, or about 1 nL or less.

[0055] Microfluidic device 220 may be coupled to the section of tubing 140 by a device input line 222. Device input line 222 may be used to flow a suspension of cells or beads into a channel (not shown) in microfluidic device 220 that is in communication with the well array (not shown).

[0056] The microfluidic device may include an array of wells for compartmentalizing a biological sample into one or more subsamples for isolating, screening, and/or retrieving single cells or biomolecules in the sample. The well may be a nanowell. The microfluidic device may include at least about 100 nanowells, at least about 1,000 nanowells, at least about 10,000 nanowells, at least about 100,000 nanowells, or at least about 1,000,000 nanowells. The microfluidic device may include at least about 100 nanowells, at least about 200 nanowells, at least about 300 nanowells, at least about 400 nanowells, at least about 500 nanowells, at least about 600 nanowells, at least about 700 nanowells, at least about 800 nanowells, or at least about 900 nanowells. The microfluidic device may include at least about 1,000 nanowells, at least about 2,000 nanowells, at least about 3,000 nanowells, at least about 4,000 nanowells, at least about 5,000 nanowells, at least about 6,000 nanowells, at least about 7,000 nanowells, at least about 8,000 nanowells, or at least about 9,000 nanowells. The microfluidic device may include at least about 10,000 nanowells, at least about 20,000 nanowells, at least about 30,000 nanowells, at least about 40,000 nanowells, at least about 50,000 nanowells, at least about 60,000 nanowells, at least about 70,000 nanowells, at least about 80,000 nanowells, or at least about 90,000 nanowells. The microfluidic device may include at least about 100,000 nanowells, at least about 200,000 nanowells, at least about 300,000 nanowells, at least about 400,000 nanowells, at least about 500,000 nanowells, at least about 600,000 nanowells, at least about 700,000 nanowells, at least about 800,000 nanowells, at least about 900,000 nanowells, or at least about 1,000,000 nanowells.

[0057] Each array of wells may contain identical volumes or non-identical volumes. The well may be a nanowell. The nanowell may have a volume of at most about 2 nanoliters (nL). The nanowell may have a volume of at most about 1 nL. The nanowell may have a volume of at most about 0.1 nL, at most about 0.2 nL, at most about 0.3 nL, at most about 0.4 nL, at most about 0.5 nL, at most about 0.6 nL, at most about 0.7 nL, at most about 0.8 nL, at most about 0.9 nL, at most about 1 nL, at most about 1.1 nL, at most about 1.2 nL, at most about 1.3 nL, at most about 1.4 nL, at most about 1.5 nL, at most about 1.5 nL, at most about 1.6 nL, at most about 1.7 nL, at most about 1.8 nL, at most about 1.9 nL, or at most about 2 nL. A nanowell may have a volume of about 1 nL.

[0058] Acoustic transducer

[0059] The present disclosure provides a system for redistributing one or more particles in a sample. The one or more particles in said sample may be further processed by an acoustic transducer. The acoustic transducer may be used to apply an acoustic beam to a microfluidic device comprising the sample comprising one or more particles. The acoustic transducer may be mounted externally to a microfluidic device on a motorized stage, moveable in x, y, z directions to allow real time adjustment of the position of the acoustic transducer laterally and/or vertically relative to the microfluidic device.

[0060] The acoustic transducer may be used to apply a focused acoustic beam to an individual nanowell in microfluidic device. The acoustic transducer may be mounted externally to the microfluidic device, such that no integration of the acoustic transducer within the microfluidic device is required. This setup may simplify and reduce the cost of fabrication of the microfluidic device. Further, external application of the acoustic beam the microfluidic device may be contactless and thereby limit the introduction of contaminants into the microfluidic device.

[0061] The acoustic transducer may be configured such that the propagation direction of the acoustic beam is perpendicular to the plane of microfluidic device. In some cases, a focused acoustic beam may be delivered from the acoustic transducer at a distance from the microfluidic device. For example, the focused acoustic beam may be delivered from the acoustic transducer at a distance of about 0.25 centimeters (cm) to about 2 cm from the microfluidic device. The focused acoustic beam may be delivered from the acoustic transducer at a distance of at least about 0.1 cm, at least about 0.2 cm, at least about 0.3 cm, at least about 0.4 cm, at least about 0.5 cm, at least about 0.6 cm, at least about 0.7 cm, at least about 0.8 cm, at least about 0.9 cm, at least about 1 cm, at least about 1.1 cm, at least about 1.2 cm, at least about 1.3 cm, at least about 1.4 cm, at least about 1.5 cm, at least about 1.6 cm, at least about 1.7 cm, at least about 1.8 cm, at least about 1.9 cm, at least about 2 cm, or more.

[0062] The acoustic transducer may be configured to operate at a frequency of about 1 to about 150 megahertz (MHz). The acoustic transducer may be configured to operate at a frequency of about 15 to about 25 MHz. The acoustic transducer may be configured to operate at a frequency of at least about 1 MHz, at least about 2 MHz, at least about 3 MHz, at least about 4 MHz, at least about 5 MHz, at least about 6 MHz, at least about 7 MHz, at least about 8 MHz, at least about 9 MHz, at least about 10 MHz, or more. The acoustic transducer may be configured to operate at a frequency of at least about 10 MHz, at least about 20 MHz, at least about 30 MHz, at least about 40 MHz, at least about 50 MHz, at least about 60 MHz, at least about 70 MHz, at least about 80 MHz, at least about 90 MHz, at least about 100 MHz, at least about 110 MHz, at least about 120 MHz, at least about 130 MHz, at least about 140 MHz, at least about 150 MHz, or more.

[0063] The acoustic transducer may be configured to apply the focused acoustic beam having a spot size in the range of about 25 micrometers (pm) to about 200 pm. The acoustic transducer may be configured to apply a focused acoustic beam having a spot size of at least about 25 pm, at least about 60 pm, at least about 70 pm, at least about 80 pm, at least about 90 pm, at least about 100 pm, at least about 110 pm, at least about 120 pm, at least about 130 pm, at least about 140 pm, at least about 150 pm, at least about 160 pm, at least about 170 pm, at least about 180 pm, at least about 190 pm, at least about 200 pm, or more. The spot size of a focused acoustic beam may be selected based on the density of nanowell features in a nanowell array of a microfluidic device.

[0064] The acoustic transducer may be coupled to a microfluidic device by immersion in a coupling medium that is acoustically low-absorbing. The coupling medium may be water. [0065] Optical Imaging System

[0066] Systems as described herein may further comprise an optical imaging device to image the sampel comprising one or more particles. An optical imaging device may incorporate bright field and fluorescence microscopy capabilities. The optical imaging device may be used to analyze a compartmentalized sample in one or more nanowells of the nanowell array. The optical imaging device may be configured with high magnification capabilities for high resolution imaging of single cells.

[0067] For example, the optical imaging system may be a fluorescence microscope with the capability to image a well array over a range of fluorescent wavelengths and in brightfield. For example, the microscope may include multiple illumination wavelengths and filter cubes to work at different fluorescent conditions. The microscope may include multiple objectives to provide magnifications in the range of at least about 2 to at least about 20X. The microscope may provide a magnification of at least about 2X, at least about 3X, at least about 4X, at least about 5X, at least about 6X, at least about 7X, at least about 8X, at least about 9X, at least about 10X, at least about 1 IX, at least about 12 X, at least about 13X, at least about 14X, at least about 15X, at least about 16X, at least about 17X, at least about 18X, at least about 19X, at least about 20X, or more. [0068] The microscope may include a scientific CMOS camera to collect high resolution images over a large field of view at fast frame rates. The microscope may include fast and precise stages to move the microfluidic device and hence produce high resolution stitched images of the entire well array.

[0069] Computer

[0070] FIG. 5 shows a computer system 501 that is programmed or otherwise configured to control acoustic retrieval processes as disclosed herein. The computer system 501 can regulate various aspects of flow homogenization processes of the present disclosure. For example, the computer may be electronically coupled to various components of the present disclosure, such as the imaging device and acoustic transducer. The computer may be programmed to control various aspects of the acoustic retrieval processes as disclosed herein, such as imaging, focusing, processing image data, interpreting image data, generation of acoustic beams and pulses, properties of beams and pulses, flow of in the microfluidic device.

[0071] The computer system 501 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0072] The computer system 501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 501 may also include a memory or memory location 510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 515 (e.g., hard disk), communication interface 520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 525, such as cache, other memory, data storage and/or electronic display adapters. The memory 510, storage unit 515, interface 520 and peripheral devices 525 are in communication with the CPU 505 through a communication bus (solid lines), such as a motherboard. The storage unit 515 can be a data storage unit (or data repository) for storing data. The computer system 501 can be operatively coupled to a computer network (“network”) 530 with the aid of the communication interface 520. The network 530 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 530 in some cases is a telecommunication and/or data network. The network 530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 530, in some cases with the aid of the computer system 501, can implement a peer-to- peer network, which may enable devices coupled to the computer system 501 to behave as a client or a server. [0073] The CPU 505 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 510. The instructions can be directed to the CPU 505, which can subsequently program or otherwise configure the CPU 505 to implement methods of the present disclosure. Examples of operations performed by the CPU 505 can include fetch, decode, execute, and writeback.

[0074] The CPU 505 can be part of a circuit, such as an integrated circuit. One or more other components of the system 501 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0075] The storage unit 515 can store files, such as drivers, libraries and saved programs. The storage unit 515 can store user data, e.g., user preferences and user programs. The computer system 501 in some cases can include one or more additional data storage units that are external to the computer system 501, such as located on a remote server that is in communication with the computer system 501 through an intranet or the Internet.

[0076] The computer system 501 can communicate with one or more remote computer systems through the network 530. For instance, the computer system 501 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 501 via the network 530. [0077] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 501, such as, for example, on the memory 510 or electronic storage unit 515. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 505. In some cases, the code can be retrieved from the storage unit 515 and stored on the memory 510 for ready access by the processor 505. In some situations, the electronic storage unit 515 can be precluded, and machine-executable instructions are stored on memory 510.

[0078] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0079] Aspects of the systems and methods provided herein, such as the computer system 501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0080] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0081] The computer system 501 can include or be in communication with an electronic display 535 that comprises a user interface (UI) 540 for providing, for example, information on the position or actuation parameters of an acoustic beam. The UI may provide information on the frequency of an acoustic beam. The UI may provide information on the sequence of pulses. The UI may provide information on the pulse period. The UI may provide information on the duration of pulses. The UI may provide information on the position of an acoustic beam. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0082] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 505.

[0083] Systems and methods as disclosed herein may further comprise one or more computer processors individually or collectively programmed to implement a method for acoustic retrieval. The one or more computer processors may be connected to an acoustic transducer. The one or more processors may be operatively coupled to the acoustic transducer, and may affect the acoustic transducer to supply one or more pulses to a sample, resulting in actuation of the sample. The one or more computer processors may receive optical imaging data from an optical imaging device. The imaging data may be interpreted to assess a content of one or more wells in an array of wells. A well may be selected for content actuation based at least part on the interpretation of the data. An acoustic transducer may then apply an acoustic beam to the well, thereby actuating the contents of the well.

Particle Loading into a Microfluidic Device

[0084] The present disclosure provides systems and methods for redistributing one or more particles in one or more samples. The system may comprise a motor assembly configured to supply one or more cycles. The motor assembly may be attached to a rotor. The rotor may have one or more side. The rotor may be circular. The system may further comprise an actuating arm attached to the rotor at an attachment point such that the actuating arm is free to rotate about the attachment point when the actuating arm is in motion. The system may further comprise a section of tubing, wherein the section of tubing is threaded through the end of the actuating arm that is distal to the rotor. The section of tubing may contain the sample comprising one or more particles.

[0085] A particle suspension that flows through a tubing from a source to a collection point is generally not uniformly distributed during the fluidic transport. The nonuniformity of the particles flowing through the tubing may arise due to inhomogeneity of the particle density in the source container, or forces (e.g., gravitational forces) on the particles during transport. [0086] The present disclosure provides a microfluidic device loading system for uniformly distributing a suspension of particles flowed from particle suspension source into a microfluidic device.

[0087] As depicted in FIG. 2, a volume of sample liquid 212 from sample source 210 may be flowed into tubing 140 via sample input line 214. Motor 120 may be activated and operated in pulse width modulation mode using power supply 230 and driver board 235 controlled by computer 240. The pulse width may, for example, be set by computer 240 to a value to obtain a rotational velocity in the range of from about 5 rpm to about 20 rpm. As motor 120 is activated, rotor 122 rotates, thereby both pulling actuating arm 130 in and out, and moving it up and down. This produces an aggressive shaking motion of tubing 140 in the two directions normal to the axis of the tubing. The shaking motion redistributes the particles (e.g., cells) in sample liquid 212 uniformly in the tubing as they flow through the tubing section between the anchor points 142. The shaking motion of tubing 140 homogenizes the particles (e.g., cells) in sample liquid 212 with respect to the flow inside tubing 140. Because of this shaking motion, there is a uniform distribution of particles (e.g., cells) distributed within the flow streams injected into microfluidic device 220 via device input line 222.

[0088] The microfluidic device loading system may be used for homogenizing the flow of a particle suspension injected into a microfluidic device. When the particle suspension is injected into the microfluidic device, the homogeneity translates to a uniform distribution of the particles in the microfluidic device.

[0089] The invention provides a method for uniformly loading a particle suspension into a well array of a microfluidic device. In one embodiment, the particle suspension is a biological sample that includes one or more targets of interest for downstream analysis.

[0090] FIG. 3 is a flow diagram illustrating an example of a method 300 of using microfluidic device loading system 200 to populate an array of wells in a microfluidic device. Method 300 may include, but is not limited to, the following steps.

[0091] In a step 310, a microfluidic device loading system that includes a mechanical actuation device coupled to a sample source and a microfluidic device is provided. For example, microfluidic device loading system 200 that includes sample source 210 and microfluidic device 220 is provided. Sample source 210 may contain a sample liquid that includes a suspension of particles such as cell or beads (e.g., capture beads with a biomolecule of interest bound thereon). [0092] In a step 315, a volume of sample liquid is flowed from the sample source into the mechanical activation device for homogenizing the flow of the particle suspension. For example, a volume of sample liquid 212 may be flowed from sample source 210 via sample input line 214 and into tubing 140. Motor 120 may be activated and operated in pulse width modulation mode using power supply 230 and driver board 235 controlled by computer 240. The pulse width may, for example, be set by computer 240 to a value to obtain a rotational velocity in the range of from about 5 rpm to about 20 rpm. As motor 120 is activated, rotor 122 rotates, thereby both pulling actuating arm 130 in and out, and moving it up and down to produce a shaking motion of tubing 140 in the two directions normal to the axis of the tubing. The shaking motion uniformly redistributes the particles (e.g., cells) in sample liquid 212 in the tubing 140 the liquid flows through the tubing section between the anchor points 142. The shaking motion of tubing 140 homogenizes the particles (e.g., cells) in sample liquid 212 with respect to the flow inside tubing 140.

[0093] In a step 320, the sample liquid is partitioned uniformly into the wells of the microfluidic device. For example, the sample liquid in tube 140 is flowed into microfluidic device 220 via device input line 222 and into a channel of the microfluidic device that is in communication with the well array. Because of the shaking motion of tubing 140, there is a uniform distribution of particles (e.g., cells) within the flow stream injected into microfluidic device 222 and loaded into the wells of the microfluidic device. Because of the uniformity in delivery of the particles in the sample liquid injected into the microfluidic device, the particles are uniformly distributed in the well array and the entire well is filled with maximum efficiency.

[0094] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

[0095] A challenge in loading a particle suspension into a well array of a microfluidic device using a liquid flow approach (i.e., via flowing through a tubing) is to load the particles uniformly into the well array. The device, system, and method of the invention may be used to uniformly load a particle suspension into a well array (e.g., a nanowell array) of a microfluidic device. For example, FIG. 4 shows a schematic diagram of a comparison of loading a particle suspension into a well array of a microfluidic device using a conventional flow approach vs a mechanical actuation-assisted flow process of the invention.

[0096] Referring now to panel (a) of FIG. 4, in a conventional well-array loading approach, any inhomogeneity of the particles in the tubing maps to an inhomogeneity during injection of the particles into the well array. For example, when a sample liquid is flowed via a tubing from a sample source and into a flow channel of a microfluidic device, a relatively large portion of the particles in the sample liquid may be localized to one side or the other of the channel, i.e., the particles do not mix, but stay in their streamlines. This nonuniformity in delivery of the particles in the sample liquid results in an inefficiency that leaves a large section of the well array unpopulated, i.e., unused.

[0097] Referring now to panel (b) of FIG. 4, the mechanical actuation-assisted process of the invention described in this disclosure eliminates the inhomogeneities and produces a uniform distribution of particles through the well array. In this case, the entire well array is filled with maximum efficiency.

[0098] The mechanical actuation-assisted process of the invention may be used to deliver a uniform distribution of particles into microfluidic flow channels of different widths. For example, the width of a flow channel consisting of a well array may be about 25 mm or less. In one example, the width of a flow channel consisting of a well array may be from about 3 mm to about 7 mm.

[0099] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the disclosure be limited by the specific examples provided within the specification. While the disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. Furthermore, it shall be understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of disclosure and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.