Massively parallel cell analysis and sorting apparatus and method

Abstract

A massively parallel microfluidic chip is provided having multiple sections stacked or layered along a stacking direction to form multiple microchannels at least partially oriented for flow along the stacking direction. The multiple sections can include a transfer section for introducing a sample fluid containing particles, a particle focusing section configured to focus particles in the sample fluid, and an actuation section including multiple interrogation regions and multiple actuators. Each interrogation region and actuator is associated with at least one microchannel within the multiple microchannels. The arrangement of the microfluidic channels along the stacking direction allows for a very high packing density of channels and interrogation regions on a single chip, providing massively parallel processing of particles.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 419,852, filed October 27, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Microfluidic devices have been used to analyze populations of particles and sort the particles according to the results of the analysis, which is traditionally accomplished by flowing the particles through a region where the analysis occurs, such as a region where optical interrogation and detection is performed. Summary of the Invention

[0003] A microfluidic chip is disclosed. In some embodiments, the microfluidic chip includes multiple sections stacked or layered in a stacking direction to form multiple microchannels at least partially oriented for flow along the stacking direction. The multiple sections include a transfer section for introducing a sample fluid containing particles. The multiple sections include a particle focusing section configured to focus particles in the sample fluid. The multiple sections include an actuation section including multiple interrogation regions and multiple actuators. Each of the multiple interrogation regions and each of the multiple actuators is associated with at least one microchannel in the multiple microchannels.

[0004] A particle processing system is disclosed. In some embodiments, the particle processing system includes a microfluidic chip having a plurality of sections stacked or layered in a stacking direction to form a plurality of microchannels at least partially oriented to flow along the stacking direction. The plurality of sections includes a transfer section for introducing a sample fluid containing particles. The plurality of sections includes a particle focusing section configured to focus particles in the sample fluid. The plurality of sections includes an actuation section including a plurality of interrogation regions and a plurality of actuators. Each of the plurality of interrogation regions and each of the plurality of actuators is associated with at least one microchannel within the plurality of microchannels. In some embodiments, the particle processing system includes an electromagnetic source system for illuminating the plurality of interrogation regions. In some embodiments, the particle processing system includes a detection system for receiving light from the plurality of interrogation regions. In some embodiments, the particle processing system includes a computing system operably connected to the detection system and the actuation section of the microfluidic chip. In some embodiments, the computing system is configured to control actuation of the plurality of particle deflectors based on signals received from the detection system.

[0005] A method for assembling a microfluidic chip is disclosed. In some embodiments, the method includes aligning a moving section with a plurality of alignment holes in a converging section using a plurality of alignment posts. The method includes bonding the moving section to the converging section. The method includes aligning the actuating section to the converging section by aligning a plurality of alignment holes in the actuating section with the alignment posts. The method includes bonding the actuating section to the converging section.

[0006] In some embodiments, a method for sorting particles using a microfluidic chip is disclosed. The method includes flowing a sample stream containing particles through a plurality of microchannels formed by a plurality of sections stacked or layered in a stacking direction to form the microfluidic chip. The plurality of microchannels are at least partially oriented to flow along the stacking direction. The method includes focusing the particles into each of the plurality of microchannels using a converging section of the plurality of sections. The method includes detecting particle characteristics of particles flowing through a plurality of interrogation regions within an actuation section of the plurality of sections. Each interrogation region is associated with a microchannel. The method includes sorting the particles using an actuation device associated with each microchannel in response to the detected particle characteristics. [Brief explanation of the drawings]

[0007] It should be noted that the various features and combinations of features described below and illustrated in the drawings can be arranged and / or organized differently to result in embodiments that are still within the spirit and scope of the present disclosure. Additionally, the components in the drawings are not necessarily drawn to scale or proportionally, emphasis instead being placed on clearly illustrating the principles involved. Moreover, for ease of illustration, various features may not be shown in certain drawings. Furthermore, various components or elements may be illustrated as transparent, or the use of cross-hatching or other standard drawing techniques may be used, for purposes of describing or showing items behind layers or other elements, or to generally simplify the display of certain drawings thereof. To assist those skilled in the art in making and using the disclosed systems, assemblies, and methods, reference is made to the accompanying drawings.

[0008] [Figure 1] 1 illustrates a massively parallel microfluidic chip having stacked or layered sections along a stacking direction according to various embodiments taught herein. [Figure 2]1 illustrates a cross-sectional view through a schematic diagram of a microfluidic chip according to the present disclosure engaged with optical illumination and detection elements. [Figure 3A] 1 illustrates a cross-sectional view through a schematic diagram of one embodiment of a microfluidic chip engaged with optical illumination and detection elements. [Figure 3B] 3B illustrates a top view of the microfluidic chip of FIG. 3A. [Figure 4A] 1 illustrates a cross-sectional view through a schematic diagram of one embodiment of a microfluidic chip including electrical sensor elements engaged with optical illumination and detection elements. [Figure 4B] 1 illustrates a cross-sectional view through a schematic diagram of one embodiment of a microfluidic chip engaged with optical illumination and detection elements, where the interrogation region is located in a horizontal portion of a microchannel where all or most of the fluid flows horizontally (i.e., transverse to the stacking direction). [Figure 5] 1 illustrates an embodiment of a microfluidic chip in which an actuator is located on or integrated into a cover layer according to some embodiments taught herein. [Figure 6] 6 illustrates a partial cross-sectional view of the chip of FIG. 5 showing an actuator that diverts particles into a branch channel. [Figure 7A] 1A-1C illustrate partial cross-sectional views of a single microfluidic channel and multiple microfluidic channels, respectively, of a microfluidic chip including a nozzle, according to some embodiments taught herein. [Figure 7B] 1A-1C illustrate partial cross-sectional views of a single microfluidic channel and multiple microfluidic channels, respectively, of a microfluidic chip including a nozzle, according to some embodiments taught herein. [Figure 8] 1 illustrates an isolated section of a microfluidic chip including alignment holes and alignment posts according to some embodiments taught herein. [Figure 9] 1 illustrates a microfluidic chip having a central exhaust channel according to some embodiments taught herein. [Figure 10A]1 illustrates a schematic diagram of a fluid manifold in a microfluidic chip taught herein, according to various embodiments. [Figure 10A] 1 illustrates a schematic diagram of a fluid manifold in a microfluidic chip taught herein, according to various embodiments. [Figure 11] 1 illustrates a particle processing system including a microfluidic chip according to the present disclosure having sections stacked or layered along a stacking direction according to various embodiments taught herein. [Figure 12] 12 illustrates a side view of the particle processing system of FIG. 11 including an electromagnetic source system, an optical separation system, and a detection system according to various embodiments taught herein. [Figure 13A] 1A-1D illustrate side views of various electromagnetic source systems according to embodiments taught herein. [Figure 13B] 1A-1D illustrate side views of various electromagnetic source systems according to embodiments taught herein. [Figure 13C] 1A-1D illustrate side views of various electromagnetic source systems according to embodiments taught herein. [Figure 13D] 1A-1D illustrate side views of various electromagnetic source systems according to embodiments taught herein. [Figure 13E] 1A-1D illustrate side views of various electromagnetic source systems according to embodiments taught herein. [Figure 13F] 1A-1D illustrate side views of various electromagnetic source systems according to embodiments taught herein. [Figure 14A] 1A-1D illustrate side views of various detection systems according to embodiments taught herein. [Figure 14B] 1A-1D illustrate side views of various detection systems according to embodiments taught herein. [Figure 14C] 1A-1D illustrate side views of various detection systems according to embodiments taught herein. [Figure 14D] 1A-1D illustrate side views of various detection systems according to embodiments taught herein. [Figure 14E]1 illustrates an embodiment of a particle processing system in which a single lens combines light from multiple interrogation regions into a detection system, according to some embodiments taught herein. [Figure 15] FIG. 1 is a block diagram of a computing device suitable for use with embodiments of the present disclosure. [Figure 16] 1 shows a flowchart of a method for fabricating a microfluidic chip according to some embodiments taught herein. [Figure 17] 1 shows a flowchart of a method for processing particles using a microfluidic chip according to some embodiments taught herein. [Figure 18A] 1 illustrates a cross-sectional view through a schematic diagram of a microfluidic chip according to the present disclosure. [Figure 18B] 1 illustrates a cross-sectional view through a schematic diagram of a microfluidic chip according to the present disclosure in conjunction with optical illumination and detection elements. [Figure 18C] 1A-1D illustrate cross-sectional views through a schematic diagram of a microfluidic chip with actuators at various locations according to the present disclosure. [Figure 18D] 1A-1D illustrate cross-sectional views through a schematic diagram of a microfluidic chip with actuators at various locations according to the present disclosure. [Figure 18E] 1A-1D illustrate cross-sectional views through a schematic diagram of a microfluidic chip with actuators at various locations according to the present disclosure. [Figure 19A] 1 illustrates an exploded view through a schematic diagram of a microfluidic chip according to the present disclosure. [Figure 19B] 1 illustrates a top view through a three-dimensional (3D) schematic diagram of a microfluidic chip according to the present disclosure. [Figure 19C] FIG. 19C illustrates a perspective view through the 3D schematic of the microfluidic chip of FIG. 19B. [Figure 19D] FIG. 19C illustrates a partial side view of the microfluidic chip of FIG. 19B. [Figure 20] 10 illustrates stack images from a top view of a stack section of a fabricated microfluidic chip during operation along the stack direction. [Figure 21A]FIG. 1 shows a perspective view of a simulation for hydrodynamic focusing in the focusing region disclosed herein. [Figure 21B] 21B illustrates a top view of the simulation of FIG. 21A. [Figure 21C] 21B illustrates a side view of the simulation of FIG. 21A. [Figure 22A] 1 illustrates a schematic diagram of a microfluidic chip with a 2x2 layout of particle processing units according to various embodiments. [Figure 22B] 22B illustrates a schematic diagram of the particle processing unit of FIG. 22A. [Figure 23] 1 illustrates an exploded view through a schematic diagram of a microfluidic chip with a 2x2 layout of particle processing units according to the present disclosure. [Figure 24A] FIG. 1 illustrates a schematic diagram of a microfluidic chip with a main fluid path layout for feeding a 2×2 layout of particle processing units according to the present disclosure. [Figure 24B] FIG. 1 illustrates a schematic diagram of a microfluidic chip with a main fluid path layout for feeding a 4×4 layout of particle processing units according to the present disclosure. [Figure 24C] FIG. 1 illustrates a schematic diagram of a microfluidic chip with a main fluid path layout for feeding an 8×8 layout of particle processing units according to the present disclosure. [Figure 24D] FIG. 1 illustrates a schematic diagram of a microfluidic chip with a main fluid path layout to serve a 16×16 layout of particle processing units according to the present disclosure. [Figure 25A] 1 illustrates a partial side view of a microfluidic chip according to the present disclosure. [Figure 25B] 25B illustrates an isometric view of the microfluidic chip of FIG. 25A. [Figure 25C] 25B illustrates a top view of the microfluidic chip of FIG. 25A. [Figure 25D] 25B illustrates a bottom view of the microfluidic chip of FIG. 25A. [Figure 26A] 1 illustrates the layout of the main fluid paths of a microfluidic chip with a 16x16 layout of particle processing units. [Figure 26B] 26B illustrates the layout of the measurement and actuation sections of the 16×16 layout of the particle processing unit of FIG. 26A. [Figure 27A] 1 illustrates an exploded view through a schematic diagram of a disk-shaped microfluidic chip with an m×n (m>1, n>1) layout of particle processing units according to the present disclosure. [Figure 27B] FIG. 27B illustrates an isometric view of the assembled disk-shaped microfluidic chip of FIG. 27A. [Figure 27C] 27B illustrates a front view of the disk-shaped microfluidic chip of FIG. 27A. [Figure 27D] 27B illustrates a rear view of the disk-shaped microfluidic chip of FIG. 27A. [Figure 27E] 27B illustrates a side view of the disk-shaped microfluidic chip of FIG. 27A. [Figure 28A] 1 illustrates various embodiments of a particle processing system for simultaneous illumination and detection according to the present disclosure. [Figure 28B] 1 illustrates various embodiments of a particle processing system for simultaneous illumination and detection according to the present disclosure. [Figure 29A] 1 illustrates an illumination scheme at the fluid plane of a particle processing system. [Figure 29B] 1 illustrates a light projection scheme imaged onto a detection surface of a particle processing system. DETAILED DESCRIPTION OF THE INVENTION

[0009] The systems and methods taught herein employ a "massively parallel" stack design of microfluidic structures by utilizing sections in a stacked or layered relationship (i.e., one section on top of the other) that combine to form multiple microfluidic channels oriented primarily along a stacking direction that is perpendicular to the plane defining the sections. Massively parallel microfluidic chips enable the use of microfluidic chips to perform highly parallel particle analysis, processing, or sorting operations (or any mixture thereof). Specifically, the use of sections that are stacked or layered along the stacking direction enables the use of non-planar microfluidic features that can be more densely packed on the chip, thereby enabling more simultaneous particle operations and faster overall throughput. Sections may be defined by function and operation, as discussed below. In some embodiments, a section may include multiple functions. In some embodiments, a section may include one or more layers. In some embodiments, one or more layers may provide different functions. For example, as described herein, an actuating section may include one or more layers, and layers may be classified to provide focusing, actuation, etc. As described below, in some embodiments, the convergence section and actuation section may be combined into a single section, providing the convergence and actuation functions and operations in a single section; in this manner, the actuation section may include multiple convergence and interrogation regions.

[0010] In some embodiments of the systems and methods taught herein, fluid paths are configured to be primarily aligned to flow in a stacking direction along the thickness of the microfluidic chip. In some embodiments, the flow direction of particles according to the present disclosure coincides with the optical axis used by an illumination or detection system at or near the interrogation point. In some embodiments, the flow direction of particles according to the present disclosure within the interrogation region is perpendicular to the optical axis used by an illumination or detection system at or near the interrogation point. In conventional systems, fluid flow is primarily across a single plane (i.e., generally parallel to the longitudinal or transverse axis of the microfluidic chip). Thus, despite significant efforts to reduce the cross-sectional area occupied by each collection of particle processing elements (e.g., main and branch microfluidic channels, sorting actuators, particle focusing features), conventional microfluidic chips that use flow-on-chip that is primarily end-to-end within the plane of the chip limit the number of parallel sorting, processing, and analysis operations that can be performed on a single chip. In the massively parallel microfluidic chips taught herein, the third (e.g., thickness) dimension is utilized to greatly increase the density of parallel particle processing elements on a single chip while still providing the characteristics necessary for successful particle sorting and processing (channel dimensions may ultimately be the limiting factor).

[0011] In conventional systems, microfluidic chip designs transport fluids in a two-dimensional (2D) fashion, with microchannels, reservoirs, mixing regions, sensing regions such as interrogation regions, focusing regions, and other regions laid out on a single, planar 2D layer. In 2D designs, fluids are delivered to microfluidic channels within a thin microfluidic chip. The microfluidic channels are positioned (e.g., etched) within the plane of the chip so that fluids and particles flow the length of the chip from one end to the other along the chip's length. Each system for manipulating particles requires space along the chip length and access to microchannels, e.g., manifold or branching regions, detection regions, and sorting regions, with the combining region located at a different location along the chip's length. Thus, the length of the chip increases as additional systems are added, and paralleling fluid flow by adding microchannels along the width (or transverse) direction increases the width of the chip to accommodate the extra microchannels. Historically, this design choice has been driven by many reasons, including cost, simplicity, basic operation, designer philosophy, ease of fabrication, the desire to use a single material, and the need to mimic or minimize manufacturing steps. Specifically, the placement of flow channels parallel to the plane defined by the chip is driven by the long-standing practice of etching elongated trenches into the substrate, which can often be implemented using one or very few fabrication steps and has been widely applied to glass and silicon substrates. However, traditional 2D approaches can have significant drawbacks, such as low feature density, complex fluid branching schemes, limited substrate functionality, low sample throughput, and limitations on various physical, chemical, or biological interfaces (e.g., thermal, optical, chemical, biological, biocomposite, mechanical, and electrical interfaces cannot be implemented within or along a single substrate).

[0012] The systems and methods taught herein improve upon traditional 2D devices (e.g., substantially planar devices in which one dimension of the device, such as thickness, is several orders of magnitude smaller than the other dimensions) by enabling the relocation of non-optical devices, fluidic pathways, actuators, hydrodynamic focusing elements, fluidic manifolds, and other features associated with processing particles within multiple microchannels from the highly competitive physical area of ​​the optical zone (i.e., the plane near the top surface of the microfluidic chip that is accessible to optical interrogation and detection devices). Instead, these features and elements can be provided in sections that are stacked or layered below the optical zone in the stacking direction. Because these features are removed from the plane where optical interrogation and detection occurs, the space within the optical interrogation zone previously occupied by these features and elements can instead be used for additional microchannels for particle processing. As a result, the density of particle processing features can be significantly increased using massively parallel microfluidic chips as taught herein.

[0013] Furthermore, the implementation of three-dimensional (3D) microfluidic structures as taught herein significantly increases the feature density of the microfluidic, optical, and actuation (one or a combination), significantly increasing the particle throughput of such devices, for example, when the majority of the fluid flow at any one time travels along or parallel to the excitation and / or detection optical axis (not necessarily measurement, minimizing the ratio). In some embodiments, the fluid flow is transverse to the optical axis for purposes of interrogation.

[0014] The systems and methods taught herein, in various embodiments, can include densities of particle processing units, particle interrogation regions, particle focusing regions, or particle sorting actuators greater than 1 per cm, 5 per cm, 10 per cm, 20 per cm, 50 per cm, 100 per cm, 250 per cm, or 500 per cm. The systems and methods taught herein have a reduced cross-sectional area per individual particle processing unit at the viewing or imaging surface of the electromagnetic source system or detection system. The reduced cross-section reduces the amount of “real area” dedicated to or occupied by the portion of the particle processing unit that interfaces with external components, such as the electromagnetic source system and detection system. In other words, by reducing the area of ​​the particle processing unit at the imaging surface of the electromagnetic source system, the detection system, or both, to approximately the size of the interrogation or sorting monitoring region, more particle processing units can be placed on a microfluidic chip substrate of a given chip size. Similarly, a desired throughput can be achieved with a smaller-sized microfluidic chip compared to conventional microfluidic chips attempting to achieve the same level of throughput. Another advantage of the three-dimensional stacking or layering approach is the ability to fit more particle processing units or microfluidic chips onto a single wafer (or other component with size constraints) during fabrication. This approach can also reduce the cost per device per occupied unit area. Finally, this approach can reduce the total fluid path traversed by particles, which may improve particle outcomes such as cell viability, as well as the total fluid volume required to operate the system, compared to traditional planar chip architectures.

[0015] As used herein, the term "massively parallel" microfluidic chip is defined as a chip in which the microfluidic channels carrying particles are organized such that their longitudinal portions are positioned and oriented at least partially parallel to the height or thickness of the chip at locations on the chip where particle operations (e.g., particle processing, focusing, analysis, or sorting operations) occur.

[0016] As used herein, a "planar" substrate is a material that has a first dimension (e.g., thickness) that is significantly smaller (e.g., at least two orders of magnitude smaller) than the other two dimensions (e.g., length and width).

[0017] As used herein, the term "stacking direction" refers to the direction perpendicular to the planar layers or sections that form the microfluidic chip.

[0018] As used herein, the term "hydrodynamic focusing" refers to narrowing, accelerating, and positioning a sample using a sheath fluid to create a laminar flow. For example, but not limited to, hydrodynamic focusing is often used to position particles at the center of a microfluidic channel so that they can be reliably probed by an optical system focused at or slightly off-center in the channel. In some embodiments, hydrodynamic focusing is achieved by "squeezing" the sample stream by introducing a sheath fluid into a microchannel through which the sample fluid is flowing. In some embodiments, hydrodynamic focusing is achieved by narrowing the dimensions of the microchannel after the sample stream and sheath fluid co-flow, for example, using a slope, taper, or step. Hydrodynamic focusing can be distinguished from sample injection (where a sample stream is introduced into a channel already flowing with sheath fluid) because injection does not accelerate particles in the sheath fluid.

[0019] As used herein, the term "particle" includes, but is not limited to, cells (e.g., platelets, white blood cells, tumor cells, germ cells, stem cells, sperm, etc.), organelles, and multicellular organisms. Particles may include liposomes, proteoliposomes, yeast, bacteria, viruses, pollen, algae, etc. Furthermore, particles may include genetic material, RNA, DNA, fragments, proteins, etc. Particles may also refer to non-biological particles. For example, particles may include metals, minerals, polymeric materials, glass, ceramics, composite materials, etc. Particles may be naturally occurring or man-made. Particles may also refer to synthetic beads (e.g., polystyrene), such as beads provided with fluorescent dye-conjugated antibodies. Particles may be platforms selected by sex for sex selection in mammals, or by therapeutic or clinical value for monitoring disease in humans or other animals, or in one or more drug development applications.

[0020] As used herein, the term "microfluidic system" refers to a system or device that includes at least one fluid channel having microscale dimensions. A microfluidic system may be configured to handle, process, detect, analyze, eject, and / or sort fluid samples and / or particles within the fluid samples.

[0021] As used herein, the term "channel" refers to a path formed in or through a medium or substrate that allows the movement of fluids, such as liquids and gases.

[0022] The term "microchannel" refers to a channel formed within a microfluidic system or device, preferably having a cross-sectional dimension ranging from about 1.0 μm to about 2000 μm, preferably from about 25 μm to about 500 μm, and most preferably from about 50 μm to about 300 μm. One of ordinary skill in the art would be able to determine the appropriate volume and length of a microchannel for a desired application. The above ranges are intended to include the values ​​recited above as upper or lower limits. Generally, microchannels can have any selected cross-sectional shape, e.g., U-shaped, D-shaped, rectangular, triangular, oval / elliptical, circular, square, trapezoidal, etc., cross-sectional geometric shape. The geometric shape can be constant or can vary along the length of the microchannel. Furthermore, microchannels can have any selected arrangement or configuration, including linear, nonlinear, merging, branching, looping, twisting, stepped, etc. configurations. A microfluidic system or device, e.g., a microfluidic chip or chip-substrate stack, can include any suitable number of microchannels for transporting fluids. The microfluidic chip may be provided as part of a disposable cartridge for removable engagement with a microfluidic device. Additionally, the microfluidic chip may be provided as part of a disposable cartridge, which is a fully enclosed and sealed or sealable fluid channel system. Additionally, the microfluidic chip or portions of the chip may be removable, replaceable, and / or irreversibly bonded / fused.

[0023] As used herein, a "particle processing unit" is a unit that contains features for performing particle processing functions (such as particle sorting) and can be replicated multiple times across a chip to increase total parallel processing throughput. Such features can include fluid focusing elements, interrogation regions, sorting or sorting actuators, and microfluidic channels for fluid or particle input or output. In some examples below, a "particle processing unit" includes a microfluidic input channel (which can accept fluid from a pool or from an inlet that feeds channels with multiple units), at least two microfluidic output channels (which can deliver sorted or processed particles and waste particles to pools or outlets that feed into or from multiple units), an interrogation region, and associated actuators. As described in more detail below, the microfluidic chips taught herein contain multiple particle processing units operating in parallel. By including N particle processing units on a single microfluidic chip, the total throughput of particles that can be processed by the chip is increased by at least N-fold.

[0024] As used herein, "interrogation" refers to probing a particle to determine its properties, and in some cases, its class, type, or identity. In the case of optical systems, interrogation involves detecting light emitted or otherwise scattered from, or the absence of light due to interaction with, an illuminated particle to determine the value of another particle property, such as size, shape, morphology, orientation, fluorescence intensity or wavelength, optical scattering intensity or wavelength, geometry, volume, surface area, ellipticity, refractive index, particle size, porosity, conductivity, identity, type, phenotype, protein or molecular expression, genetic content, live / dead status, velocity, etc.

[0025] As used herein, "processing" a particle refers to acting in response to determining one or more particle properties of the particle, including, but not limited to, actuating an actuator to sort the particle, isolating a particle population, purifying a particle population, concentrating a particle population, optically tweezing a particle (e.g., a cell), or zapping (i.e., damaging, impairing, rendering non-functional, or killing) the particle.

[0026] Generally, the present disclosure relates to measuring and sorting particles, droplets, and / or fluids in microfluidic devices. More specifically, the present disclosure relates to manipulating particles, droplets, and / or fluids within microfluidic devices in a massively parallel manner through the integration of new device designs and methods. Such manipulation may include zapping or altering particles, droplets, and / or fluids. Such manipulation may be the result of particle interrogation. Such manipulation may include elongation, binding, or other changes in the physical, chemical, or biological properties of particles.

[0027] Particle separation is of great interest for many biological and biomedical applications. As the demand for sorted or analyzed particle populations for biomedical applications increases, a need arises to increase processing throughput to provide the ability to process larger numbers of samples more quickly. Various approaches have been attempted, including arranging multiple channels and sorting devices on a single planar microfluidic chip. Traditionally, particle motion is performed as particles flow in-plane within a flow channel; for example, the direction of in-plane fluid flow in a conventional microfluidic channel is in a plane approximately perpendicular to the optical detection axis through the chip.

[0028] The microfluidic chips and associated systems taught herein are capable of analyzing, processing, and / or selecting particles based on their intrinsic properties determined by the particle's interaction with electromagnetic radiation or light (e.g., scattering (forward, backward, or side), reflectance, and / or autofluorescence), independent of protocols and required reagents. According to some embodiments, the microfluidic system uses a closed, sterile, disposable cartridge containing the microfluidic chip so that all surfaces in contact with the sample fluid are isolated from the user and / or non-disposable equipment. The microfluidic system analyzes and / or processes particles at high speeds. A microfluidic sorting system using the microfluidic chip 100 as taught herein delivers sorted particles with high yield, high purity, high enrichment, and high efficacy, or other predetermined / desired population attributes.

[0029] FIG. 1 illustrates a schematic exploded view of a massively parallel microfluidic chip 100 according to various embodiments taught herein. The massively parallel microfluidic chip 100 includes multiple sections that are stacked to form the chip 100. The microfluidic chip 100 may include a translation section 110, a particle focusing section 120, and an actuation section 130 that includes a cover layer 131. Each of the translation section 110, the particle focusing section 120, and the actuation section 130 may be formed from a single layer of material or multiple layers of material. Those skilled in the art will recognize that some or all of the sections may be formed by additive manufacturing, lithography, bonding, or molding, or some or all of the sections may be formed individually and later assembled into the microfluidic chip 100. The sections of the microfluidic chip are stacked or layered in a stacking direction 107 (i.e., the thickness of the microfluidic chip). When sections of chip 100 are stacked or layered, a plurality of fluid microchannels are formed through one or more of the sections that are primarily oriented to allow flow along stacking direction 107. The plurality of microfluidic channels and associated particle processing elements form a particle processing unit that is arranged in pattern 144.

[0030] A fluid containing particles is introduced into one of the microchannels in the transfer section 110. Some embodiments may not include a transfer section. The fluid containing particles flows along the fluid input path 104 through the stacked or layered sections, either upward or downward (i.e., from the transfer section to or through the particle focusing section), depending on the stacking direction within the microchannel. The particles are focused in the particle focusing section 120 and enter the actuation section 130. The actuation section 130 can separate desired particles from undesired particles based on measured particle characteristics. In some embodiments, the actuation section 130 includes an interrogation section or region. In some embodiments, the interrogation section is a differentiation section separate from the actuation section 130. The desired particles flow back along the first outlet path 106 (sometimes referred to as the "retention" path) through the stacked or layered sections within the microchannel, either downward or upward (i.e., back toward the transfer section 110), depending on the stacking direction. Undesired particles flow along the second exhaust path 108 (sometimes referred to as the "waste" path) through the stacked or layered sections in the microchannel, either downward or upward depending on the stacking direction (i.e., back towards the transfer section 110). The particles in the first exhaust path 106 and the second exhaust path 108 can be separately extracted from the chip 100 at the transfer section 110.

[0031] The cross-sectional view of FIG. 2 facilitates explanation of features of a massively parallel microfluidic chip 100 according to some embodiments. FIG. 2 illustrates a cross-sectional view through a schematic diagram of the microfluidic chip 100 during engagement with optical illumination / detection elements and fluids and particles flowing therethrough. For ease of explanation, FIG. 2 illustrates a single particle processing unit 101 as taught herein. However, the microfluidic chip 100 may include 10-1000 particle processing units 101, as described with respect to FIG. 2. In the particle processing unit 101, one or more sample fluid input paths 104 are joined by one or more sheath fluid input paths 105a, 105b to surround the sample fluid with sheath fluid, and in some embodiments, to focus the sample fluid. For example, a sample fluid is injected into the main microchannel 114 through the transfer section 110, while sheath fluids on the sheath fluid input paths 105a, 105b are injected into the respective sheath channels 115a, 115b through the transfer section 110. The sheath channels 115a, 115b intersect with the main microchannel 114 in the focusing section 120, where the sheath fluid contacts the sample fluid. In some embodiments that do not use a sheath fluid, there is no sheath fluid input path 105, and only the sample fluid flows through the microchannel 114 and up the sample fluid input path 104. The sheath fluid and sample fluid containing the particles 50, 60 meet within the microchannel 114 at a particle focusing region 123 of the particle focusing section 120. The particle focusing region 123 is where the sheath fluid acts to focus the particles 50, 60, as shown by the streamlines within the particle focusing region 123, which indicate compression of the sample fluid by the sheath fluid in the region of the fluid intersection. The particles 50, 60 then enter the interrogation region 113 within the actuation section 130. Each particle 50, 60 can be interrogated by light directed by a lens 222 through the cover section 131 onto the particle. Light is emitted, scattered, absorbed, or lost by the particles according to the particle's properties. Detection of the emitted or scattered light, or changes in the level of existing light, through lens 222 allows for identification of particle properties.For example, specific characteristics of particles, such as size, morphology, fluorescence, light scattering, and other properties, can be identified by detecting emitted or scattered light or changes in existing light levels (e.g., extinction). Based on the identification of particle properties, the actuator 112 of the actuation section 130 can be actuated to divert desired particles 60 (i.e., particles having desired properties) or undesired particles 50 (i.e., particles not having desired properties) from the normal flow path 191 and onto the bypass flow path 192. In FIG. 2 , particles 60 subject to action from the actuator 112 flow through the microchannel 118 on the second discharge path 108 onto the bypass flow path 192, while particles not acted upon by the actuator 112 flow through the microchannel 116 on the first discharge path 106. In some embodiments, lens 224 may monitor the results of sorting (i.e., the results of operation) as particles pass through first sorting monitoring region 161 in first discharge flow path 106, or lens 226 may monitor the results of sorting as particles pass through second sorting monitoring region 162 in second discharge flow path 108. Light emitted from particles through lenses 224 and 226 passes through cover section 131 and may be detected to verify whether sorting occurred as expected.

[0032] While lenses 222, 224, and 226 are illustrated to aid in describing features of microfluidic chip 100, it is understood that the lenses are part of a particle processing system separate from the microfluidic chip and are not a feature of the microfluidic chip. In other embodiments, the cover section 131 of the chip may include integrated optical features such as a lenticular array, a metalens array, or other optical properties configured to simplify alignment and / or optics and chip design. The integrated optical features may be, by way of non-limiting example, molded elements that comprise the cover section 131 made from glass or plastic. Embodiments of particle processing systems for use with the microfluidic chip 100 taught herein are described in more detail below, beginning with the description of FIG. 6 . In some embodiments, individual lenses 222, 224, and 226 may be single units within an array of lenses or microlenses.

[0033] The transfer section 110 can transport fluids into and out of multiple microfluidic channels within the microfluidic chip 100. The transfer section 110 can include inlet ports to allow the introduction of fluids into the microfluidic chip. For example, the transfer section 110 can include an inlet port for introducing a sample fluid containing particles for processing or sorting. The transfer section 110 can also include one or more inlet ports for introducing a sheath fluid used to modify the sample fluid flow, for example, to focus particles. The transfer section 110 can also include one or more outlet ports for extracting fluids and particles from the microfluidic chip 100. In one embodiment, a first outlet port allows the extraction of “retain” pathway particles and fluid, while a second outlet port allows the extraction of “waste” pathway particles. The transfer section 110 can include one or more manifolds for distributing fluids from the ports to one or more microchannels within the microfluidic chip. For example, a sample inlet manifold can distribute sample fluid to each of multiple microfluidic channels. Similarly, a sheath inlet manifold can distribute sheath fluid to each of multiple microfluidic channels. In some embodiments, the manifold can perform aggregation of targeted or non-targeted particles from multiple microfluidic channels by combining the outputs from multiple output paths and channeling them into a single stream through an outlet port. For example, the manifold in the transfer section 110 can combine fluids from all of the first output paths 106 or all of the second output paths 108 for extraction from the chip 100.

[0034] In some embodiments, the transfer section 110 can include one or more filters. Filters can ensure that large particles, debris, particle agglomerates, and other large items do not enter the microfluidic chip 100. Large items can lead to undesirable effects such as blockage of the microfluidic channels or inaccuracies in measurement and sorting, among other problems.

[0035] The particle focusing section 120 can entrain (e.g., focus, align, separate, stabilize, orient, etc.) particles upstream of the interrogation region 113 to optimize or enhance the particle interrogation process. Specifically, the particle focusing section 120 can enable particle focusing within the microfluidic channel 114. Particle focusing can result in fluidically well-behaved particles, particles moving in single file, particles confined to a defined region of the microfluidic channel (e.g., a central fluid core), particles that are randomly spaced or evenly spread so that they can be reliably measured and accurately sorted, or any combination of the above. Focusing can be achieved using a variety of techniques. In one example, hydrodynamic focusing can be achieved by a change in the geometry of the microfluidic channel 114, such as a slope or taper, which contracts the fluid volume and accelerates and narrows the flow within the channel. Alternatively or additionally, a sheath fluid can be injected into the microfluidic channel 114 via sheath channels 115a, 115b that intersect the microfluidic channel 114. The sheath channels 115a, 115b can be positioned on either side of the microchannel 114 to create countervailing forces at the same intersection. In other embodiments, the intersections of different sheath fluid channels 115a, 115b with the microfluidic channel 114 can occur at different longitudinal positions along the length of the microfluidic channel. As shown in FIG. 2, the sheath channels can flow parallel to the microfluidic channel 114. However, the sheath channels can also flow perpendicularly or at any angle to the intersection with the microfluidic channel 114, and of course, additional sheath fluid channels can flow in adjacent planes not shown in the cross-sectional view of FIG. 2 (i.e., the sheath channels can be "behind" or "in front of" the microfluidic channel 114 shown in the cross-sectional plane of FIG. 2).

[0036] In some embodiments, a sheath fluid can be used as a transport fluid to reduce clogging. The sheath fluid can be used to mechanically or chemically affect particles within the microfluidic channel 114, including through biochemical interactions between sheath fluid components and the particles. In various embodiments, the sheath fluid can be used to wash, dilute, unify, align, orient, deflect, accelerate, decelerate, or focus particles in the sample fluid.

[0037] The particle focusing section 120 can also use alternative techniques to hydrodynamic techniques for particle focusing, including acoustic waves (including surface acoustic waves), electrophoresis, magnetic, optical, or other techniques. Surface acoustic waves can be generated using interdigital transducers (IDTs) located on or within the layers of the particle focusing section 120. In embodiments that do not use hydrodynamic focusing, the sheath fluid can be reduced in volume or eliminated entirely as unnecessary. In some embodiments, alternative focusing techniques can be used along with or in conjunction with hydrodynamic focusing. In some embodiments, passive particle focusing methods that rely on structural changes in the microfluidic channel can be used. For example, Dean inertial flow techniques can be used in the particle focusing section 120 to focus particles that contain spirals or serpentines within the flow channel. In some embodiments, the particle focusing section 120 can ensure reliable cell singlet (i.e., positioning of cells in a "single file" along the length of the microfluidic channel) to enable accurate or reproducible measurements of particle properties, for example, using optical measurements. The particle focusing section 120 can separate the cells using hydrodynamic methods, mechanical methods such as acoustic forces, or optical methods such as optical tweezers.

[0038] In some embodiments, particles may not be completely focused within particle focusing section 120. Some focusing of particles may occur, for example, at or within boundary or junction regions between sections. Specifically, hydrodynamic forces and effects may not be maintained individually within a single section, but may persist downstream to some extent such that a focusing effect can still be said to occur after particles exit particle focusing section 120.

[0039] The actuation section 130 includes actuators 112 associated with one or more of the microfluidic channels 114 downstream of the interrogation region 113 associated with one or more of the microfluidic channels 114. For ease of explanation, a single actuator 112 associated with a microfluidic channel 114 is illustrated and described. However, the microfluidic chip 100 may include 10-2000 actuators 112, as described with respect to FIG. 2 and other figures disclosed herein. Those skilled in the art will appreciate that each particle processing unit 101 includes or is associated with at least one actuator 112. The actuation section 130 provides a suitable means for diverting targeted particles, non-targeted particles, or both targeted and non-targeted particles into one or more separate fluid paths 116, 118 downstream of the actuators 112. The properties of targeted or non-targeted particles can be directed, diverted, switched, etc., into selected flow paths 106, 108. In some embodiments, the process of directing particles to selected channels 106, 108 may be performed on a particle-by-particle basis. The actuator 112 can use any of a variety of techniques to achieve separation of targeted particles from non-targeted particles. Techniques include, but are not limited to, mechanical, optical, chemical, thermal, bubble-based, dielectrophoretic, piezoelectric, acoustic, acoustic wave-based, magnetic, valve-based, or membrane-based forces. One or more layers of the actuation section 130 may be formed at least in part from lithium niobate (LiNbO), lithium tantalate, lead zirconate titanate (LZT), zinc oxide (ZnO), aluminum nitride, quartz, polyvinylidene fluoride (PVdF), or another piezoelectric material. In some embodiments, the piezoelectric layer may be formed as a thin film on top of a different layer.

[0040] In some embodiments, the actuation section 130 may be formed from individual layers. The actuation section 130 may include an actuator layer 132 that includes one or more actuators 112 formed therein or thereon. For example, an acoustic wave actuator may be formed on the actuator layer 132 using conventional cleanroom etching and deposition techniques. The actuator layer 132 may, in some embodiments, be sandwiched between adjacent layers 133, 134. In some embodiments, the adjacent layers 133, 134 may, for example, help guide acoustic waves to the microchannels 114. The actuator section 130 may include a treated particulate layer 136 immediately below the cover layer 131. The treated particulate layer 136 may have a lateral (i.e., flowing perpendicular to the stacking direction 107) portion of the exhaust channels 116, 118 etched therein. In some embodiments, the actuator section 130 does not include a fluid distribution layer. In such embodiments, the treated particulate layer 136 is considered a separate section from the actuation section.

[0041] In some embodiments, the actuator 112 in the actuation section 130 can include a switching surface acoustic wave actuator. The acoustic wave actuator (such as an interdigital transducer or IDT) can be coupled to an acoustic wave generator to generate acoustic energy that is coupled into the microfluidic channel 114 to divert particles into a selected outlet stream or channel. The IDT actuator can be configured to generate a moving or flowing surface acoustic wave (TSAW) or pressure pulse in the fluid in the microfluidic channel 114. This pressure pulse can be used to drive a slug of fluid into a selected region or direction of the microfluidic channel 114, or into a selected exhaust channel 116, 118. Alternatively, a pair of IDTs can be provided, one on each side of the microfluidic channel 114 at the switching site. Examples of surface acoustic wave generators, IDTs, actuators, and arrangements of these elements with respect to microchannels suitable for use in the present invention are described in more detail in U.S. Pat. No. 10,646,870, entitled "MICROFLUIDIC DEVICE AND SYSTEM USING ACOUSTIC MANIPULATION," issued May 12, 2020, the entire contents of which are incorporated herein by reference.

[0042] In some embodiments, the actuation section 130 can include one or more pressure pulse channels 138. The pressure pulse channel 138, such as a fluid buffer region, can be located on the opposite side of the actuator 112 in the microfluidic channel 114 to buffer or reduce the effect of the pressure pulse from the actuator 112. This absorption of the pulse can then reduce the perturbation experienced by the fluid flowing in the microfluidic channel 114, allowing for faster re-establishment of laminar flow and therefore faster switching times. In some embodiments, the actuation section 130 can include acoustic damping elements positioned between adjacent particle processing units 101. The acoustic damping elements can absorb or reduce acoustic energy from the actuator 112 in a first particle processing unit 101 so that this energy does not disrupt or affect fluid flow in the different particle processing units 101. In this way, the acoustic damping elements can acoustically isolate actuators in different particle processing units 101 on the same microfluidic chip. In some embodiments, the damping elements can include air gaps.

[0043] In some embodiments, the cover layer 131 of the actuation section 130 can include an optical interface layer. The cover layer 131 can allow for observation, detection, or both observation and detection of particles flowing through the device. In some embodiments, the cover layer 131 can include a window or window-like layer to provide a transparent optical interface to the fluid below the cover layer 131. The cover layer 131 can act as a liquid-proof barrier, i.e., the cover layer 131 can prevent the transfer of water, oil, or other liquids from the inside to the outside of the chip 100, or vice versa. In some embodiments, the cover layer 131 can form part of one or more fluid channel paths through which fluids flow. Other features, such as electrodes, can also be applied to, incorporated into, or included on the cover layer 131. The cover layer 131 can include the actuation device 112 in some embodiments, as described in more detail below. The cover layer 131 may be formed of one or more materials, such as glass or plastic, configured to provide high transmission of wavelengths of electromagnetic radiation of interest for a particular application (e.g., excitation light, scattered light, or fluorescent light wavelengths). In exemplary embodiments, the cover layer 131 may enable transmission of light in a range representing wavelengths ranging from ultraviolet (UV) to near-infrared (IR) wavelengths, or a subportion of the UV to near-IR range depending on the design intent. In some embodiments, the cover section 131 may include integrated optical features, such as a lenticular array, a metalens array, or other optical properties configured to simplify alignment and / or optical and chip design. The integrated optical features may be, by way of non-limiting example, molded elements that comprise the cover section 131 made from glass or plastic.

[0044] In some embodiments, the total thickness of the massively parallel microfluidic chip 100 can be in the range of 0.5 mm to 10 mm. In some embodiments, the thickness of an individual section (i.e., the translation section 110, the particle focusing section 120, or the actuation section 130) can be in the range of 10 micrometers to 1000 micrometers.

[0045] In the embodiment illustrated in FIGS. 2-4B , desired particles 60 are diverted by the action of actuator 112. However, it should be understood that the system can act to divert undesired particles 50 while leaving desired particles 60 largely undisturbed. Such an operation is referred to as “anti-sorting,” and additional descriptions and embodiments of particle processing systems and chips utilizing anti-sorting in a manner consistent with the present teachings can be found in U.S. patent application Ser. No. 17 / 723,236, filed April 18, 2022, the entire contents of which are incorporated herein by reference. In some embodiments, undesired particles 50 can be modified, for example, physically, thermally, or chemically, to damage or destroy the undesired particles 50. Modification can be used instead of, or in addition to, physical isolation of undesired particles 50 from desired particles 60.

[0046] Within the chip stack, and in some embodiments, particles may flow toward (or away from) the imaging system (e.g., parallel or anti-parallel along the optical axis 260 of the imaging system). In some embodiments, the interrogation region 113 and the actuator 112 are both located within the actuation section 130. In some embodiments, the actuator 112 may operate within the interrogation region 113. Of course, the interrogation region 113 and the actuator 112 may be located at different points within the chip stack or in different sections or layers. Furthermore, particle flow and measurement using the microfluidic chip 100 taught herein is not limited to a direction toward or away from the imaging system, but may occur along other paths, such as a horizontal flow path.

[0047] The pattern 144 of the particle processing unit 101 in the microfluidic chip 100 can be selected to optimize functionality within the microfluidic chip 100. For example, the pattern 144 can be configured to reduce or minimize the total flow path of all fluids (or a subset of fluids, such as only the sample fluid or only the sheath fluid) through some or all of the microchannels in the chip 100. Other examples include optimization for mechanical design or constraints, isolation or placement of electrical elements (such as a surface acoustic wave generator), thermal isolation, avoidance of crosstalk, whether mechanical, optical, electrical, or thermal, and avoidance of interference. In some embodiments, the pattern 144 of the particle processing unit 101 can be selected to match the placement of optical elements, such as a particular excitation source spacing (e.g., an array of laser beams) or detector spacing. For example, the excitation source can be a vertical cavity surface emitting laser (VCSEL) that outputs a square grid array of laser beams. In various embodiments, the pattern 144 can be triangular, square, rectangular, hexagonal (e.g., corresponding to a maximum packing density metric), random, two-dimensional, axisymmetric grid, radial, concentric, other interspersed polygonal, or crystalline. In some embodiments, the arrangement of the pattern 144 or the geometric design of the microfluidic chip 100, including the elements of each particle processing unit 101, can be selected to reduce autofluorescence from particles or from other materials in the environment, including the materials and structure of the microfluidic chip 100 itself. In some embodiments, spatial filtering methods can be used in the detection system 220, as described below.

[0048] In some embodiments, the density of interrogation regions 113, sorting monitoring regions 161 / 162, particle focusing regions 123, or particle sorting regions (i.e., actuators 112) in pattern 144 is in the range of 1 per cm to 500 per cm. In some embodiments, the systems and methods taught herein involve a small area of ​​the chip occupied by each individual particle processing unit, as viewed or imaged by electromagnetic source system 210 or detection system 220. The reduced cross-section reduces the amount of "real area" dedicated to or occupied by the portions of the particle processing unit that interface with external components, such as electromagnetic source system 210 and detection system 220. In other words, reducing the area of ​​particle processing unit 101 in the imaging plane of electromagnetic source system 210, detection system 220, or both, to approximately the size of interrogation region 113 or sorting monitoring regions 161 / 162 allows more particle processing units to be placed on a microfluidic chip substrate of a given size. In some embodiments, the number of interrogation regions 113, sorting monitoring regions 161 / 162, particle focusing regions 123, or particle sorting regions on a single microfluidic chip can be in the range of 100-1000, 100-500, 250-750, 500-750, or 500-1000.

[0049] In some embodiments, the channel geometry of microfluidic channel 114 can be varied over the path of the channel to produce a desired effect on particles flowing therein or to reduce the effects of undesired effects. For example, one or more of width, height, cross-section, or other measurable parameters can be varied over the path of channel 114 to affect particle velocity, aligned / oriented particle positioning, particle concentration or dilution, temperature control, pressure, change in flow direction, enable exposure to chemicals, enable exposure to light, enable electrical conductance or impedance measurements, or provide a screening monitoring layer.

[0050] The location of the interrogation region 113 within the actuation section 130 or another section can be selected to ensure reliable interrogation of microfluidic channel contents, such as particles. The location can, in some embodiments, be measured as a distance from the cover layer 131. The location of the interrogation region 113 can correspond to the focal depth of an illumination or detection system interfaced with the microfluidic chip 100. In some embodiments, the degree of isolation of individual microchannels and objects (e.g., particles) within the microchannels (e.g., spacing between microchannels) can be selected to ensure reliable interrogation of microfluidic channel contents, such as particles. The appropriate location of the interrogation region 113 within a single microchannel can be selected to ensure that a target (e.g., single) particle of interest is suitably isolated and can be measured independently of other particles. In one embodiment, the location of the interrogation region 113 allows for measurement of a single particle. In some embodiments, measuring multiple particles may be desirable. In some embodiments, the location of the interrogation region 113 allows for the measurement of a single "event," where an event is characterized by the receipt of an optical signal from one particle or multiple particles within a specified period or coincidence interval.

[0051] In some embodiments, sections of the microfluidic chip 100 can be fabricated separately and combined or assembled to form the microfluidic chip 100. To create sections with very high feature density, numerous manufacturing or fabrication methods can be used to create each section, such as lithography, additive manufacturing (e.g., three-dimensional printing), sputtering, deposition, molding, embossing, imprinting, subtractive manufacturing (e.g., machining, milling, chemical etching, ion beam etching, electrical discharge machining), or other methods known to those skilled in the art for fabricating structures with materials, features, functionality, and dimensions suitable for a desired purpose. In some embodiments, sections can be fabricated using a combination of different materials. Optically or biologically functional layers can also be provided through various fabrication processes. A substrate-plus-additive approach, such as conductive electrodes on a non-conductive material, can be used. The selection of materials and coatings can be made according to a particular application (e.g., cell sorting), according to Good Manufacturing Practices (GMP), according to a desired method of sterilization or cleaning-in-place program (e.g., gamma irradiation, gas, steam, or other cleaning and sterilization considerations), or any combination thereof. Individual layers or sections can be fabricated from one or more materials, including silicone, glass (e.g., UV-fused silica, quartz, or borofloat), polymer (e.g., polydimethylsiloxane [PDMS], polymethylmethacrylate [PMMA], thermoplastic elastomers [TPE], including styrenic TPE, or cyclic olefin copolymer [COC]), metal, ceramic, alloy, or crystalline material. Materials can be selected for specific properties, such as electrical conductance or impedance or acoustic transparency. The material or surface of one or more sections can be prepared or treated to be suitable for cell measurement or sorting so that cells are not adversely affected as they move through the system. In some embodiments, the material or surface of a section can be treated to strengthen cells in some way as they move through the assembly.In some embodiments, layers or sections closer to the optical interrogation and detection system (e.g., closer to the top surface of the chip) are made of a material that is transparent, while layers or sections farther from the optical interrogation and detection system (e.g., closer to the bottom surface of the chip) are made of an opaque material (which may be cheaper or easier to fabricate) or a reflective material. In this way, the chip 100 maintains a high level of optical access where it is important (i.e., near the interrogation region 113), while benefiting from the ease and low cost of manufacturing for sections that do not necessarily require high optical access, such as the translation section 110. In some embodiments, high feature density can be achieved by having a small area of ​​the microfluidic chip 100 occupied by each particle processing unit 101, as measured on the top surface of the microfluidic chip 100 or as measured on the top surface of the cover layer 131.

[0052] In some embodiments, the microfluidic chip 100 includes sections that can be configured to be disassembled (i.e., reversibly assembled) again into component parts (e.g., separate sections). In some embodiments, one or more bonding, adhesive, fusing, or contacting processes can be used to permanently bond the sections to form a microfluidic chip 100 that cannot be disassembled. The microfluidic chip 100 can be wholly or partially cleanable, sterilizable, or reusable. For example, the exemplary microfluidic chip 100 can be disassembled, and certain sections (e.g., the particle focusing section 120 or the translation section 110) can be discarded and replaced with new sections, while other sections (e.g., the actuator section 130, together with or separate from the cover layer 131) are configured to be sterilized and reused. Certain sections may be more likely to be reused due to their exotic or expensive materials (e.g., glass or plastic) or because additional components are incorporated into the section (e.g., surface acoustic wave electrodes deposited on a layer of the actuator section 130).

[0053] In some embodiments, two or more sections or all of the sections may be formed in a single process, for example, using a three-dimensional printing process.

[0054] Microfluidic chip 100 can include additional sections or layers beyond those illustrated in FIGS. 1-4B. For example, microfluidic chip 100 can include layers or sections that passively allow materials (e.g., fluids or particles) to flow through the layers or sections unaltered or unaffected. These layers or sections can perform "via-like" functions (similar to vias in a multilayer printed circuit board, including hollow holes or openings extending therethrough) to facilitate access to or connection of plumbing / fluid connections, electrical contacts, optical conduits, mechanical actuation elements, or other aspects of the chip. In some embodiments, certain layers can be shared between stacked or layered sections of microfluidic chip 100 to provide shared commonalities, such as shared functionality, shared materials, shared electrical connections, shared optical conduits or connections, shared thermal properties, etc., to facilitate particle flow, focusing, detection, or sorting operations.

[0055] Any of the transfer section 110, particle focusing section 120, or actuation section 130 can include one or more layers of material. The layers can include suitable materials, coatings, or chemical treatments to achieve the desired functionality for flowing particles and fluids, enabling measurements such as optical or electrical measurements, manipulating fluids or particles, further processing fluids through manifolds, and collecting, transporting, or controlling fluids so that desired processes can be performed. The vertical stacking of layers and sections can be designed or fabricated so that optical, mechanical, electrical, or other signals can be transmitted through the layers in a controlled manner as needed. The materials used in a given layer can be optically transparent or opaque, spectrally selective, or polarization-sensitive, as needed. Materials can have specific refractive indices or light absorption, reflection, or refraction properties. Certain layers (such as the cover layer 131) can include means for generating or detecting light. The layers of the massively parallel microfluidic chip 100 can be electrically conductive, electrically insulating, or semiconducting in different embodiments. The layers of the massively parallel microfluidic chip 100 can have material properties or embedded or attached mechanical elements that allow for the transmission, attenuation, or blocking of energy, such as acoustic energy.

[0056] 2 illustrates a "left-right" particle sorting scheme in which the exhaust channels 106, 108 are shown in this cross-sectional view as being parallel in the plane of the chip 100 (i.e., as viewed from above) and separated by 180 degrees. However, it will be appreciated that the orientation of the exhaust paths 106, 108 is not limited to the 180-degree layout shown in FIG. 2, but rather the exhaust paths may branch off from the microfluidic channel 114 in any direction.

[0057] 3A illustrates one embodiment of the microfluidic chip 100 that may be described as a "right-right" particle sorting scheme. In this embodiment, a sample fluid containing particles 50, 60 flows upward through the transfer section 110 on the first flow path 104 into the main microchannel 114, while sheath fluid flows through the transfer section 110 on the sheath fluid flow input paths 105a, 105b into the respective sheath channels 115a, 115b. The sample fluid containing particles contacts the sheath fluid and is focused by the sheath fluid in the particle focusing region 123 of the particle focusing section 120. The particles flow into the interrogation region 113 where they can be detected using a detection system, such as an optical detection system using lens 222. Based on signals detected from the particles in the interrogation region 113 (e.g., optical fluorescence, scattering, emission, or quenching), a control system, such as a computing device, can identify whether the particles are desired particles 60 or undesired particles 50. If a desired particle 60 is detected, the actuator 112 is actuated to divert the particle to the first exhaust path 106. If an undesired particle 50 is detected, the actuator 112 may take no action, allowing the undesired particle 50 to follow its natural flow path to the second exhaust channel 108. In this embodiment, both exhaust microchannels 116, 118 are located in the same direction relative to the microfluidic channel 114, but are "stacked" one on top of the other in the stacking direction 107. In other words, one exhaust channel can pass above, below, or cross the other exhaust (or input) channel along the stacking direction (i.e., a line drawn through the chip in the stacking direction can pass through more than one microchannel).

[0058] In the chip embodiment shown in FIG. 3A, the exhaust channels pass directly beneath each other with a zero-degree relative angle between them when viewed from the top of the chip (i.e., zero degrees of rotation around the stacking direction 107). However, it should be appreciated that the exhaust channels 116, 118 can branch off from the microfluidic channel 114 at any relative angle between them between zero and 360 degrees. An example of a similar "right-to-right" microfluidic chip 100 including angled branching channels is illustrated by the top view of FIG. 3B. In this example, the four particle processing units 101 are shown when viewed downward through the cover layer 131. In other words, the main microchannel 114 flows "off the page" at the viewer along the stacking direction 107. The second exhaust microchannel 118 branches off from the main microchannel 114 at an angle 333 relative to the direction in which the first exhaust microchannel 116 branches off from the main microchannel 114. Furthermore, the orientation of particle processing units 101 on the same chip can be rotated relative to the orientation of other particle processing units 101. Such relative rotation can improve packing density in some embodiments by positioning common microfluidic channels closer together, e.g., by positioning the sample input paths 104 or output channels 116, 118 of several particle processing units 100 closer to each other, thereby simplifying the manifold paths.

[0059] In some embodiments, the arrangement of channels in the microfluidic chip allows for sorting monitoring, where particles 50, 60 can be measured downstream of the actuator 112. Sort monitoring is a process that provides confirmation of the success or failure of a particular sorting or actuation operation. Once a sorting decision is made and an actuation occurs, a sorting monitoring device (e.g., as part of a detection system) can measure the actual path of the particle relative to an expected or desired path. In FIG. 2, lens 224 provides sorting monitoring for particles in the first outlet channel 116, while lens 226 provides sorting monitoring for particles in the second outlet channel 118.

[0060] FIG. 4A illustrates one embodiment of a microfluidic chip 100, providing different mechanisms for sorting monitoring. In FIG. 4A, the microfluidic chip 100 includes an input electrical sensor 135 associated with the microfluidic channel 114 and output electrical sensors 137, 139 associated with the respective output channels 116, 118. The input electrical sensor 135 can detect the presence of particles 50, 60 in the microfluidic channel 114. The output electrical sensors 137, 139 can detect the presence of particles in their respective output channels. The electrical sensors 135, 137, 139 can operate to detect the presence of particles using the Coulter principle, measured conductivity (or interruptions / disruptions in conductivity), electrical resistivity, or other methods. The electrical sensors 135, 137, 139 can be configured to provide massively parallel particle measurements using conductive traces distributed throughout the chip stack. The conductive traces can be fed from the microfluidic chip 100 at one or more edges of the chip and thereby connected to a control system.

[0061] Signals from the electrical sensors 135, 137, and 139 can be received by a control system. The control system can compare the detected position of the particle in the discharge channel (based on signals from the discharge electrical sensors 137 and 139) to the particle's expected position based on previous sorting decisions and actuations. This information can provide valuable feedback to the control system regarding sorting success rates and can be the basis for real-time or delayed adjustments to sorting or detection parameters to improve sorting success rates. The control system can receive signals from the input electrical sensor 135 that can identify particle characteristics on which sorting decisions can be based. This information can be obtained by the electrical sensor 135 instead of, or in addition to, using optical measurement techniques. In some embodiments, the signal from the input electrical sensor 135 can be used as a validation system to measure the success of measurements using a different detection system (e.g., an optical detection system).

[0062] In some embodiments, the input electrical sensor 135 is co-located with the interrogation region 113 such that particles are detected by the input electrical sensor 135 and any other detection systems, such as an optical detection system, at approximately the same time. In other embodiments, the input electrical sensor 135 can be located at a different location on the flow path from the interrogation region 113. In the drawings, the electrical sensors 135, 137, 139 are illustrated as being in the actuation section 130. However, the electrical sensors can be located in different layers within the same section of the microfluidic chip 100 or in different sections in other embodiments.

[0063] 4B illustrates one embodiment of a microfluidic chip 100 including a particle processing unit 101 featuring a horizontal segment 325 of a microchannel that runs parallel to the cover layer 131. The interrogation region 113 can be located in the horizontal segment 325, where all or most of the fluid in such embodiments flows horizontally (i.e., transverse to the stacking direction), such that optical interrogation and detection occurs as particles move perpendicular to the stacking direction 107 and perpendicular to the optical axis of any illumination or detection system that is probing the particles. The use of the horizontal segment 325 can be advantageous in some embodiments by making particle measurements more uniform when particles flow transverse to the optical axis of the optical detection and illumination system rather than along the optical axis. Note that even though the interrogation region 113 is located in a portion of the microfluidic channel 114 that moves transverse to the stacking direction 107, fluid movement of particles throughout the chip is still primarily along the stacking direction 107. Because flow is still primarily along the stack direction 107, the microfluidic chip still takes advantage of massive parallelization by allowing many particle processing units 101 to be mounted on a single chip.

[0064] 5 and 6 illustrate an embodiment of a microfluidic chip 100 in which an actuator 112 is located in or on a cover layer 131 of an actuation section 130, according to various embodiments taught herein. For example, the actuator 112 may include an IDT or other structural feature that generates acoustic energy. As shown in the partial cross-section of FIG. 6 , the actuator 112, in contact with the cover layer 131, can couple acoustic energy near the junction between the input microchannel 114 and the exhaust channels 116, 118. The actuator 112 can selectively divert or direct the particles 50, 60 to the appropriate exhaust channels 116, 118 by applying acoustic energy to the particles 50, 60.

[0065] Returning to FIG. 5 , in some embodiments, the microfluidic chip 100 is formed from a circular substrate. In various embodiments, the microfluidic chip 100 can have a rectangular or square form factor (as in FIG. 1 ), a circular or oval form factor (as in FIG. 5 ), or any other suitable form factor that meets application-specific considerations. The transfer section 110 includes a sample inlet 124 for inputting a particle-containing sample fluid. In some embodiments, the sample fluid is collected in an on-chip sample fluid reservoir 111, from which the sample fluid can enter each of the multiple microchannels 114. Similarly, the particle focusing section 120 can include a sheath fluid inlet 125 that allows input of sheath fluid into the microfluidic chip 100. In some embodiments, the sheath fluid enters an on-chip sheath fluid reservoir 122, from which the sheath fluid can be introduced into the multiple microchannels 114 to focus particles in the sample fluid stream. In some embodiments, the sheath fluid inlet 125 is coupled to a manifold that distributes the sheath fluid to each of the converging sections 120 of the particle processing unit 101. The converging sections 120 may include a plurality of nozzles 312 to facilitate the introduction of the sheath fluid into the sample fluid stream within the microchannel 114. In some embodiments, the nozzles may generate a jet-like flow of the sample fluid into the surrounding volume of the sheath fluid.

[0066] 7A and 7B illustrate close-up views of an embodiment of the transfer section 110 and the particle focusing section 120, which includes multiple particle focusing regions 123. As seen in FIG. 7A, the focusing regions 123 can be integral with a layer within the particle focusing section 120. In some embodiments, the particle focusing region 123 can include a nozzle-like feature 312 formed by a protrusion 313. In some embodiments, the particle focusing region 123 can include an orientation feature that induces a change in particle orientation (e.g., asymmetric particles, such as sperm cells, can be oriented in a preferred direction). As shown in FIG. 7A, sample fluid flows upward from the on-chip sample reservoir 111 through the interior of the layer 127 of the particle focusing section 120, which forms the nozzle 312, and is ejected through the nozzle 312 into the sheath fluid within the on-chip sheath fluid reservoir 122. In some embodiments, the sample fluid flows upward from a manifold (not shown) through the interior of the layer 127 of the particle focusing section 120, which forms the nozzle 312, and is ejected through the nozzle 312 into the sheath fluid. The sheath fluid is under pressure to flow into the microchannels 114, thus acting on the sample fluid to focus particles in the flow within the microfluidic channels 114. Figure 7B illustrates a cross-sectional view of a microfluidic chip 110 in which a particle focusing region 123 feeds the sample into multiple parallel microchannels 114. In this drawing, streamlines are illustrated for each sample stream as it narrows and converges under the action of the pressurized sheath fluid. Each sample stream within each microchannel 114 is focused by sheath fluid in an on-chip sheath fluid reservoir 122.

[0067] In some embodiments taught herein, the exhaust channels 116, 118 flow downward back through the chip and exit the chip in or through the transfer section 110. However, in some embodiments, such as the chip 100 of FIG. 5, the actuation region 130 can include a retention outlet 128 connected to the first (or retention) exhaust channel 118 and a waste outlet 126 connected to the second (or waste) exhaust channel. The outlets 126, 128 can, in some embodiments, pass through a sidewall 130a of the actuation section 130. The sidewall 130a can be the outer wall or perimeter of the chip 100 that is perpendicular to the top surface (e.g., the outer surface of the cover layer 131) where optical interrogation and detection occurs.

[0068] The sections of the microfluidic chip 100 can be prepared or manufactured separately. In some embodiments, each section (e.g., the translation section 110, the particle focusing section 120, and the actuation section 130) can include one or more alignment holes 315. As shown in FIG. 8 , the alignment holes allow the separate sections 110, 120, and 130 to be aligned and assembled into the final microfluidic chip 100. For example, alignment posts 310 can be inserted through the alignment holes 315 in one or more of the sections. While the posts 310 are illustrated as passing through all of the sections, the posts 310 can be permanently affixed to or manufactured as part of one or more of the sections. In this situation, the remaining sections can be positioned with the alignment holes 315 aligned with the posts 310 and slid onto the posts to join the sections and form the chip 100. In some embodiments, the posts 310 can be removable from the sections of the microfluidic chip 100 before assembly of the final microfluidic chip 100. For example, post 310 may be removed after insertion through alignment hole 315. Post 310 may break off the underlying section during assembly of microchip 100, for example, after the sections have been joined.

[0069] 9 illustrates one embodiment of a microfluidic chip 100 that includes a central exhaust channel 118 connected to the exhaust channels from each individual particle processing unit 101 of the microfluidic chip. In some embodiments, the interrogation regions and actuators are positioned at different azimuthal positions surrounding the center of the chip 100. The actuators 112 divert desired particles to the exhaust channels 118 that feed toward the center of the chip 100. At the center of the chip 100, the exhaust channels 118 combine into a single exhaust channel 118a that flows downward through the actuation section 130, particle focusing section 120, and transfer section 110. The sorted particles in the exhaust channel 118 then exit the chip through a retention outlet 128. In some embodiments, the retention outlet 128, sample inlet 124, and sheath inlet 125 are illustrated as passing through the bottom of the microfluidic chip. In other embodiments, one or more of these inlets and outlets may be located on the sidewalls or top surface of chip 100 (ie, through cover layer 131).

[0070] 10A and 10B schematically illustrate various fluid manifold or tubing embodiments suitable for the microfluidic chip 100 taught herein. The fluid manifold can extend through one or more of each of the above sections to provide fluidic connections to each of the sections. The fluid manifolds 400, 400′ can include a microfluidic channel assembly configured to receive particles from a particle source supply (i.e., the off-chip sample fluid reservoir 244) via one or more input fluidic connection elements and deliver particles to a collection system (e.g., including the off-chip collection reservoirs 216, 218) via one or more output fluidic connection elements. FIG. 10A illustrates possible layering of chip substrates that can be used to enable manifolding between layers to achieve desired fluid flow (and particle transport) throughout the system. In FIGS. 10A and 10B, black boxes indicate locations where fluid from a single path splits into multiple paths or where fluid from multiple paths combine into a single path (i.e., fluidic junctions). Fluid paths that intersect in this schematic but do not have black boxes at the intersections do not intersect to form fluidic junctions in the actual chip. The microfluidic chip 100 is plumbed in such a manner that each of a plurality of particle processing units 101 receives particle-laden sample fluid from a sample fluid reservoir 244 (either on-chip or off-chip) along an input fluid path 104 via a suitable manifolding means and into an interrogation region 113 where the particles 50, 60 of interest are interrogated and characterized. In some embodiments, particles 50, 60 may be individually diverted to one of a plurality of exit paths 106, 108 via a suitable sorting mechanism, such as an actuator 112.

[0071] The fluid manifold 400 can include distribution layers 117a-e that facilitate the division or combination of fluid paths. For example, distribution layer 117a can divide the input fluid path 104 from the reservoir 244 into multiple input fluid paths 104a, 104b that travel to individual particle processing units 101 within the distribution layer 117. Similarly, distribution layer 117b can combine fluid from outlet paths 108a, 108b from different particle processing units 101 into a single outlet path 108 that flows to the collection reservoir 218. Distribution layer 117c combines fluid from outlet paths 106a, 106b from different particle processing units 101 into a single outlet path 106 that flows to the collection reservoir 216. Distribution layer 117d divides the fluid after passing through the actuator 112 into first outlet paths 106a, 106b and second outlet paths 108a, 108b. Distribution layers 117a-e may be formed within any individual section, including the transfer section 110, the particle focusing section 120, or the actuation section 130. Distribution layers 117a-e may also span across sections or provide bridges between different sections. In some embodiments, diverted (and non-diverted) fluid from each microfluidic cytometer element may be pooled using manifolds within the chip stack and collected in a suitable container (or connected to another process).

[0072] FIG. 10B illustrates a fluid manifold 400′ that also uses sheath fluid from the sheath fluid reservoir 215 as a means to aid sample transport, provide hydrodynamic focusing, reduce contact between particles and internal channel walls, and to singulate, entrain, or otherwise reliably distribute particles for purposes such as transport, measurement, sorting, concentration / dilution, etc. (including combinations of two or more of these functions). Additionally, the sheath fluid may be used to support or modify the biochemical state of the sample. The addition of the sheath fluid path 105 to the chip 100 of FIG. 10B is facilitated by a distribution layer 117e, which splits the sheath fluid path 105 into separate sheath streams 105a, 105b that flow to each individual particle processing unit 101 within the chip 100. The distribution layer 117a in FIG. 10B also serves as a location where the sheath and sample fluids intersect, where effects such as hydrodynamic focusing can occur. It should be noted that the distribution layers 117a-e can comprise a single physical layer or patterned substrate, or can comprise a stack of multiple layers or substrates. Specifically, the distribution layer 117a of Figure 10B can comprise several stacked or layered substrate layers to allow for contouring of the microfluidic channels 114 formed therein to provide focusing or directing fluid as needed.

[0073] 10A and 10B illustrate an embodiment having a single sample fluid reservoir 244 and a single sheath fluid reservoir 215. However, it should be understood that the microfluidic chip 100 can, in some embodiments, interface with multiple fluid reservoirs 244, 215. For example, the microfluidic chip 100 can include sub-manifolds, each of which is associated with its own sample fluid reservoir 244 and sheath fluid reservoir 215. In some embodiments, the microfluidic chip 100 can engage a fluid cartridge that includes the sample fluid reservoir 244 and the sheath fluid reservoir 215. The combined chip and cartridge can be enclosed and sealed (or selectively sealable) from the external environment and configured for removable engagement with the particle processing system 200, as described below. In some embodiments, the fluidic system (i.e., the fluidic microchannels and manifold elements for fluid transport) may be provided as a sealable cartridge or chip that can operably enclose all of the fluid contacting surfaces used during particle processing (i.e., the sealable cartridge or sealable chip can be operably sealed during any particle processing operation such that fluid does not enter or exit the chip during the particle processing operation). The cartridge may also include exhaust reservoirs 216, 218 on-board.

[0074] In some embodiments, the microfluidic chip may be disposable. This may provide benefits combined with sealable fluid-contact surfaces in that operators can avoid contact with fluids within the chip (to improve biosafety) and fluids are prevented from becoming contaminated by external factors during processing. Samples can be removed from selected sample chambers on the chip after processing, and the chip can be discarded to avoid cross-contamination of samples or sheaths from different experimental runs. In other embodiments, the microfluidic chip 100 may be formed of materials that can be sterilized so that the microfluidic chip 100 can be reused. For example, materials may be selected to withstand sterilization processes including one or more of ethylene oxide, ultraviolet light, or high heat and pressure. The microfluidic chip 100 may be removable and engageable with a particle processing system, as described below.

[0075] A microfluidic particle analysis and / or sorting system 200 that may include or be operatively coupled to a massively parallel microfluidic chip 100 according to embodiments of the present disclosure may have a wide variety of uses, such as as a cell sorting platform for sex selection in mammals by sorting sperm by sex, as a therapeutic medical device to enable cell-based therapy, in clinical diagnostics to aid in the monitoring of human or other animal diseases, or in one or more drug development applications.

[0076] 11 , an ultra-high-throughput particle processing system 200 suitable for implementing exemplary embodiments of the present disclosure is illustrated schematically. The particle processing system 200 includes a microfluidic chip 100, an electromagnetic source system 210, a detection system 220, an optical separation system 205, and a computing device 150. One or more sample reservoirs 244 can supply a particle-containing sample fluid to the transfer section 110, where the sample fluid is split into multiple fluid input paths 104, each corresponding to a particle processing unit. Along each fluid input path 104, particles in the fluid converge within the particle focusing section 120 and reach an interrogation region. In some embodiments, the interrogation region is located within the actuation section 130. The electromagnetic source system 210 is configured to illuminate the interrogation region of each of the multiple microchannels. The detection system 220 is configured to receive light emitted or scattered from particles within the interrogation region, which is indicative of the particle's characteristics or identity. The detection system 220 simultaneously receives and processes light from each of the multiple particle processing units in the chip 100. The light separation system 205 can include one or more spectrally selective elements, such as a dichroic beam splitter or reflector, to allow portions of the illumination light path from the electromagnetic source system 210 to overlap with the detection light path of the detection system 220. In some embodiments, the light separation system 205 can include a low-pass filter with a cutoff frequency of 480 nm. The computing device 150 receives signals from the detection system 220 and controls operation of actuators in the actuation section 130 to selectively direct particles into the first fluid discharge path 106 or the second fluid discharge path 108. Particles in the first fluid discharge path 106 flow into the first discharge reservoir 216 (sometimes referred to as the "holding reservoir"), while particles in the second fluid discharge path 108 flow into the second discharge reservoir 218 (sometimes referred to as the "waste reservoir").

[0077] Computing device 150 can monitor, measure, calculate, characterize, and perform the necessary steps to command and control certain components in the system to change their state and / or change the path of one or more particles. Computing device 150 can include a computer with a processing unit or can include another electronic device and can communicate with one or more other similar or different processors to perform the necessary functions. In some embodiments, computing device 150 can utilize one or more sensors to operate reliably, predictably, accurately, and reproducibly. Computing device 150 can be partially or fully integrated into a microfluidic chip-substrate stack in various embodiments.

[0078] Particle processing system 200 may be configured, sized, or adapted to analyze, sort, and / or process (e.g., purify, measure, isolate, detect, monitor, and / or concentrate) particles (e.g., cells, microparticles, nanoparticles, molecules, etc.). For example, system 200 may be a cytometer, a cell purification system, etc., although the present disclosure is not limited thereto. Rather, it should be noted that system 200 may take a variety of forms, and the systems and methods described may be applied to other particle processing systems.

[0079] 12 illustrates a side schematic view of particle processing system 200 with particular focus on electromagnetic source system 210, detection system 220, and optical separation system 205. Electromagnetic source system 210 is used for precise illumination of particles within multiple channels of massively parallel microfluidic chip 100. Electromagnetic source system 210 can include one or more light sources 212 and one or more beam-shaping optics 214, 217. For example, light source 212 can be a single source that outputs light across a large aperture, or multiple individual sources that individually output light. Similarly, beam-shaping optics 214, 217 can include a single monolithic optical element, such as a fixed array of microlenses or a single macrolens (e.g., a microscope objective), or can include multiple individual optics, such as separate microlenses that can be individually positioned laterally relative to the optical axis, or multiple optics arranged in series along the optical axis. The light source 212 is focused onto multiple locations within the microfluidic chip, including each of the multiple interrogation regions 113, and in embodiments using selective monitoring, onto the multiple selective monitoring regions 161 / 162.

[0080] In some embodiments, particles within microchannel 114 are illuminated in an epi-illumination fashion. The illumination light interacts with the particles to generate optical signals based on one or more of fluorescence, reflectance, scattering, or extinction, which can be measured by detection system 220. As described in more detail below in FIGS. 13A-13F, electromagnetic source system 210 can provide an illumination beam or multiple illumination light beams that can be flood-illuminated (i.e., a broad beam of relatively uniform intensity), split (e.g., through multiple elements such as multiple beam splitters), scanned / moving, multi-source (e.g., multiple sources split, multiple laser sources such as one or more vertical-cavity surface-emitting lasers (VCSELs)), switched on or off as needed, periodically or otherwise (e.g., randomly) modulated, or any combination of the above. Excitation sources of different wavelengths can be used as needed for specific applications, where certain attributes of the particles of interest can be interrogated and measured. Excitation of particles within the microfluidic chip 100 can occur using one or more transmissive or reflective optical elements, such as one or more lenses, mirrors, optical fibers, tapered optical elements, diffractive elements, spectral elements, plasmonic elements, tapered optical elements, or the like, in any combination. The beam-shaping optics 217 can include a large-format lens (such as those described in U.S. Pat. No. 10,215,995, issued February 26, 2019, and incorporated herein by reference in its entirety), a microscope objective (such as that manufactured by Mitutoyo Corporation), or a multi-lens array (such as the microlens array and system described in U.S. Pat. No. 10,190,960, issued January 29, 2019, and incorporated herein by reference in its entirety). The beam-shaping optics 217 can focus light onto the interrogation region 113 or sorting monitoring region 161 / 162 of the microfluidic chip 100 and can receive light from the device such that an appropriate field of view is achieved to meet the need for multiple microfluidic measurement sites within the device.Additional beam-shaping optics 217 (not shown) can include an optical aperture, pinhole, stencil, or mask that allows only certain portions of the top surface of cover layer 131 to be optically observed. The optical aperture, in some embodiments, can provide spatial filtering. The optical aperture can be fabricated on-chip or formed as a separate layer attached to or mated with chip 100. In some embodiments, the optical aperture can be provided in a separate imaging plane. In some embodiments, beam-shaping optics 214, 217 can include one or more lenses (including microlenses), mirrors, or filters, including optical elements that can segment or split the beam into beamlets.

[0081] Detection system 220 includes one or more detectors 223 and one or more of beam-shaping optics 217, 225, 224, 226. Detection system 220 collects light reflected, scattered, fluoresced, or extinguished from particles (i.e., optical signals reduced by the presence of particles) and projects the optical signals through appropriate optical elements (including spectrally selective, spatially selective, or both spectrally and spatially selective elements) onto a sensor in detector 223. It should be noted that some optical elements, such as beam-shaping optics 217 of FIG. 12, may be common elements to both electromagnetic source system 210 and detection system 220 in some embodiments.

[0082] The light separation system 205 can include one or more spectrally selective elements, such as dichroic mirrors that transmit or reflect light depending on the wavelength of the light.

[0083] 12 illustrates the components of the electromagnetic source system 210 and the detection system 220 as separate components separate from the microfluidic chip 100. However, in some embodiments, certain components of these subsystems may be incorporated directly into the stacked or layered configuration of the microfluidic chip 100. For example, the beam-shaping optics 217 may be formed directly on the top surface of the cover layer 131 in some embodiments.

[0084] In the embodiment illustrated in FIG. 12 , the light source 212 includes a laser or broadband light source, and the optical element is a microlens array 214. The beam from the laser or broadband source fills the collection aperture of the entire microlens array, which splits the beam into beamlets. The beamlets are reflected from a spectrally selective reflector of the light separator system 205 and directed toward a second microlens array 217. The second microlens array 217 focuses the illumination light onto the interrogation region 113 or the sorting monitoring region 161 / 162 within the microfluidic chip 100. In various embodiments, the light source 212 can be provided as another monochromatic light source, one or more polychromatic light sources, or a mixture of monochromatic and polychromatic light sources. While the beam-shaping optics 217 are illustrated in FIG. 12 as a microlens array, the beam-shaping optics 217 can include one or more lenses, mirrors, and filters, including optical elements capable of segmenting or splitting the beam into beamlets.

[0085] Particles flowing through the microchannel interact with the illumination light, generating an optical signal. The optical signal is collected by the microlens array 217 and projected toward the optical separation system 205. The light travels through a spectrally selective reflector to other beam-shaping optics of the detection system 220, including an optical filter 225 that performs spectral selection (or "cleanup") on the optical signal, and a light collector 224, such as a microlens array or diffractive element, that focuses the light for reception by the detector 223. In some embodiments, the optical filter 225 can include a bandpass filter centered at 530 nm. The detection system 220, in some embodiments, can perform spectral selection and detection using an array of elements. Examples of optical signals that can be generated in optical particle analysis, cytometry, and / or sorting when a beam intersects a particle include, but are not limited to, optical extinction, angle-dependent light scattering (forward and / or side scattering), and fluorescence. Optical extinction refers to the amount of electromagnetic radiation or light that a particle loses, absorbs, or blocks. Angular-dependent light scatter refers to the proportion of electromagnetic radiation that is scattered or bent at each angle away from or toward an incident electromagnetic radiation beam. Fluorescent electromagnetic radiation is electromagnetic radiation that is absorbed and / or scattered by molecules associated with particles or cells and re-emitted at different wavelengths. In some instances, fluorescence detection can be performed using endogenous fluorescent molecules.

[0086] 12, the microlens array 224, in the form of a bundle or array of optical fibers 226, focuses the light onto separate beam-shaping optics of the detection system. In some embodiments, a separate optical fiber in the array 226 is associated with each element of the microlens array 224. The optical fiber array 226 carries the light to a sensor in the detector 223. In some embodiments, the detector 223 may include a charge-coupled device (CCD), and light from individual interrogation areas 113 or sorting monitoring areas 161 / 162 may be directed to different pixels or groups of pixels in the CCD. In some embodiments, the detector 223 may include photomultiplier tubes (PMTs), such as silicon PMs, arranged in a one- or two-dimensional array such that light from individual interrogation areas 113 or sorting monitoring areas 161 / 162 is directed to different PMs in the array. In some embodiments, optical scrambling or other crosstalk reduction techniques, such as those described in U.S. Pat. No. 9,335,247 to Sharpe et al., issued May 10, 2016, the entire contents of which are incorporated herein by reference, may be used to reduce the amount of unwanted light reaching each sensor of detector 223.

[0087] In some embodiments, the electromagnetic source system 210 and the detection system 220 can achieve optical excitation and detection through interfacing planar layers and a preceding number of optical elements for transmitting light for excitation and / or detection. For example, the light source 212 can include a VCSEL substrate having multiple excitation sources (i.e., VCSELs) positioned generally adjacent to the top surface of the cover layer 131 of the microfluidic chip 100. The detector substrate is also positioned such that excitation light can be delivered to the chip 100 and detection light can be received from the chip using suitable optical "transparency." In some embodiments, the substrate having elements corresponding to both the electromagnetic source system 210 and the detection system 220 can be assembled and positioned in a manner that allows particle measurements from within the chip 100.

[0088] 13A-13F illustrate side views of various embodiments of an electromagnetic source system 210 interacting with a microfluidic chip 100 according to the present disclosure. The embodiments are presented to provide an overview of several non-limiting ways in which precise illumination of locations within the microfluidic chip (such as the interrogation region 113 or the sorting monitoring regions 161 / 162) can be achieved. In FIG. 13A, the light source 212 of the electromagnetic source system 210 includes an array of lasers. The array of lasers can be arranged as a one-dimensional or two-dimensional array. In some embodiments, the array of lasers can include vertical-cavity surface-emitting lasers (VCSELs) combined with collimation and focusing optics (i.e., beam-shaping optics 214) to match the illumination spatial profile to the spatial geometry of the pattern 144 of particle processing units within the microfluidic chip 100. The array of lasers can, in some embodiments, be mounted on a single module. In one embodiment, the array of lasers may include a VCSEL laser array having gallium nitride (GaN) sources arranged in a 16x16 square grid (256 sources total), such as the array described in the scientific journal article entitled "Watt-class blue vertical-cavity surface-emitting laser arrays" by Masaru Kuramoto et al., Applied Physics Express, 12, 091004, 2019, the entire contents of which are incorporated herein by reference.

[0089] 13B illustrates a light source 212 that includes multiple individual lasers arranged in an array. The lasers can be packaged separately and brought into close proximity for placement in a pattern that complements the pattern 144 of the particle processing units.

[0090] 13C illustrates a light source including a shaped laser beam 212a. The laser beam 212a illuminates beam-shaping optics 214, which in this embodiment is in the form of a segmented mirror. The segmented mirror can be a static optical element that generates an array of laser beamlets in a one- or two-dimensional pattern that complements the pattern 144 of particle processing units in the microfluidic chip. In some embodiments, the beam-shaping optics 214 can be a segmented refractive element (e.g., a prism or grating) rather than a mirror.

[0091] Figure 13D illustrates an arrangement similar to Figure 12, but omitting the beam-shaping optics 214 disposed between the light source 212 and the light separation system 205. In this embodiment, the light source 212 may include a collimated laser beam that is not separated into beamlets until it passes through beam-shaping optics 217 in the form of a lenslet array. The lenslet array generates converging beamlets in either one or two dimensions to illuminate the interrogation region 113 or the sorting monitoring region 161 / 162.

[0092] In Figure 13E, the light source includes an array of illumination optical fibers 212b. Each illumination optical fiber 212b is associated with an element of beam-shaping optics 214, such as a single microlens in a microlens array. The beam-shaping optics 214, in some embodiments, can collimate the light from the illumination optical fibers 212b. The illumination optical fibers 212b can perform the illumination function with collimation, reflection, and illumination characteristics similar to the embodiment shown in Figure 13A. The illumination light reflects from the light separation system 205 and is directed through the beam-shaping optics 217, which can include a microlens array. The light from each fiber 212b is focused to illuminate a respective interrogation region 113 or screening monitoring region 161 / 162.

[0093] FIG. 13F illustrates an embodiment in which the light source includes a shaped light beam 212a, such as a shaped laser beam. For example, the light source 212a can include a laser beam expanded to fill the aperture of the beam-shaping optics 214. The beam-shaping optics 214 can include a dynamic beam splitter that splits the shaped light beam into segments corresponding to individual interrogation regions 113 or sorting monitoring regions 161 / 162 within the microfluidic chip 100. The dynamic beam splitter (such as a dynamic laser splitting element) can shape or direct portions of the light source 212 toward the microfluidic chip stack. The beam splitter can utilize dynamic technologies such as digital light processing (DLP), digital micromirrors, microelectromechanical systems (MEMS), or other dynamic diffraction, refraction, or reflection techniques to split the shaped light beam into segments. In addition to splitting or segmenting the light source 212 into beamlets, the dynamic beam-shaping optics 214 can move the beamlets to aid in alignment, particle tracking, focusing, or other methods to enhance measurement accuracy and precision.

[0094] 14A-14D illustrate various embodiments of a detection system 220 that are compatible with the systems and methods taught herein. In each of Figures 14A-14D, detected light from a particle reaches the bottom surface of a spectrally selective element 225 from a light isolation device 205. The spectrally selective element 225 cleans up the light by removing stray light of wavelengths that are not of interest, including, for example, the wavelength of the illumination light in some embodiments.

[0095] In FIG. 14A , light emitted or reflected from the interrogation region of the microfluidic chip passes through one or more beam-shaping optics 224 where it is focused. The beam-shaping optics 224 may include a microlens array or a diffractive element. The focused light then passes through a spatially selective element 228. The spatially selective element 228 may include a pinhole, knife edge, or other optical element that accepts the desired light (or blocks a portion of the undesired received light). The spatially selective element 228 may ensure clean coupling of the light into a first end of an optical fiber bundle or array 229. The second end of the optical fiber 226 is coupled to a detector 223 (not shown). The detector 223 may include multiple detecting elements, such as individual photomultiplier tubes (PMTs) or individual pixels in a CCD. In some embodiments, the fibers 226 may be associated one-to-one with the individual detecting elements. In some embodiments, each fiber 226 can transmit light to multiple detection elements, but each detection element can be associated with one fiber 226 to improve isolation between received light from different particle processing units.

[0096] 14B illustrates a detection system 220 in which light received from the interrogation region of the microfluidic chip 100 passes through a spectrally selective element 225 and beam-shaping optics 224. The beam-shaping optics 224 focuses the light directly onto the individual detection elements (which may also be referred to as a sensor array) of the detector 223. A spatially selective element 228 is not shown in FIG. 14B, although such an element may be used in this embodiment.

[0097] In Figure 14C, the received light passes through a spectrally selective element 225, which blocks undesired wavelengths, before passing through a spectral separator 221, such as a grating or prism. The spectral separator 221 can disperse the light according to wavelength so that different wavelengths of received light are directed to spatially separate detector elements or to different locations on the same detector element. In this way, the spectral separator 221 can act as a combined spectral and spatially selective element to direct specific portions of the light received from a particle to the appropriate detector element. As shown in Figure 14C, the light beam is split into two foci on the detector 223 such that the locations of the two foci are separated on corresponding detector elements with enlarged receiving areas.

[0098] FIG. 14D illustrates one embodiment of a detection system 220 that includes a separate detector 223. Light received from the interrogation region 113 or the screening monitoring region 161 / 162 is directed to a beam separator 227. The beam separator 227 can split each received light beam into two beams traveling in different directions. In some embodiments, the beam separator 227 can separate the received light based on the spectral characteristics of the light (e.g., transmitting light below a wavelength cutoff frequency and reflecting light above the cutoff frequency or wavelength). In some embodiments, the beam separator 227 can split the light based on characteristics such as polarization. In some embodiments, the beam separator 227 can split the received light regardless of the characteristics of the light, such as in the case of a 50 / 50 partially reflective beam splitter that splits the received light into two beams of equal intensity. One split beam of the received light passes through the first beam shaping optics 224a and converges along a first path onto the first detector 223a, while the other split beam of the received light passes through the second beam shaping optics 224b and converges onto the second detector 223b.

[0099] In some embodiments, the detection system 220 can separately detect light emanating from a single interrogation region 113 or sorting monitoring region 161 / 162 at different angles. For example, the angular light collection geometry can differ between scatter and fluorescence measurements. This fact can be exploited to identify particle characteristics (and ultimately particle identity or type) based on the difference in scattered versus fluorescent light measured from different angular positions, according to light-particle interactions described by Mie or Rayleigh theory.

[0100] As described above, the beam shaping optics 217 can include a microlens array, where the microlenses of the array are associated one-to-one with the interrogation region 113, the sorting monitoring region 161 / 162, or both. In some embodiments, the microlenses of the array can be associated with two or more interrogation regions 113, the sorting monitoring region 161 / 162, or both. For example, each microlens can be associated with two, three, four, five, six, seven, or eight light emitting regions, such as the interrogation region 113 and the sorting monitoring region 161 / 162. In some embodiments, the beam shaping optics 217 can include lenses appropriately formatted to collect light from multiple interrogation regions 113, the sorting monitoring region 161 / 162, or both, as shown in FIG. 14E. The lenses can function to accomplish one or more of collection, collimation, and focusing of light received from particles within the multiple interrogation regions 113 or the sorting monitoring region 161 / 162. The lenses can focus light onto individual elements of the detector 223 (e.g., individual pixels or individual photomultiplier tubes). In some embodiments, additional lenses or other beam shaping optics can be used to direct light from the chip 100 to the detector 223. Techniques for crosstalk mitigation can be used as described above. Beam shaping optics 217 including large lenses for collecting light from multiple locations on the chip are suitable for use in other embodiments described with respect to Figures 14A-14D. In some embodiments, the beam shaping optics 217 can focus illumination light from the electromagnetic source system 210 onto separate interrogation regions 113, sorting monitoring regions 161 / 162, or both.

[0101] 15 is a block diagram of a computing device 150 suitable for use with embodiments of the present disclosure. Computing device 150 may be, but is not limited to, a smartphone, a laptop, a tablet, a desktop computer, a server, or a network appliance. In some embodiments, computing device 150 may include a field programmable gate array (FPGA). In some embodiments, computing device 150 may include an application specific integrated circuit (ASIC).

[0102] Computing device 150 includes one or more non-transitory computer-readable media for storing one or more computer-executable instructions or software for implementing various embodiments taught herein. Non-transitory computer-readable media may include, but are not limited to, one or more types of hardware memory (e.g., memory 156), non-transitory tangible media (e.g., storage device 526, one or more magnetic storage disks, one or more optical disks, one or more flash drives, one or more solid-state disks), etc. For example, memory 156 included in computing device 150 may store computer-readable and computer-executable instructions 560 or software for performing operations of computing device 150 (e.g., instructions for processing particles in method 1100 described below). Computing device 150 also includes a configurable and / or programmable processor 155 and associated core 504, and optionally one or more additional configurable and / or programmable processors 502′ and associated cores 504′ (e.g., in the case of a computer system having multiple processors / cores), for executing computer-readable and computer-executable instructions or software stored in memory 156 and other programs for implementing embodiments of the present disclosure. Processor 155 and processor 502′ may each be a single-core processor or a multi-core (504 and 504′) processor. Either or both of processor 155 and processor 502′ may be configured to execute one or more of the instructions described in connection with computing device 150.

[0103] Virtualization may be used in computing device 150 so that infrastructure and resources within computing device 150 can be dynamically shared. Virtual machines 512 may be provided to handle processes running on multiple processors so that the processes appear to be using only one computing resource rather than multiple computing resources. Multiple virtual machines may also be used on a single processor.

[0104] The memory 156 may include computer system memory or random access memory such as DRAM, SRAM, EDO RAM, etc. The memory 156 may also include other types of memory or combinations thereof.

[0105] A user may interact with computing device 150 through a visual display device 514, such as a computer monitor, which may display one or more graphical user interfaces 516. A user may interact with computing device 150 using a multi-point touch interface 520 or a pointing device 518.

[0106] Computing device 150 may also include one or more computer storage devices 526, such as a hard drive, CD-ROM, or other computer-readable medium, for storing data and computer-readable instructions 560 and / or software (e.g., applications) that implement exemplary embodiments of the present disclosure. For example, exemplary storage device 526 may include instructions 560 or software routines to enable data exchange with, or operational control of, detector 223 or light source 212. Storage device 526 may also include instructions 560 or software routines for performing particle processing methods, such as method 1100.

[0107] Computing device 150 may include a communications interface 554 configured to interface with one or more networks, e.g., a local area network (LAN), a wide area network (WAN), or the Internet, via one or more network devices 524, through various connections, including, but not limited to, a standard telephone line, a LAN or WAN link (e.g., 802.11, T1, T3, 56 kb, X.25), a broadband connection (e.g., ISDN, Frame Relay, ATM), a wireless connection, a controller area network (CAN), or some combination of any or all of the above. In an exemplary embodiment, computing device 150 may include one or more antennas 522 to facilitate wireless communications (e.g., via the network interface) between computing device 150 and a network and / or between computing device 150 and components of a system, such as electromagnetic source system 210, detection system 220, or a pump operably connected to fluid reservoir 215 / 215 / 216 / 218. Communications interface 554 may include an internal network adapter, a network interface card, a PCMCIA network card, a card bus network adapter, a wireless network adapter, a USB network adapter, a modem, or any other device suitable for interfacing computing device 150 to any type of network that enables communication and for performing the operations taught herein.

[0108] Computing device 150 may execute operating system 510, such as versions of the Microsoft® Windows® operating system, different releases of Unix® and Linux® operating systems, versions of MacOS® for Macintosh computers, embedded operating systems, real-time operating systems, open source operating systems, dedicated operating systems, or other operating systems capable of executing on computing device 150 and performing the operations taught herein. In an exemplary embodiment, operating system 510 may execute in native mode or in an emulated mode. In an exemplary embodiment, operating system 510 may execute on one or more cloud machine instances.

[0109] 16 illustrates a flowchart of a method 1100 of fabricating a microfluidic chip according to some embodiments taught herein. The method 1100 includes aligning the translation section 110 with a plurality of alignment holes 315 in the particle focusing section 120 using a plurality of alignment posts 310 (step 1102). The method 1100 includes bonding the translation section 110 to the particle focusing section 120 (step 1104). The method 1100 includes aligning the actuation section 130 to the particle focusing section 120 by aligning a plurality of alignment holes 315 in the actuation section 130 with the alignment posts 310 (step 1106). The method 1100 includes bonding the actuation section 130 to the particle focusing section 120.

[0110] 17 illustrates a flowchart of a method 1200 for processing particles using a microfluidic chip 100 according to some embodiments taught herein. The method 1200 includes flowing a sample stream containing particles 50, 60 through a plurality of microchannels 114 formed by a plurality of sections 110, 120, 130 stacked or layered in a stacking direction 107 to form the microfluidic chip 100 (step 1202). The plurality of microchannels 114 are at least partially oriented to flow along the stacking direction 107. The method 1200 includes focusing the particles 50, 60 into each of the plurality of microchannels using a particle focusing section 120 of the plurality of sections 110, 120, 130 (step 1204). The method 1200 also includes detecting particle characteristics of the particles 50, 60 flowing through a plurality of interrogation regions 113, which in some embodiments are located within the actuation section 130 of the plurality of sections (step 1206). Each interrogation region 113 is associated with a microchannel 114. For example, detecting the particle characteristic may include illuminating the particles 50, 60 within the interrogation region 113 using an electromagnetic source system 210 that focuses light through the cover layer 131 of the actuation section 130. The particles 50, 60 then emit or scatter light, or dissipate or absorb the illuminating light, which generates an optical signal corresponding to the particle characteristic that is detectable by the detection system 220. In some embodiments, the particles 50, 60 may flow through multiple microsorters in series (on one layer) with multiple sequential actuators 112. The method 1200 includes sorting the particles in response to the detected particle characteristic using the actuators 112 associated with each microchannel 114 (step 1208). For example, the computing system 150 may receive a signal from the detection system 220 and determine whether the signal corresponds to the presence or absence of the particle characteristic in the particles 50, 60. If the detected particle characteristics indicate that the particle is a desired particle 60 to be sorted into the retention channel 118, the computing system 150 controls the actuator 112 to divert the particle 60 into the retention channel 118.

[0111] FIG. 18A illustrates a cross-sectional view through a schematic diagram of a microfluidic chip 100 according to the present disclosure. Hydrodynamic focusing occurs partially during the transition from a first direction relative to the optical illumination and detection elements. The microfluidic chip 100 is one embodiment of a "right-to-right" particle sorting scheme. As illustrated in FIG. 18A, a sample fluid containing particles 50, 60 flows upward through a transfer section 110 on a fluid input channel 104 into a main microchannel 114, while sheath fluid flows through transfer sections 110 on sheath fluid flow input paths 105a, 105b into respective sheath channels 115a, 115b. The sample fluid containing particles contacts and is focused by the sheath fluid within a particle focusing region 123′. Hydrodynamic focusing occurs partially during the transition from a first direction (e.g., vertical, parallel to the optical axis 260, and / or along the stacking direction 107) to a second direction (e.g., horizontal, perpendicular to the optical axis 260, and / or along the stacking direction 107), and partially due to the addition of sheath fluid via the sheath channel 115b after the particles have transitioned to the second direction. The actuator section 130 can include a processed particle layer 136 directly below the cover layer 131. The fluid distribution layer 136 can form horizontal channels (i.e., flow perpendicular to the stacking direction 107) and is fluidly coupled to the exhaust channels 116, 118. The flow direction of particles within the processed particle layer 136 is perpendicular to the optical axis 260 used by an illumination or detection system at or near the interrogation region 113. The particles flow into the interrogation region 113 where they can be detected using a detection system, such as an optical detection system using lens 222. Based on the signal (e.g., optical fluorescence, scattering, emission, or extinction) detected from the particle within the interrogation region 113, a control system such as a computing device 150 can identify whether the particle is a desired particle 60 or an undesired particle 50. If a desired particle 60 is detected, an actuator 112 (e.g., as illustrated in FIG. 22B) may take no action, allowing the desired particle 60 to follow its natural flow path to the first exhaust channel 106.If an undesired particle 50 is detected, the actuator 112 (e.g., as illustrated in FIG. 22B ) is actuated to divert the particle to the second exhaust channel 108. In some embodiments, the second exhaust channel 108 can be connected to the natural flow path, and the first exhaust channel 106 can be a bypass flow channel. The actuator 112 (e.g., as illustrated in FIG. 22B ) can be actuated to divert the desired particle 60 to the first exhaust channel 106. The actuator 112 can take no action, such that the undesired particle 50 passes through the natural flow path to the second exhaust channel 108.

[0112] 18B illustrates a cross-sectional view through a schematic diagram of a microfluidic chip 100 according to the present disclosure in association with optical illumination and detection elements. As illustrated in FIG. 18B, compared to the microfluidic chip 100 of FIG. 18A, the microfluidic chip 100 includes a particle focusing section 120. The particle focusing section 120 allows hydrodynamic focusing to occur within a particle focusing region 123 along the stacking direction 107 and then transition from a vertical direction to a horizontal direction, where all or most of the fluid flows horizontally (i.e., transverse to the stacking direction). In some embodiments, during the transition from the vertical direction to the horizontal direction, additional hydrodynamic focusing can occur in a particle focusing region 123′.

[0113] 18C-18E illustrate cross-sectional views through a schematic diagram of a microfluidic chip 100 having actuators 112 in various locations according to the present disclosure. One or more actuators 112 can be located throughout the actuation section 130. In some embodiments, the actuators 112 can be located above, on, or between layers, below the top layer (e.g., cover layer 131), or some combination thereof. When above, within, or just below the top layer (such that this layer can be optically transparent or partially transparent), a mechanism can be used that can take the form of an electrode layer positioned to allow acoustic energy to interact with particles from within selected microfluidic channels (e.g., horizontal channel subsection 146) of the actuation section 130. See, for example, FIG. 5.

[0114] If below the substrate, one or more actuators 112 may interact with particles (e.g., in horizontal channel subsection 146) by communicating with them and / or by using vias or other channels that allow connections through multiple layers. The actuators 112 may be piezoelectric actuators, such as piezoelectric drive pins, that interface with a membrane layer, which may be sufficiently flexible to allow deflection of the actuators 112 to impart pulses onto the channels of actuation section 130, thereby allowing the particles to deflect. Such membrane layers may be the same or different material as microfluidic chip 100, such as a polymer, glass, metal, or other hybrid or combination material layer.

[0115] There are many other ways in which particles may be actuated (i.e., sorted, or for that matter, de-sorted), which may include, by non-limiting example, valves, pumps, acoustic elements, thermal elements, expansion elements, bubble generators, vibration devices, etc. These devices may be located on, within, or through multiple layers of microfluidic chip 100. Additionally, actuator 112 may be part of microfluidic chip 100 or may interface with microfluidic chip 100 as desired for a particular application and / or where cost and complexity are considerations.

[0116] 19A illustrates an exploded view through a schematic diagram of a microfluidic chip 100 according to the present disclosure. The microfluidic chip 100 includes a movement section 110, a particle focusing section 120, and an actuation section 130. Sandwiching the movement section 110, the focusing section 120, and the actuation section 130 are cover sections 131. One cover section 131a is adjacent to or in direct or indirect contact with the movement section 110, and the other cover section 131b is adjacent to or in direct or indirect contact with the actuation section 130. The cover sections 131 can act to seal the microfluidic chip 100. The cover sections 131 can also be formed of a suitable material to avoid interference with optical measurement, optical detection, and optical interrogation of particles within the microfluidic chip 100.

[0117] The transfer section 110 can transport fluid in and out of multiple microfluidic channels in the microfluidic chip 100. For example, within the particle focusing section 120 are multiple particle focusing regions 123 for focusing particles, e.g., at least one particle focusing region for each of the particle processing units 101. One exemplary particle focusing region 123 is illustrated in FIG. 19 , and further details of the particle focusing region 123 are discussed below in conjunction with FIGS. 21A-21C . The particle focusing region 123 includes a sample inlet port 124 for introducing particle-containing sample fluid from the sample channel 114 into the particle focusing region 123 in the focusing section 120. The particle focusing region 123 also includes sheath inlets 125 a, 125 b, which are fluidly coupled to the sheath inlets 115 a, 115 b, respectively. Downstream of particle focusing region 123 is outlet 128 fluidly coupled to discharge channel 118, e.g., a waste or retention discharge channel, and outlet 126 fluidly coupled to discharge channel 116, e.g., a waste or retention discharge channel.

[0118] The actuation section 130 includes a processed particle layer 136. In some embodiments, the processed particle layer 136 includes a horizontal channel subsection 146 downstream of each of the converging regions 123. The horizontal channel subsections 146 include an interrogation region 113 where the particles are interrogated as discussed herein. Downstream of the interrogation region 113 are exhaust channels 116, 118 that are fluidly coupled to the horizontal channel subsections 146 via outlets 126, 128.

[0119] Each cover section 131 can allow for observation, detection, or both observation and detection of particles flowing within the chip 100. In some embodiments, each cover section 131 can include a window or window-like layer to provide a transparent optical interface to the fluid below the corresponding cover section 131. The cover section 131 can act as a liquid-proof barrier, i.e., the cover section 131 can prevent the movement of water or oil from the inside to the outside of the chip 100 or vice versa. In some embodiments, the cover section 131 can form part of one or more fluid channel paths through which fluid flows. Other features, such as electrodes, can also be applied to, incorporated into, or included on the cover section 131. In some embodiments, the cover section 131b near the actuation section 130 can include the actuator 112, as described in more detail above. The cover section 131 can be formed of one or more materials configured to provide high transmission of wavelengths of electromagnetic radiation of interest for a particular application (e.g., wavelengths of excitation light, scattered light, or fluorescence). In some embodiments, the cover section 131 can allow transmission of light in a range representing wavelengths in the ultraviolet (UV) to near-infrared (IR) wavelength range, or a subportion of the UV to near-IR range depending on the design intent.

[0120] FIG. 19B illustrates a top view through a three-dimensional (3D) schematic diagram of a microfluidic chip 100 according to the present disclosure. FIG. 19C illustrates a perspective view through the 3D schematic diagram of the microfluidic chip 100 of FIG. 19B. FIG. 19D illustrates a partial side view of the microfluidic chip 100 of FIG. 19B. As illustrated in FIGS. 19B-19D, the translation section 110, the particle focusing section 120, and the actuation section 130 are stacked along a stacking direction 107. The actuation section 130 is fluidly coupled to a sample input channel 104, a sheath input channel 105, and an outlet channel 108. The translation section 110 and the particle focusing section 120 have a sample input channel 114, a sheath input channel 115, and outlet channels 116, 118 (as illustrated in FIGS. 25A-25D). In some embodiments, the translation section 100 can include the particle focusing section 120. In some embodiments, the actuation section 130 can include a particle focusing section 120 (e.g., as illustrated in FIG. 18A). In some embodiments, the particle focusing section 120 is in a separate section (e.g., as illustrated in FIG. 18A).

[0121] 20 illustrates a stacked image from a top view of stacked sections of a fabricated microfluidic chip 100 during operation along stacking direction 107. Microfluidic chip 100 includes a translation section 110 having a sample channel 114, a sheath channel 115, and exhaust channels 116 and 118, a particle focusing section 120 having a particle focusing region 123, and an actuation section 130 having a processed particle layer 136. Particle focusing region 123 includes a sample inlet port 124 for introducing particle-containing sample fluid from sample channel 114 into particle focusing region 123. Particle focusing region 123 also includes sheath inlets 125a and 125b, which are fluidically coupled to sheath inlets 115a and 115b, respectively. Downstream of particle focusing region 123 are outlet 128 fluidly coupled to exhaust channel 118, e.g., a waste or retention exhaust channel, and outlet 126 fluidly coupled to exhaust channel 116, e.g., a waste or retention exhaust channel. The processed particle layer 136 includes a horizontal channel subsection 146 downstream of the focusing region 123. The horizontal channel subsection 146 includes an interrogation region 113 where the particles are interrogated as discussed herein. Downstream of the interrogation region 113 are exhaust channels 116, 118 that are fluidly coupled to the horizontal channel subsection 146 via outlets 126, 128. The sample fluid with the particles flows into the sample channel 114 and then into the particle focusing region 123. The sheath fluid flows into the sheath channel 115 and then into the particle focusing region 123. Hydrodynamic focusing occurs within the particle focusing region 123. The particle focusing allows the particles to be fluidically well controlled, move in single file, and be confined to the fluid core 142 (e.g., central fluid core).

[0122] Figure 21A shows a perspective view of a simulation for hydrodynamic focusing in the focusing region disclosed herein. In the illustrated hydrodynamic focusing configuration, focusing of particles in the sample stream occurs during and after the transition of the sample stream from a vertical to a horizontal orientation with the vertical addition of sheath fluid. Figure 21B illustrates a top view of the simulation of Figure 21A.

[0123] 21C illustrates a side view of the simulation. Hydrodynamic focusing can create laminar flow 148 by narrowing, accelerating, and positioning the sample fluid from the sample channel 114 and using sheath fluid from the sheath channel 115 to "squeeze" the sample fluid, for example, by introducing sheath fluid into which the sample fluid is flowing.

[0124] As illustrated in Figures 21A-21C, the particle-laden sample fluid flows vertically through sample channel 114 into particle focusing region 123. Similarly, sheath fluid flows vertically through sheath channels 115a and 115b into particle focusing region 123. In particle focusing region 123, the sheath and sample vertical flow channels transition from a vertical to a horizontal direction. As illustrated, sheath fluid from channel 115a prevents the sample from contacting the channel walls and focusing the sample in a first vertical direction as the sample transitions from vertical to horizontal flow. Sheath fluid from channel 115b focuses the sample in a second vertical direction. In some embodiments, the width of the flow channel can be reduced to provide horizontal hydrodynamic focusing, e.g., horizontal tapered regions, and horizontal control using side sheath channels.

[0125] Thus, the sample fluid containing the particles contacts and is focused by the sheath fluid in the particle focusing region 123. Hydrodynamic focusing occurs during the transition from a first direction (e.g., vertical) to a second direction (e.g., horizontal) to create a laminar flow 148 into the distribution layer 136.

[0126] FIG. 22A illustrates a schematic diagram of a microfluidic chip 100 having 2×2 particle processing units 101 according to various embodiments. FIG. 22A is exemplary and intended to help illustrate the massively parallel teachings taught herein and is not intended to limit the microfluidic chip 100 to a 2×2 matrix. The transfer section 110 of the microfluidic chip 100 can include a fluid manifold 400 (as illustrated in FIG. 23 ) having a microfluidic channel assembly configured to receive particles from a particle source supply (i.e., an off-chip sample fluid reservoir 244) via a sample fluid input path 104 and sheath from a sheath fluid reservoir 215 via a sheath fluid input path 105. The microfluidic chip 100 can further include a distribution layer 136 to facilitate the division or combination of fluid paths, as further described with respect to FIG. 22B . The fluid manifold 400 lays out the various microchannels in a manner that equally supplies (i.e., uniform fluid pressure and fluid flow) all particle processing units 101. The main fluid path layout 410 can include multiple microchannels in the same or different distribution layers to direct fluid from the sample input path 104, sheath fluid input path 105, and output paths 106, 108, respectively, into each particle processing unit 101. Examples of main fluid path layouts 410 are further described with respect to Figures 24A-24D.

[0127] FIG. 22B illustrates a schematic diagram of a single particle processing unit 101 of FIG. 21A. A sample fluid containing particles flows through sample channel 114 into particle focusing region 123, while a sheath fluid flows through sheath channel 115 into particle focusing region 123. In some embodiments, the sample fluid can enter particle focusing region 123 through a nozzle-like feature 312. Compared to the microfluidic chip 100 of FIGS. 18-21, sheath channel 115 has a more complex structure. As illustrated in FIG. 22B, sheath channel 115a is divided into sheath sub-channels 115b and 115c. Sheath channel 115b is divided into sheath sub-channels 115f and 115e. The sample fluid containing particles contacts the sheath fluid from sheath sub-channels 115c-115e. The sample fluid can be focused by the sheath fluid via hydrodynamic focusing. The focused particles flow into a distribution layer 136 having an interrogation region 113. The particles in the interrogation region 113 can be detected using a detection system, such as an optical detection system using lens 222. Based on the signal (e.g., optical fluorescence, scattering, emission, or extinction) detected from the particles in the interrogation region 113, a control system, such as computing device 150, can identify whether the particle is a desired particle 60 or an undesired particle 50. If a desired particle 60 is detected, actuator 112a can be actuated to divert the desired particle 60 to the first discharge channel 116 via outlet 126. If an undesired particle 50 is detected, actuator 112b can be actuated to divert the particle to the second discharge channel 118 via outlet 128. Of course, distribution layer 136 can have other configurations. For example, only one actuator 112 is required. If a desired particle 60 is detected, the actuator 112 may take no action, allowing the desired particle 60 to follow its natural flow path to the first exhaust channel 106. If an undesired particle 50 is detected, the actuator 112 operates to divert the particle to the second exhaust path 108.In some embodiments, the second outlet channel 108 can be connected to the natural flow path, and the first outlet channel 106 can be a bypass flow channel. The actuator 112 can be actuated to divert the desired particles 60 to the first outlet channel 106. The actuator 112 can take no action, so that the undesired particles 50 pass through the natural flow path to the second outlet channel 108.

[0128] The other particle processing units 101 can perform similar operations. Particles from the first exhaust channel 116 of each particle processing unit 101 can further flow into the first exhaust path 106 to the exhaust reservoir 216. Particles from the second exhaust channel 118 of each particle processing unit 101 can further flow into the first exhaust path 108 to the exhaust reservoir 218.

[0129] FIG. 23 illustrates an exploded view through a schematic diagram of a microfluidic chip 100 having 2×2 particle processing units according to the present disclosure. The microfluidic chip 100 includes a moving section 110, a particle focusing section 120, and an actuating section 130. In some embodiments, the moving section 110, the focusing section 120, and the actuating section 130 are sandwiched between a cover section 131, as illustrated in FIG. 19. The particle focusing section 120 includes a sample inlet port 124, a sheath inlet port 125, and outlet ports 126, 128 (e.g., as illustrated in FIG. 19) for each particle processing unit 101. The particle focusing section 120 connects the actuating section 103 with the moving section 110 and can change the flow direction between the actuating section 103 and the moving section 110. In some embodiments, the particle focusing section 120 can be non-parallel to the actuating section 130. For example, the microchannels in the particle focusing section 120 can be non-parallel to the distribution layer 136 with the interrogation region 113. The transfer section 110 includes a fluid manifold that includes a distribution layer 117 that facilitates the splitting or combining of the microfluidic channel assemblies described above and with respect to Figures 10A-10B.

[0130] FIG. 24A illustrates a schematic diagram of a microfluidic chip 100 having a main fluid path layout 410 for supplying a 2×2 layout of particle processing units 101 according to the present disclosure. FIG. 24B illustrates a schematic diagram of a microfluidic chip 100 having a main fluid path layout 410 for supplying a 4×4 layout of particle processing units 101 according to the present disclosure. FIG. 24C illustrates a schematic diagram of a microfluidic chip 100 having a main fluid path layout 410 for supplying an 8×8 layout of particle processing units 101 according to the present disclosure. FIG. 24D illustrates a schematic diagram of a microfluidic chip 100 having a main fluid path layout 410 for supplying a 16×16 layout of particle processing units 101 according to the present disclosure. Those skilled in the art will recognize that the scale of this approach can be further increased. The main fluid path layout 410 can supply all particle processing units 101 equally (i.e., with uniform pressure and fluid flow). In some embodiments, the main fluid path layout 410 can include multiple microchannels in the same or different distribution layers to direct fluid from the sample input path 104, the sheath fluid input path 105, respectively, into each particle processing unit 101. For example, Figure 26A illustrates the layout of the main fluid path layout 410 of one layer of a microfluidic chip 100 having a 16x16 layout of particle processing units 101. Figure 26B illustrates the layout of the measurement and actuation section 130 of the 16x16 layout of particle processing units of Figure 26A.

[0131] FIG. 25A illustrates a partial front view of a microfluidic chip 100 having multiple particle processing units 101 according to the present disclosure. While FIG. 25A illustrates a 2×2 layout of the multiple particle processing units 101, this is done to facilitate explanation of the concepts taught herein for those skilled in the art. FIG. 25B illustrates an isometric view of the microfluidic chip 100 of FIG. 25A. FIG. 25C illustrates a top view of the microfluidic chip 100 of FIG. 25A. FIG. 25D illustrates a bottom view of the microfluidic chip 100 of FIG. 25A. As illustrated in FIGS. 25A-25D, each particle processing unit 101 includes a transfer section 110, a particle focusing section 120, and an actuation section 130. The transfer section 110 and the particle focusing section 120 have a sample input channel 114, a sheath input channel 115, and output channels 116 and 118. An actuation section 130 is also illustrated, including a processed particle layer 136. In some embodiments, the actuation section 130 can include a particle focusing section 120 (e.g., as illustrated in FIG. 18A). In some embodiments, the transfer section 100 can include a particle focusing section 120. In some embodiments, the particle focusing section 120 is in a separate section (e.g., as illustrated in FIG. 18A). The sample fluid input path 104 has a main fluid path in the vertical direction and branches horizontally into multiple channels at some point, for example, to supply each particle processing unit. In some embodiments, there may be two or more sample fluid input paths 104 depending on the number of particle processing units in the microfluidic chip. For ease of illustration, in FIGS. 25A-25D, the sample fluid input path 104 branches horizontally into four branched fluid paths (as illustrated in FIG. 25C), one for each particle processing unit 101. Each branched fluid path connects a sample input channel 114 that directs sample fluid flow to a focusing structure, which in the illustrated embodiment is one or more layers within the actuation section 130. Each of the sheath fluid input paths 105 has a main fluid path that is initially vertical and transitions to a horizontal direction at the focusing structure (as illustrated in FIG. 25D). The actuation section 130 also includes exhaust paths 106, 108 downstream of the focusing structure.Exemplary diagrams of focusing structures suitable for use in Figures 25A-25D can be seen in Figures 20-21C.

[0132] FIG. 27A illustrates an exploded view through a schematic diagram of a disk-shaped microfluidic chip 100 having an m×n (m>1, n>1) layout of particle processing units 101 according to the present disclosure. FIG. 27B illustrates an isometric view of the disk-shaped microfluidic chip 100 of FIG. 27A. FIG. 27C illustrates a front view of the disk-shaped microfluidic chip 100 of FIG. 27A. FIG. 27D illustrates a rear view of the disk-shaped microfluidic chip 100 of FIG. 27A. FIG. 27E illustrates a side view of the disk-shaped microfluidic chip 100 of FIG. 27A.

[0133] As illustrated in Figures 27A-27E, the microfluidic chip 100 includes a fluid manifold 400 having an outlet manifold 420 and an inlet manifold 430, a particle focusing section 120, and an actuation section 130. The outlet manifold 420 can direct particles from the actuation section 130 through the particle focusing section 120 to an exhaust reservoir. The inlet manifold 430 can direct sample fluid and / or sheath fluid through the particle focusing section 120 into the actuation section 130. The actuation section 130 includes multiple actuation subsections, such as m x n actuation subsections. In some embodiments, the actuation section 130 includes multiple focusing subsections, such as m x n focusing subsections or regions, as described herein. Each actuation subsection is used for a specific particle processing unit 101. As illustrated in FIG. 27E, the microfluidic chip 100 can be illuminated and / or detected in an illumination / detection direction 440 to illuminate and / or detect particles within the working section 130, for example, an interrogation region of the working section 130.

[0134] 28A-28B illustrate various embodiments of illumination and detection systems for simultaneous illumination and detection according to the present disclosure. Each embodiment of the illumination and detection systems presented in FIGS. 28A-28B includes an electromagnetic source system 210, a detection system 220, one or more optical separation systems 205, and other associated optical elements. Examples of the electromagnetic source system 210, the detection system 220, and the optical separation system 205 are described above with respect to FIGS. 11-14E. In some embodiments, the detection system 220 can include one or more silicon photomultiplier tube arrays, multi-pixel photon counter (MPPC) arrays, multi-anode photomultiplier tube assemblies, and / or other suitable detector arrays. Examples of the microfluidic chip 100 are described above with respect to at least FIGS. 1-10 and 18-27.

[0135] In some embodiments, particle processing system 200 provides ultra-high throughput particle processing by using wide-field illumination, partially in epi-illumination mode, to simultaneously illuminate and / or detect signals from multiple particle processing units 101. For example, as illustrated in FIG. 28A , electromagnetic source system 210 simultaneously illuminates multiple particle processing units 101 (e.g., the interrogation region of each of multiple microchannels in each particle processing unit 101) within microfluidic chip 100. Light separation system 205 is used to direct illumination light to illuminate each particle processing unit 101 within microfluidic chip 100. In some embodiments, light separation system 205 is also used to direct backscattered light from particle processing units 101 to detection surface 270a via optical filter 225a. Scattered light (e.g., back-, side-, and forward-scattered light) and light emitted from particles from each particle processing unit 101 are detected by detector planes 270b-270d via optical filters 225b and 225c, respectively. It should be appreciated that particle processing system 200 can have more detection planes than those illustrated in FIG. 28A to detect scattered light (back-scattered light, side-scattered light, and / or other suitable scattered light) and light emitted from particles at different wavelengths (e.g., autofluorescence, fluorescence). As illustrated in FIG. 28B, electromagnetic source system 210 includes spatial light modulator (SLM) 214 and light source 212. SLM 214 converts a single laser beam from light source 212 into a two-dimensional (2D) array of m × n beamlets that simultaneously illuminate each particle processing unit 101 within microfluidic chip 100. A single light separation system 205 is used to direct illumination light from the SLM 214 to illuminate each particle processing unit 101 in the microfluidic chip 100. A first lens assembly 610 collects light emitted or scattered from particles in each particle processing unit 101 and directs it to the detection system 220 via a second lens assembly 620, which directs the light from each particle processing unit 101 to a corresponding detection spot on the detection system 220.The detection system 220 simultaneously receives and processes light emitted or scattered from particles in each particle processing unit 101 .

[0136] 29A illustrates a fluid surface 710 of the particle processing system 200. The fluid surface 710 includes 8x8 illumination spots 712. Each illumination spot 712 is focused onto a respective particle processing unit 101 (e.g., interrogation region 113).

[0137] 29B illustrates the detector plane 270 of the particle processing system 200. The detector plane 270 includes 8×8 detection spots 272. Each detection spot 272 detects scattered light and / or light emitted from a respective particle processing unit 101. Of course, a particle detection system can have more than 8×8 illumination spots and 8×8 detection spots as illustrated.

[0138] As can be understood from the foregoing, the concepts of the present disclosure can be embodied in a variety of ways. Accordingly, the embodiments or elements disclosed by this description or shown in the accompanying drawings of this application are not intended to be limiting, but rather to illustrate the numerous and varied embodiments encompassed by this disclosure generally, or the equivalents encompassed with respect to any particular element thereof. Additionally, the specific description of an embodiment or element may not explicitly describe every possible embodiment or element, and many alternatives are implicitly disclosed by the description and drawings.

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0140] As will be apparent to those skilled in the art upon reading this disclosure, each of the embodiments described and illustrated herein has individual components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0141] All numerical values ​​herein are assumed to be modified by the term "about," whether or not explicitly indicated. For purposes of this disclosure, ranges may be expressed as from "about" one particular value to "about" another particular value. It will be understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

Claims

1. A microfluidic chip comprising: a plurality of sections stacked or layered in a stacking direction to form a plurality of microfluidic channels at least partially oriented for flow along the stacking direction, the plurality of sections comprising: a transfer section for introducing a sample fluid containing particles; and a second section including a measurement section or the measurement section and an actuation section including a plurality of interrogation regions, each of the plurality of interrogation regions associated with at least one microfluidic channel in the plurality of microfluidic channels.

2. The microfluidic chip of claim 1 , wherein the plurality of sections further comprises a particle focusing section configured to focus the particles in the sample fluid.

3. The microfluidic chip of claim 2 , wherein the particle focusing section includes a plurality of nozzles for combining a sample fluid with a sheath fluid.

4. The microfluidic chip of claim 1 , wherein the actuation section comprises a plurality of actuators.

5. The microfluidic chip of claim 4 , wherein each of the plurality of actuators comprises an interdigital transducer that generates surface acoustic waves to deflect particles within the microfluidic channel.

6. The microfluidic chip of claim 5 , wherein the actuation section further comprises one or more acoustic damping elements for acoustically isolating the actuator.

7. The microfluidic chip of claim 1 , wherein the actuation section comprises a plurality of particle focusing regions.

8. The microfluidic chip of claim 1 , wherein the actuation section includes a cover layer configured to provide optical access to the plurality of interrogation regions along the stacking direction.

9. The microfluidic chip of claim 1 , further comprising a plurality of guide elements for aligning the sections along the stacking direction.

10. 10. The microfluidic chip of claim 1, wherein the areal density of the plurality of interrogation regions ranges from 1 to 500 regions per cm.

11. 10. The microfluidic chip of claim 1, wherein particle flow within each microfluidic channel is primarily along the stacking direction within the respective interrogation region.

12. The microfluidic chip of claim 11 , wherein the particle flow transitions from a predominantly vertical to a predominantly horizontal direction due to focusing.

13. 12. The microfluidic chip of claim 11, wherein the particle flow transitions from a primarily vertical to a primarily horizontal direction for interrogation.

14. The microfluidic chip of claim 1 , wherein at least some of the sections are separable from one another.

15. 15. The microfluidic chip of claim 14, wherein at least one of the plurality of sections is interchangeable based on a desired result or based on a characteristic of a population of particles to be processed by the microfluidic chip.

16. The microfluidic chip of claim 1 , wherein the sections are permanently attached or fused together.

17. 2. The microfluidic chip of claim 1, wherein the transfer section includes a sample input port and a sheath input port, and the transfer section transports sample fluid from the sample input port and sheath fluid from the sheath input port to the plurality of microfluidic channels.

18. 10. The microfluidic chip of claim 1, wherein the transfer section includes a first outlet port for enabling extraction of desired particles from the microfluidic chip and a second outlet port for enabling extraction of undesired particles from the microfluidic chip.

19. 10. The microfluidic chip of claim 1, wherein the actuation section further comprises a plurality of pressure pulse dampers, each pressure pulse damper disposed along an associated microfluidic channel opposite a respective actuation device.

20. 1. A particle processing system comprising:

1. A microfluidic chip comprising a plurality of sections stacked or layered in a stacking direction to form a plurality of microfluidic channels at least partially oriented for flow along the stacking direction, the plurality of sections comprising: a transfer section for introducing a sample fluid containing particles; and a microfluidic chip including a second section including a measurement section or the measurement section and an actuation section including a plurality of interrogation regions, each of the plurality of interrogation regions associated with at least one microfluidic channel in the plurality of microfluidic channels; an electromagnetic source system for illuminating the plurality of interrogation regions; a detection system for receiving light from the plurality of interrogation regions; a computing system operably connected to the detection system and the actuation section of the microfluidic chip, the computing system configured to control actuation of a plurality of particle deflectors based on signals received from the detection system.

21. 21. The particle processing system of claim 20, wherein the plurality of sections further comprises a particle focusing section configured to focus the particles in the sample fluid.

22. 22. The particle processing system of claim 21, wherein the particle focusing section includes a plurality of nozzles for combining a sample fluid with a sheath fluid.

23. 21. The particle processing system of claim 20, wherein the actuation section comprises a plurality of actuators.

24. 24. The particle processing system of claim 23, wherein each of the plurality of actuators comprises an interdigital transducer that generates surface acoustic waves to deflect particles within the microfluidic channel.

25. 25. The particle processing system of claim 24, wherein the actuation section further comprises one or more acoustic damping elements for acoustically isolating the actuator.

26. 21. The particle processing system of claim 20, wherein the actuation section comprises a plurality of particle focusing regions.

27. 21. The particle processing system of claim 20, wherein the electromagnetic source system comprises a plurality of vertical cavity surface emitting lasers (VCSELs).

28. 26. The particle processing system of claim 25, further comprising an optical isolation system.

29. 21. The particle processing system of claim 20, wherein the detection system comprises a microlens array and a detector, each microlens in the microlens array collecting light from a respective interrogation region in the plurality of interrogation regions and delivering the light to the detector.

30. 21. The particle processing system of claim 20, wherein the actuation section includes a cover layer configured to provide optical access to the plurality of interrogation regions along the stacking direction.

31. 21. The particle processing system of claim 20, further comprising a plurality of guide elements for aligning the sections along the stacking direction.

32. 21. The particle processing system of claim 20, wherein the areal density of the plurality of interrogation regions ranges from 1 to 500 regions per cm.

33. 21. The particle processing system of claim 20, wherein particle flow within each microfluidic channel is primarily perpendicular to the stacking direction within the respective interrogation region.

34. 21. The particle processing system of claim 20, wherein at least some of the sections are separable from one another.

35. 35. The particle processing system of claim 34, wherein at least one section of the plurality of sections is interchangeable based on a desired result or based on a characteristic of a population of particles to be processed by the microfluidic chip.

36. 21. The particle processing system of claim 20, wherein the sections are permanently attached or fused together.

37. 21. The particle processing system of claim 20, wherein the transfer section includes a sample input port and a sheath input port, the transfer section transporting sample fluid from the sample input port and sheath fluid from the sheath input port to the plurality of microfluidic channels.

38. 21. The particle processing system of claim 20, wherein the transfer section includes a first outlet port for enabling extraction of desired particles from the microfluidic chip and a second outlet port for enabling extraction of undesired particles from the microfluidic chip.

39. 21. The particle processing system of claim 20, wherein the actuation section further comprises a plurality of pressure pulse dampeners, each pressure pulse dampener being positioned along an associated microfluidic channel opposite a respective actuation device.

40. 1. A method for assembling a microfluidic chip, comprising: aligning the moving section with the plurality of alignment holes in the converging section using a plurality of alignment posts; joining the moving section to the converging section; aligning a measurement section, or a second section including the measurement section and the working section, with the converging section by aligning a plurality of alignment holes in the working section with the alignment posts; and joining the second section to the converging section.

41. 1. A method for sorting particles using a microfluidic chip, comprising: flowing a sample stream comprising particles through a plurality of microfluidic channels formed by a plurality of sections stacked or layered in a stacking direction to form the microfluidic chip, the plurality of microfluidic channels being at least partially oriented for flow along the stacking direction; focusing particles into each of the plurality of microfluidic channels using a converging section of the plurality of sections; detecting particle characteristics of particles flowing through a plurality of interrogation regions within a working section of the plurality of sections, each interrogation region associated with a microfluidic channel; and sorting the particles using an actuator associated with each microfluidic channel in response to the detected particle characteristic.

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