Large-scale parallel cell analysis and sorting apparatus and methods

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 419,852, filed on October 27, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Microfluidic devices have been used to analyze populations of particles and sort the particles based on the results of the analysis. Conventionally, this is achieved by flowing the particles through an area where analysis occurs, such as an area where optical interrogation and detection occurs. Summary of the Invention

[0004] A microfluidic chip is disclosed. In some embodiments, the microfluidic chip includes a plurality of segments stacked or layered in a stacking direction to form a plurality of microchannels, the plurality of microchannels being at least partially oriented to flow along the stacking direction. The plurality of segments include a transfer segment for introducing a sample fluid including particles. The plurality of segments include a particle focusing segment configured to focus the particles in the sample fluid. The plurality of segments include an actuation segment including a plurality of interrogation regions and a plurality of actuators. Each interrogation region in the plurality of interrogation regions and each actuator in the plurality of actuators is associated with at least one microchannel in the plurality of microchannels.

[0005] A particle processing system is disclosed. In some embodiments, the particle processing system includes a microfluidic chip having a plurality of segments stacked or layered in a stacking direction to form a plurality of microchannels, the plurality of microchannels being at least partially oriented to flow along the stacking direction. The plurality of segments includes a transfer segment for introducing a sample fluid including particles. The plurality of segments includes a particle focusing segment configured to focus the particles in the sample fluid. The plurality of segments includes an actuation segment including a plurality of interrogation regions and a plurality of actuators. Each interrogation region in the plurality of interrogation regions and each actuator in the plurality of actuators is associated with at least one microchannel in 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 segment of the microfluidic chip. In some embodiments, the computing system is configured to control actuation of a plurality of particle deflectors based on signals received from the detection system.

[0006] A method for assembling a microfluidic chip is disclosed. In some embodiments, the method includes aligning a transfer segment with a plurality of alignment holes in a focusing segment using a plurality of alignment posts. The method includes bonding the transfer segment to the focusing segment. The method includes aligning an actuation segment with the focusing segment by aligning a plurality of alignment holes in an actuation segment with the alignment posts. The method includes bonding the actuation segment to the focusing segment.

[0007] In some embodiments, a method for sorting particles using a microfluidic chip is disclosed. The method includes passing a sample stream including particles through a plurality of microchannels, the plurality of microchannels being formed by a plurality of segments, the plurality of segments being 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 particles in each of the plurality of microchannels using a focusing segment in the plurality of segments. The method includes detecting particle characteristics of particles flowing through a plurality of interrogation regions in an actuation segment in the plurality of segments. Each interrogation region is associated with a microchannel. The method includes sorting the particles using an actuator associated with each microchannel in response to the detected particle characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] It should be noted that the various features and combinations of features described below and illustrated in the accompanying drawings may be arranged and / or organized in different ways to produce embodiments that remain within the spirit and scope of the present disclosure. In addition, the components in the accompanying drawings are not necessarily drawn to scale or presented to scale, with emphasis instead on clearly illustrating the relevant principles. Furthermore, various features may not be shown in certain of the accompanying drawings to simplify the illustrations. Additionally, various components or elements may be shown in a transparent manner or may be presented using cross-hatching or other standard drawing techniques to describe or illustrate objects between layers or behind other elements, or to generally simplify the views of certain of the accompanying drawings. To assist those of ordinary skill in the art in making and using the disclosed systems, assemblies, and methods, reference is made to the accompanying drawings.

[0009] Figure 1

[00146] Massively parallel microfluidic chips with segments stacked or layered along a stacking direction according to various embodiments taught herein are presented.

[0010] Figure 2 A cross-sectional view schematically illustrating a microfluidic chip in conjunction with optical illumination and detection elements according to the present disclosure is shown.

[0011] Figure 3A A cross-sectional view schematically illustrating an embodiment of a microfluidic chip interfaced with optical illumination and detection elements is shown.

[0012] Figure 3B Shown Figure 3A Top view of the microfluidic chip.

[0013] Figure 4A A cross-sectional view schematically illustrating an embodiment of a microfluidic chip coupled with optical illumination and detection elements and including electrical sensor elements is shown.

[0014] Figure 4B A cross-sectional view schematically showing an embodiment of a microfluidic chip interfaced with optical illumination and detection elements is shown, wherein the interrogation zone is located in a horizontal portion of a microchannel where all or most of the fluid flows horizontally (ie, transverse to the stacking direction).

[0015] Figure 5 Embodiments of microfluidic chips are presented where actuators are located on or integrated within a cover layer according to some embodiments taught herein.

[0016] Figure 6 Shown Figure 5 A partial cross-sectional view of the chip shows the actuators deflecting particles into branch channels.

[0017] Figure 7A and 7B Partial cross-sectional views of a single microfluidic channel and a plurality of microfluidic channels of a microfluidic chip including nozzles according to some embodiments taught herein are shown, respectively.

[0018] Figure 8 Shown is a separation section of a microfluidic chip comprising alignment wells and alignment posts according to some embodiments taught herein.

[0019] Figure 9 Shown is a microfluidic chip with a central output channel according to some embodiments taught herein.

[0020] Figures 10A-10B Schematic diagrams of fluid manifolds within a microfluidic chip as taught herein are shown, according to various embodiments.

[0021] Figure 11

[0014] Presented are particle handling systems according to various embodiments taught herein that include a microfluidic chip according to the present disclosure having segments stacked or layered along a stacking direction.

[0022] Figure 12 A system including an electromagnetic source system, a light separation system, and a detection system according to various embodiments taught herein is presented. Figure 11 Side view of the particle handling system.

[0023] Figures 13A-13FShown are side views of various electromagnetic source systems according to embodiments taught herein.

[0024] Figures 14A-14D Shown are side views of various detection systems according to embodiments taught herein.

[0025] Figure 14E

[00146] An embodiment of a particle processing system according to some embodiments taught herein is presented wherein a single lens couples light from multiple interrogation regions to a detection system.

[0026] Figure 15 is a block diagram of a computing device suitable for use with embodiments of the present disclosure.

[0027] Figure 16 A flow chart illustrating a method of fabricating a microfluidic chip according to some embodiments taught herein is shown.

[0028] Figure 17 A flow chart illustrating a method of processing particles using a microfluidic chip according to some embodiments taught herein.

[0029] Figure 18A Shown are cross-sectional views of schematic representations of microfluidic chips according to the present disclosure.

[0030] Figure 18B A cross-sectional view schematically representing a microfluidic chip in accordance with the present disclosure in relation to optical illumination and detection elements is shown.

[0031] Figures 18C-18E Shown are cross-sectional views schematically representing a microfluidic chip with actuators at different locations according to the present disclosure.

[0032] Figure 19A An exploded view schematically representing a microfluidic chip according to the present disclosure is shown.

[0033] Figure 19B A three-dimensional (3D) schematic representation of a top view of a microfluidic chip according to the present disclosure is shown.

[0034] Figure 19C Shows a 3D schematic representation Figure 19B Perspective view of the microfluidic chip.

[0035] Figure 19D Shown Figure 19B A partial side view of the microfluidic chip is shown.

[0036] Figure 20 Stacked images showing a top view of an upward stacking section of a fabricated microfluidic chip in operation along the stacking direction.

[0037] Figure 21AA perspective view of a fluid dynamic focusing simulation in a focusing region as disclosed herein is shown.

[0038] Figure 21B Shown Figure 21A A top view of the simulation.

[0039] Figure 21C Shown Figure 21A Side view of the simulation.

[0040] Figure 22A Schematic diagrams of a microfluidic chip with a 2 x 2 layout of particle handling units according to various embodiments are shown.

[0041] Figure 22B Shown Figure 22A Schematic diagram of the particle processing unit.

[0042] Figure 23 An exploded view schematically showing a microfluidic chip with a 2 x 2 layout of particle handling units according to the present disclosure is shown.

[0043] Figure 24A A schematic diagram of a microfluidic chip with a main fluid path layout to supply particle processing units in a 2 x 2 layout according to the present disclosure is shown.

[0044] Figure 24B A schematic diagram of a microfluidic chip with a main fluid path layout to supply particle processing units in a 4 x 4 layout according to the present disclosure is shown.

[0045] Figure 24C A schematic diagram of a microfluidic chip with a main fluid path layout to supply particle processing units in an 8 x 8 layout according to the present disclosure is shown.

[0046] Figure 24D A schematic diagram of a microfluidic chip according to the present disclosure is shown with a main fluid path layout to supply particle handling units in a 16 x 16 layout.

[0047] Figure 25A A partial side view of a microfluidic chip according to the present disclosure is shown.

[0048] Figure 25B Shown Figure 25A Isometric view of a microfluidic chip.

[0049] Figure 25C Shown Figure 25A Top view of the microfluidic chip.

[0050] Figure 25D Shown Figure 25A Bottom view of the microfluidic chip.

[0051] Figure 26A The layout of the main fluidic paths of a microfluidic chip with particle handling units in a 16 x 16 layout is shown.

[0052] Figure 26B Shown Figure 26A Layout of the measurement and actuation sections of a particle handling unit in a 16 x 16 configuration.

[0053] Figure 27A An exploded view of a disk-shaped microfluidic chip schematically illustrating a particle handling unit having an mxn (m>1, n>1) layout according to the present disclosure is shown.

[0054] Figure 27B Shown Figure 27A Isometric view of the assembled disk-shaped microfluidic chip.

[0055] Figure 27C Shown Figure 27A Front view of a disk-shaped microfluidic chip.

[0056] Figure 27D Shown Figure 27A Rear view of the disk-shaped microfluidic chip.

[0057] Figure 27E Shown Figure 27A Side view of a disk-shaped microfluidic chip.

[0058] Figures 28A-28B Various embodiments of a particle processing system for synchronized illumination and detection according to the present disclosure are presented.

[0059] Figure 29A Demonstrates the lighting scheme of the particle handling system at the fluid plane.

[0060] Figure 29B A scheme for light projection onto a detector plane for imaging in a particle handling system is presented. DETAILED DESCRIPTION

[0061] The systems and methods taught herein adopt a "massively parallel" stacking design of a microfluidic structure by utilizing segments in a stacked or layered relationship (i.e., one segment is located on top of another segment), wherein the segments are combined to form a plurality of microfluidic channels oriented primarily in a stacking direction perpendicular to the plane defining the segments. The massively parallel microfluidic chip can be used to implement analysis, processing, or sorting operations (or any combination of these operations) of high-density parallel particles using the microfluidic chip. Specifically, the use of segments stacked or layered along the stacking direction enables the use of non-planar microfluidic features, which can be more densely stacked on the chip, thereby achieving a greater number of synchronous particle operations and a faster total throughput. As discussed below, the segments can be defined by function and operation. In some embodiments, the segments can include multiple functions. In some embodiments, the segments can include one or more layers. In some embodiments, the one or more layers can provide different functions. For example, as described herein, an actuation segment can include one or more layers, and the layers can be grouped to provide focusing, actuation, etc. As described below, in some embodiments, the focusing segment and the actuation segment can be combined into a single segment and provide focusing and actuation functions and operations in a single segment, such that the actuation segment can include multiple focusing regions and interrogation regions.

[0062] In some embodiments of the systems and methods taught herein, the fluid path is configured so that it is mainly aligned to flow in the stacking direction along the thickness of the microfluidic chip. In some embodiments, the flow direction of the particles according to the present disclosure is consistent with the optical axis used by the illumination or detection system at or near the inquiry point. In some embodiments, the flow direction of the particles according to the present disclosure in the inquiry area is perpendicular to the optical axis used by the illumination or detection system at or near the inquiry point. In conventional systems, fluid flow mainly spans a single plane (that is, substantially parallel to the longitudinal or transverse axis of the microfluidic chip). Therefore, although significant efforts have been made to reduce the cross-sectional area occupied by each set of particle processing elements (e.g., main microfluidic channels, branch microfluidic channels, sorting actuators, particle focusing features), the number of parallel sorting, processing and analysis operations that can be performed on a single chip using on-chip flow, mainly end-to-end in the chip plane, is still limited. In the massively parallel microfluidic chips taught herein, utilization of the third (e.g., thickness) dimension enables a significant increase in the density of parallel particle processing elements on a single chip while still providing the features required for successful particle sorting and processing (where channel size may ultimately be the limiting factor).

[0063] In conventional systems, microfluidic chip design is presented in a two-dimensional (2D) format, wherein microchannels, reservoirs, mixing areas, sensing areas (such as interrogation areas, focusing areas and other areas) are all arranged on a single plane 2D layer. In a 2D design, fluid is supplied to the microfluidic channels in a thin microfluidic chip. The microfluidic channels are positioned (e.g., etched) in the plane of the chip so that fluid and particles flow downward from one end of the chip to the other end of the chip length along the chip length. Each system for manifold manipulation requires space along the chip length and access to the microchannels, such as manifolds or branching areas, detection areas and sorting areas and combination areas are located at different positions along the chip length. Therefore, as other systems are added, the chip length increases, and the parallelization of fluid flow by adding microchannels along the width (or lateral) direction increases the chip width to accommodate additional microchannels. Historically, this design choice has been driven by multiple reasons, including cost, simplicity, basic operation, designer mindset, ease of manufacture, desire to use a single material and to mimic or minimize manufacturing steps. Specifically, the arrangement of flow channels parallel to the plane defined by the chip is driven by the long-standing practice of etching elongated trenches in substrates, which can typically be performed using one or only a few fabrication steps and has been widely applied to glass and silicon substrates. However, conventional 2D approaches can have significant disadvantages, 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, biocombined, mechanical, and electrical cannot be achieved within or along a single substrate (material)).

[0064] The systems and methods taught herein improve upon conventional 2D devices (e.g., substantially planar devices where one dimension of the device, such as thickness, is orders of magnitude smaller than the other dimensions) by enabling non-optical devices, fluid pathways, actuators, fluid dynamic focusing elements, fluid manifolds, and other features associated with processing particles in multiple microchannels to be moved out of the highly competitive region of the optical area (i.e., one or more planes near the top surface of a microfluidic chip accessible to optical interrogation and detection devices). Instead, these features and elements can be disposed in sections that are stacked or layered below the optical area in the stacking direction. Because these features are removed from the plane where optical interrogation and detection occur, the space in the optical interrogation area that would otherwise be occupied by these features and elements can be used instead for additional particle processing microchannels. Thus, the density of particle processing features can be substantially increased using massively parallel microfluidic chips as taught herein.

[0065] Furthermore, the implementation of three-dimensional (3D) microfluidic structures as taught herein substantially increases the density of microfluidic, optical, and actuation (one or a combination) features, and substantially increases the particle throughput of such devices, e.g., where the majority of the fluid flow is at some point along or parallel to the optical axis of excitation and / or detection (not necessarily a measurement result, but to minimize the ratio). In some embodiments, for interrogation purposes, the fluid flow is transverse to the optical axis.

[0066] The systems and methods taught herein may include a density of particle handling units, particle interrogation regions, particle focusing regions, or particle sorting actuators that, in various embodiments, exceeds 1 particle / cm 2 , 5 pieces / cm 2 , 10 / cm 2 , 20 pieces / cm 2 , 50 pieces / cm 2 , 100 pieces / cm 2 , 250 pieces / cm 2 or 500 pieces / cm 2 . The systems and methods taught herein reduce the cross-sectional area of ​​each individual particle processing unit in the observation or imaging plane of the electromagnetic source system or the detection system. The reduction in cross-section reduces the amount of "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 the detection system). In other words, by reducing the area of ​​the particle processing unit in the imaging plane of the electromagnetic source system, the detection system, or both to approximately the size of the interrogation area or the sorting-monitoring area, more particle processing units can be arranged on a microfluidic chip substrate of a given chip size. Similarly, compared to conventional microfluidic chips that attempt to achieve the same level of flux, a smaller microfluidic chip can achieve the desired flux. Another advantage of the three-dimensional stacking or layering approach is that more particle processing units or microfluidic chips can be accommodated on a single wafer (or other size-limited components) during manufacturing. This approach can also reduce the cost of each device per unit area occupied. Finally, compared to conventional planar chip architectures, this approach can reduce the total fluid path for particle movement, which can improve particle results such as cell survival, and also reduce the total fluid volume required to operate the system.

[0067] As used herein, the term "massively parallel" microfluidic chip is defined as a chip that is organized such that the microfluidic channels carrying particles are oriented with longitudinal portions positioned at least partially parallel to the height or thickness direction of the chip at locations where particle operations (e.g., particle processing, focusing, analysis, or sorting operations) occur on the chip.

[0068] As used herein, the term "planar" substrate is a material having a first dimension (eg, thickness) that is significantly smaller (eg, at least two orders of magnitude smaller) than its other two dimensions (eg, length and width).

[0069] As used herein, the term "stacking direction" refers to a direction perpendicular to the planar layers or sections forming the microfluidic chip.

[0070] As used herein, the term "hydrodynamic focusing" refers to the use of a sheath fluid to narrow, accelerate, and position a sample to generate laminar flow. For example, hydrodynamic focusing is typically, but not exclusively, used to position particles to the center of a microfluidic channel so that they can be reliably detected by an optical system focused at or near the center of the channel. In some embodiments, hydrodynamic focusing is achieved by introducing a sheath fluid into a microchannel in which the sample fluid is flowing to "squeeze" the sample stream. In some embodiments, hydrodynamic focusing is achieved by narrowing the size of the microchannel at a location after the sample and sheath fluids have flowed together, using, for example, a ramp, a cone, or a step. Hydrodynamic focusing is distinguished from sample injection (wherein the sample stream is introduced into a channel in which a sheath fluid is already flowing) because injection does not accelerate particles in the sheath fluid.

[0071] As used herein, the term "particle" includes, but is not limited to, cells (e.g., platelets, leukocytes, tumor cells, embryonic cells, stem cells, sperm, etc.), organelles, and multicellular organisms. Particles can include liposomes, proteoliposomes, yeast, bacteria, viruses, pollen, algae, etc. Additionally, particles can include genetic material, RNA, DNA, fragments, proteins, etc. Particles can also refer to non-biological particles. For example, particles can include metals, minerals, polymeric substances, glass, ceramics, composite materials, etc. Particles can be naturally occurring or artificial. Particles can also refer to synthetic beads (e.g., polystyrene), such as beads with antibodies conjugated to fluorescent dyes. Particles can be sorted by sex on a platform for sex preselection of mammals, or sorted by therapeutic or clinical value for monitoring diseases in humans or other animals, or diseases in one or more drug development applications.

[0072] As used herein, the term "microfluidic system" refers to a system or device comprising at least one fluid channel having microscale dimensions. The microfluidic system can be configured to handle, process, detect, analyze, eject, and / or sort a fluid sample and / or particles in a fluid sample.

[0073] As used herein, the term "channel" refers to a passageway formed in or through a medium or substrate that allows for the movement of fluids such as liquids and gases.

[0074] The term "microchannel" refers to a channel, which is preferably formed in a microfluidic system or device, with a cross-sectional size 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 will be able to determine the appropriate volume and length of microchannel for desired application. The above range is intended to include the value listed above as an upper or lower limit. Generally speaking, microchannel can have any selected cross-sectional shape, such as U-shaped, D-shaped, rectangular, triangle, elliptical / oval, circular, square, trapezoidal and other cross-sectional geometries. The geometry can be constant or can vary along the length of microchannel. In addition, microchannel can have any selected arrangement or configuration, including configurations such as linear, nonlinear, convergent, branched, annular, twisted, stepped. Microfluidic system or device (such as, microfluidic chip or chip substrate stacking) can include any suitable number of microchannels for conveying fluid. The microfluidic chip can be provided as part of a disposable cartridge for removable engagement with a microfluidic instrument. Furthermore, the microfluidic chip can be provided as part of a disposable cartridge, wherein the disposable cartridge is a completely enclosed and sealed or sealable fluid channel system. Furthermore, the microfluidic chip or portions of the chip can be removable, replaceable, and / or irreversibly bonded / fused.

[0075] As used herein, a "particle processing unit" is a unit that includes features that perform particle processing functions (such as particle sorting) and can be replicated multiple times across the chip to increase the total parallel processing throughput. Such features can include fluid focusing elements, interrogation areas, sorters 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 receive fluid from a pool or from an inlet of a channel that supplies multiple units), at least two microfluidic output channels (which can deliver sorted or processed particles and waste particles to a pool or outlet that supplies multiple units or is supplied by multiple units), an interrogation area, and associated actuators. As described in more detail below, the microfluidic chip taught herein includes multiple particle processing units operating in parallel. By including N particle processing units on a single microfluidic chip, the total throughput of particles that the chip can process is increased by at least N times.

[0076] As used herein, "interrogation" refers to probing particles to determine characteristics of the particles, and in some cases, to determine the classification, type, or nature of the particles. With respect to optical systems, interrogation includes detecting light emitted or otherwise scattered from an illuminated particle, or the absence of light due to interaction with the particle, to determine the value of one or more particle characteristics, such as size, shape, morphology, orientation, fluorescence intensity or wavelength, light scattering intensity or wavelength, geometry, volume, surface area, ellipticity, refractive index, granularity, porosity, electrical conductivity, nature, type, phenotype, protein or molecule expression, genetic content, live / dead status, velocity, and the like.

[0077] As used herein, "processing" a particle means taking an action in response to a determination of one or more particle characteristics of the particle, including but not limited to activating an actuator to sort particles, isolate a population of particles, purify a population of particles, enrich a population of particles, optically tweeze particles (e.g., cells), or inactivate (i.e., damage, disable, deactivate, or kill) particles.

[0078] Generally speaking, the present disclosure relates to the measurement and sorting of particles, droplets, and / or fluids in microfluidic devices. More specifically, the present disclosure relates to the manipulation of particles, droplets, and / or fluids in microfluidic devices in a massively parallel manner by integrating novel device designs and methods. Such manipulations can include deactivating or modifying particles, droplets, and / or fluids. Such manipulations can be the result of interrogating particles. Such manipulations can include stretching, bonding, or other changes in the physical, chemical, or biological properties of particles.

[0079] Particle separation is of great importance for many biological and biomedical applications. As the demand for sorting or analyzing particle populations for biomedical applications grows, the need to increase processing throughput to provide the ability to process more samples faster increases. Various approaches have been attempted, including providing multiple channels and sorters on a single planar microfluidic chip. Conventionally, particle manipulation is performed while the particles flow along a plane within a flow channel, such as in a conventional microfluidic channel, where the direction of fluid flow along the plane is substantially perpendicular to the optical detection axis through the chip.

[0080] The microfluidic chips and associated systems taught herein are capable of analyzing, processing and / or selecting particles based on determined intrinsic properties, such as by the interaction of electromagnetic radiation or light with the particles (e.g., (forward, backward or side) scattering, reflection and / or autofluorescence), without relying on protocols and necessary reagents. According to some embodiments, the microfluidic system uses a closed, sterile, disposable box including the microfluidic chip so that all surfaces that contact the sample fluid are isolated from the user and / or from non-disposable instruments. The microfluidic system analyzes and / or processes particles at high speed. 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 predefined / desired population attributes.

[0081] Figure 1 A schematic exploded view of a massively parallel microfluidic chip 100 according to various embodiments taught herein is shown. The massively parallel microfluidic chip 100 includes a plurality of stacked segments forming the chip 100. The microfluidic chip 100 may include a transfer segment 110, a particle focusing segment 120, and an actuation segment 130 including a cover layer 131. Each of the transfer segment 110, the particle focusing segment 120, and the actuation segment 130 may be formed from a single material layer or multiple material layers. One of ordinary skill in the art will recognize that some or all of the segments may be formed by additive manufacturing, photolithography, bonding, or molding, or that some or all of the segments may be formed separately and subsequently assembled into the microfluidic chip 100. The segments of the microfluidic chip are stacked or layered in a stacking direction 107 (i.e., the thickness of the microfluidic chip). When the segments of the chip 100 are stacked or layered, a plurality of fluid microchannels are formed that pass through one or more of the segments, the plurality of fluid microchannels being primarily oriented to flow along the stacking direction 107. The plurality of microfluidic channels and associated particle handling elements form a particle handling unit arranged in pattern 144 .

[0082] A fluid containing particles is introduced into one microchannel from the plurality of microchannels in the transfer section 110. Some embodiments may not include a transfer section. The fluid containing particles flows upward or downward in the microchannel along the fluid input path 104 (i.e., from the transfer section to or through the particle focusing section) through the stacked or layered sections, depending on the stacking direction. 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 distinct section separate from the actuation section 130. The desired particles flow back downward or upward in the microchannel along the first output path 106 (sometimes referred to as the "retentate" path) through the stacked or layered sections, depending on the stacking direction (i.e., back toward the transfer section 110). Undesirable particles flow back through the stacked or layered sections along a second output path 108 (sometimes referred to as a "waste" path) in the microchannel, either downward or upward, depending on the stacking direction (i.e., back toward the transfer section 110). Particles in the first output path 106 and the second output path 108, respectively, can be extracted from the chip 100 at the transfer section 110.

[0083] Figure 2 The cross-sectional view of FIG. 1 facilitates describing features of the massively parallel microfluidic chip 100 according to some embodiments. Figure 2 A cross-sectional view of a schematic representation of a microfluidic chip 100 is shown during engagement with optical illumination / detection elements and with fluid and particle flow therein. Figure 2 For ease of illustration, a single particle processing unit 101 as taught herein is shown. However, as described with respect to Figure 2As described, the microfluidic chip 100 includes between 10 and 1000 particle processing units 101. In the particle processing unit 101, one or more sample fluid input paths 104 can be combined with one or more sheath fluid input paths 105a, 105b to surround the sample fluid with sheath fluid, and in some embodiments, focus the sample fluid. For example, the sample fluid is injected into the main microchannel 114 through the transfer section 110, and the sheath fluid on the sheath fluid input paths 105a, 105b is injected into the corresponding 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 where sheath fluid is not used, the sheath fluid input path 105 is not present, and only the sample fluid flows through the microchannel 114 along the sample fluid input path 104. The sheath fluid and sample fluid, including particles 50, 60, intersect in microchannel 114 at particle focusing region 123 of particle focusing section 120. Particle focusing region 123 is the area where the sheath fluid acts on the focused particles 50, 60, as indicated by the streamlines in particle focusing region 123, which illustrate the compression of the sample fluid by the sheath fluid in the fluid intersection region. Particles 50, 60 then enter interrogation region 113 in actuation section 130. Each particle 50, 60 can be interrogated by light directed through cover section 131 and onto the particle by lens 222. The light is emitted, scattered, absorbed, or extinguished by the particle, depending on its characteristics. Detecting the emitted or scattered light, or changes in the level of existing light, through lens 222 enables identification of particle characteristics. For example, by detecting changes in the level of emitted or scattered light, or existing light (e.g., extinction), specific characteristics of the particle, such as size, morphology, fluorescence, light scattering, and other characteristics, can be identified. Based on the identification of the particle characteristics, the actuator 112 of the actuation section 130 can be actuated to divert the desired particle 60 (i.e., a particle having the desired characteristics) or the undesired particle 50 (i.e., a particle not having the desired characteristics) away from the normal flow path 191 and into the deflected flow path 192. Figure 2 In the embodiment of the present invention, particles 60 acted upon by actuator 112 flow along second output path 108 through microchannel 118 and onto deflected flow path 192, while particles not acted upon by actuator 112 flow along first output path 106 through microchannel 116. In some embodiments, the particles are monitored by lens 224 as they pass through first sorting monitoring region 161 in first output flow path 106, or by lens 226 as they pass through second sorting monitoring region 162 in second output flow path 108, and the sorting results (i.e., the actuation results) can be monitored. Light emitted from the particles through lenses 224 and 226 can pass through cover section 131 and be detected to verify whether sorting occurred as expected.

[0084] While lenses 222, 224, 226 are depicted to aid in illustrating features of the microfluidic chip 100, it should be understood that the lenses are part of a particle handling system that is separate from the microfluidic chip and are not features of the microfluidic chip. In other embodiments, the cover section 131 of the chip may include integrated optical features, such as a lens array, a metalens array, or other optical properties, to simplify alignment and or optical and chip design. As a non-limiting example, the integrated optical features may be molded elements made of glass or plastic that comprise the cover section 131. Figure 6

[0046] Beginning with a description of the present invention, embodiments of a particle handling system for use with the microfluidic chip 100 taught herein will be described in greater detail below. In some embodiments, the individual lenses 222, 224, 226 can be single units in a lens or microlens array.

[0085] The transfer section 110 can transport fluids into and out of multiple microfluidic channels in the microfluidic chip 100. The transfer section 110 can include an inlet port capable of introducing fluids into the microfluidic chip. For example, the transfer section 110 can include an inlet port for introducing a sample fluid containing particles to be processed or sorted. The transfer section 110 can also include one or more inlet ports for introducing sheath fluids to modify the sample fluid flow, such as for focusing 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, the first outlet port can extract "retained" path particles and fluids, while the second outlet port can extract "waste" path 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, the sample inlet manifold can distribute the sample fluid to each microfluidic channel in the multiple microfluidic channels. Similarly, the sheath inlet manifold can distribute the sheath fluid to each microfluidic channel in the multiple microfluidic channels. In some embodiments, a manifold can be used to aggregate target or non-target particles from multiple microfluidic channels by combining and directing the outputs from multiple output paths into a single stream through an outlet port. For example, the manifold of the transfer section 110 can combine fluids from all first output paths 106 or fluids from all second output paths 108 for extraction from the chip 100.

[0086] In some embodiments, the transfer section 110 may include one or more filters. The filters can prevent large particles, debris, particle aggregates, and other large objects from entering the microfluidic chip 100. Large objects can cause undesirable effects, such as clogging of microfluidic channels or inaccurate measurements and sorting.

[0087] The particle focusing section 120 can manipulate (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 achieve focusing of particles in the microfluidic channel 114. Particle focusing causes the particles to: behave well in the fluid; travel in a single file; be confined to a defined region of the microfluidic channel (e.g., a central fluid core); be randomly spaced or evenly distributed, allowing for reliable measurement and accurate particle sorting; or any combination thereof. Focusing can be achieved using a variety of techniques. In one example, hydrodynamic focusing can be achieved by changing the geometry of the microfluidic channel 114 (e.g., a ramp or taper), which constricts the fluid volume and accelerates and narrows the flow in the channel. Alternatively or additionally, a sheath fluid can be injected into the microfluidic channel 114 through sheath channels 115a, 115b that intersect the microfluidic channel 114. The sheath channels 115a, 115b can be positioned on opposite sides of the microchannel 114 to generate counteracting forces at the same intersection. In other embodiments, the intersections of different sheath fluid channels 115a, 115b with the microfluidic channel 114 may occur at different longitudinal positions along the length of the microfluidic channel. Figure 2 As shown, the sheath channel can flow parallel to the microfluidic channel 114. However, the sheath channel can also flow from a perpendicular direction or any angle to the intersection with the microfluidic channel 114, and it should be understood that additional sheath fluid channels can be Figure 2 Flow in adjacent planes not shown in the cross-sectional view of FIG (ie, the sheath channel may be located Figure 2 14). The “behind” or “front” of the microfluidic channel 114 is shown in the cross-sectional plane of FIG.

[0088] In some embodiments, the sheath fluid can be used as a transport fluid to reduce clogging. The sheath fluid can be used to mechanically or chemically affect particles in the microfluidic channel 114, including through biochemical interactions of sheath fluid components with the particles. In various embodiments, the sheath fluid can be used to wash, dilute, single-particle, align, orient, deflect, accelerate, decelerate, or center particles in the sample fluid.

[0089] The particle focusing section 120 may also employ techniques that are alternatives to fluid dynamics techniques for particle focusing, including acoustic wave (including surface acoustic wave) techniques, electrophoresis techniques, magnetic techniques, optical techniques, or other techniques. Surface acoustic waves may be generated using interdigital transducers (IDTs) located on or within a layer of the particle focusing section 120. In embodiments where fluid dynamics focusing is not employed, the volume of the sheath fluid may be reduced or eliminated entirely where unnecessary. In some embodiments, alternative focusing techniques may be used together or in conjunction with fluid dynamics focusing. In some embodiments, passive particle focusing methods that rely on structural changes in the microfluidic channel may be used. For example, the particle focusing section 120 may employ Dean inertial flow techniques (including spirals or serpentines in the flow channel) to focus particles. In some embodiments, the particle focusing section 120 may ensure reliable singulation of cells (i.e., positioning cells "in a single file" along the length of the microfluidic channel), thereby enabling accurate or repeatable measurement of particle properties using, for example, optical measurements. The particle focusing section 120 can use fluid dynamic methods, mechanical methods (such as acoustic forces), or optical methods (such as optical tweezers) to separate cells.

[0090] In some embodiments, particles may not be completely focused within the particle focusing section 120. For example, focusing of some particles may occur at or within a boundary or connecting region between sections. Specifically, fluid dynamic forces and effects may not be discretely maintained within a single section, but may extend downstream to a certain extent, such that focusing effects may be considered to be still occurring after the particles have left the particle focusing section 120.

[0091] The actuation section 130 includes an actuator 112 associated with one or more of the microfluidic channels 114, the one or more microfluidic channels being located downstream of an interrogation zone 113 associated with the one or more microfluidic channels 114. For ease of illustration, a single actuator 112 associated with the microfluidic channel 114 is shown and described. However, as disclosed herein with respect to Figure 2As described in the other figures, the microfluidic chip 100 includes between 10 and 2000 actuators 112. Those skilled in the art will understand that each particle processing unit 101 includes at least one actuator 112 or is associated with it. The actuation section 130 provides a suitable means to deflect target particles, non-target particles, or both target and non-target particles into one or more separation fluid paths 116, 118 downstream of the actuator 112. The characteristics of the target or non-target particles can be directed, deflected, switched, etc. to selected flow paths 106, 108. In some embodiments, the process of directing particles to selected flow paths 106, 108 can be performed on a particle-by-particle basis. The actuator 112 can use any of a variety of technologies to achieve separation of target particles from non-target particles. The various technologies include, but are not limited to, mechanical force, optical force, chemical force, thermal force, bubble-based force, dielectrophoretic force, piezoelectric force, acoustic force, sound wave-based force, magnetic force, valve-based force, or membrane-based force. One or more layers of the actuation section 130 can be formed at least in part from lithium niobate (LiNbO3), lithium tantalate, lead zirconium titanate (LZT), zinc oxide (ZnO), aluminum nitride, quartz, polyvinylidene fluoride (PVdF), or another piezoelectric material. In some embodiments, the piezoelectric layer can be formed as a thin film on top of a different layer.

[0092] In some embodiments, the actuation section 130 can be formed from a separate layer. The actuation section 130 can include an actuator layer 132, which includes one or more actuators 112 formed therein or thereon. For example, the acoustic wave actuators can be formed on the actuator layer 132 using conventional cleanroom etching and deposition techniques. In some embodiments, the actuator layer 132 can be sandwiched between adjacent layers 133 and 134. In some embodiments, for example, the adjacent layers 133 and 134 can help guide the acoustic waves to the microchannels 114. The actuator section 130 can include a treated particle layer 136 immediately below the cover layer 131. The treated particle layer 136 can have a transverse portion (i.e., perpendicular to the stacking direction 107) of the output channels 116 and 118 etched therein. In some embodiments, the actuator section 130 does not include a fluid distribution layer. In such embodiments, the treated particle layer 136 is considered a separate segment from the actuation section.

[0093] In some embodiments, the actuator 112 in the actuation section 130 may include a switching surface acoustic wave actuator. An acoustic wave actuator (such as an interdigital transducer or IDT) may be connected to an acoustic wave generator to generate acoustic energy coupled to the microfluidic channel 114, thereby deflecting particles into a selected outlet stream or channel. The IDT actuator may be configured to generate a traveling or streaming surface acoustic wave (TSAW) or pressure pulse in the fluid of the microfluidic channel 114. This pressure pulse may be used to drive a fluid slug into a selected region or direction of the microfluidic channel 114, or into selected output channels 116, 118. Alternatively, a pair of IDTs may be disposed on both sides of the microfluidic channel 114 at a switching site in a one-side manner. Examples of surface acoustic wave generators, IDTs, actuators, and arrangements of these elements relative to microchannels suitable for use in the present invention are described in more detail in U.S. Patent No. 10,646,870, entitled “MICROFLUIDIC DEVICE AND SYSTEM USING ACOUSTIC MANIPULATION,” published on May 12, 2020, the entire contents of which are incorporated herein by reference.

[0094] In some embodiments, the actuation section 130 may include one or more pressure pulse channels 138. The pressure pulse channel 138, such as a fluid buffer, may be located in the microfluidic channel 114 at a position opposite the actuator 112 to buffer or reduce the effect of the pressure pulse from the actuator 112. In turn, the absorption of this pulse can reduce the disturbance experienced by the fluid flow in the microfluidic channel 114 and can allow for faster laminar flow reconstruction and, therefore, faster switching times. In some embodiments, the actuation section 130 may include an acoustic attenuation element positioned between adjacent particle processing units 101. The acoustic attenuation element can absorb or attenuate the acoustic energy from the actuator 112 in the first particle processing unit 101 so that this energy does not interfere with or affect the fluid flow in a different particle processing unit 101. In this way, the acoustic attenuation element can acoustically isolate the actuators in different particle processing units 101 on the same microfluidic chip. In some embodiments, the attenuation element may include an air gap.

[0095] In some embodiments, the cover layer 131 of the actuation section 130 may include an optical interface layer. The cover layer 131 may enable observation, detection, or both of particles flowing within the device. In some embodiments, the cover layer 131 may include a window or window-like layer, thereby providing a transparent optical interface for the fluid beneath the cover layer 131. The cover layer 131 may act as a liquid barrier, i.e., the cover layer 131 may prevent water, oil, or other liquids from migrating from the interior of the chip 100 to the exterior, or vice versa. In some embodiments, the cover layer 131 may form part of one or more fluidic channel pathways through which fluids flow. Other features, such as electrodes, may also be applied to, integrated into, or included on the cover layer 131. As described in more detail below, in some embodiments, the cover layer 131 may include the actuator 112. The cover layer 131 may be formed from one or more materials, such as glass or plastic, configured to provide high transmittance of electromagnetic radiation wavelengths of interest (e.g., wavelengths of excitation light, scattered light, or fluorescence) for a particular application. In one exemplary embodiment, the cover layer 131 may allow transmission of light having wavelengths ranging from ultraviolet (UV) to near infrared (IR), or a range representing a sub-portion of the UV to near IR range, depending on design intent. In some embodiments, the cover segment 131 may include integrated optical features, such as a lens array, a metalens array, or other optical properties, to simplify alignment and / or optical and chip design. As a non-limiting example, the integrated optical features may be molded elements made of glass or plastic that comprise the cover segment 131.

[0096] In some embodiments, the total thickness of the massively parallel microfluidic chip 100 can range from 0.5 mm to 10 mm. In some embodiments, the thickness of an individual segment (i.e., the transfer segment 110, the particle focusing segment 120, or the actuation segment 130) can range from 10 microns to 1000 microns.

[0097] exist Figure 2-4B In the illustrated embodiment, the desired particles 60 are deflected by the action of the actuator 112. However, it will be understood that the system can act to deflect the undesired particles 50 while leaving the desired particles 60 substantially undisturbed. Such activity is referred to as "counter-sorting," and further descriptions and embodiments of particle processing systems and chips utilizing counter-sorting methods compatible with the present teachings can be found in U.S. patent application No. 17 / 723,236 filed on April 18, 2022, the entire contents of which are incorporated herein by reference. In some embodiments, the undesired particles 50 can be modified, for example, physically, thermally, or chemically to damage or destroy the undesired particles 50. The modification can be an alternative to or in addition to physically isolating the undesired particles 50 from the desired particles 60.

[0098] Within the chip stack, and in some embodiments, particles can flow toward (or away from) the imaging system (e.g., parallel or antiparallel along the optical axis 260 of the imaging system). In some embodiments, both the interrogation region 113 and the actuator 112 are located within the actuation section 130. In some embodiments, the actuator 112 can operate within the interrogation region 113. It should be understood that the interrogation region 113 and the actuator 112 can be located at different points in the chip stack or in different sections or layers. In addition, the particle flow and measurement performed using the microfluidic chip 100 taught herein are not limited to a direction toward or away from the imaging system, but can occur along another path, such as a horizontal flow path.

[0099] The pattern 144 of the particle processing units 101 within 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) flowing through some or all of the microchannels in the chip 100. Other examples include: optimization around mechanical design or constraints; isolation or placement of electrical components (such as surface acoustic wave generators); thermal isolation; avoiding mechanical, optical, electrical, or thermal crosstalk; and avoiding interference. In some embodiments, the pattern 144 of the particle processing units 101 can be selected to match the arrangement of optical components, such as a specific excitation source spacing (e.g., a laser beam array) 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 indicator), random, two-dimensional, axisymmetric, radial, concentric, other staggered polygons, or crystalline. In some embodiments, the geometric design of the microfluidic chip 100 (including the arrangement of the pattern 144 or the elements of each particle processing unit 101) can be selected to reduce autofluorescence from particles or other materials in the environment (including the materials and structures of the microfluidic chip 100 itself). In some embodiments, spatial filtering methods can be used in the detection system 220, as described below.

[0100] In some embodiments, the density of the interrogation region 113, the sorting monitoring regions 161 / 162, the particle focusing region 123, or the particle sorting region (i.e., the actuator 112) in the pattern 144 is in the range of 1 / cm 2 Up to 500 pieces / cm 2In some embodiments, the systems and methods taught herein include a chip with a small area occupied by each individual particle processing unit during observation or imaging, such as by the electromagnetic source system 210 or the detection system 220. The reduction in cross-section reduces the amount of "area" dedicated to or occupied by the portion of the particle processing unit that interfaces with external components, such as the electromagnetic source system 210 and the detection system 220. In other words, by reducing the area of ​​the particle processing unit 101 in the imaging plane of the electromagnetic source system 210, the detection system 220, or both, to approximately the size of the interrogation region 113 or the sorting-monitoring region 161 / 162, more particle processing units can be arranged 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 range from 100 to 1000, 100 to 500, 250 to 750, 500 to 750, or 500 to 1000.

[0101] In some embodiments, the channel geometry of the microfluidic channel 114 can be varied along the course of the channel to produce a desired effect on particles flowing therein or to reduce the effects of undesired impacts. For example, one or more of the width, height, cross-section, or other measurable parameters can be varied along the course of the channel 114 to affect particle velocity, aligned / directional particle positioning, particle concentration or dilution, temperature control, pressure, flow direction changes, enable chemical exposure, enable light exposure, enable conductivity or impedance measurements, or provide a separation monitoring layer.

[0102] The position of the interrogation region 113 in the actuation section 130 or another section can be selected to ensure reliable interrogation of microfluidic channel contents, such as particles. In some embodiments, the position can be measured as a distance from the cover layer 131. The position of the interrogation region 113 can correspond to the depth of focus 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., the spacing between microchannels) can be selected to ensure reliable interrogation of microfluidic channel contents, such as particles. The appropriate position of the interrogation region 113 within a single microchannel can be selected to ensure that the target (e.g., a single) particle of interest is properly isolated and can be measured independently of other particles. In one embodiment, the position of the interrogation region 113 enables measurement of a single particle. In some embodiments, it may be desirable to measure multiple particles. In some embodiments, the position of the interrogation region 113 enables measurement of a single "event," wherein the event is characterized by receiving an optical signal from one or more particles within a specified time span or coincidence interval.

[0103] In some embodiments, the segments of the microfluidic chip 100 can be produced individually and combined or assembled to form the microfluidic chip 100. To produce segments with very high feature density, a variety of production or manufacturing methods can be used to produce each segment, such as photolithography, additive manufacturing (e.g., 3D printing), sputtering, deposition, molding, embossing, printing, subtractive manufacturing (e.g., machining, milling, chemical etching, ion beam etching, electrical discharge machining), or other methods known to those of ordinary skill in the art to produce structures with materials, features, functions, and dimensions suitable for the desired purpose. In some embodiments, segments can be manufactured using a combination of different materials. Layers with optical or biological functions can also be provided by various manufacturing methods. Substrate-plus-additional-material methods can be used, such as conductive electrodes on non-conductive materials. Materials and coatings can be selected based on the specific application (e.g., for cell sorting), Good Manufacturing Practice (GMP), the desired sterilization or clean-in-place protocol (e.g., gamma irradiation, gas, steam, or other cleaning and sterilization methods), or any combination of these factors. Individual layers or segments can be made of one or more materials, including silicone, glass (e.g., UV fused silica, quartz, or borofloat), polymers (e.g., polydimethylsiloxane [PDMS], polymethyl methacrylate [PMMA], thermoplastic elastomers [TPEs], including styrenic TPEs or cyclic olefin copolymers [COCs]), metals, ceramics, alloys, or crystalline materials. Materials can be selected based on specific properties, such as electrical conductivity or impedance or acoustic transmittance. The material or surface of one or more segments can be prepared or treated to be suitable for cell measurement or sorting so that cells are not adversely affected as they travel through the system. In some embodiments, the material or surface of a segment can be treated to enhance the cells as they move through the assembly. In some embodiments, layers or segments closer to the optical interrogation and detection system (e.g., closer to the top surface of the chip) are made of a transparent material, while layers or segments farther away 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 manufacture) or a reflective material. In this way, the chip 100 maintains a high level of optical access in critical areas (i.e., near the interrogation region 113), while also benefiting from ease of production and lower costs in areas that do not necessarily require high optical access, such as the transfer section 110. In some embodiments, a high feature density can be achieved by having each particle processing unit 101 occupy a small area of ​​the microfluidic chip 100, such as the top surface of the measurement microfluidic chip 100 or the top surface of the measurement cover layer 131.

[0104] In some embodiments, the microfluidic chip 100 includes segments that can be configured to be disassembled (i.e., reversibly assembled) back into component parts (e.g., separate segments). In some embodiments, one or more bonding, bonding, fusion, or contact methods can be used to permanently connect segments to form a microfluidic chip 100 that cannot be disassembled. The microfluidic chip 100 can be cleanable, sterilizable, or reusable in whole or in part. For example, the example microfluidic chip 100 can be disassembled, wherein certain segments (e.g., the particle focusing segment 120 or the transfer segment 110) are discarded and replaced with new segments, while other segments (e.g., the actuator segment 130, which is together with or separated from the cover layer 131) can be configured to be sterilized and reused. Certain segments are more likely to be reused due to special or expensive materials (e.g., glass or plastic) or because additional components are integrated into the segment (e.g., surface acoustic wave electrodes deposited on a layer of the actuator segment 130).

[0105] In some embodiments, more than one segment or all segments may be formed in a single process using, for example, three-dimensional printing methods.

[0106] The microfluidic chip 100 may include more than Figure 1-4B . For example, the microfluidic chip 100 can include layers or sections that passively allow materials (e.g., fluids or particles) to flow through the layers or sections without change or influence. These layers or sections can function as "through-hole-like" (similar to through-holes in multi-layer printed circuit boards, including hollow channels or pores extending therethrough) to facilitate access or connection of pipes / fluid connections, electrical contacts, light pipes, mechanical actuators, or other chip aspects. In some embodiments, certain layers can be shared between stacked or layered sections of the microfluidic chip 100 to create shared commonalities, such as shared functions, shared materials, shared electrical connections, shared light pipes or connections, shared thermal properties, and more commonalities, thereby facilitating particle flow, focusing, detection, or sorting operations.

[0107] Any of the transfer section 110, the particle focusing section 120, or the actuation section 130 may include one or more material layers. The layers may include suitable materials, coatings, or chemical treatments to achieve the desired functions of the flowing particles and fluids, thereby enabling measurements such as optical or electrical measurements, manipulation of fluids or particles, further processing of fluids through manifolds, and collection, transport, or control of fluids so that the desired method can be performed. The vertical stack of layers and sections can be designed or manufactured so that optical, mechanical, electrical, or other signals can be transmitted through the layers in a controlled manner as desired. The material used for a given layer can be optically transparent or opaque, spectrally selective, or polarization-sensitive as desired. The material can have a specific refractive index or light absorption, reflection, or refraction properties. Certain layers (such as the cover layer 131) may include devices for generating or detecting light. The layers of the massively parallel microfluidic chip 100 can be 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 can transmit, attenuate, or block energy such as acoustic energy.

[0108] Figure 2 A 'left-right' particle sorting scheme is shown, wherein the output flow paths 106, 108 are shown in this cross-sectional view as being parallel and separated by 180 degrees in the plane of the chip 100 (i.e., when viewed from above). However, it should be understood that the orientation of the output paths 106, 108 is not limited to Figure 2 A 180-degree layout is shown, but the output paths may branch from the microfluidic channel 114 in any direction.

[0109] Figure 3AAn embodiment of a microfluidic chip 100 is shown, which can be described as a 'right-right' particle sorting scheme. In this embodiment, a sample fluid including particles 50, 60 flows upward along a first flow path 104 into a main microchannel 114 through a transfer section 110, while a sheath fluid flows along sheath fluid flow input paths 105a, 105b through the transfer section 110 into corresponding sheath channels 115a, 115b. The sample fluid including the particles contacts the sheath fluid and is focused by the sheath fluid in a particle focusing region 123 of a particle focusing section 120. The particles flow to an interrogation region 113, where they can be detected using a detection system, such as an optical detection system using a lens 222. Based on the detected signal (e.g., optical fluorescence, scattering, emission, or extinction) from the particles in the interrogation region 113, a control system, such as a computing device, can identify whether the particle is a desired particle 60 or an undesired particle 50. When a desired particle 60 is detected, an actuator 112 is actuated to deflect the particle to a first output path 106. When an undesired particle 50 is detected, the actuator 112 may take no action, allowing the undesired particle 50 to follow its natural flow path through the second output channel 108. In this embodiment, the two output microchannels 116, 118 are located in the same orientation relative to the microfluidic channel 114, but are "stacked" one above the other in the stacking direction 107. In other words, one output channel can pass over or under other output (or input) channels in the stacking direction (i.e., a line running through the chip in the stacking direction can pass through two or more microchannels).

[0110] In the Figure 3A In the embodiment of the chip of FIG. 1 , the output channels pass directly underneath each other as viewed from above the chip, where the relative angle between the output channels is zero degrees (i.e., zero degrees rotated about the stacking direction 107). However, it should be understood that the output channels 116, 118 can branch off from the microfluidic channel 114 with some relative angle between the channels ranging from zero degrees to 360 degrees. Figure 3Bshows an example of a similar "right-right" microfluidic chip 100 including angled branching channels. In this example, the four particle handling units 101 shown are visible looking down through the cover layer 131. In other words, the main microchannel 114 "flows out of the page" toward the viewer in the stacking direction 107. The second output microchannel 118 branches off from the main microchannel 114 at an angle 333 relative to the direction of branching from the first output microchannel 116. In addition, the orientation of the particle handling units 101 on the same chip can be rotated relative to the orientation of other particle handling units 101. In some embodiments, such relative rotation can simplify the manifold paths and thereby improve the packing density by positioning common microfluidic channels closer together, for example, by positioning the sample input paths 104 or output channels 116, 118 of several particle handling units 100 closer to each other.

[0111] 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. Sorting monitoring is a process that provides confirmation of the success or failure of a particular sorting or actuation operation. Once a sorting decision has been made and actuation has occurred, a sorting monitoring device (e.g., as part of a detection system) can measure the actual path of the particle relative to the expected or desired path. Figure 2 , lens 224 provides sorting monitoring for particles in the first output channel 116 , while lens 226 provides sorting monitoring for particles in the second output channel 118 .

[0112] Figure 4A An embodiment of a microfluidic chip 100 is shown, and a different sorting monitoring mechanism is provided. Figure 4A In the embodiment of the present invention, the microfluidic chip 100 includes an input electrical sensor 135 associated with a microfluidic channel 114 and an output electrical sensor 137, 139 associated with corresponding 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 sensor 137, 139 can detect the presence of particles in its corresponding output channel. The electrical sensor 135, 137, 139 can be operated to detect the presence of particles using the Coulter principle, measured conductivity (or interruption / interference of conductivity), resistivity or other methods. Electrical sensors 135, 137, 139 can be formed to use conductive traces arranged to be stacked throughout the chip, thereby realizing large-scale parallel particle measurement. The conductive traces can be supplied from the chip at one or more edges of the microfluidic chip 100 and thus connected to a control system.

[0113] The signals from the electrical sensors 135, 137, 139 can be received by a control system. The control system can compare the detected position of the particles in the output channel (based on the signals from the output electrical sensors 137, 139) with the expected position of the particles based on the previous sorting decision and actuation. This information can provide valuable feedback about the sorting success rate for the control system, and can become the basis for adjusting the sorting or detection parameters in real time or delayed to improve the sorting success rate. The control system can receive signals from the input electrical sensor 135, which can identify particle characteristics that can be used as the basis for sorting decisions. This information can be obtained by the electrical sensor 135 without using optical measurement technology, or can be obtained in addition to using optical measurement technology. In some embodiments, the signal from the input electrical sensor 135 can be used as a verification system to measure the measurement success rate using different detection systems (e.g., optical detection systems).

[0114] In some embodiments, the input electrical sensor 135 is co-located with the interrogation zone 113, allowing particles to be detected nearly simultaneously by the input electrical sensor 135 and any other detection system, such as an optical detection system. In other embodiments, the input electrical sensor 135 can be located at a different location on the flow path than the interrogation zone 113. In the figures, the electrical sensors 135, 137, 139 are depicted as being present in the actuation section 130. However, in other embodiments, the electrical sensors can be located in different layers within the same section or in different sections of the microfluidic chip 100.

[0115] Figure 4B An embodiment of a microfluidic chip 100 is shown that includes a particle handling unit 101 having a horizontal section 325 of a microchannel running parallel to the cover layer 131. In such an embodiment, the interrogation region 113 can be located at the horizontal section 325, where all or most of the fluid flows horizontally (i.e., transverse to the stacking direction), so that optical interrogation and detection are performed when the particles move perpendicular to the stacking direction 107 and perpendicular to the optical axis of any illumination system or detection system that detects the particles. In some embodiments, particle measurements are more consistent when the particles flow transverse to the optical axis of the optical detection and illumination system rather than along the optical axis, so using the horizontal section 325 may be advantageous. Note that although the interrogation region 113 is located in a portion of the microfluidic channel 114 that travels transversely to the stacking direction 107, the fluid travel of the particles throughout the chip is still primarily along the stacking direction 107. Because the flow is still primarily along the stacking direction 107, the microfluidic chip still has the advantages of massive parallelization by fitting a large number of particle handling units 101 onto a single chip.

[0116] Figure 5 and 6An embodiment of a microfluidic chip 100 is shown in which the actuator 112 is located in or on the cover layer 131 of the 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. Figure 6 As shown in the partial cross-sectional view of FIG, the actuator 112 in contact with the cover layer 131 can couple acoustic energy into the vicinity of the connection point between the input microchannel 114 and the output channels 116, 118. The actuator 112 can selectively deflect or direct the particles 50, 60 to the correct output channels 116, 118 by applying acoustic energy to the particles 50, 60.

[0117] return Figure 5 In some embodiments, the microfluidic chip 100 is formed from a circular substrate. In various embodiments, the microfluidic chip 100 may have a rectangular or square form factor (e.g., Figure 1 ), round or oval morphological factors (such as Figure 5 ) or any other suitable form factor that meets the considerations of a specific application. The transfer section 110 includes a sample inlet 124 for inputting a sample fluid including particles. In some embodiments, the sample fluid is collected in an on-chip sample fluid reservoir 111, from which the sample fluid can enter each microchannel in the plurality of microchannels 114. Similarly, the particle focusing section 120 may include a sheath fluid inlet 125, which enables the input of sheath fluid into the microfluidic chip 100. In some embodiments, the sheath fluid enters the on-chip sheath fluid reservoir 122, from which the sheath fluid can be introduced into the plurality of microchannels 114 to focus the particles in the sample fluid flow. In some embodiments, the sheath fluid inlet 125 is coupled to a manifold that distributes the sheath fluid to the focusing section 120 of each particle processing unit in the particle processing unit 101. The focusing section 120 may include a plurality of nozzles 312 to facilitate the introduction of sheath fluid into the sample fluid flow in the microchannel 114. In some embodiments, the nozzle can generate a jet-like flow of sample fluid into a surrounding sheath fluid volume.

[0118] Figure 7A and 7B An enlarged view of an embodiment of a transfer section 110 and a particle focusing section 120 including a plurality of particle focusing regions 123 is shown. Figure 7A As shown, the particle focusing region 123 can be integrally formed 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 protrusions 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 into a preferred orientation). Figure 7AAs shown, the sample fluid flows upward from the on-chip sample reservoir 111 through the interior of the layer 127 of the particle focusing section 120 forming the nozzle 312, and is discharged into the sheath fluid in the on-chip sheath fluid reservoir 122 through the nozzle 312. 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 forming the nozzle 312, and is discharged into the sheath fluid through the nozzle 312. The sheath fluid flows into the microchannel 114 under pressure and thereby acts on the sample fluid to focus particles in the flow within the microfluidic channel 114. Figure 7B A cross-sectional view of a microfluidic chip 110 is shown, wherein a particle focusing region 123 supplies sample to a plurality of parallel microchannels 114. In this figure, the streamlines of each sample stream are shown, each sample stream being narrowed and focused by pressurized sheath fluid. Each sample stream in each microchannel 114 is focused by sheath fluid in an on-chip sheath fluid reservoir 122.

[0119] In some embodiments taught herein, the output channels 116, 118 flow back down through the chip and exit the chip within or through the transfer section 110. However, in some embodiments, such as Figure 5 In the embodiment of the chip 100, the actuation region 130 may include a retentate outlet 128 connected to the first (or retentate) output channel 118 and a waste outlet 126 connected to the second (or waste) output channel. In some embodiments, the outlets 126, 128 may pass through a sidewall 130a of the actuation section 130. The sidewall 130a may be an outer wall or periphery of the chip 100 perpendicular to the top surface (e.g., the outer surface of the cover layer 131) through which optical interrogation and detection are performed.

[0120] The segments of the microfluidic chip 100 can be prepared or manufactured separately. In some embodiments, each segment (e.g., the transfer segment 110, the particle focusing segment 120, and the actuation segment 130) can include one or more alignment holes 315. Figure 8As shown, the alignment holes enable the separated segments 110, 120, 130 to be aligned and assembled into the final microfluidic chip 100. For example, the alignment post 310 can be inserted into the alignment hole 315 in one or more segments. Although the post 310 shown passes through all segments, the post 310 can be permanently fixed to one or more of the segments or produced as part thereof. In this case, the remaining segments can be positioned so that the alignment holes 315 are aligned with the post 310 and slid onto the post to connect the segments to form the chip 100. In some embodiments, the post 310 can be detachable from the segment of the microfluidic chip 100 before the assembly of the final microfluidic chip 100. For example, the post 310 can be disassembled after being inserted into the alignment hole 315. For example, after the segments are connected, the post 310 may break off from the underlying segment during the assembly of the microchip 100.

[0121] Figure 9 An embodiment of a microfluidic chip 100 is shown that includes a central output channel 118 connected to the output channels of each individual particle processing unit 101 of the microfluidic chip. In some embodiments, the interrogation area and the actuator are positioned at different azimuthal positions around the center of the chip 100. The actuator 112 deflects the desired particles into the output channel 118 that feeds toward the center of the chip 100. At the center of the chip 100, the output channels 118 merge into a single output channel 118a that flows downward through the actuation section 130, the particle focusing section 120, and the transfer section 110. The sorted particles in the output channel 118 then leave the chip through the retentate outlet 128. In some embodiments, the retentate outlet 128, the sample inlet 124, and the sheath inlet 125 are shown as passing through the bottom surface of the microfluidic chip. In other embodiments, one or more of these inlets and outlets can be positioned on the sidewall or top surface of the chip 100 (i.e., through the cover layer 131).

[0122] Figure 10A and 10B Schematically illustrates various fluid manifolds or tubing embodiments suitable for the microfluidic chip 100 taught herein. The fluid manifold can extend through one or more of each of the segments described above to provide fluid coupling to each segment. The fluid manifolds 400, 400' may include a microfluidic channel assembly configured to receive particles from a particle source supply (i.e., an off-chip sample fluid reservoir 244) via one or more input fluid communication elements and to send the particles to a collection system (including, for example, an off-chip collection reservoir 216, 218) via one or more output fluid communication elements. Figure 10A Possible layering of chip substrates that can be used to implement interlayer manifolds to achieve the desired fluid flow (and particle transport) throughout the system is demonstrated. Figure 10A and 10B In the figure, the black box indicates the position (i.e., fluid connection point) where the fluid is divided into multiple paths from a single path or where the fluid is merged into a single path from multiple paths. In this schematic diagram, the fluid paths that intersect but do not have black boxes at the intersection will not intersect in the actual chip to form fluid connection points. The microfluidic chip 100 is pipelined in this way, that is, each particle processing unit in the multiple particle processing units 101 receives the particle-carrying sample fluid from the sample fluid reservoir 244 (located on the chip or separated from the chip) along the input fluid path 104 by appropriate manifolding means, and sends the particles 50, 60 to the interrogation area 113 where they are interrogated and characterized. In some embodiments, the particles 50, 60 can be deflected to one of the multiple outlet paths 106, 108 on a one-by-one basis by a suitable sorting mechanism such as an actuator 112.

[0123] The fluid manifold 400 may include distribution layers 117a-e that facilitate the segmentation or merging of fluid pathways. For example, the distribution layer 117a can segment the input fluid path 104 from the reservoir 244 into multiple input fluid paths 104a, 104b that travel within the distribution layer 117 to separate particle processing units 101. Similarly, the distribution layer 117b can merge the fluid from the output paths 108a, 108b of different particle processing units 101 into a single output path 108 that flows to the collection reservoir 218. The distribution layer 117c merges the fluid from the output paths 106a, 106b of different particle processing units 101 into a single output path 106 that flows to the collection reservoir 216. The distribution layer 117d segments the fluid after it passes through the actuator 112 into a first output path 106a, 106b and a second output path 108a, 108b. The distribution layers 117a-e can be formed within any individual segment, including the transfer segment 110, the particle focusing segment 120, or the actuation segment 130. The distribution layers 117a-e can also span segments or act as bridges between different segments. In some embodiments, the deflected (and undeflected) fluid from each microfluidic cytometry element can be collected using a manifold within the chip stack and collected in a suitable container (or connected to another process).

[0124] Figure 10B A fluid manifold 400' is shown that also uses sheath fluid from the sheath fluid reservoir 215 as a means of assisting in the following functions: sample transport; providing hydrodynamic focusing; reducing contact of particles with the inner walls of the channel; reliably singulating / manipulating or otherwise distributing particles for purposes of transport, measurement, sorting, concentration / dilution, etc., including combinations of more than one of these functions. In addition, the sheath fluid can be used to support or modify the biochemical state of the sample. Figure 10BA sheath fluid path 105 is added to the chip 100 , and the distribution layer divides the sheath fluid path 105 into separate sheath flows 105 a , 105 b flowing to each individual particle processing unit 101 in the chip 100 . Figure 10B The distribution layer 117a in the also serves as the location where the sheath fluid meets the sample fluid and acts (e.g., hydrodynamic focusing can occur). Note 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, Figure 10B The distribution layer 117a in may include several stacked or layered substrate layers to enable contouring of the microfluidic channels 114 formed therein to provide focusing or directing of the fluid as desired.

[0125] Figure 10A and 10B An embodiment is shown having a single sample fluid reservoir 244 and a single sheath fluid reservoir 215. However, it should be understood that in some embodiments, the microfluidic chip 100 can interface with multiple fluid reservoirs 244, 215. For example, the microfluidic chip 100 can include sub-manifolds each associated with its own sample fluid reservoir 244 and sheath fluid reservoir 215. In some embodiments, the microfluidic chip 100 can be coupled to a fluid cartridge comprising 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 can be configured for removable engagement with a particle processing system 200 as described below. In some embodiments, the fluid system (i.e., the fluid microchannels and manifold elements for fluid transport) can be provided as a sealable cartridge or chip that can operably seal all fluid contact surfaces used during particle processing (i.e., the sealable cartridge or sealable chip can be operably sealed during any particle processing operation so that fluid does not enter or leave the chip during the particle processing operation). The cassette may also include onboard output reservoirs 216 , 218 .

[0126] In some embodiments, the microfluidic chip can be disposable. This can provide benefits in combination with a sealable fluid contact surface, i.e., the operator can avoid contacting the fluid in the chip (in order to improve biosafety), and the fluid avoids contamination from external factors during processing. The sample can be taken out from the sorted sample chamber on the chip after processing, and the chip can be discarded to avoid cross contamination of samples or sheaths caused by different experimental runs. In other embodiments, the microfluidic chip 100 can be formed by a material that can be sterilized so that the microfluidic chip 100 can be reused. For example, a material that can withstand sterilization treatment can be selected, including one or more of ethylene oxide, ultraviolet light, or high temperature and high pressure. As described below, the microfluidic chip 100 can be detachable and engageable with a particle handling system.

[0127] According to an embodiment of the present disclosure, a microfluidic particle analysis and / or sorting system 200 including or operatively coupled to a massively parallel microfluidic chip 100 may have a wide range of applications as a cell sorting platform: for sex preselection of mammals by sex-based sperm sorting; as a therapeutic medical device for implementing cell-based therapies; for assisting clinical diagnosis in monitoring human or other animal diseases; or for one or more drug development applications.

[0128] exist Figure 11 , an ultra-high throughput particle processing system 200 suitable for implementing an illustrative embodiment of the present disclosure is schematically shown. 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 sample fluid including particles to the transfer section 110, wherein the sample fluid is divided into a plurality of fluid input paths 104, each corresponding to a particle processing unit. Along each fluid input path 104, the particles in the fluid are focused in the particle focusing section 120 and reach an interrogation region. In some embodiments, the interrogation region is located in the actuation section 130. The electromagnetic source system 210 is configured to illuminate the interrogation region in each microchannel of the plurality of microchannels. The detection system 220 is configured to receive light that has been emitted or scattered from the particles in the interrogation region, the light indicating a characteristic or identity of the particles. The detection system 220 simultaneously receives and processes light from each of the plurality of particle processing units in the chip 100. The light separation system 205 can include one or more spectrally selective elements, such as dichroic beam splitters or reflectors, and can overlap portions of the illumination light path from the electromagnetic source system 210 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 the operation of the actuators in the actuation section 130 to selectively direct particles into the first fluid output path 106 or the second fluid output path 108. Particles in the first fluid output path 106 flow into the first output reservoir 216 (sometimes referred to as the "retentate" reservoir), while particles in the second fluid output path 108 flow into the second output reservoir 218 (sometimes referred to as the "waste" reservoir).

[0129] The computing device 150 can monitor, measure, calculate, characterize, and take the necessary steps to command and control certain components within the system to change their state and or change the path of one or more particles. The computing device 150 may include a computer with a processing unit, or may include another electronic device and may communicate with one or more other similar or different processors to perform the necessary functions required. In some embodiments, the computing device 150 may utilize one or more sensors to enable reliable, predictable, accurate, and repeatable actions. In various embodiments, the computing device 150 may be partially or fully integrated into the microfluidic chip substrate stack.

[0130] The particle processing system 200 can be configured, sized, or adapted to analyze, sort, and / or process (e.g., purify, measure, isolate, detect, monitor, and / or enrich) particles (e.g., cells, microparticles, nanoparticles, molecules, etc.). For example, the system 200 can be a cell counter, a cell purification system, etc., although the present disclosure is not limited thereto. Rather, the system 200 can take a variety of forms, and it should be noted that the systems and methods described can be applied to other particle processing systems.

[0131] Figure 12 A schematic side view of a particle processing system 200 is shown, specifically focusing on an electromagnetic source system 210, a detection system 220, and an optical separation system 205. The electromagnetic source system 210 is used to precisely illuminate particles within multiple channels of a massively parallel microfluidic chip 100. The electromagnetic source system 210 can include one or more light sources 212 and one or more beam shaping optics 214, 217. For example, the light source 212 can be a single source that outputs light at a large aperture, or multiple discrete sources that individually output light. Similarly, the beam shaping optics 214, 217 can include a single, integral optical element, such as a fixed microlens array or a single macrolens (e.g., a microscope objective), or can include multiple discrete optical devices, such as individual microlenses that can be individually positioned transverse to the optical axis or multiple optical devices positioned 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 zones 113 and, in embodiments employing sorting monitoring, the multiple sorting monitoring zones 161 / 162.

[0132] In some embodiments, the particles in the microchannel 114 are reliably illuminated in an epi-illumination manner. The illumination light interacts with the particles to generate an optical signal that can be measured by the detection system 220 based on one or more of fluorescence, reflection, scattering, or extinction. As described below in Figures 13A-13FAs described in more detail in , the electromagnetic source system 210 can provide an illumination beam or multiple illumination beams that can be flood illuminating (i.e., a wide beam of relatively uniform intensity), split (e.g., through multiple elements, such as multiple beamsplitters), scanned / moved, multi-source (e.g., multiple laser sources, such as one or more vertical cavity surface emitting lasers (VCSELs) where multiple sources can be split), turned on or off as required, modulated periodically or otherwise (e.g., randomly), or any combination of the foregoing. Excitation sources of different wavelengths can be used as needed for a specific application, where specific properties of the particles of interest can be interrogated and measured. Excitation of particles in the microfluidic chip 100 can be achieved using one or more transmissive or reflective optical elements, such as any combination of one or more lenses, mirrors, optical fibers, tapered optical elements, diffraction elements, spectral elements, plasmonic elements, tapered optical elements. The beam shaping optics 217 may include a large format lens (such as the lens described in U.S. Patent No. 10,215,995, issued on February 26, 2019, and incorporated herein by reference in its entirety), a microscope objective (such as from Mitutoyo Corporation), or a multi-lens array (such as the microlens array and system described in U.S. Patent No. 10,190,960, issued on January 29, 2019, and incorporated herein by reference in its entirety). The beam shaping optics 217 can focus light onto the interrogation area 113 or the sorting monitoring area 161 / 162 of the microfluidic chip 100 and can receive light from the device to achieve an appropriate field of view to meet the needs of multiple microfluidic measurement sites within the device. Additional beam shaping optics 217 (not shown) may include an optical aperture, pinhole, template, or mask that allows only specific portions of the top surface of the cover layer 131 to be optically observed. In some embodiments, the optical aperture can provide spatial filtering. The optical aperture can be fabricated on the chip or formed as a separate layer attached to or mated with the chip 100. In some embodiments, the optical aperture can be provided in a separate imaging plane. In some embodiments, the 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 sub-beams.

[0133] The detection system 220 includes one or more detectors 223 and one or more beam shaping optics 217, 225, 224, 226. The detection system 220 collects light reflected, scattered, fluorescent, or quenched (i.e., light signals attenuated by the presence of particles) from the particles and projects the light signals onto the sensors of the detectors 223 through appropriate optical elements (including spectrally selective elements, spatially selective elements, or both spectrally and spatially selective elements). Note that in some embodiments, some optical elements, such as Figure 12The beam shaping optics 217 in FIG. 2 may be a common element of both the electromagnetic source system 210 and the detection system 220 .

[0134] The light separation system 205 may include one or more spectrally selective elements. For example, the spectrally selective element may include a dichroic mirror that transmits or reflects light depending on the wavelength of the light.

[0135] Figure 12 The components of the electromagnetic source system 210 and the detection system 220 are shown as discrete components separate from the microfluidic chip 100. However, in some embodiments, certain components of these subsystems can be integrated directly into a stacked or layered configuration of the microfluidic chip 100. For example, in some embodiments, the beam shaping optics 217 can be formed directly on the top surface of the cover layer 131.

[0136] exist Figure 12 In the embodiment shown in , the light source 212 includes a laser or a broadband light source, and the optical element is a microlens array 214. The beam from the laser or broadband source fills the entire collective aperture of the microlens array, and the microlens array splits the beam into sub-beams. The sub-beams are reflected from the spectrally selective reflector of the light separation system 205 and directed to the second microlens array 217. The second microlens array 217 focuses the illumination light onto the interrogation area 113 or the sorting monitoring area 161 / 162 in the microfluidic chip 100. In various embodiments, the light source 212 can be provided as one or more monochromatic light sources, one or more polychromatic light sources, or a mixture of monochromatic and polychromatic light sources. Although Figure 12 The beam shaping optics 217 are depicted as a microlens array, but the beam shaping optics 217 may include one or more lenses, mirrors, filters, including optical elements that may segment or split the beam into sub-beams.

[0137] Particles flowing in the microchannel interact with the illumination light and generate a light signal. This light signal is collected by a microlens array 217 and projected to a light separation system 205. The light passes through a spectrally selective reflector to reach the other beam-shaping optics of a detection system 220, including an optical filter 225 that spectrally selects (or "cleans") the light signal and a light collector 224, such as a microlens array or diffraction element, that focuses the light in preparation for reception by a detector 223. In some embodiments, the optical filter 225 may comprise a bandpass filter centered at 530 nm. In some embodiments, the detection system 220 may utilize an array of elements for spectral selection and detection. When a beam intersects particles, examples of light signals that may be generated in optical particle analysis, cell counting, and / or sorting include, but are not limited to, light extinction, angle-dependent light scatter (forward and / or side scatter), and fluorescence. Light extinction refers to the amount of electromagnetic radiation or light extinguished, absorbed, or blocked by a particle. Angle-dependent light scatter refers to the fraction of electromagnetic radiation that is scattered or bent away from or toward the incident electromagnetic radiation beam at each angle. Fluorescent electromagnetic radiation is electromagnetic radiation that is absorbed and / or scattered by molecules associated with a particle or cell and re-emitted at a different wavelength. In some examples, fluorescence detection can be performed using intrinsically fluorescent molecules.

[0138] exist Figure 12 In the illustrated embodiment, microlens array 224 focuses light into another beam-shaping optical element of the detection system in the form of a fiber bundle or array 226. In some embodiments, each individual fiber in array 226 is associated with each element of microlens array 224. Fiber array 226 carries light to the sensors of detector 223. In some embodiments, detector 223 may comprise a charge-coupled device (CCD), and light from individual interrogation regions 113 or sorted monitoring regions 161 / 162 may be directed to different pixels or groups of pixels within the CCD. In some embodiments, detector 223 may comprise photomultiplier tubes (PMTs), such as silicon PMTs, arranged in a one-dimensional or two-dimensional array such that light from individual interrogation regions 113 or sorted monitoring regions 161 / 162 is directed to different PMTs in the array. In some embodiments, fiber scrambling or other crosstalk reduction techniques may be employed to reduce the amount of unwanted light reaching each sensor of detector 223, such as the crosstalk mitigation techniques described in U.S. Patent No. 9,335,247 to Sharpe et al., issued May 10, 2016, which is incorporated herein by reference in its entirety.

[0139] In some embodiments, the electromagnetic source system 210 and the detection system 220 can achieve optical excitation and detection by interfacing planar layers and eliminating a large number of optical elements to relay light for excitation and or detection. For example, the light source 212 can include a VCSEL substrate having multiple excitation sources (i.e., VCSELs) that is positioned adjacent to the top surface of the cover layer 131 of the microfluidic chip 100. The detector substrate is also positioned so that the excitation light can be delivered to the chip 100 and the detection light can be received from the chip using appropriate optical 'transparency'. In some embodiments, a 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 enables particle measurement from within the chip 100.

[0140] Figures 13A-13F Side views of various embodiments of an electromagnetic source system 210 interacting with a microfluidic chip 100 according to the present disclosure are shown. The embodiments are presented to provide an overview of various non-limiting ways to perform precise illumination of various locations within a microfluidic chip, such as the interrogation area 113 or the sorting monitoring areas 161 / 162. Figure 13A In the embodiment of the present invention, the light source 212 of the electromagnetic source system 210 includes a laser array. The laser array can be arranged into a one-dimensional or two-dimensional array. In some embodiments, the laser array can include a combination of a vertical cavity surface emitting laser (VCSEL) and a collimating and focusing optical device (i.e., a beam shaping optical device 214) to match the spatial geometry of the pattern 144 of the particle processing unit in the microfluidic chip 100 to the illumination space profile. In some embodiments, the laser array can be installed in a single module. In one embodiment, the laser array can include a VCSEL laser array arranged in a 16x 16 square grid (a total of 256 sources) using a gallium nitride (GaN) source, such as described in Masaru Kuramoto et al., entitled "Watt-class blue vertical-cavity surface-emitting laser arrays", "Applied Physics Express", 12, 091004, 2019, the entire content of which is incorporated herein by reference.

[0141] Figure 13B A light source 212 is shown comprising a plurality of individual lasers arranged in an array. The lasers may be individually packaged and arranged in close proximity to form a pattern that complements the pattern 144 of the particle processing unit.

[0142] Figure 13CA light source is shown that includes a shaped laser beam 212a. In this embodiment, the laser beam 212a illuminates a beam shaping optical device 214 in the form of a segmented mirror. The segmented mirror can be a static optical element that produces an array of laser beamlets in a one-dimensional or two-dimensional pattern that complements the pattern 144 of the particle processing unit in the microfluidic chip. In some embodiments, the beam shaping optical device 214 can be segmented into a refractive element (e.g., a prism or grating) rather than a mirror.

[0143] Figure 13D Shows a Figure 12 A similar arrangement is shown, but omitting the beam shaping optics 214 placed between the light source 212 and the light separation system 205. In this embodiment, the light source 212 may comprise a collimated laser beam that is not separated into beamlets before passing through the beam shaping optics 217 in the form of a lenslet array. The lenslet array generates one- or two-dimensional focused beamlets for illuminating the interrogation region 113 or the sorting monitoring regions 161 / 162.

[0144] exist Figure 13E In the embodiment, the light source includes an array of illumination fibers 212b. Each illumination fiber 212b is associated with an element of beam shaping optics 214 (e.g., a single microlens in a microlens array). In some embodiments, the beam shaping optics 214 can collimate the light from the illumination fibers 212b. The illumination fibers 212b can be aligned with the beam shaping optics 214. Figure 13A The illumination functions with similar collimation, reflection, and illumination characteristics to the illustrated embodiment. The illumination light is reflected from the light separation system 205 and directed through the beam shaping optics 217, which may include a microlens array. The light from each optical fiber 212b is focused to illuminate the corresponding interrogation region 113 or sorting monitoring region 161 / 162.

[0145] Figure 13FAn embodiment is shown in which the light source includes a shaped light beam 212a, such as a shaped laser beam. For example, the light source 212a may include a laser beam that has been expanded to fill the aperture of the beam shaping optics 214. The beam shaping optics 214 may include a dynamic beam splitter that divides the shaped light beam into segments corresponding to the individual interrogation regions 113 or sorted monitoring regions 161 / 162 in the microfluidic chip 100. The dynamic beam splitter, such as a dynamic laser beam splitting element, may shape or direct the portions of the light source 212 toward the microfluidic chip stack. The beam splitter may utilize dynamic technologies, such as digital light processing (DLP), digital micromirrors, microelectromechanical systems (MEMS), or other dynamic diffraction, refraction, or reflection technologies, to divide the shaped light beam into segments. In addition to splitting or segmenting the light source 212 into beamlets, the dynamic beam shaping optics 214 may move the beamlets to assist with alignment, particle tracking, focusing, or other methods for improving measurement accuracy and precision.

[0146] Figures 14A-14D Various embodiments of a detection system 220 that is compatible with the systems and methods taught herein are shown. Figures 14A-14D In each of the figures, detection light from particles travels from light separation device 205 to the bottom surface of spectrally selective element 225. Spectrally selective element 225 cleans the light by cleaning stray light that is not of wavelengths of interest, including, for example, the wavelength of illumination light in some embodiments.

[0147] exist Figure 14A In the embodiment of the present invention, light emitted or reflected from the interrogation area of ​​the microfluidic chip passes through one or more beam shaping optical devices 224 where it is focused. The beam shaping optical device 224 may include a microlens array or a diffraction element. The focused light then passes through a spatial selection element 228. The spatial selection element 228 may include a pinhole, a knife edge, or other optical element that accepts the desired light (or blocks a portion of the received undesired light). The spatial selection element 228 can ensure that the light is cleanly coupled into the first end of the fiber bundle or array 229. The second end of the optical fiber 226 is coupled to the detector 223 (not shown). The detector 223 may include multiple detection elements, such as individual photomultiplier tubes (PMTs) or individual pixels in a CCD. In some embodiments, the optical fibers 226 can be associated with individual detection elements on a one-to-one basis. In some embodiments, each optical fiber 226 can transmit light to multiple detection elements, although each detection element can be associated with one optical fiber 226 to improve the isolation between the received light from different particle processing units.

[0148] Figure 14BA detection system 220 is shown in which light received from an interrogation region of a microfluidic chip 100 passes through a spectrally selective element 225 and beam shaping optics 224. The beam shaping optics 224 focus the light directly onto individual detection elements of a detector 223 (which may also be referred to as a sensor array). Figure 14B The spatial selection element 228 is not shown, but such an element may be used in this embodiment.

[0149] exist Figure 14C In the embodiment of the present invention, the received light passes through a spectral selective element 225 that blocks undesired wavelengths and then passes through a spectral separator 221, such as a grating or a prism. The spectral separator 221 can disperse the light according to wavelength so that received light of different wavelengths is spatially directed to separate detection elements or different locations on the same detection element. In this way, the spectral separator 221 can act as a combined spectral and spatial selective element because it directs specific portions of the received light from the particle to the appropriate detector element. Figure 14C As shown, the light beam is split into two focal points on the detector 223 such that the positions of the two focal points are separated on corresponding detection elements having extended receiving areas.

[0150] Figure 14D An embodiment of a detection system 220 including a separate detector 223 is shown. Received light from the interrogation zone 113 or the sorting monitoring zones 161 / 162 is directed to a beam splitter 227. The beam splitter 227 can split each received light beam into two beams traveling in different directions. In some embodiments, the beam splitter 227 can separate the received light based on the spectral properties of the light (e.g., transmitting light below a wavelength cutoff frequency and reflecting light at or above the cutoff frequency or wavelength). In some embodiments, the beam splitter 227 can split the light based on properties such as polarization. In some embodiments, the beam splitter 227 can split the received light without regard to the properties of the light, such as in the case of a 50 / 50 partially reflecting beam splitter that splits the received light into two beams of equal intensity. One split beam of the received light passes through a first beam shaping optic 224a and is focused along a first path onto a first detector 223a, while the other split beam of the received light passes through a second beam shaping optic 224b and is focused onto a second detector 223b.

[0151] In some embodiments, the detection system 220 can detect light emitted at different angles from a single interrogation region 113 or sorting monitoring region 161 / 162, respectively. For example, the angular light collection geometry may differ between scattering and fluorescence measurements. This fact can be used to identify particle properties (and ultimately the identity or type of the particle) based on the difference between scattered light relative to fluorescence, as measured from different angular positions, according to the light-particle interaction described by Mie or Rayleigh theory.

[0152] As described above, the beam shaping optics 217 may include an array of microlenses, wherein the microlenses of the array are associated one-to-one with the interrogation zone 113, the sorting monitoring zone 161 / 162, or both. In some embodiments, the microlenses of the array may be associated with more than one interrogation zone 113, the sorting monitoring zone 161 / 162, or both. For example, each microlens may be associated with two, three, four, five, six, seven, or eight light emitting zones, such as the interrogation zone 113 and the sorting monitoring zone 161 / 162. In some embodiments, the beam shaping optics 217 may include lenses that are appropriately formatted to collect light from multiple interrogation zones 113, the sorting monitoring zones 161 / 162, or both. Figure 14E As shown. The lens can function to accomplish one or more of the following: collect, collimate, and focus light received from particles in multiple interrogation regions 113 or sorting monitoring regions 161 / 162. The lens can focus light onto individual elements (e.g., individual pixels or individual photomultiplier tubes) of the detector 223. In some embodiments, additional lenses or other beam shaping optics can be used to relay light from the chip 100 to the detector 223. As described above, crosstalk mitigation techniques can be used. The beam shaping optics 217, which includes large lenses for collecting light from multiple locations on the chip, are suitable for use with devices related to Figures 14A-14D In some embodiments, beam shaping optics 217 can focus illumination light from electromagnetic source system 210 onto separate interrogation region 113, sorting monitoring regions 161 / 162, or both.

[0153] Figure 15 1 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 device. 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).

[0154] The 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), and the like. For example, the memory 156 included in the computing device 150 may store computer-readable and computer-executable instructions 560 or software (e.g., instructions for processing particles in method 1100 as described below) for implementing the operations of the computing device 150. The computing device 150 also includes a configurable and / or programmable processor 155 and an associated core 504, and optionally, one or more additional configurable and / or programmable processors 502' and associated cores 504' (e.g., in the case where the computer system has multiple processors / cores) for executing the computer-readable and computer-executable instructions or software stored in the 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. One or both of processor 155 and processor 502' may be configured to execute one or more instructions described in relation to computing device 150.

[0155] Virtualization can be employed in computing device 150 so that infrastructure and resources in computing device 150 can be dynamically shared. A virtual machine 512 can 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 can also be used with one processor.

[0156] 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 a combination thereof.

[0157] A user may interact with computing device 150 through a visual display device 514 (eg, a computer monitor) that may display one or more graphical user interfaces 516. A user may interact with computing device 150 using a multi-touch interface 520 or a pointing device 518.

[0158] The 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) for implementing exemplary embodiments of the present disclosure. For example, the exemplary storage device 526 may include instructions 560 or software routines to enable data exchange with or operational control of the detector 223 or the light source 212. The storage device 526 may also include instructions 560 or software routines to perform particle processing methods such as method 1100.

[0159] The computing device 150 may include a communication interface 554 configured to interact 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 over 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 foregoing. In an example embodiment, the computing device 150 may include one or more antennas 522 to facilitate wireless communication (e.g., via a network interface) between the computing device 150 and the network and / or between the computing device 150 and system components (e.g., electromagnetic source system 210, detection system 220, or a pump operatively connected to fluid reservoirs 215 / 215 / 216 / 218). Communications interface 554 may include a built-in network adapter, a network interface card, a PCMCIA network card, a cardbus 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 capable of communicating and performing the operations taught herein.

[0160] The computing device 150 may run an operating system 510, such as various versions of Operating systems, different distributions and Operating system, versions for Macintosh computers An embedded operating system, real-time operating system, open source operating system, proprietary operating system, or other operating system capable of running on computing device 150 and performing the operations taught herein. In an exemplary embodiment, operating system 510 can run in native mode or emulated mode. In an exemplary embodiment, operating system 510 can run on one or more cloud machine instances.

[0161] Figure 16A flow chart of a method 1100 for fabricating a microfluidic chip according to some embodiments taught herein is shown. The method 1100 includes aligning a transfer section 110 with a plurality of alignment holes 315 in a particle focusing section 120 using a plurality of alignment posts 310 (step 1102). The method 1100 includes bonding the transfer section 110 to the particle focusing section 120 (step 1104). The method 1100 includes aligning the actuation section 130 with 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.

[0162] Figure 17 A flow chart of a method 1200 for processing particles using a microfluidic chip 100 according to some embodiments taught herein is shown. Method 1200 includes flowing a sample stream including particles 50, 60 through a plurality of microchannels 114 formed by a plurality of segments 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. Method 1200 includes focusing particles 50, 60 in each of the plurality of microchannels using a particle focusing segment 120 in the plurality of segments 110, 120, 130 (step 1204). Method 1200 also includes detecting particle characteristics of the particles 50, 60 flowing through a plurality of interrogation zones 113, which, in some embodiments, are located in an actuation segment 130 in the plurality of segments (step 1206). Each interrogation zone 113 is associated with a microchannel 114. For example, detecting the particle characteristic may include illuminating the particles 50, 60 in 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 in turn emit or scatter light, or extinguish or absorb the illuminating light, and this produces an optical signal corresponding to the particle characteristic that can be detected by the detection system 220. In some embodiments, the particles 50, 60 can flow through a plurality of microsorters (on a layer) in series with a plurality of actuators 112 in series. The method 1200 includes sorting the particles using the actuator 112 associated with each microchannel 114 in response to the detected particle characteristic (step 1208). For example, the computing system 150 can 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 that should be sorted into the retentate channel 118 , the computing system 150 controls the actuator 112 to deflect the particle 60 into the retentate channel 118 .

[0163] Figure 18AA cross-sectional view schematically illustrating a microfluidic chip 100 according to the present disclosure is shown. Hydrodynamic focusing occurs in part during a transition from a first orientation relative to the optical illumination and detection elements. The microfluidic chip 100 is an embodiment of a 'right-right' particle sorting scheme. Figure 18A As shown, a sample fluid including particles 50, 60 flows upwardly along a fluid input path 104 through a transfer section 110 into a main microchannel 114, while a sheath fluid flows through the transfer section 110 along sheath fluid flow input paths 105a, 105b into respective sheath channels 115a, 115b. The sample fluid including particles contacts the sheath fluid and is focused by the sheath fluid in a particle focusing region 123'. Hydrodynamic focusing occurs in part during a transition from a first direction (e.g., a vertical direction, a direction parallel to the optical axis 260, and / or a direction along the stacking direction 107) to a second direction (e.g., a horizontal direction, a direction perpendicular to the optical axis 260, and / or a direction along the stacking direction 107) and is in part due to the addition of sheath fluid to the sheath channel 115b after the particles transition to the second direction. The actuator section 130 may include a treated particle layer 136 immediately 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 fluidically coupled to the output channels 116, 118. In the treated particle layer 136, the flow direction of the particles is perpendicular to the optical axis 260 used by the illumination or detection system at or near the interrogation area 113. The particles flow to the interrogation area 113, where they can be detected using a detection system, such as an optical detection system using a lens 222. Based on the detected signal (e.g., optical fluorescence, scattering, emission, or extinction) from the particles in the interrogation area 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. When a desired particle 60 is detected, an actuator 112 (e.g., such as a Figure 22B 106) may take no action and allow the desired particles 60 to follow a natural flow path through the first output channel 106. When an undesirable particle 50 is detected, the actuator 112 (e.g., Figure 22B 106) is actuated to deflect the particles to the second output path 108. In some embodiments, the second output channel 108 can be connected to the natural flow path and the first output channel 106 can be a deflected flow channel. Figure 22B 1 (shown) can be actuated to deflect desired particles 60 to first output channel 106. Actuator 112 can remain inactive, allowing undesired particles 50 to flow through second output channel 108 along a natural flow path.

[0164] Figure 18B1 shows a schematic cross-sectional view of a microfluidic chip 100 according to the present disclosure in relation to optical illumination and detection elements. Figure 18B What is shown, and Figure 18A Compared to the microfluidic chip 100 of FIG. 1 , the microfluidic chip 100 includes a particle focusing section 120. The particle focusing section 120 allows hydrodynamic focusing to occur in a particle focusing region 123 along the stacking direction 107 and then transition from a vertical direction to a horizontal direction in which 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 the particle focusing region 123′.

[0165] Figures 18C-18E A cross-sectional view schematically showing a microfluidic chip 100 having actuators 112 at various locations in accordance with the present disclosure is shown. One or more actuators 112 can be located throughout the actuation section 130. In some embodiments, the actuators 112 can be located above a layer, on or between layers, below a top layer (e.g., a cover layer 131), or some combination thereof. For locations above, within, or immediately below a 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 so that acoustic energy can interact with particles in selected microfluidic channels from the actuation section 130 (e.g., a horizontal channel sub-segment 146). For example, see Figure 5 .

[0166] For the case of being located below the substrate, one or more actuators 112 can interact with the particles (e.g., in the horizontal channel sub-segment 146) by communicating and / or by using through-holes or other channels that allow connection through multiple layers. The actuator 112 can be a piezoelectric actuator, such as a piezoelectric drive pin that interfaces with a membrane layer, and the membrane layer can be flexible enough to allow deflection of the actuator 112, thereby applying a pulse to the channel of the actuation section 130, thereby allowing the particles to be deflected. Such a membrane layer can be the same or different from the material of the microfluidic chip 100, such as a polymer, glass, metal or other mixed or combined material layer.

[0167] There are many other ways in which particles can be actuated (i.e., sorted or, for that matter, de-sorted), and such ways can include the non-limiting examples of valves, pumps, acoustic elements, thermal elements, expansion elements, bubble generators, vibration devices, etc. These devices can be located on, within, or throughout multiple layers of the microfluidic chip 100. Furthermore, the actuator 112 can be part of the microfluidic chip 100 or can interface with the microfluidic chip 100 depending on the desires of a particular application and / or applications where cost and complexity are a concern.

[0168] Figure 19A An exploded view schematically showing a microfluidic chip 100 according to the present disclosure is shown. The microfluidic chip 100 includes a transfer section 110, a particle focusing section 120, and an actuation section 130. Sandwiching the transfer section 110, the focusing section 120, and the actuation section 130 is a cover section 131. One cover section 131a is adjacent to or in direct or indirect contact with the transfer section 110, and another cover section 131b is adjacent to or in direct or indirect contact with the actuation section 130. The cover section 131 can serve to seal the microfluidic chip 100. The cover section 131 can also be formed of a suitable material to avoid interfering with optical measurement, optical detection, and optical interrogation of particles in the microfluidic chip 100.

[0169] The transfer section 110 can transfer fluids to and from the plurality of microfluidic channels in the microfluidic chip 100. For example, within the particle focusing section 120 are a plurality of particle focusing regions 123 for focusing particles, such as at least one particle focusing region for each particle processing unit in the particle processing unit 101. FIG19 illustrates an example particle focusing region 123, and FIG20 illustrates an example particle focusing region 123. Figures 21A-21C Details of the particle focusing region 123 are discussed further. The particle focusing region 123 includes a sample inlet port 124 for introducing a sample fluid including particles 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 125a, 125b, which are fluidically coupled to the sheath inlets 115a, 115b, respectively. Downstream of the particle focusing region 123 is an outlet 128 fluidically coupled to the output channel 118 (e.g., a waste or retentate output channel) and an outlet 126 fluidically coupled to the output channel 116 (e.g., a waste or retentate output channel).

[0170] The actuation section 130 includes a treated particle layer 136. In some embodiments, the treated particle layer 136 includes a horizontal channel sub-segment 146 downstream of each of the focal regions 123. The horizontal channel sub-segment 146 includes an interrogation region 113, where particles are interrogated, as described herein. Downstream of the interrogation region 113 are output channels 116, 118 fluidically coupled to the horizontal channel sub-segment 146 via outlets 126, 128.

[0171] Each cover segment 131 can enable observation, detection, or both observation and detection of particles flowing within the chip 100. In some embodiments, each cover segment 131 can include a window or a window-like layer to provide a transparent optical interface for the fluid below the corresponding cover segment 131. The cover segment 131 can act as a liquid-proof barrier, that is, the cover segment 131 can prevent water or oil from moving from the inside of the chip 100 to the outside, or vice versa. In some embodiments, the cover segment 131 can form part of a fluid channel path in which one or more fluids flow. Other features such as electrodes can also be applied to the cover segment 131, integrated therein, or included thereon. In some embodiments, the cover segment 131b near the actuation segment 130 can include an actuator 112 as described in more detail above. The cover segment 131 can be formed of one or more materials that are configured to provide high transmittance of electromagnetic radiation wavelengths of interest (e.g., wavelengths of excitation light, scattered light, or fluorescence) for a particular application. In some embodiments, depending on design intent, the cover segment 131 may allow transmission of light with wavelengths in the ultraviolet (UV) to near infrared (IR) wavelength range, or a range representing a sub-portion of the UV to near IR range.

[0172] Figure 19B A three-dimensional (3D) schematic representation of a top view of a microfluidic chip 100 according to the present disclosure is pictorially shown. Figure 19C Shows a 3D schematic representation Figure 19B A perspective view of the microfluidic chip 100 is shown. Figure 19D Shown Figure 19B A partial side view of the microfluidic chip 100 is shown. Figures 19B-19D As shown, the transfer section 110, the particle focusing section 120, and the actuation section 130 are stacked along a stacking direction 107. The actuation section 130 is fluidically coupled to the sample input channel 104, the sheath input channel 105, and the output channel 108. 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, 118 (e.g., Figures 25A-25D In some embodiments, the transfer section 100 can include a particle focusing section 120. In some embodiments, the actuation section 130 can include a particle focusing section 120 (e.g., Figure 18A In some embodiments, the particle focusing section 120 is located in a separation section (e.g., as shown). Figure 18A shown).

[0173] Figure 20A stacked image shows a top view of upwardly stacked sections of a fabricated microfluidic chip 100 in operation along a stacking direction 107. The microfluidic chip 100 includes a transfer section 110 having a sample channel 114, a sheath channel 115, and output channels 116, 118; a particle focusing section 120 having a particle focusing region 123; and an actuation section 130 having a processed particle layer 136. The particle focusing region 123 includes a sample inlet port 124 for introducing a sample fluid containing particles from the sample channel 114 into the particle focusing region 123. The particle focusing region 123 also includes sheath inlets 125a, 125b, which are fluidically coupled to the sheath inlets 115a, 115b, respectively. Downstream of the particle focusing region 123 are an outlet 128 fluidically coupled to the output channel 118 (e.g., a waste or retentate output channel) and an outlet 126 fluidically coupled to the output channel 116 (e.g., a waste or retentate output channel). The treated particle layer 136 includes a horizontal channel sub-segment 146 downstream of the focusing region 123. The horizontal channel sub-segment 146 includes an interrogation region 113 where particles are interrogated, as described herein. Downstream of the interrogation region 113 are output channels 116, 118 fluidically coupled to the horizontal channel sub-segment 146 via outlets 126, 128. A sample fluid with particles flows into the sample channel 114 and then into the particle focusing region 123. A sheath fluid flows into the sheath channel 115 and then into the particle focusing region 123. Hydrodynamic focusing occurs in the particle focusing region 123. The focusing of the particles allows the particles to be well fluidically controlled, to travel in single file, and to be confined to a fluid core 142 (e.g., a central fluid core).

[0174] Figure 21A A perspective view of a hydrodynamic focusing simulation in a focusing region as disclosed herein is shown. In the illustrated hydrodynamic focusing configuration, focusing of particles in a sample stream occurs during a transition of the sample stream from a vertical to a horizontal direction, and sheath fluid is added in the vertical direction after the transition. Figure 21B Shown Figure 21A A top view of the simulation.

[0175] Figure 21C A side view of the simulation is shown. Hydrodynamic focusing can use sheath fluid from the sheath channel 115 to narrow, accelerate, and position sample fluid from the sample channel 114 to generate laminar flow 148, such as by introducing sheath fluid at the sample fluid flow to "squeeze" the sample fluid.

[0176] like Figures 21A-21CAs shown, the sample fluid including particles flows vertically in the sample channel 114 into the particle focusing region 123. Similarly, the sheath fluid flows vertically in the sheath channels 115a, 115b into the particle focusing region 123. At the particle focusing region 123, the vertical flow path of the sheath and sample transitions from a vertical direction to a horizontal direction. As shown, the sheath fluid from channel 115a prevents the sample from contacting the channel walls, thereby focusing the sample in a first vertical direction when the sample transitions from vertical flow to horizontal flow. The 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, resulting in horizontal hydrodynamic focusing, such as a horizontal tapered region, and horizontal control using side sheath channels.

[0177] Thus, the sample fluid including the particles contacts the sheath fluid 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 direction) to a second direction (e.g., horizontal direction) to generate a laminar flow 148 into the distribution layer 136.

[0178] Figure 22A A schematic diagram of a microfluidic chip 100 with 2 x 2 particle handling units 101 is shown according to various embodiments. Figure 22A This is intended to be schematic to help illustrate the massively parallel concepts taught herein and is not intended to limit the microfluidic chip 100 to a 2 x 2 matrix. The transfer section 110 of the microfluidic chip 100 may include a fluid manifold 400 (e.g., Figure 23 ), the fluid manifold has 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 to receive sheath from a sheath fluid reservoir 215 via a sheath fluid input path 105. The microfluidic chip 100 may further include a distribution layer 136 that facilitates segmentation or merging of fluid pathways, as described with respect to FIG. Figure 22B As further described. The fluid manifold 400 arranges the various microchannels in a manner that supplies all particle processing units 101 equally (i.e., uniform fluid pressure and fluid flow). The main fluid path layout 410 may include multiple microchannels located in the same distribution layer or different distribution layers for respectively directing fluids from the sample input path 104, the sheath fluid input path 105, and the output paths 106, 108 to each particle processing unit 101. An example of the main fluid path layout 410 will be further described. Figures 24A-24D Provide a description.

[0179] Figure 22B Shown Figure 21ASchematic diagram of a single particle processing unit 101 of FIG. The sample fluid including particles flows into the particle focusing region 123 through the sample channel 114, and the sheath fluid flows into the particle focusing region 123 through the sheath channel 115. In some embodiments, the sample fluid can enter the particle focusing region 123 through the nozzle-like feature 312. Compared with the microfluidic chip 100 of FIG. 18-21, the sheath channel 115 has a more complex structure. Figure 22B As shown, the sheath channel 115a is divided into sheath sub-channels 115b and 115c. The sheath channel 115b is divided into sheath sub-channels 115f and 115e. The sample fluid including the particles contacts the sheath fluid from the sheath sub-channels 115c-115e. The sample fluid can be focused by the sheath fluid through fluid dynamic focusing. The focused particles flow into the distribution layer 136 having the interrogation area 113. A detection system, such as an optical detection system using a lens 222, can be used to detect the particles in the interrogation area 113. Based on the detected signal (e.g., optical fluorescence, scattering, emission or extinction) of the particles in the interrogation area 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. When the desired particle 60 is detected, the actuator 112a can be actuated to deflect the desired particle 60 to the first output channel 116 through the outlet 126. When an undesired particle 50 is detected, actuator 112b can be actuated to deflect the particle through outlet 128 to second output channel 118. It should be understood that distribution layer 136 can have other configurations. For example, only one actuator 112 is required. When a desired particle 60 is detected, actuator 112 may not take any action, allowing the desired particle 60 to pass through first output channel 106 along the natural flow path. When an undesired particle 50 is detected, actuator 112 is actuated to deflect the particle to second output channel 108. In some embodiments, second output channel 108 can be connected to the natural flow path, and first output channel 106 can be a deflected flow channel. Actuator 112 can be actuated to deflect the desired particle 60 to first output channel 106. Actuator 112 may not take any action, allowing the undesired particle 50 to pass through second output channel 108 along the natural flow path.

[0180] Other particle processing units 101 may perform similar operations. Particles from the first output channel 116 of each particle processing unit 101 may further flow into the first output path 106 to reach the output reservoir 216. Particles from the second output channel 118 of each particle processing unit 101 may further flow into the first output path 108 to reach the output reservoir 218.

[0181] Figure 23An exploded view schematically illustrates a microfluidic chip 100 having 2 x 2 particle processing units according to the present disclosure. The microfluidic chip 100 includes a transfer section 110, a particle focusing section 120, and an actuation section 130. In some embodiments, the transfer section 110, the particle focusing section 120, and the actuation section 130 are sandwiched by a cover section 131, as shown in FIG19. The particle focusing section 120 includes a sample inlet port 124, a sheath inlet port 125, and outlet ports 126, 128 for each particle processing unit 101 (e.g., as shown in FIG19). The particle focusing section 120 can connect the actuation section 103 with the transfer section 110 and change the flow direction between the actuation section 103 and the transfer section 110. In some embodiments, the particle focusing section 120 can be non-parallel to the actuation section 130. For example, the microchannel in the particle focusing section 120 can be non-parallel to the distribution layer 136 having the interrogation region 113. The transfer section 110 includes a fluid manifold. The fluid manifold includes the microfluidic channel assembly as described above and the microfluidic channel assembly as described above. Figures 10A-10B The distribution layer 117 promotes the division or merging of fluid pathways.

[0182] Figure 24A A schematic diagram of a microfluidic chip 100 having a main fluid path layout 410 to feed a 2 x 2 layout of particle handling units 101 according to the present disclosure is shown. Figure 24B A schematic diagram of a microfluidic chip 100 having a main fluid path layout 410 to feed a 4 x 4 layout of particle handling units 101 according to the present disclosure is shown. Figure 24C A schematic diagram of a microfluidic chip 100 having a main fluid path layout 410 to feed particle handling units 101 in an 8 x 8 layout according to the present disclosure is shown. Figure 24D A schematic diagram of a microfluidic chip 100 having a main fluid path layout 410 to supply a 16 x 16 layout of particle processing units 101 according to the present disclosure is shown. Those skilled in the art will appreciate that the scale of this method can be further expanded. The main fluid path layout 410 can supply all particle processing units 101 in an equivalent manner (i.e., uniform pressure and fluid flow). In some embodiments, the main fluid path layout 410 can include multiple microchannels located in the same distribution layer or different distribution layers for respectively directing fluids from the sample input path 104 and the sheath fluid input path 105 to each particle processing unit 101. For example, Figure 26A The layout of the main fluid path layout 410 of one layer of the microfluidic chip 100 having particle handling units 101 in a 16 x 16 layout is shown. Figure 26B Shown Figure 26A Layout of the measurement and actuation section 130 of the particle handling unit in a 16 x 16 layout.

[0183] Figure 25A FIG1 shows a partial front view of a microfluidic chip 100 having a plurality of particle processing units 101 according to the present disclosure. Figure 25A A 2 x 2 layout of multiple particle processing units 101 is shown, however, for ease of illustrating the concepts taught herein. Figure 25B Shown Figure 25A An isometric view of the microfluidic chip 100 is shown. Figure 25C Shown Figure 25A A top view of the microfluidic chip 100 is shown. Figure 25D Shown Figure 25A The bottom view of the microfluidic chip 100 is shown. Figures 25A-25D As shown, 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, 118. The actuation section 130 is also shown and includes a processed particle layer 136. In some embodiments, the actuation section 130 may include the particle focusing section 120 (e.g., Figure 18A In some embodiments, the transfer section 100 can include a particle focusing section 120. In some embodiments, the particle focusing section 120 is located in the separation section (e.g., Figure 18A ). The sample fluid input path 104 has a main fluid passage in the vertical direction and branches into multiple channels at a certain point (for example, in the horizontal direction) to supply each particle processing unit. In some embodiments, there may be more than one sample fluid input path 104 depending on the number of particle processing units in the microfluidic chip. For ease of illustration, Figures 25A-25D In the embodiment, the sample fluid input path 104 branches into four branch fluid paths in the horizontal direction (eg Figure 25C As shown, each particle processing unit in the particle processing unit 101 has a branch fluid path. Each branch fluid path connects to the sample input channel 114, which directs the sample fluid into the focusing structure, which in the embodiment shown is one or more layers in the actuation section 130. Each sheath fluid input path 105 initially has a vertical main fluid path and changes to a horizontal direction in the focusing structure (as shown). Figure 25D The actuation section 130 also includes output paths 106, 108 downstream of the focusing structure. Figures 25A-25D An example illustration of the focusing structure can be found in Figure 20-21C Found in.

[0184] Figure 27AAn exploded view of a disk-shaped microfluidic chip 100 schematically illustrating a particle handling unit 101 having an mxn (m>1, n>1) layout according to the present disclosure is shown. Figure 27B Shown Figure 27A An isometric view of a disk-shaped microfluidic chip 100 is shown. Figure 27C Shown Figure 27A A front view of a disk-shaped microfluidic chip 100 is shown. Figure 27D Shown Figure 27A 1. Rear view of the disk-shaped microfluidic chip 100. Figure 27E Shown Figure 27A 1 is a side view of a disk-shaped microfluidic chip 100.

[0185] like Figures 27A-27E As shown, 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 output 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 a plurality of actuation sub-segments, such as mxn actuation sub-segments. In some embodiments, the actuation section 130 includes a plurality of focusing sub-segments, such as mxn focusing sub-segments or regions as described herein. Each actuation sub-segment is used for a specific particle processing unit 101. As Figure 27E As shown, the microfluidic chip 100 can be illuminated and / or detected in an illumination / detection direction 440 to illuminate and / or detect particles in the actuation section 130 , such as one or more interrogation regions of the actuation section 130 .

[0186] Figures 28A-28B Various embodiments of an illumination system and a detection system for synchronized illumination and detection according to the present disclosure are presented. Figures 28A-28B Each embodiment of the illumination system and detection system presented in the present invention includes an electromagnetic source system 210, a detection system 220, one or more light separation systems 205, and other related optical components. Examples of electromagnetic source systems 210, detection systems 220, and light separation systems 205 are described above with respect to Figure 11-14E In some embodiments, the detection system 220 may 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. The examples of the microfluidic chip 100 are described above at least with respect to Figure 1-1 0 and 18-27 are described.

[0187] In some embodiments, particle processing system 200 provides ultra-high throughput particle processing, in part, by simultaneously illuminating multiple particle processing units 101 using wide-field illumination in an epi-illumination manner and / or simultaneously detecting signals from particle processing units 101. For example, Figure 28A As shown, the electromagnetic source system 210 simultaneously illuminates multiple particle processing units 101 in the microfluidic chip 100 (e.g., the interrogation area of ​​each microchannel in the multiple microchannels of each particle processing unit 101). The light separation system 205 is used to direct the illumination light to illuminate each particle processing unit 101 in the microfluidic chip 100. In some embodiments, the light separation system 205 is also used to direct the backscattered light from the particle processing unit 101 to the detection plane 270a through the optical filter 225a. The scattered light (e.g., backscattered light, side scattered light, forward scattered light) and particle emission light from each particle processing unit 101 are detected by the detector planes 270b-270d through the optical filters 225b and 225c, respectively. It should be understood that the particle processing system 200 can have more than Figure 28A More detector planes are provided to detect scattered light (backscattered light, side-to-forward scattered light and / or other suitable scattered light) and particle emission light (e.g., autofluorescence, fluorescence) of different wavelengths. Figure 28B As shown, electromagnetic source system 210 includes a spatial light modulator (SLM) 214 and a light source 212. SLM 214 converts a single laser beam from light source 212 into a two-dimensional (2D) array of mxn beamlets that simultaneously illuminate each particle processing unit 101 in microfluidic chip 100. A single light separation system 205 is used to direct illumination light from SLM 214 to illuminate each particle processing unit 101 in microfluidic chip 100. A first lens assembly 610 collects and directs light emitted or scattered by particles in each processing unit 101 to reach detection system 220 through a second lens assembly 620, which collects light emitted or scattered by particles in each particle processing unit 101 and directs the light from each particle processing unit 101 to a corresponding detection point of detection system 220. Detection system 220 simultaneously receives and processes light emitted or scattered by particles in each particle processing unit 101.

[0188] Figure 29A Shown is a fluid plane 710 of the particle processing system 200. The fluid plane 710 includes 8x8 illumination points 712. Each illumination point 712 is focused on each particle processing unit 101 (eg, interrogation region 113).

[0189] Figure 29BDetector plane 270 of particle processing system 200 is shown. Detector plane 270 includes 8 x 8 detection points 272. Each detection point 272 detects scattered light and / or emitted light from each particle processing unit 101. It should be understood that the particle detection system can have more than the 8 x 8 illumination points and 8 x 8 detection points shown.

[0190] As will be appreciated from the foregoing, the concepts of the present disclosure can be embodied in a variety of ways. Therefore, the embodiments or elements disclosed in the specification or illustrated in the accompanying drawings are not intended to be limiting, but rather to illustrate the multiple and different embodiments generally encompassed by the present disclosure or the equivalents encompassed with respect to any specific element thereof. Furthermore, the specific description of an embodiment or element may not explicitly describe all possible embodiments or elements; many alternatives are implicitly disclosed by the specification and drawings.

[0191] 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 should also be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

[0192] As will be apparent to those skilled in the art upon reading this disclosure, each of the embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0193] Regardless of whether explicitly stated, all numerical values ​​herein are assumed to be modified by the term "about." For purposes of this disclosure, ranges may be expressed as "about" one particular value to "about" another particular value. It will be understood that the endpoints of each range within the range are significant both relative to the other endpoint and independently of the other endpoint. When a value is expressed as an approximation 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 segments stacked or layered in a stacking direction to form a plurality of microchannels, the plurality of microchannels being at least partially oriented for flow along the stacking direction, the plurality of segments comprising: a transfer section for introducing a sample fluid including particles; as well as A second section comprising a measurement section or the measurement section and an actuation section, the actuation section comprising a plurality of interrogation regions, each interrogation region of the plurality of interrogation regions being associated with at least one microchannel of the plurality of microchannels. 2 . The microfluidic chip according to claim 1 , wherein the plurality of segments further comprise a particle focusing segment configured to focus the particles in the sample fluid.

3. The microfluidic chip of claim 2, wherein the particle focusing section comprises a plurality of nozzles to merge the sample fluid with the sheath fluid. The microfluidic chip according to claim 1 , wherein the actuation section comprises a plurality of actuators. 5 . The microfluidic chip according to 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. The microfluidic chip according to claim 5 , wherein the actuation section further comprises one or more acoustic attenuation elements to acoustically isolate the actuator. The microfluidic chip according to claim 1 , wherein the actuation section comprises a plurality of particle focusing regions.

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

9. The microfluidic chip according to claim 1, further comprising a plurality of guide elements to align the segments along the stacking direction.

10. The microfluidic chip of claim 1 , wherein the area density of the plurality of interrogation regions is in the range of 1 region / cm 2 Up to 500 areas / cm 2 . The microfluidic chip according to claim 1 , wherein the flow of particles in each microchannel is mainly performed along the stacking direction in the corresponding interrogation area.

12. The microfluidic chip of claim 11, wherein the flow of the particles is switched from a primarily vertical direction to a horizontal direction for focusing.

13. The microfluidic chip of claim 11, wherein the flow of the particles is switched from a primarily vertical direction to a horizontal direction for interrogation. The microfluidic chip according to claim 1 , wherein at least some of the plurality of segments are separable from each other.

15. The microfluidic chip of claim 14, wherein at least one of the plurality of segments is exchangeable based on a desired outcome or based on a characteristic of a particle population to be processed by the microfluidic chip.

16. The microfluidic chip of claim 1, wherein the plurality of segments are permanently attached or fused to each other. 17 . The microfluidic chip according to claim 1 , wherein the transfer section comprises a sample input port and a sheath input port, and the transfer section transports the sample fluid from the sample input port and the sheath fluid from the sheath input port to the plurality of microchannels.

18. The microfluidic chip of claim 1, wherein the transfer section comprises a first outlet port to enable extraction of desired particles from the chip, and the transfer section comprises a second outlet port to enable extraction of undesired particles from the chip.

19. The microfluidic chip of claim 1, wherein the actuation section further comprises a plurality of pressure pulse dampeners, each pressure pulse dampener being disposed along an associated microfluidic channel opposite a corresponding actuator.

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

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

22. The particle processing system of claim 21, wherein the particle focusing section comprises a plurality of nozzles to merge the sample fluid with the sheath fluid.

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

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

25. The particle handling system of claim 24, wherein the actuation section further comprises one or more acoustic attenuation elements to acoustically isolate the actuator.

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

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

28. The particle processing system of claim 15, further comprising a light separation system.

29. 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 corresponding interrogation region of the plurality of interrogation regions and delivering the light to the detector.

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

31. The particle handling system of claim 20, further comprising a plurality of guide elements to align the segments along the stacking direction.

32. The particle processing system of claim 20, wherein the area density of the plurality of interrogation zones is in the range of 1 zone / cm 2 Up to 500 areas / cm 2 .

33. The particle processing system of claim 20, wherein the flow of particles in each microchannel is primarily perpendicular to the stacking direction in the corresponding interrogation region.

34. The particle handling system of claim 20, wherein at least some of the plurality of segments are separable from one another.

35. The particle processing system of claim 34, wherein at least one of the plurality of segments is capable of being exchanged based on a desired outcome or based on a characteristic of a particle population to be processed by the microfluidic chip.

36. The particle handling system of claim 20, wherein the plurality of segments are permanently attached or fused to one another.

37. The particle processing system of claim 20, wherein the transfer section comprises a sample input port and a sheath input port, the transfer section delivering sample fluid from the sample input port and sheath fluid from the sheath input port to the plurality of microchannels.

38. The particle handling system of claim 20, wherein the transfer section includes a first outlet port to enable extraction of desired particles from the chip, and the transfer section includes a second outlet port to enable extraction of undesired particles from the chip.

39. The particle handling system of claim 20, wherein the actuation section further comprises a plurality of pressure pulse dampeners, each pressure pulse dampener disposed along an associated microfluidic channel opposite a respective actuator.

40. A method of assembling a microfluidic chip, the method comprising: aligning the transfer section with the plurality of alignment holes in the focusing section using the plurality of alignment posts; bonding the transfer segment to the focusing segment; aligning a second section comprising a measurement section or the measurement section and an actuation section with the focusing section by aligning a plurality of alignment holes in the actuation section with the alignment posts; as well as The second segment is bonded to the focusing segment.

41. A method for sorting particles using a microfluidic chip, the method comprising: allowing a sample stream including particles to flow through a plurality of microchannels, the plurality of microchannels being formed by a plurality of segments stacked or layered in a stacking direction to form the microfluidic chip, the plurality of microchannels being at least partially oriented to flow along the stacking direction; focusing particles in each microchannel of the plurality of microchannels using a focusing segment of the plurality of segments; detecting particle characteristics of particles flowing through a plurality of interrogation regions in an actuation segment of the plurality of segments, each interrogation region being associated with a microchannel; as well as In response to the detected characteristics of the particles, the particles are sorted using an actuator associated with each microchannel.

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