Micro-fluidic chip equipment for carrying out light force measurement and cell imaging by utilizing micro-fluidic chip configuration and dynamics

Abstract

The invention relates to a micro-fluidic chip device for optical force measurement and cell imaging by utilizing micro-fluidic chip configuration and dynamics. A microfluidic chip arrangement in which the injection is performed in a vertically upward direction with a fluid container located below the chip to minimize particle sedimentation at and before the analysis portion of the chip channel. The input and fluid flow upward through the bottom of the chip, using a manifold in one aspect, which avoids orthogonal redirection of fluid dynamics. The contents contained in the vial are located below the chip and are directly pumped vertically upward into the first channel of the chip. The long channel extends from the bottom of the chip to the vicinity of the top of the chip, and a short horizontal bend is turned at the position. The fluid is pumped upward to a horizontal analysis section, which is the highest channel / fluidic point in the chip, thus close to the top of the chip, which makes the imaging clearer. In the analysis process, the laser can also suspend the cells or particles in the channel.

Description

This application is a divisional application of Chinese patent application 2017800983141 (PCT / US2017 / 068373) filed on December 23, 2017 and entitled “Microfluidic chip device for photoforce measurement and cell imaging using microfluidic chip configuration and dynamics”. Technical Field

[0001] The present invention generally relates to apparatus and methods for particle analysis and imaging of particles or cells in fluids, and more particularly to apparatus and methods for imaging particles in fluids using pressure, fluid dynamics, electrodynamics, and optical forces.

[0002] The present invention is directed to a microfluidic chip in which injection occurs in a vertical upward direction with fluid vials located below the chip to minimize particle settling at and before the analytical portion of the chip channels.

[0003] The implementation of the present invention requires changes to existing microfluidic chip designs. For example, in order to align the vials vertically with the chip, a different chip interface than the prior art needs to be established. Specifically, the present invention allows inputs and fluids to pass from the bottom of the chip upward, using a manifold in one aspect, which avoids horizontal redirection of fluids / fluid dynamics, rather than having input tubes engage the chip in an orthogonal manner via ports (typical practice in microfluidic chip lab systems), the ports are attached to the largest face of the chip, and then the fluid is pumped first across the chip and then over the chip.

[0004] According to the present invention, the contents of the vial are located below the chip and are pumped directly vertically upward into the first channel of the chip. The long channel extends from the bottom of the chip to near the top of the chip. The channel then makes a short horizontal bend, but the new channel is so short that it almost offsets any effects of cell sedimentation caused by gravity and zero flow velocity at the wall. Then, contrary to the prior art, the fluid is pumped upward to the analysis section. Therefore, the horizontal analysis section is the highest channel / fluidic point in the chip, so the horizontal analysis section is close to the top of the chip, which makes the chip material (such as glass) between the microscope / camera and the sample less than in the prior art, and the imaging is clearer. During the analysis process, the laser also suspends the cells in this channel to prevent them from settling. Background Art

[0005] Related technical notes According to the prior art, a microfluidic chip vial containing cells or particles to be separated and / or analyzed is placed on the side and pumped horizontally into a channel in the microfluidic chip. First, the contents of the vial (e.g., particles or cells) are pumped vertically upward, then turn a U-turn downward, and then are pumped horizontally into the chip (see, e.g., U.S. Pat. No. 9,594,071).

[0006] The horizontal connection to the chip, coupled with the dead volume in the connection (empty space in the fluidic connection, which is unavoidable to some extent), causes significant additional sedimentation under the action of gravity. This configuration also necessitates the relatively large diameter channel required for the connection, which, in addition to the dead volume, also creates a relatively low velocity area, further exacerbating the problem of particle sedimentation. The chip according to the present invention not only eliminates the need for a large horizontal input channel, but also eliminates the need for a more abrupt change from the large horizontal input channel to the relatively thin upwelling flow in the first vertical chip channel. This configuration eliminates horizontal sedimentation and unnecessary changes in direction that cause sedimentation. Having cells enter the bottom edge of the chip also solves the problem of sedimentation in the dead volume by orienting it perpendicular to gravity so that cells or particles cannot settle at the bottom of the horizontal channel, but instead they are constantly guided upward by the flow. This is not intuitive, and a lot of experimentation was required to understand the problem before designing the solution currently presented. In contrast to the present invention, currently available microfluidic devices include custom or commercially available connections on polished surfaces and larger areas of glass, which generally force any particles such as cells contained in the sample stream to turn and travel horizontally immediately upon entering the chip.

[0007] In addition, in the prior art, cells or particles make several horizontal moves on the microfluidic chip before reaching the analysis channel, which causes sedimentation. When the contents of the vial enter the chip and the channel in the chip, the contents are pumped horizontally relative to the vertical chip channel. The channel then flows upward and turns a long horizontal bend, at which time the cells tend to settle at the bottom of the channel due to gravity, and at the same time, due to laminar flow conditions, the cells tend to have lower speeds at the walls. In essence, due to the parabolic velocity profile, the flow rate is the largest in the middle of the channel and drops to zero at or near the channel wall. After the first horizontal chip channel, the fluid turns downward before the analysis channel, where particle imaging or separation is performed. Due to this configuration, there is a relatively long distance between the microscope / camera and the analysis channel. In this typical prior art configuration, the particles are forced downward and eventually leave the bottom of the chip.

[0008] Furthermore, due to limitations in the prior art, multiple horizontal moves are required so that cells settle in multiple places in the channel. This in turn results in a degradation of the image quality because of the need to image through additional material at the edge of the chip. The prior art channel of the chip has to pump vertically upward, then horizontally, and then zigzag to get enough cells or particles to suspend, then down and off the chip. The present invention avoids the zigzag channel.

[0009] There are also prior art for 3D image rendering of cells or particles in fluids. For example, M. Habaza, M. Kirschbaum, C. Gent-Marschner, G. Dadikman, I. Banea, R. Korenstein, C. Dushel, NT Shaked in Advanced Science (2017, 4, 1600205) taught capturing cells, rotating the cells at high speed, and using interferometry to measure the refractive index distribution in the cells. Interferometry has also been used to analyze cells in microfluidic channels (see, for example, Y. Song et al., Applied Physics Review (Physical Review Applied), February 27, 2014; 1: 014002). However, the present invention requires that multiple images be taken for cells or particles as they travel in a fluid stream and pass through the focal plane of (one or more) imaging devices, thereby eliminating the need to capture cells in order to obtain 3D images. Other techniques have been taught, such as using mechanical displacement platforms to move cells or particles (e.g., N. Lu et al., Optics Express, 2008 Sep 29;16(20):16240-6), but none of these techniques use brightfield imaging as described in the present invention, nor do they utilize fluid flow to provide cell positioning relative to the image focal plane.

[0010] All prior art references cited herein are hereby incorporated by reference in their entirety. Summary of the invention

[0011] The present invention is directed to a microfluidic chip in which injection is performed and the sample vial is located below the chip in order to minimize particle settling. Thus, the contents of the vial are located below the chip and are pumped directly vertically upward into the channel of the chip. The long channel extends from the bottom of the chip to near the top of the chip. The channel then makes a short horizontal bend, but the new channel is too short to make any effect on cell settling, which is caused by zero flow rate at the channel wall, insignificant. Then, in contrast to the prior art, the sample is pumped upward to the analysis section. Therefore, the horizontal analysis section is the highest channel / fluidic point in the chip, so the horizontal analysis section is close to the top of the chip, which allows less glass between the microscope / camera than in the prior art, and clearer imaging. The distance between the analysis channel and the top of the chip can be between 100 microns and 2 mm, but can also be as long as 100 mm, such as from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, etc. In one embodiment, after passing through the analysis portion of the chip, the sample (eg, fluid, cells, and / or particles) is pumped down to the bottom of the chip and forced to move outward.

[0012] The present invention is further directed to a microfluidic chip in which horizontal movement is minimized, especially when fluid enters the chip channels. Prior art chips contain about 13 mm horizontal channels (non-analytical portion), of which about 2 mm are injection ports with a much larger diameter, exacerbating the sedimentation caused by low speed. The chip described in the present invention has a (non-analytical) horizontal channel of about 0.2 to 3.0 mm in a preferred embodiment, although the length of the horizontal channel can be between 0.01 and 100.0 mm, such as from 0.01 mm to 0.02 mm, from 0.02 mm to 0.03 mm, from 0.03 mm to 0.04 mm, and so on. This depends on the channel system of the invention, which is different from the prior art in terms of order of magnitude, which can improve flow and eliminate cell / particle sedimentation.

[0013] Another aspect of the present invention is directed to a microfluidic chip in which imaging and analysis are performed based on or near the corners of the chip, because this makes the glass between the camera and the analysis channel less and the distance shorter to improve the imaging from multiple viewpoints. This also enables the use of higher numerical objective lenses to improve fine imaging by increasing the order of magnitude. This design improvement reduces image distortion caused by glass (or other substances constituting the chip, such as plastic or any transparent or translucent material) and distance (for example, due to defects in the glass). The distance between the imaging device and the analysis channel can be between 100 microns and 2 mm, but can also be as long as 100 mm, such as from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, and so on.

[0014] In addition, the present invention is directed to a microfluidic classification chip, which is separated from the downstream of the analysis channel, allowing the use of pressure and / or laser (or other optical force) to activate the classification function simultaneously or sequentially. On the one hand, in order to perform the classification function, the flow will continue from the analysis channel. For example, the particles will be directed into the vertical channel and then directed to the horizontal classification channel. In one embodiment, optical force and / or pressure are applied in the flow direction of the classification channel to push the particles through the channel. Particles that are not directly acted upon by optical force will be diverted to alternative channels due to, for example, gravity, electrodynamic force, magnetic force, laminar flow line, streamline, reduced flow rate, orthogonal optical force, or vacuum (vacuum is applied to suck particles into alternative channels). On the other hand related to the analysis channel after classification, the present invention allows guiding optical force from the back side of the chip (laser or optical force is oriented in the same flow direction), and allows decomposition of the main laser in some aspects. In some embodiments, optical force and / or pressure can be applied in the opposite direction of the material moving through the channel, such as countercurrent, or can be applied in the same direction of the material moving in the channel, such as downstream. Cell or particle sorting can be performed on a single device or separate chips. Figure 1BIn the embodiment, the fifth channel before the outlet pipe 145 contains one or more branches to achieve single or multiple classification areas.

[0015] In another aspect of the invention, a manifold is connected to a vial in a manner such that tubing passes through the manifold and is connected to a vial or other container on another side that is in contact with a substance (e.g., a fluid) in the vial. The manifold allows the vial to be connected to the microfluidic chip but stored below the chip, and / or the manifold allows the contents of the vial to be injected from the bottom of the chip, alleviating several problems faced by the prior art, such as cell or particle sedimentation where the vial or tubing from the vial is connected to or communicates with the microfluidic chip.

[0016] In another aspect, the present invention is directed to a microfluidic chip holder comprising a structure for directing a light source, the structure comprising an integrated prismatic cavity, the integrated prismatic cavity being equipped with a prism so that light is emitted at an angle relative to the chip, which is a preferred method for illuminating a confined geometric configuration. In some embodiments, the light source includes, but is not limited to, a fiber optic device or a collimated or focused light source. In particular, the light source is precisely aligned or directed to, or directed into, an analysis channel.

[0017] In another aspect of the invention, the device includes a second imaging device, which is oriented orthogonal to the first camera and the channel view. The reasons for setting the second camera vary. On the one hand, the reason for setting the second imaging device is to help analyze the visual alignment of the laser or light force in the channel. On the other hand, using the method described in the present invention, data from the first camera can be recorded. With the second camera, the data can be combined with the data from the first camera, bringing additional data that can be used to more accurately infer the cell position, size, shape, volume, etc. This additional information about the same cell (or particle) improves accuracy and expands the scope of measurement and analysis. On the other hand, the second camera combined with the first camera can achieve a three-dimensional reconstruction of the cell or particle, or a group of cells or particles, which is imaged by an orthogonal camera and a camera in the flow or a camera positioned toward the side of the chip. Using the algorithm described in the present invention or other algorithms, including reversing or slowing the flow and taking one or more images for a specific cell or particle, or a group of cells or particles, the present invention allows the analysis and processing of multiple images, thereby allowing the determination of features / characteristics / quantitative measurements, such as cell volume, cell shape, nuclear position, nuclear volume, organelle or inclusion body position, etc. In another aspect of the invention, the camera is oriented to image on an axis in the direction of flow.

[0018] In one aspect, the present invention does not require a serpentine or zigzag channel to maintain proper suspension of particles, which is preferred in the prior art. Due to the vertical nature of the fluid and particles or cells injected into the chip, the vertically integrated pumped particles can directly go up through the channel to the first horizontal channel (referred to as the second channel in the present invention), thereby avoiding the zigzag channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings illustrate certain aspects of some embodiments of the present invention and are not intended to limit or define the present invention. Together with the description, the accompanying drawings are used to explain certain principles of the present invention.

[0020] Figure 1A is a schematic diagram depicting a global view of the device configuration, including the chip, manifold, and vials. Figure 1B is a schematic diagram showing some depiction of the chip channel orientation and locations.

[0021] Figure 2A and Figure 2B Contains a schematic diagram depicting a microfluidic chip holder according to the present invention.

[0022] Figure 3A and Figure 3B Schematic diagram depicting angles and aspects of a chip holder according to the present invention.

[0023] Figure 4A and Figure 4B is a schematic diagram showing an example of a cell path and imaging configuration associated with the chip. Figure 4C Schematic diagram showing alternative cellular pathways.

[0024] Figure 5 A schematic diagram showing on-chip multiplanar imaging and how it can be used to render 3D images and information.

[0025] Fig. 6A and Figure 6B A schematic diagram showing on-chip multiplanar imaging in a fluid flow and how it can be used to render 3D images.

[0026] Figure 7 is a schematic diagram showing possible ways to capture cells and / or equilibrate cells within the analysis portion of the chip, as well as possible ways to image cells from multiple angles.

[0027] Figure 8 is a schematic showing how the camera and illumination source can be placed to align with the laser and flow direction so that the particles are directed away from the camera.

[0028] Fig. 9is a schematic showing how the camera and illumination source can be placed to align with the laser and flow direction so that the particles move toward the camera. DETAILED DESCRIPTION

[0029] The present invention has been described with reference to specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations may be made in the practice of the present invention without departing from the scope or spirit of the present invention. Those skilled in the art will recognize that these features may be used alone or in any combination based on the requirements and specifications of a given application or design. Embodiments containing various features may also consist of or consist essentially of these various features. Other embodiments of the present invention will be apparent to those skilled in the art upon consideration of the specification and practice of the present invention. The description of the present invention provided is merely exemplary or explanatory in nature, and therefore variations that do not depart from the essence of the present invention are intended to be included within the scope of the present invention.

[0030] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited to its application in the component structure and configuration details set forth in the following description or shown in the following drawings. The present invention can have other embodiments or can be practiced or implemented in a variety of ways. In addition, it should be understood that the words and terms used in the present invention are for illustration only and are not limiting.

[0031] Now go to Figure 1A , Figure 1A An overall view of the device taught by the present invention is shown. Sample vials 130 are located below the microfluidic chip 100 (also generally referred to as substrates in the present invention), and the number or size of vials is not limited. The vials are configured to hold any type of sample that can be moved through the device, such as one or more substances, including but not limited to one or more fluids, liquids, gases, plasma, serum, blood, cells, platelets, particles, etc., or a combination thereof. In the context of this specification, the terms fluid or sample may be used to refer generally to such one or more substances. The vials are directly or indirectly connected, for example indirectly using air pipe fittings 110, to be operably connected to the underside of the chip. As Figure 1A As further shown, vials may also be connected via manifold 120 . Figure 1B A preferred embodiment of a microfluidic chip 100 is shown, wherein substances, fluids, particles and / or cells are injected into an outer surface of the microfluidic chip, such as an edge (e.g., Figure 1B in the XZ plane). Figure 1BInjection along one of the minimum distance shared planes (e.g., XZ plane or XY plane) is shown. In this particular embodiment, the length of side length X160 is less than the length of side length Y170 and the length of side length Z180. This configuration allows the sample to have minimal deviation after entering the channel on the chip (e.g., no right turn is required, which is usually required in the prior art).

[0032] Figure 1A A manifold 120 is shown connecting one or more vials 130 to a microfluidic chip 100 so that substances, fluids, particles and / or cells can be injected or pumped vertically upward into the microfluidic chip. The manifold allows injection of substances from the bottom of the chip while also placing electronic equipment, flow sensors and tubing (liquid and air) in a manner that minimizes cell sedimentation, thereby optimizing throughput. The air tubing provides pressure or vacuum, and when sealed, provides a closed system within the scope of the manifold device. In one embodiment, there is a distance between (one or more) vials and the manifold, indicating that there is no seal and the pressure of the internal volume is atmospheric pressure. This allows substances to be pumped from containers open to the atmosphere or to such containers (vacuum required for pumping from open containers). The manifold places electronic equipment, flow sensors and tubing (liquid and air) in a manner that minimizes cell sedimentation, thereby optimizing throughput.

[0033] By injecting the contents from the bottom of the chip, the present invention minimizes horizontal movement in the inlet and outlet tubing 140, 145, which can lead to problems such as settling of particles or cells in the channel(s) 150. In this example, the outlet tubing is offset from the inlet tubing in the Z and X dimensions ( Figure 1B In some embodiments, the outlet tubing is offset, un-offset, straight, aligned, or angled relative to the inlet tubing. This configuration avoids the need for a serpentine or zigzag vertical channel because the current configuration solves sedimentation problems that occur in the prior art, such as when a fluid combined with cells and / or particles is injected or pumped into the chip from the side in a horizontal direction and then must change direction and fluid dynamics to be pushed upward. The manifold and injection from the bottom of the chip also allow for the placement of additional components, parts, mechanical devices, or hardware below the chip (see Figure 1A ).

[0034] The working principle of manifold 120 is to regulate the air pressure above the contents of the vial and provide the correct geometry for the flow sensor and electronics. Pipes such as fluid or air pipes pass through the manifold and are connected to the vials on the other side. Pressurized air passes through one side of the manifold, forming a closed pressurized system within the manifold. By adjusting the pressure in the closed area, the system allows parameters such as flow rate and fluid dynamics to be changed. The pressurized area is both in (one or more) vials 130 and in the manifold 120. On the other hand, the vial does not require any air connection because it is open to the ambient atmosphere. This allows sampling of a wider variety of containers and sources. In this embodiment, a vacuum is applied to one or more other vials, thereby creating a pressure difference to drive the fluid flow from the open container.

[0035] In one embodiment, pressure-based sample injection is used. The vial is filled with the sample in the fluid and sealed with a lid or a tube connected to the chip. Before being attached to the lid, the vial can be open to the air or sealed with a diaphragm or other airtight device. In one aspect, the lid may contain two connectors, one for fluids such as gas and the other for liquid. Optionally, the method embodiment also includes providing a sample inlet line tip in communication with the sample inlet line, the sample inlet line tip being in communication with the first channel.

[0036] In another embodiment, vacuum-based sample injection is used. The vial is filled with the sample in the fluid and sealed with a lid or a tube connected to the chip. Before being attached to the lid, the vial can be open to the air or sealed with a diaphragm. In one aspect, the lid may contain two connectors, one for fluids such as gas and the other for liquid. Optionally, fluid can be sucked out of the vial open to the atmosphere by applying vacuum pressure to one or more other vials. Optionally, the method embodiment also includes providing a sample inlet line tip connected to the sample inlet line, and the sample inlet line tip is connected to the first channel.

[0037] Figure 2A-2B and Figure 3A-3BA chip holder 200, 300 is shown. The chip holder includes a structure to guide a light source 210 (e.g., a fiber optic light source, a light emitting diode, or a laser) and an integrated prism cavity 220, 320 to which a prism can be mounted. The light source is guided or arranged to the desired location by an integrated structure or channel 240 within the chip holder. The built-in space for the fiber optic light source allows illumination, such as an illumination cone 250, even in a restricted geometric environment (e.g., on a microfluidic chip 230, 330 or a channel in a microfluidic chip). In a preferred aspect, the light source is particularly precisely aligned, directed, or focused on an analysis channel 260. In a preferred embodiment, the chip holder includes built-in space for prisms 220, 320 and fiber optic light sources 210, allowing illumination in a restricted geometric configuration. The chip also includes a hole or opening 350 at the bottom of the holder to precisely arrange the fluidic tubing described in the present invention. In one embodiment, the adjustable screw is integrated with the threaded hole 360 ​​on one or more faces for proper positioning.

[0038] Figure 4A 4 is a preferred embodiment of the microfluidic chip 400 described in the present invention. As shown in the figure, the fluid first moves vertically upward through the first channel 410 and then communicates with the second horizontal channel 420. Figure 4AAs shown, another vertical channel 430 brings the fluid closer to the top of the chip, at which point the fourth channel 440 is horizontal and includes an analysis channel. These channels are operably connected to each other to allow samples to move through the system from one channel to another. In some embodiments, the sample can flow from the first channel to the second channel, then to the third channel, and then to the fourth channel, or flow in reverse, or a combination of the two. A pump and / or a vacuum device can be provided to provide positive and / or negative pressure at the opening and / or outlet of the channel so that the substance can move through the channel. The analysis channel is close to one or more outer surfaces of the substrate, such as the face, edge or side of the chip. For example, the analysis channel according to this configuration is close to the top and side of the chip, thereby improving the imaging and analysis of the substance through the microfluidic chip. In a preferred embodiment, the distance from the analysis channel to the top and side of the chip is between about 1mm and about 2mm. However, the distance from the analysis channel to the top of the chip can be between 0.1mm and 100mm, such as from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, from 0.3mm to 0.4mm, and so on. In other words, the analysis channel may be arranged within the top 50%, 33%, 25%, 10% or 5% of the substrate. The length of the horizontal analysis channel according to the present invention can be between about 250 microns and about 10 mm. However, the length of the analysis channel can be between 100 microns and 100 mm, for example, from 0.1 mm to 0.2 mm, from 0.2 mm to 0.3 mm, from 0.3 mm to 0.4 mm, etc. In other words, the length of the analysis channel can be about 75% or less of the height, width or length of the substrate / chip, for example, 50% or less, 33% or less, 25% or less, 10%, or 5% or less of the height, width or length of the substrate / chip.

[0039] Figure 4B Two imaging devices 450, such as machine vision cameras, are shown in FIG. In one embodiment, the camera may be located above the chip and oriented orthogonal to the analysis channel. In another embodiment, the camera may be located on the side of the chip and oriented orthogonal to the flow direction or diagonal to the flow direction (e.g., above the channel, below the channel, or at an angle to the side of the channel). In some embodiments, the camera may be placed in a position where imaging can be performed at any angle to one or more material flows, such as orthogonal to or at 90 degrees to one or more material flows, or such as from 0 to 90 degrees, or from 10 to 80 degrees, or from 30 to 60 degrees, and so on. In another embodiment, two or more cameras may be used to photograph cells or particles in the analysis channel. For example, a camera may be above the chip and oriented orthogonal to the analysis channel. A second camera may be located on the side of the chip and oriented orthogonal to the flow direction or diagonal to the flow direction (e.g., above the channel, below the channel, or at an angle to the side of the channel). As Figure 4BAs shown in , one or more light sources 460 can be used to illuminate the analysis channel 440, and such light sources can be located below the chip and illuminate upward, to the side of the chip, and in the flow direction or against the flow direction, or above the chip and illuminate downward, diagonally toward the analysis channel.

[0040] Alternatively, a dichroic mirror 840 or other suitable optical element may be used to selectively redirect light of a particular wavelength range while allowing light of other wavelength ranges to pass, e.g. Figure 8 This will allow the camera 810 to be placed in alignment with the analysis channel. There are several embodiments of this, including placing the optical laser 830 and the camera 810 at the same end or at opposite ends of the analysis area, as shown in Figures 8 and 9. It may also be necessary to have an illumination source 860 for the camera, such as Figure 8 and 9 As shown, the illumination source 860 may be oriented in several ways. The light source may be a broad spectrum light source such as one or more LEDs, or a narrow light source such as a laser. The camera may be used in the form of a single camera, or as part of a multi-camera system combined with other viewpoints as described in the present invention.

[0041] Figure 4A Also shown is that a light source 480 (e.g., a laser) can be used to affect the cell flow. The laser can be placed in alignment with the cell flow or can be placed opposite to the cell flow. The laser can also be placed and / or oriented to be orthogonal or diagonal to the cell flow.

[0042] Embodiments of the present invention include apparatus for particle analysis. (See e.g. Figure 4A , Figure 4B , Figure 4C , Figure 5 , Fig. 6A , Figure 6B , Figure 7-9) Embodiments of the invention include at least one camera 450 for capturing images of particles or cells in a microfluidic channel (e.g., 440). In one embodiment, a laser or other optical force 480 is included, such as a parallel light source operable to generate at least one parallel light source beam. The at least one parallel light source beam includes at least one beam cross section. Embodiments of the invention include a substrate with a first channel 410 extending in a vertical direction in the substrate such that a first plane substantially crosses the first channel 410 along its length, whereby a fluid sample is injected into the substrate / chip from the bottom of the chip and is forced upward under positive or negative pressure. Embodiments of the invention include a second channel 420 orthogonal to the first channel, such that the second channel 420 is arranged horizontally in the substrate such that a second plane substantially crosses the second channel 420 along its length, and the second plane is arranged orthogonal to the first plane. The second channel is in a horizontal direction of the chip. The second channel is directly or indirectly connected to the first channel. The second channel is directly or indirectly connected to a short vertically upward third channel 430, which brings the channel network closer to the top of the chip. The third channel is directly or indirectly connected to the fourth horizontal channel 440 located near the top and / or corner of the chip. In a preferred embodiment, the fourth channel is the channel closest to the top of the chip. In one embodiment, the fourth channel is an analysis channel. In one aspect, the camera 450 is oriented to be orthogonal to the flow direction in the fourth channel. An embodiment of the present invention includes a focusing particle jet nozzle operably connected to the first channel. In another aspect of the present invention, the second channel is resized and passed through a nozzle before being connected to the third channel.

[0043] Figure 4CAnother embodiment of the sample path of the microfluidic chip is shown. As shown, the fluid first travels in the chip in a vertical upward direction through a first channel 410, which is then directly or indirectly communicated with a second horizontal channel 420, which is located at the top of the chip in this example and includes an analysis channel in this embodiment. The analysis channel according to this configuration is close to the top of the chip, thereby improving the imaging and analysis of the material passing through the microfluidic chip. In a preferred embodiment, the distance from the analysis channel to the top and side of the chip is between about 1mm and about 2mm. However, the distance from the analysis channel to the top of the chip can be between 0.1mm and 100mm, such as from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, from 0.3mm to 0.4mm, etc. In other words, the analysis channel may be arranged in 50%, 33%, 25%, 10% or 5% of the top of the substrate. The length of the horizontal analysis channel according to the present invention can be between about 250 microns and about 10mm. However, the length of the analysis channel can be between 100 microns and 100 mm, such as from 0.1 mm to 0.2 mm, from 0.2 mm to 0.3 mm, from 0.3 mm to 0.4 mm, etc. In other words, the length of the analysis channel can be about 75% or less of the height, width or length of the substrate / chip, such as 50% or less, 33% or less, 25% or less, 10%, or 5% or less of the height, width or length of the substrate / chip. In some embodiments, the substrate may include one or more analysis channels, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 analysis channels.

[0044] Figure 4C Also shown is a light source 480 (e.g., a laser) that can be used to affect a material flow such as a cell flow. The laser can be placed in alignment with the cell flow or can be placed opposite to the cell flow. The laser can also be placed and / or oriented to be orthogonal or diagonal to the cell flow.

[0045] like Figure 1A-1B and Figure 4A-4CAs shown, the fluid flow containing cells or particles is directed to pass vertically through the first channel. One or more substances (fluid, cells and / or particles) enter through the bottom of the chip at the opening of the first channel and enter the substrate in a vertical direction. The length of the first vertical channel is between 100 microns and 100mm, for example, from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, etc. The first channel is followed by a second orthogonal / horizontal channel, and in a preferred embodiment, the second orthogonal / horizontal channel is shorter than the first channel. The length of the second channel can be between 250 microns and 100mm, for example, from 0.25mm to 0.5mm, from 0.5mm to 0.75mm, from 0.75mm to 1.0mm, etc. The third channel extends vertically and is parallel to the first channel. The length of the third channel can be between 50 microns and 100mm, for example, from 0.05mm to 0.1mm, from 0.1mm to 0.15mm, from 0.15mm to 0.2mm, etc. The channels are arranged to be operatively connected, directly or indirectly, to allow one or more substances to move through the multiple channels. The typical direction of fluid flow is given by Figure 4A and 4C The flow direction is given by the arrows in the figure, but the flow can be reversed.

[0046] In a preferred embodiment, the fourth channel comprises an analytical channel, and the fourth channel is a channel with a length between 250 microns and 100 mm, such as from 0.25 mm to 0.5 mm, from 0.5 mm to 0.75 mm, from 0.75 mm to 1.0 mm, etc. In this embodiment, the fourth channel is the channel closest to the top of the chip. The distance between the fourth channel and the top of the chip measured vertically can be between 100 microns and 2 mm, but can also be as long as 100 mm, such as from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, etc. In other words, the fourth channel may be arranged within 50%, 33%, 25%, 10% or 5% of the top of the chip. An imaging device such as a camera may be oriented orthogonal to the fourth channel, and its distance from the fourth channel may be between about 100 microns and 2 mm, but the distance from the fourth channel may be as long as 100 mm, for example, from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, and so on.

[0047] In one embodiment, a laser or other light source has a focusing lens element. Figure 4A The present invention describes that when the laser 480 is in operation, a laser beam is emitted and directed through a focusing lens element into the fourth flow channel 440. Since the gradient force attracts particles toward the area with the highest laser intensity, the particles are aligned within the laser beam. The laser scattering force pushes the particles (e.g., Figure 4A from left to right).

[0048] In another embodiment, in addition to the camera or camera oriented orthogonally to the fourth channel, a second camera or camera (see 450) is oriented toward the side of the analysis (or fourth) channel and is aimed orthogonally to the fourth channel or at any angle to the fourth channel; for example, Figure 4B Taking an orthogonal view image for each cell allows calculation of multiple cell properties in two dimensions, such as size and shape, increasing the amount of information that can be obtained for each cell. This will also allow calculation of cell volumetric properties, including total volume, shape, and provide information about cell size along axes that are not parallel to the flow direction (e.g. Figure 5 The three-dimensional model and its analysis will enable more accurate analysis of the cell, such as cell size, shape, orientation, and other quantitative and qualitative measurements of one or more particles or one or more cells in the fourth channel of the microfluidic chip.

[0049] exist Figure 5 , portions of the analysis channel 540 are shown from two different planes, such as planes that can be imaged from an imaging device (see, e.g. Figure 4A-4C ). In a first plane 520, a portion of a cell or particle 510 is imaged in a certain orientation. In a second plane 530, another portion of the same cell or particle is imaged from a different perspective of another camera. This enables calculation of multiple cell properties, creates a data matrix for each cell per camera, and creates a three-dimensional representation of the cell or particle 550.

[0050] As different parts of the cell pass through the focal plane(s) of the imaging device (e.g., 630 (XZ focal plane) and 640 (YZ focal plane)), different slices of the cell can be imaged as the cell moves, for example, with the aid of fluid flow 620. This is shown in FIG. Fig. 6A In. Fig. 6A In this example, parts of a cell or particle are imaged at multiple points in time and / or space, in different orientations and from different viewing angles. In this example, as the cell moves through the focal plane and rotates, it appears at different sizes in successive images (e.g. Fig. 6A, four example images per plane from two different cameras). A more complex three-dimensional rendering of a particle or cell is achieved using a three-dimensional reconstruction algorithm 650. From this rendering, certain properties of the cell or particle can be inferred, such as cell size, volume, location and size of the nucleus and other organelles, and a measurement or overview of the cell morphology. In addition, the rotation of the cell can be measured as a function of the torque based on the optical force due to changes in biophysical or biochemical properties (including but not limited to refractive index, birefringence, or cell shape or morphology).

[0051] Figure 6B Depicted are multi-plane imaging within the chip, and multiple images of the same cell taken over time, for example, as the cell moves through the focal plane. In this case, the view of the cell is referred to in the art as a "slice" or "image slice". The image slice is actually the thickness of the optical plane being imaged. The thickness of the image plane or slice is determined by factors such as the optical magnification of the imaging system. At higher magnifications, the working distance of the objective lens is reduced, so it is necessary to bring the lens closer to the cell or particle to be imaged. In one embodiment, a laser or other optical force 670 can be used to affect the cell flow in the analysis channel. In a preferred embodiment, the cell or particle can be purposefully lured into or out of the focal plane of the channel by moving the laser and / or camera, or using fluid dynamic focusing to adjust (one or more) streams or positions for imaging. The laser source distance 660 can be moved using, for example, a piezoelectric actuator or a linear electro-optical machine stage. This may be done in a per-cell or per-group manner. The hydrodynamic focusing of the cell can be changed to affect the initial position and trajectory of the cell. For example, particles may be aligned or oriented in the focal plane within a laser beam due to gradient forces that attract particles toward the area of ​​highest laser intensity. Laser scattering forces push particles in the direction of laser beam propagation. See Figure 6B Moving the laser along the X-axis in this example enables imaging of features in different parts of the cell, as shown in disk 680. This may represent, for example, a nucleus, an organelle, an inclusion body, or other features of a cell or particle. Due to the gradient force, the laser pulls the cell to its center. It is also contemplated that two or more cameras may increase detail and accuracy.

[0052] Figure 7 The embodiment of the invention shown in FIG. 7 is a static mode, where a particle or cell 710 is stopped at a specific differential retention site due to a balance of optical forces 730 and fluidic forces 735. The optical force may be applied by, for example, a laser or collimated light source. Figure 5 and Figure 6A-6BAs shown and described, images can be taken in multiple planes. A flow sensor is used to measure the flow rate of each particle when it stops in the flow at a given laser power. Because the optical force and the fluidic force are balanced, the fluidic resistance (i.e., the resistance from the flow rate and the channel direction) is equal to the optical force. The properties of each cell can be measured in turn in this way. Although not a high-throughput measurement system, embodiments of the present invention enable close observation and imaging of captured cells, and also enable dynamic changes in optical forces caused by biochemical or biological changes in the cells. Reagent streams containing chemicals, biochemicals, cells, or other standard biopharmaceuticals can be introduced into the flow channel to interact with the captured (one or more) cells. These dynamic processes can be quantitatively monitored by measuring changes in optical forces during experiments with single or multiple cells.

[0053] In one embodiment, a camera or other imaging device is oriented and / or focused along or against the flow in the assay channel such that the camera or other imaging device is aligned and parallel to the flow (see, e.g., Figure 8 and Fig. 9 ). Figure 8 A camera 810 aligned with an analysis channel 820 and a laser or parallel light source 830 is shown. A dichroic mirror or similar device 840 reflects laser light 835 from the camera to somewhere else to prevent damage, but passes light 865 produced by an illumination source 860 to achieve imaging. The camera is oriented parallel to a fluid stream 870 so that, in one embodiment, a cell or particle 880 moves away from the camera. The illumination source is oriented orthogonal to the channel and the laser. A second dichroic mirror 845 that passes through the laser light and reflects the illumination light is used to direct the illumination light and the laser light through the channel. Another embodiment of this configuration swaps the positions of the laser and the illumination source so that the laser is orthogonal to the channel and the illumination source is parallel to the channel. The second dichroic mirror will still direct the laser light and visible light through the channel.

[0054] Fig. 9 Another embodiment is shown. In this case, the camera or imaging device 910 is oriented so that the cells or particles 980 are traveling toward the camera in the fluid stream 970. Therefore, the camera and laser 930 are located on the same side of the channel, and the illumination source 960 is located at the opposite end of the channel. Two dichroic mirrors 940 and 945 are used to direct the laser light 935 and the illumination light 965 into the channel, direct the illumination light to the camera, and divert the laser light from the illumination source to somewhere else. Another embodiment of this configuration swaps the position of the laser and the camera so that the laser is orthogonal to the channel and the illumination source is parallel to the channel. The second dichroic mirror 945 will then direct the laser light through the channel and direct the illumination light to the camera.

[0055] Optionally, an embodiment of the present invention further comprises at least one optical element, which is located between the optical force source and the fourth channel, and the at least one optical element can be operated to generate a standard TEM00 mode beam, a standard TEM01 mode beam, a standard TEM10 mode beam, a standard Hermite-Gaussian beam mode, a standard Laguerre-Gaussian beam mode, a Bessel beam, or a standard multimode beam. Optionally, at least one optical element comprises a standard cylindrical lens, a standard axis pyramid, a standard concave mirror, a standard toric mirror, a standard spatial light modulator, a standard acousto-optic modulator, a standard piezoelectric mirror array, a diffractive optical element, a standard quarter-wave plate, and / or a standard half-wave plate. Optionally, the optical force source may comprise a standard circularly polarized beam, a standard linearly polarized beam, or a standard elliptically polarized beam.

[0056] Optionally, the present invention includes an apparatus comprising a microfluidic channel, a laser light source focused into the microfluidic channel by an optical device, an electric field source connected to the microfluidic channel by an electrode during operation; flowing particles in a liquid passing through the microfluidic channel; and manipulating the laser light and the electric field to act in combination on the particles in the microfluidic channel to separate the particles based on size, shape, refractive index, charge, charge distribution, charge mobility, dielectric constant and / or deformability. In another embodiment, an apparatus includes a microfluidic channel configured to provide a DEP field to the interior of the channel through (1) an electrode system or (2) an insulator dielectrophoresis (DEP) system, and also includes a laser light source focused into the microfluidic channel by an optical device; a plurality of flowing particles in the liquid enter the microfluidic channel; and operating the laser light and the field in combination to act on the particles in the microfluidic channel to capture the particles or change their velocity, wherein the above-mentioned DEP field is linear or nonlinear. Another possible embodiment of the device includes a microfluidic channel, which includes an inlet and a plurality of outlets, and also includes a laser light source, which is focused by an optical device to pass through the microfluidic channel at a critical angle that matches the flow rate in the microfluidic channel to generate optical forces on particles and simultaneously maximize the residence time of selected particles in the laser light, thereby separating the particles into the plurality of outlets, wherein the laser light can be operated to exert forces on particles flowing through the microfluidic channel, thereby separating the particles into the plurality of outlets.

[0057] Optionally, embodiments of the present invention further include at least one particle interrogation unit in communication with one or more channels, such as (one or more) analysis channels, in particular a fourth channel. The particle interrogation unit includes a standard illuminator, standard optics, and a standard sensor. Optionally, at least one particle interrogation unit includes a standard bright field imager, a standard light scattering detector, a standard single wavelength fluorescence detector, a standard spectral fluorescence detector, a standard CCD camera, a standard CMOS camera, a standard photodiode, a standard photomultiplier tube, a standard photodiode array, a standard chemiluminescence detector, a standard bioluminescence detector, and / or a standard Raman spectroscopic detector.

[0058] At least one particle interrogation unit connected to the fourth channel includes a laser force-based device or apparatus that facilitates the identification, selection, and classification of cellular diseases. In one aspect, the unit utilizes inherent differences in optical pressure caused by changes in particle size, shape, refractive index, or morphology as a means of separating and characterizing particles. In one aspect, a near-infrared laser beam applies physical forces to cells, which are then measured. The optical forces of radiation pressure, when balanced with the fluidic drag on the particles, result in changes in particle velocity that can be used to identify different particles, or changes in particle populations based on inherent differences. Fluidic and optical force balance can also be used to change the relative position between particles based on intrinsic properties of the particles, resulting in physical separation. Another embodiment of the interrogation unit includes an apparatus for particle analysis and / or separation, such as at least one parallel light source, which can be operated to generate at least one parallel light source beam. At least one parallel light source beam includes at least one beam cross section.

[0059] Embodiments of the present invention involve the combination of several of the above-described design elements discussed above in the integrated device. Embodiments also include methods of using such devices. Figure 1A-1B An example of such an integrated device is illustrated. The illustrated embodiment of the present invention is a five-layer structure, all five layers are bonded to each other to produce a solid-state microfluidic chip, although the chip may be a structure opposite to the bonding layers. Several standard materials can be used to manufacture the chip, including but not limited to fused quartz, crown glass, borosilicate glass, soda-lime glass, sapphire glass, cycloolefin polymer (COP), poly(dimethyl)siloxane (PDMS), OSTE, polystyrene, polymethyl methacrylate, polycarbonate, other plastics or polymers. The chip allows for sample input, fluid dynamic focusing, optical interrogation, imaging, analysis, sample exit, and transparent optical access to and from the laser light area. The chip in the embodiment may also be 3D printed, molded or otherwise formed.

[0060] Optionally, at least one particle type includes a plurality of particle types. Each particle type in the plurality of particle types includes respective intrinsic properties and respective induced properties. Optionally, the intrinsic properties include size, shape, refractive index, morphology, intrinsic fluorescence and / or aspect ratio. Optionally, the induced properties include deformation, angular orientation, rotation, rotation rate, antibody-labeled fluorescence, aptamer-labeled fluorescence, DNA-labeled fluorescence, dye-labeled fluorescence, differential retention metric and / or gradient force metric. The method embodiment also includes identifying and separating a plurality of particles based on at least one of the intrinsic properties and the induced properties, according to the respective particle types. Optionally, the method embodiment also includes interrogating or manipulating the sample stream. Optionally, interrogating the sample stream includes determining at least one of the intrinsic properties and the induced properties of the particle type, and measuring the particle velocity of the plurality of particles. Measurement of at least one intrinsic property has a wide range of applications, including but not limited to: determining the viral infectivity of a cell sample (the number of functional infectious viral particles present in a particular cell population, similar to a plaque assay or endpoint dilution assay) for viral quantification, process development and monitoring, sample release assays, adventitious factor testing, clinical diagnostics, biomarker discovery; determining cell productivity for antibodies or proteins for process development and monitoring; determining the efficacy, quality, or activation state of cells produced as cell-based therapies, including CAR T and other oncology applications and stem cells; determining the effects of chemicals, bacteria, viruses, antibiotics, or antiviral drugs on a particular cell population; and determining the disease state or potential of a research or clinical cell sample. Optionally, the photoforce source includes at least one beam axis and the sample stream includes a sample stream axis. The steps of determining at least one of the intrinsic and induced properties of the particle type, and measuring the particle velocity of a plurality of particles together constitute a displacement of the beam axis from the sample stream axis. Optionally, the step of determining at least one of an intrinsic property and an induced property of a particle type, and the step of measuring particle velocity of a plurality of particles together constitute the calculation of a slope and trajectory of a particle of a plurality of particles offset from the sample flow axis to at least one beam axis.

[0061] Those skilled in the art will recognize that these features may be used alone or in any combination, or omitted, based on the requirements and specifications of a given application or design. When an embodiment talks about "comprising" certain features, it should be understood that the embodiment may be "composed of" or "essentially composed of" any one or more of the above features. Other embodiments of the present invention will be apparent to those skilled in the art after consideration of the specification and practice of the present invention.

[0062] It should be particularly noted that a range of values ​​is provided in this specification, and each value between the upper and lower limits of the range is also specifically disclosed. The upper and lower limits of these smaller ranges may also be independently included in the range or excluded from the range. Unless the context clearly provides otherwise, the singular forms "a", "an" and "the" include plural references. The description and examples should be regarded as exemplary or explanatory, and changes that do not deviate from the essence of the present invention are included within the scope of the present invention. In addition, all references cited in the present invention are incorporated herein in their entirety by reference alone, and the citation of these references is intended to provide an effective method for supplementing the enabling disclosure of the present invention, and to provide a background that describes in detail the level of ordinary technology in the art.

Claims

1. A device comprising: A substrate comprising a plurality of channels configured to transport one or more substances, wherein the plurality of channels comprises: a first channel, arranged vertically in the substrate, a second channel, operably connected to the first channel, and arranged horizontally in the substrate, a third channel, operably connected to the second channel, and arranged vertically in the substrate, a fourth channel, operably connected to the third channel and horizontally arranged in the substrate; The first, second, third and fourth channels are arranged in a manner that provides a path for the one or more substances to move through the substrate from the first channel to the second channel to the third channel to the fourth channel.

2. The device according to claim 1, characterized in that The one or more substances are injected into the first channel vertically arranged in the substrate through a bottom horizontal planar surface having an opening leading to the first channel, maintaining directional and volume continuity of the first channel in the vertical direction.

3. The device according to claim 1, characterized in that The one or more substances are injected into the first channel vertically arranged in the substrate through a bottom horizontal planar surface, and the bottom horizontal planar surface has an opening leading to the first channel, wherein the bottom horizontal planar surface has at least one length from one edge to another edge that is shorter than at least one length from one edge to another edge on the vertical planar surface of the chip, maintaining the direction and volume continuity of the first channel in the vertical direction.

4. The device according to claim 1, characterized in that The one or more substances are injected into the first channel vertically arranged in the substrate through a bottom horizontal plane surface, and the bottom horizontal plane surface has an opening leading to the first channel, wherein the bottom horizontal plane surface has a surface area smaller than or equal to the surface area on the vertical plane surface of the chip, maintaining the direction and volume continuity of the first channel in the vertical direction.

5. The device according to claim 1, characterized in that The first channel includes an opening arranged on the outer surface of the substrate, and the opening is arranged in a manner to provide a path for the one or more substances to vertically enter the substrate and move vertically in the first channel.

6. The device according to claim 1, characterized in that The one or more substances are injected into the first channel vertically arranged in the substrate through a bottom horizontal plane surface, and the bottom horizontal plane surface has an opening leading to the first channel, maintaining the direction and volume continuity of the first channel in the vertical direction, wherein the cross-section of the first channel and the cross-section of the opening have the same area, a smaller area, or a larger area.

7. The device according to claim 1, characterized in that The one or more substances are injected into the first channel vertically arranged in the substrate through a bottom horizontal plane surface, and the bottom horizontal plane surface has an opening leading to the first channel, and the direction and volume continuity of the first channel are maintained in the vertical direction, wherein the shape, size and orientation of the first channel and the opening are set in a manner to maintain the direction and volume continuity.

8. The apparatus of claim 1, further comprising a collimated or focused light source oriented to interact with particles or cells in the fourth channel.

9. The device according to claim 1, characterized in that A parallel or focused light source is oriented to propagate in, opposite to, orthogonal to, or diagonally to the direction of movement of the one or more substances in the fourth channel.

10. The apparatus of claim 1, wherein the fourth channel images and analyzes particles or cells in multiple focal planes.

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