KR20230157541A - Microfluidic chip device for optical force measurements and cell imaging using microfluidic chip configuration and dynamics - Google Patents

Abstract

The present invention relates to a microfluidic chip configuration in which injection occurs in an upward vertical direction and fluid vessels are positioned below the chip to minimize particle settling before and in the analytical portion of the channels of the chip. In one aspect, the input and fluid flows upward through the bottom of the chip using a manifold, which prevents orthogonal reorientation of the fluid dynamics. The contents of the vial are placed beneath the chip and pumped directly upward and vertically into the first channel of the chip. A long channel extends from the bottom of the chip to near the top of the chip. Additionally, the channel takes short horizontal turns which almost excludes any effects of cell settling due to gravity and zero flow velocity in the walls. Fluid is pumped to the horizontal analysis portion, which is the highest channel/fluid point on the chip and therefore closer to the top of the chip, resulting in sharper imaging. The laser can also suspend cells or particles within this channel during analysis, preventing their settling.

Description

Microfluidic chip device for optical force measurement and cell imaging using microfluidic chip configuration and dynamics {MICROFLUIDIC CHIP DEVICE FOR OPTICAL FORCE MEASUREMENTS AND CELL IMAGING USING MICROFLUIDIC CHIP CONFIGURATION AND DYNAMICS}

The present invention relates generally to devices and methods for particle analysis and imaging of particles or cells in fluids, and in particular to devices and methods using pressure, fluid dynamics, electrokinetic, and optical forces. Disclosed are devices and methods for particle imaging of fluids.

The present invention relates to a microfluidic chip, in which injection occurs in an upward vertical direction, and particles settle in and in front of the analysis portion of the channels of the chip. Fluid vials are placed under the chip to minimize settling.

In order to implement the present invention, changes must be made to existing microfluidic chip designs. For example, to maintain the vials vertically along the chip, a different interface with the chip must be established compared to the prior art. Specifically, an input tube passes through a port attached to the largest side of the chip (as is typically done in microfluidic lab-on-a-chip systems). Instead of interfacing with the chip in an orthogonal manner and then pumping the fluid first across the chip and then up, the present invention in one aspect uses a manifold to have the input and fluid come up through the bottom of the chip; This prevents horizontal re-orientation of the fluid/fluid dynamics.

According to the invention, the contents of the vial are placed beneath the chip and pumped upwards and vertically directly into the first channel of the chip. A long channel extends from the bottom of the chip to near the top of the chip. And, although the channel takes a short horizontal turn, the new channel is short enough to virtually eliminate any effect of cell settling due to gravity and zero flow velocity at the walls. Additionally, unlike the prior art, fluid is pumped up to the analysis section. Therefore, the horizontal analysis portion is the highest channel/fluid point within the chip and is therefore close to the top of the chip, which requires less chip material (e.g. glass) between the microscope/camera and the sample than in the prior art, and therefore Causes clearer imaging. The laser also suspends the cells in this channel during analysis, preventing their sedimentation.

According to the prior art, microfluidic chip vials containing cells or particles to be separated and/or analyzed are placed on the side and pumped horizontally into channels within the microfluidic chip. First, the contents of the vials (e.g., particles or cells) are pumped vertically upward, then make a U-turn and move downward, and then are pumped horizontally into the chip (e.g., US Pat. No. 9,594,071 (see issue).

The connection to the chip is horizontal, which combined with the dead volume in the connection (empty space in fluidic connections to some extent unavoidable) causes significant additional settling due to gravity. This configuration also requires a relatively large diameter channel required by the connection, which creates a relatively low velocity region in addition to dead volume, further increasing the problem of particle settling. The current chip according to the invention eliminates the need for a large horizontal input channel and the rather sharp change from the large horizontal input channel to a relatively thin upward flow in the first vertical chip channel. This configuration eliminates horizontal settling and unnecessary directional changes that cause settling. Having the cells enter the lower edge of the chip also avoids the problem of settling in the dead volume by orienting the chip perpendicular to gravity so that the cells or particles do not settle at the bottom of the horizontal channel, but rather continue to be guided upward by the flow. Solve it. This is counter-intuitive and required a lot of experimentation to recognize the problem before designing the currently implemented solution. Currently available microfluidic devices, unlike the present invention, incorporate custom or commercially available connections on the polished surface of the glass and larger areas, which typically allow for any particles, such as cells, contained within the sample stream. Have the player take the turn immediately and move horizontally when entering the chip.

Additionally, in the prior art, cells or particles have several horizontal runs on the microfluidic chip before reaching the analysis channel, causing sedimentation. At the point where the vial contents enter the chip and channels within the chip, the contents are pumped horizontally compared to the vertical in-chip channel. The channel then flows upward and takes a long horizontal turn, at which point the cells not only settle to the bottom of the channel due to gravity, but also experience lower velocities at the walls due to laminar flow conditions. There is a tendency. Essentially, due to the parabolic velocity profile, the flow is highest in the middle of the channel and decreases to zero at or near the channel walls. After the first horizontal in-chip channel, the fluid takes a downward turn before the analysis channel where particles are imaged or separated. Due to this configuration, there is a relatively large distance between the microscope/camera and the analysis channel. In this typical prior art configuration, the particles are pushed down and ultimately exit the bottom of the chip.

Moreover, due to limitations in the prior art, multiple horizontal runs are required, allowing cells to settle in multiple locations within the channels. This also causes a reduction in image quality due to the need to image through additional material at the edge of the chip. Channels within a prior art chip had to be pumped vertically upward, then horizontally, then in a zigzag fashion for proper cell or particle suspension, then down and out of the chip. Zigzag channels are excluded by the invention.

There also exists prior art regarding rendering 3D images of cells or particles in a fluid. For example, M. Habaza, M. Kirschbaum, C. Guernth-Marschner, G. Dardikman, I. Barnea, R. Korenstein, C. Duschl, N. T. Shaked, Adv. Sci. 2017, 4, 1600205 teaches capturing cells, rotating them at high speed, and measuring the refractive index distribution within the cells using interferometry. Interferometry has also been used to analyze cells within microfluidic channels (see, e.g., Y. Sung et al., Phys. Rev. Appl. 2014 Feb. 27; 1: 014002). However, the present invention seeks to take multiple images of a cell or particle as it moves in a fluid flow and passes through the focal planes of the imaging device(s), thus capturing the cell to render a 3D image. Make sure you don't have to do it at all. Other techniques, such as using a mechanical translation stage to move cells or particles (e.g., N. Lue et al., Opt. Express 2008 Sep. 29; 16(20): 16240-6 ) are taught, none of which uses bright field imaging as described herein and none of which utilize fluid flow to provide cell positioning relative to the image focal plane. No.

All prior art references cited herein are incorporated by reference in their entirety.

The present invention relates to a microfluidic chip where injection occurs and sample vials are placed beneath the chip to minimize particle settling. Accordingly, the contents of the vial are placed beneath the chip and pumped upward and vertically directly into the chip's channels. A long channel extends from the bottom of the chip to near the top of the chip. And, although the channel takes a short horizontal turn, the new channel is short enough that any effect of cell settling due to zero flow velocity at the walls of the channel is negligible. Additionally, unlike the prior art, the sample is pumped up to the analysis section. Therefore, the horizontal analysis portion is the highest channel/fluid point within the chip and is therefore close to the top of the chip, which results in less glass between the microscope/camera than prior art, and therefore sharper imaging. The distance of the analysis channel from the top of the chip can be 100 microns to 2 mm, but can be as large as 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns, etc. there is. In one embodiment, after the analysis portion of the chip, samples, such as fluids, cells and/or particles, are pumped down to the bottom of the chip and pushed out.

The invention also relates to a microfluidic chip in which horizontal runs are minimized, particularly at the point where fluid enters the chip channels. Prior art chips contain approximately 13 mm in horizontal channels (non-analysis portions), of which approximately 2 mm is for the much larger diameter injection port, exacerbating sedimentation due to low velocities. . The chip described herein has about 0.2 to 3.0 mm in horizontal channels (unanalyzed) in a preferred embodiment, but the horizontal channels may range in length from 0.01 to 100.0 mm, such as 0.01 to 0.02 mm, 0.02 to 0.03 mm. , may have a length range of 0.03 mm to 0.04 mm. This is the result of the invented channel system, which differs by a factor of 10 from the prior art, which improves flow and cell/particle sediment removal.

Another aspect of the invention relates to a microfluidic chip, wherein imaging occurs and analysis is based at or near the corners of the chip, so there is less glass and distance between the camera and analysis channels. This improves imaging from multiple viewpoints. This also allows higher numerical objective lenses to be used to improve detailed imaging by increasing magnification. This design improvement reduces distortion of the image caused by the glass (or other material containing the chip, such as plastic or any transparent or translucent material) and distance (e.g., due to defects in the glass). The distance between the imaging device and the analysis channel can be 100 micrometers to 2 mm, but can be as large as 100 mm, such as 100 micrometers to 200 micrometers, 200 micrometers to 300 micrometers, 300 micrometers to 400 micrometers, etc.

Additionally, the present invention provides a separation downstream from the analysis channel that allows for the separate use of both pressure and/or laser (or other optical forces) simultaneously or sequentially to activate a sorting function. It relates to a microfluidic sorting chip. In one aspect, flow will continue from the analysis channel for a sorting function. For example, particles may be directed into a vertical channel and then into a horizontal sorting channel. In one embodiment, optical force and/or pressure is applied in the direction of flow relative to the sorting channel to push particles through the channel. Particles that are not directly affected by optical forces, for example gravity, electrokinetic forces, magnetic forces, laminar flow lines, stream lines, reduced flow rates, orthogonal optical forces, or alternative channels The vacuum applied to suction into the alternate channel will cause it to switch to the alternate channel. In another aspect related to a sorting post-analysis channel, the present invention allows directing optical force from the backside of the chip (laser or optical force oriented in the same flow direction), and in some aspects This allows splitting the main laser. In embodiments, the optical force and/or pressure may be applied in the opposite direction of the movement of the material through the channel, such as against the flow, or in the same direction as the movement of the material within the channel, such as along the flow. may be approved. Cell or particle sorting can occur on a single device or on separate chips. For example, in Figure 1B, the fifth channel before outlet tubing 145 includes one or more bifurcations to enable single or multiple fractionation regions.

In another aspect of the invention, the manifold is connected to the vial in such a way that tubing passes through the manifold and connects to the vial or other vessel on the other side to contact the substance (e.g., fluid) within the vial. The manifold allows vials to be connected to the microfluidic chip but stored beneath the chip, and/or the manifold allows the contents of the vials to be injected from the bottom of the chip, such that the vial or tubing from the vial is It alleviates several problems experienced by prior art techniques, such as cell or particle sedimentation connected to or communicating with the microfluidic chip.

In another aspect, the present invention relates to a microfluidic chip holder, the chip holder comprising an integrated prism that allows light to exit at an angle relative to the chip when the prism is installed and is a preferred method for illuminating constrained geometries. It includes a structure for guiding a light source including an integrated prism cavity. In embodiments, the light source includes, but is not limited to, optical fibers or a collimated or focused light source. This light source is precisely aimed, oriented or especially directed into the analysis channel.

In another aspect of the invention, a device includes a first camera and a second imaging device oriented orthogonal to the channel view. The reasons for the second camera are varied. In one aspect, the reason for the second imaging device is to assist in visual alignment of the laser or optical force in the analysis channel. In another aspect, data from a first camera may be recorded using the methods described herein. With the second camera, data can be combined with data from the first camera, resulting in additional data that can be used to more accurately estimate cell location, size, shape, volume, etc. This additional information about the same cell (or particle) increases the accuracy and scope of measurements and analysis. In a further aspect, the second camera is coupled with the first camera to produce a 3D reconstruction of the cell or particle, or group of cells or particles, imaged by the orthogonal camera and the camera in the flow or the camera positioned toward the side of the chip. Allowed. Using the algorithms described herein, including reversing or slowing the flow and taking one or more images of a particular cell or particle, or group of cells or particles, the present invention allows multiple images to be analyzed and processed. Allowing for determination of characteristics/attributes/quantitative measurements such as cell volume, cell shape, nuclear position, nuclear volume, organelle or inclusion body position, etc. makes possible. In another aspect of the invention the camera is oriented to image on an axis in the direction of flow.

In one aspect, the present invention does not require tortuous or zigzag channels, as preferred according to the prior art, to keep the particles properly suspended. Because of the vertical nature of the fluid and particles or cells injected within the chip, the zigzag channel has a vertically integrated pumped channel that flows directly upward through the channel into the first horizontal channel (referred to herein as the second channel). excluded by particles.

The accompanying drawings illustrate certain aspects of some of the embodiments of the invention and should not be used to limit or define the invention. The drawings, together with the written description, serve to explain certain principles of the invention.
1A is a diagram showing a global view of the device configuration including chip, manifold and vials. FIG. 1B is a diagram showing specific depictions of chip channel orientations and positions.
2A and 2B include diagrams showing a microfluidic chip holder according to the present invention.
3A and 3B are diagrams showing angles and aspects of a chip holder according to the present invention.
4A and 4B are diagrams showing examples of cellular pathways and imaging configurations associated with the chip. Figure 4C is a diagram depicting alternative cellular pathways.
Figure 5 is a diagram illustrating on chip multi-plane imaging and how it can be used to render 3D images and information.
6A and 6B are diagrams illustrating on-chip multi-plane imaging in fluid flow and how it can be used to render 3D images.
Figure 7 is a diagram illustrating how cells can be captured and/or balanced within the analysis portion of the chip and imaged from multiple angles.
Figure 8 is a diagram showing how the camera and illumination source can be positioned along the laser and flow direction so that the particles move away from the camera.
Figure 9 is a diagram showing how the camera and illumination source can be positioned along the laser and flow direction so that particles move toward the camera.

The 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 changes may be made in the practice of the invention without departing from the scope or spirit of the 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 that include various features may also consist of or consist essentially of these various features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. The description of the invention provided is merely exemplary or explanatory in nature, and therefore changes that do not depart from the essence of the invention are intended to be within the scope of the invention.

Before describing at least one embodiment of the invention in detail, it should be understood that the invention is not limited in its application to the details of the configuration and arrangement of components presented in the following description or shown in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

Referring now to the drawings, FIG. 1A shows a global view of the device taught herein. Sample vials 130 are positioned beneath microfluidic chip 100 (which may also be generally referred to herein as a substrate), and there is no limitation as to the number or sizes of vials. Vials may contain one or more substances, including but not limited to one or more of fluids, liquids, gases, plasma, serum, blood, cells, platelets, particles, etc., or combinations thereof, through the device. It is configured to hold any type of sample that can be moved. In the context of this specification, the terms fluid or sample may be used generically to refer to one or more such substances. The vials are connected directly or indirectly, for example using air tubing 110 operably communicating with the underside of the chip. These may also be connected via manifold 120 as further shown in Figure 1A. FIG. 1B shows a preferred embodiment of a microfluidic chip 100, whereby substances, fluids, particles and/or cells are formed on one outer surface, such as an edge of the microfluidic chip (e.g., in FIG. 1B ). is injected into the XZ plane). The injection is shown in Figure 1B along one of the planes shared by a minimum distance, such as the XZ plane or the XY plane. In this particular embodiment, the length of side X (160) is less than the length of side Y (170) and the length of side Z (180). This configuration allows the sample to have minimal deviation as it enters the channels on the chip (i.e. no right turn is required, as is generally the case with the current state of the art). ).

1A, manifold 120 holds a vial or vials 130 so that substances, fluids, particles, and/or cells can be injected or pumped vertically upward into the microfluidic chip 100. ) is shown as being connected to. The manifold allows for injection of materials from the bottom of the chip, while also positioning the electronics, flow sensors, and tubing (both liquid and air) in a way that minimizes cell deposition, which optimizes throughput. do. Pneumatic tubing provides pressure or vacuum and, when sealed, provides a closed system within the confines of the manifold device. In one embodiment, there is a distance between the vial(s) and the manifold, indicating that no seal exists and the pressure in the interior volume is atmospheric. This allows pumping of substances to or from a vessel open to the atmosphere (a vacuum is required for pumping from an open vessel). The manifold positions the electronics, flow sensors, and tubing (both liquid and air) in a way that minimizes cell deposition, which optimizes throughput.

By injecting the contents from the bottom of the chip, the present invention prevents horizontal movement in the inlet tubing (140) and outlet tubing (145) causing problems such as settling of particles or cells within the channel(s) (150). minimize. The outlet tubing is offset from the inlet tubing, in this example in the Z and X dimensions (FIG. 1B). In embodiments, the outlet tubing is offset, unoffset, straight, in-line, or sloped relative to the inlet tubing. This configuration eliminates the need for tortuous or zigzag vertical channels because the current configuration does not allow fluid associated with the cells and/or particles to flow into the chip from the side in a horizontal direction, as in the prior art. This solves the settling problems that arise when the direction and fluid dynamics must change to push upward after being injected or pumped. The manifold and injection from the bottom of the chip also allow additional elements, components, machinery or hardware to be placed beneath the chip (see Figure 1A).

Manifold 120 operates by regulating the air pressure above the contents in the vial and providing the correct geometry for the flow sensor and electronics. Tubing, such as fluid or air tubing, passes through the manifold and connects to a vial on the other side. Pressurized air passes through one side of the manifold and creates a closed pressurized system within the manifold. By regulating the pressure within the enclosure, the system allows changes to parameters such as flow rate and fluid dynamics. Pressurization areas are in both vial(s) 130 and manifold 120. In another aspect, no air connection is required for the vial since it is open to the surrounding atmosphere. This allows sampling of a wider variety of containers and sources. In such embodiments, a vacuum is applied to one or more vials to create a pressure differential to drive fluid flow from the open vessel.

In one embodiment, pressure-based sample injection is used. The vials are filled with the sample in fluid and sealed with a lid or tubing connected to the chip. Before attaching the lid, the vial may be open to the air or sealed with a septum or other airtight device. In one aspect, the lid may include two connections, one for a fluid such as a gas and one for a liquid. Optionally, method embodiments further include providing a sample inlet line tip communicating with a sample inlet line communicating with the first channel.

In another embodiment, vacuum-based sample injection is used. The vials are filled with the sample in fluid and sealed with a lid or tubing connected to the chip. Before attaching the lid, the vial may be open to the air or sealed with a septum. In one aspect, the lid may include two connections, one for a fluid such as a gas and one for a liquid. Optionally, fluid can be aspirated from a vial open to atmosphere by applying vacuum pressure to one or more of the other vials. Optionally, method embodiments further include providing a sample inlet line tip in communication with a sample inlet line in communication with the first channel.

Chip holders 200 and 300 are shown in FIGS. 2A and 2B and FIGS. 3A and 3B. The chip holder includes a structure for guiding a light source 210, such as a fiber optic light source, a light emitting diode or a laser, and an integrated prism cavity 220, 320 in which a prism can be installed. The light source is guided or aligned to the desired location by an integrated structure or channel 240 within the chip holder. The built-in space for a fiber optic light source allows illumination, such as a cone light 250, even in restricted geometric environments such as the microfluidic chip 230, 330 or a channel within the microfluidic chip. This light source is, in a particularly preferred embodiment, precisely directed, oriented, or focused onto the analysis channel 260. In a preferred embodiment, the chip holder includes built-in space for prisms 220, 320 and a fiber optic light source 210, allowing illumination of limited geometries. The chip further includes holes or openings in the bottom of the holder 350 for precise alignment of fluid tubing as described herein. In one embodiment, adjustable screws are incorporated into screw holes 360 on one or more sides for proper alignment.

Figure 4A is a preferred embodiment of the microfluidic chip 400 described herein. As shown, the fluid first moves vertically upward through the first channel 410 and then communicates with the second horizontal channel 420. Another vertical channel 430 takes the fluid much closer to the top of the chip, at which point a fourth channel 440 is horizontal and contains the analysis channel, as shown in FIG. 4 . The channels operably communicate with each other to allow samples to be moved from one channel to another throughout the system. In embodiments, the sample may flow from the first channel to the second channel, to the third channel, to the fourth channel, or vice versa, or a combination thereof. Pumps and/or vacuum devices may be provided to provide positive and/or negative pressure at either or both the opening and outlet of the channels to enable movement of substances through the channels. The analysis channel is close to one or more external surfaces of the substrate, such as faces, edges, or sides of the chip. For example, the analysis channels according to this configuration are close to the top and sides of the chip, thereby improving imaging and analysis through the materials of the microfluidic chip. In a preferred embodiment, the analysis channel is about 1 mm to about 2 mm from the top and side of the chip. However, the distance of the analysis channel from the top of the chip may be 0.1 mm to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. Expressed another way, the analysis channel may be located within the top 50%, 33%, 25%, 10%, or 5% of the substrate. The length of the horizontal analysis channel according to the present invention may be from about 250 micrometers to about 10 mm. However, the length of the analysis channel may be 100 micrometers to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. Expressed another way, the length of the analysis channel is no more than about 75% of the height, width, or length of the substrate/chip, e.g., no more than 50%, no more than 33%, no more than 25% of the height, width, or length of the substrate/chip. , may be 10% or less, or 5% or less.

In FIG. 4B, two imaging devices 450, such as machine vision cameras, are shown. In one embodiment, a camera may be positioned on the chip and oriented orthogonal to the analysis channel. In other embodiments, the camera may be located on the side of the chip and oriented perpendicular to the direction of flow, or diagonal to the direction of flow (e.g., above the channel, below the channel, or at an angle to the side of the channel). . In embodiments, the camera may be configured to image at any angle relative to the flow of one or more materials, for example perpendicular or 90 degrees or 0 to 90 degrees or 10 to 80 degrees or 30 to 60 degrees to the flow of one or more materials. The position may be set to be performed in degrees, etc. In another embodiment, two or more cameras may be used to image cells or particles in the analysis channel. For example, a camera may be on the chip and oriented orthogonal to the analysis channel. The second camera may be located on the side of the chip and oriented perpendicular to the direction of flow or diagonal to the direction of flow (eg, above the channel, below the channel, or at an angle to the side of the channel). As shown in Figure 4B, one or more light sources 460 can be used to illuminate the analysis channel 440, such that these light sources can be positioned below the chip, illuminating upward against the side of the chip, or flowing over the chip. It can be illuminated either directionally or against the direction of flow, downwards or diagonally with respect to the analysis channel.

Alternatively, a dichroic mirror 840 or other suitable optical element may be used to selectively convert light in certain wavelength ranges while allowing other wavelengths to pass, as shown in FIG. 8. You can. This will allow cameras 810 to be placed along the analysis channel. As shown in FIGS. 8 and 9 , several embodiments of this occur, including placing optical force laser 830 and camera 810 at the same or opposite ends of the analysis area. An illumination source 860 for the camera may also be required and may be oriented in several ways, such as those shown in FIGS. 8 and 9. 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 as a single camera or in combination with other viewpoints as part of a multi-camera system as described herein.

As shown in Figure 4A, a light source 480, such as a laser, can be used to affect cell flow. The laser can be placed along or against the cell flow. The laser may be placed and/or oriented orthogonal or diagonal to the cell flow.

One embodiment of the present invention includes a device for particle analysis (see, for example, FIGS. 4-9). An embodiment of the invention includes at least one camera 450 for capturing images of particles or cells within microfluidic channels (e.g., 440). In one embodiment, a laser or other optical force 480, such as a collimated light source, operable to produce at least one collimated light source beam is included. At least one collimated light source beam includes at least one beam cross section. Embodiments of the present invention include a substrate having a first channel 410 extending perpendicularly in the substrate, such that the first plane intersects the first channel 410 substantially along its length, thereby forming a The sample is injected into the substrate/chip from the bottom of the chip and pushed upward by positive or negative pressure. Embodiments of the invention include a second channel 420 orthogonal to the first channel, and thus disposed horizontally to the substrate, such that the second plane intersects the second channel 420 substantially along its length. , the second plane is arranged orthogonal to the first plane. This second channel is in the horizontal direction of the chip. The second channel communicates directly or indirectly with the first channel. The second channel communicates directly or indirectly with a short upwardly vertical third channel 430 that takes the channel network closer to the top of the chip. The third channel communicates directly or indirectly with a 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, camera 450 is oriented orthogonal to the direction of flow in the fourth channel. Embodiments of the invention include a focused particle stream nozzle operably connected to a first channel. In another aspect of the invention, the second channel undergoes a change in size and passes through the nozzle before passing into the third channel.

Figure 4C shows another embodiment of a sample path in a microfluidic chip. As shown, fluid first moves vertically upward in the chip through a first channel 410 which then connects directly or indirectly with a second horizontal channel 420 in this example at the top of the chip. , and in this embodiment includes an analysis channel. The analysis channel according to this configuration is close to the top of the chip, thereby improving imaging and analysis through the materials of the microfluidic chip. In preferred embodiments, the analysis channel is about 1 mm to about 2 mm from the top and side of the chip. However, the distance of the analysis channel from the top of the chip may be 0.1 mm to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. Expressed another way, the analysis channel may be located within the top 50%, 33%, 25%, 10%, or 5% of the substrate. The length of the horizontal analysis channel according to the present invention may be from about 250 micrometers to about 10 mm. However, the length of the analysis channel may be 100 micrometers to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. Expressed another way, the length of the analysis channel is no more than about 75% of the height, width, or length of the substrate/chip, e.g., no more than 50%, no more than 33%, no more than 25% of the height, width, or length of the substrate/chip. , may be 10% or less, or 5% or less. In 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.

Also, as shown in Figure 4C, a light source 480, such as a laser, may be used to influence material flow, such as cell flow. The laser can be placed along or against the cell flow. The laser may also be placed and/or oriented orthogonal or diagonal to the cell flow.

1 and 4 the fluid flow containing cells or particles is directed 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 first vertical channel is between 100 micrometers and 100 mm in length, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, etc. The first channel is, in a preferred embodiment, followed by a second orthogonal/horizontal channel that is shorter than the first channel. The second channel may be 250 micrometers to 100 mm in length, such as 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, 0.75 mm to 1.0 mm, etc. The third channel runs perpendicular and parallel to the first channel. The third channel may be 50 micrometers to 100 mm in length, such as 0.05 mm to 0.1 mm, 0.1 mm to 0.15 mm, 0.15 mm to 0.2 mm, etc. The channels are arranged to be operably communicated, either directly or indirectly, in a manner that allows one or more substances to move through the plurality of channels. The normal direction of fluid flow is given by the flow arrows in FIGS. 4A and 4C, but can be reversed.

In preferred embodiments, the fourth channel comprises an analysis channel, which is a channel having a length between 250 micrometers and 100 mm, for example between 0.25 mm and 0.5 mm, between 0.5 mm and 0.75 mm, between 0.75 mm and 1.0 mm, etc. In this embodiment, the fourth channel is the channel closest to the top of the chip. The distance of the fourth channel from the top of the chip measured vertically may be 100 micrometers to 2 mm, but may be as small as 100 mm, such as 100 micrometers to 200 micrometers, 200 micrometers to 300 micrometers, 300 micrometers to 400 micrometers, etc. It can be big. Expressed another way, the fourth channel may be located within the top 50%, 33%, 25%, 10%, or 5% of the chip. The imaging device, such as a camera, is positioned perpendicular to the fourth channel and about 100 micrometers to 2 mm from the fourth channel, but between 100 micrometers and 200 micrometers, 200 micrometers and 300 micrometers, and 300 micrometers and 400 micrometers. It may be oriented approximately 100 mm from the fourth channel, such as the like.

In one embodiment, a laser or other light source is present with the focusing lens element. Figure 4A illustrates the present invention with a laser 480 operative to emit a laser beam and direct the beam through a focusing lens element into a fourth flow channel 440. Particles are aligned within the laser beam due to a gradient force that pulls the particles toward the area of highest laser intensity. The laser scatter force propels the particles in the direction of laser beam propagation (e.g., left to right in FIG. 4A).

In another embodiment, the second camera or image capturing device (see 450) is, in addition to the camera or image capturing device oriented orthogonally to the fourth channel, here oriented to the side of the analysis (or fourth) channel; For example, as shown in FIG. 4B, it is oriented orthogonal to the fourth channel or at an arbitrary angle. Taking one image of each cell in orthogonal views allows multiple cell properties, such as size and shape, to be calculated in two dimensions, increasing the amount of information that can be captured for each cell. increase This also allows calculation of volume properties of cells including total volume, shape, and provides insight into cells that are not symmetrical about an axis parallel to the direction of flow (Z-axis as shown in Figure 5). Using two or more cameras, the underlying 3D model of the cell 550 can be reconstructed using existing 3D reconstruction algorithms, such as, for example, the diffraction-theory method or the illumination rotation-method. It can be constructed by combining orthogonal images using reconstruction algorithms. The 3D model and analysis of the 3D model may include cell size, shape, orientation, and other quantitative and qualitative measurements regarding the particle(s) or cell(s) within the fourth channel of the microfluidic chip. It will allow more accurate analysis of cells.

In Figure 5, a portion of the analysis channel 540 is shown from two different planes, such as planes that can be imaged from an imaging device (see, eg, Figure 4). In the first plane 520, a portion of a cell or particle 510 is imaged at a given orientation. In the second plane 530, different parts of the same cell or particle are imaged from different perspectives from different cameras. This allows calculation of multiple cell properties, creating a matrix of data per cell per camera and a 3D rendition of the cell or particle 550.

As different portions of the cell pass through the focal planes of the imaging device(s) (e.g., 630 (XZ focal plane) and 640 (YZ focal plane)), the cell is moved, for example, by fluid flow 620. As it moves, different slices of the cell can be imaged. This is shown in Figure 6a. In Figure 6A, parts of a cell or particle are imaged at multiple viewpoints and/or points in space, in different orientations and from different perspectives. In this example, as the cells move and rotate through the focal plane, they appear at different sizes in successive images (such as four example images per plane from two different cameras as shown in Figure 6A). Using 3D reconstruction algorithms, this allows for more complex 3D rendering 650 of particles or cells. From this rendering, certain attributes of the cell or particle are estimated, such as cell size, volume, location and size of the nucleus as well as other organelles, and measurement or outlining of the cellular topography. It can be. Cell rotation can also be measured as a function of optical force-based torque due to changes in biophysical or biochemical properties, including but not limited to refractive index, birefringence, or cell shape or morphology.

Figure 6b shows on-chip multi-plane imaging with multiple images of the same cell taken over time, for example as the cell moves through the focal plane. In these cases, the view of the cells 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 or slice of the image plane is particularly governed by the optical magnification of the imaging system. At higher magnifications, the working distance of the objective lens decreases, creating the need for the lens to be closer to the cell or particle to be imaged. In one embodiment, a laser or other optical force 670 may be used to influence the flow of cells in the analysis channel. In a preferred embodiment, cells or particles are brought into or out of the focal plane of the channel for imaging by moving the laser and/or camera, manipulating the flow(s), or positioning using hydrodynamic focusing. It can be intentionally induced externally. For example, the laser source can be moved a distance 660 using, for example, a piezo electric actuator or a linear electric optomechanical stage. This can be done cell-by-cell or population-by-population. The hydrodynamic focusing of cells can be altered to affect their initial position and trajectory. For example, particles can be aligned or oriented at the focal plane within the laser beam due to the longitudinal force pulling the particles toward the area of highest laser intensity. The laser scattering force propels the particles in the direction of laser beam propagation (see Figure 6b). Moving the laser in this example in the X-axis allows imaging of features in different parts of the cell illustrated by disk 680. This may represent, for example, the nucleus, organelles, inclusion bodies, or other features of a cell or particle. The laser pulls the cell to its center as a result of the longitudinal force. Additionally, it is contemplated that more than two cameras may increase detail and accuracy.

The embodiment of the invention shown in FIG. 7 is a static mode in which particles or cells 710 are stationary at a specified differential retention location by balancing optical forces 730 and fluidic forces 735. (static mode). The optical force may be applied, for example, by a laser or collimated light source. Images may be taken in multiple planes such as those shown and described for FIGS. 5 and 6A-B. A flow sensor is used to measure the flow rate at which each particle stops flowing for a given laser power. Because the optical and fluid forces are balanced, the fluidic drag force (i.e., the force from flow rate and channel dimension) is equal to the optical force. The properties of each cell can be measured sequentially in this way. Although not a high-throughput measurement system, this embodiment of the invention allows close observation and imaging of captured cells, and also dynamic changes in optical force resulting from biochemical or biological changes in the cells. Reagent streams containing chemicals, biochemicals, cells, or other standard biological agents can be introduced into the flow channels to interact with the captured cell(s). These dynamic processes can be monitored quantitatively by measuring changes in optical force during experiments on a single cell or cells.

In one embodiment, the camera or other imaging device is oriented and/or focused within or against the flow of the analysis channel, such that it follows and parallel to the flow (see, e.g., FIGS. 8 and 9). 8 shows a camera 810 following an analysis channel 820 and a laser or collimated light source 830. A dichroic mirror or similar device 840 reflects the laser light 835 away from the camera to prevent damage but allows the light 865 generated by the illumination source 860 to pass through to enable imaging. The camera, in one embodiment, is oriented parallel to the fluid flow 870 so that the cells or particles 880 move away from the camera. The illumination source is oriented orthogonal to the channel and laser. A second dichroic mirror 845, which passes the laser light and reflects the illumination light, is used to direct both the illumination light and the laser light through the channel. An alternative embodiment of this configuration switches the positions of the laser and illumination source such that the laser is orthogonal to the channel and the illumination source is parallel to the channel. The second dichroic mirror will still direct both laser light and visible light through the channel.

An alternative embodiment is shown in Figure 9. In this case, the camera or imaging device 910 is oriented so that the cells or particles 980 move toward the camera in the fluid flow 970. Accordingly, the camera and laser 930 are on the same side of the channel, while the illumination source 960 is on opposite ends of the channel. Two dichroic mirrors 940 and 945 are used to direct the laser light 935 and illumination light 965 into the channel, direct the illumination light to the camera, and divert the laser light away from the illumination source. An alternative embodiment of this configuration switches the positions of the laser and camera such that the laser is orthogonal to the channel and the illumination source is parallel to the channel. A second dichroic mirror 945 will then direct the laser light through the channel and the illumination light to the camera.

Optionally, embodiments of the present invention further comprise at least one optical element between the source of optical force and the fourth channel, wherein the at least one optical element is configured to select a standard TEM ₀₀ mode beam, a standard TEM ₀₁ mode beam, a standard TEM To generate a _10- mode beam, a standard Hermite-Gaussian beam mode, a standard Laguerre-Gaussian beam mode, a Bessel beam, or a standard multimodal beam. It is possible to operate. Optionally, the at least one optical element is a standard cylindrical lens, a standard axicon, a standard concave mirror, a standard toroidal mirror, a standard spatial light modulator, or a standard acousto-optic modulator. (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 source of optical force may include a standard circularly polarized beam, a standard linearly polarized beam, or a standard elliptically polarized beam.

Optionally comprising a microfluidic channel, a laser light source focused into the microfluidic channel by optics, and a source of electric field operably coupled to the microfluidic channel via electrodes; flowing particles in a liquid through a microfluidic channel; and manipulating the laser light and electric field to jointly act on the particles within the microfluidic channel to determine their size, shape, refractive index, electrical charge, electrical charge distribution, charge mobility, and permittivity. A device that separates particles based on permittivity and/or deformability is implemented. In another embodiment, the device comprises a microfluidic channel configured to supply a dielectrophoretic (DEP) field to the interior of the channel via (1) an electrode system or (2) an insulator dielectrophoretic (DEP) system, and an optical system. It includes a laser light source focused into the channel; flowing a plurality of particles in a liquid into a microfluidic channel; Laser light and a field are jointly actuated on particles within a microfluidic channel to trap them or modify their velocity, and the DEP field can be linear or non-linear. Another possible embodiment of the device includes a microfluidic channel comprising an inlet and a plurality of outlets, and matching the flow rate within the microfluidic channel to generate an optical force on the particles while maximizing the residence time of selected particles in the laser light. A laser light source focused by an optical system to separate particles into a plurality of outlets by crossing the microfluidic channel at a critical angle, wherein the laser light applies a force to the particles flowing through the microfluidic channel to separate the particles into the plurality of outlets. It can be operated to separate within.

Optionally, embodiments of the invention further comprise at least one particle interrogation unit communicating with one or more of the analysis channel(s), particularly the fourth channel. The particle interrogation unit includes a standard illuminator, standard optics, and a standard sensor. Optionally, the at least one particle interrogation unit includes a standard bright field imager, a standard light scatter detector, a standard single wavelength fluorescent detector, or a standard spectroscopic fluorescent detector. detector, standard CCD camera, standard CMOS camera, standard photodiode, standard photomultiplier tube, standard photodiode array, standard chemiluminescent detector, standard bioluminescent detector and/or standard Includes a Raman spectroscopy detector.

At least one particle interrogation unit communicating with the fourth channel includes a laser force based device or device that facilitates cellular disease identification, selection and classification. In one aspect, the unit utilizes inherent differences in optical pressure resulting from changes in particle size, shape, refractive index, or morphology as a means to separate and characterize particles. In one aspect, a near-infrared laser beam exerts a physical force on the cells, and this force is then measured. Optical forces through radiation pressure, when balanced against the fluid drag on the particles, are based on changes in particle velocity or intrinsic differences that can be used to distinguish between different particles. Causes changes in populations of particles. Flow and optical force balances can also be used to cause physical separations by changing the relative position of particles with respect to each other based on their intrinsic properties. Another embodiment of the interrogation unit comprises a device for particle analysis and/or separation, such as at least one collimated light source operable to generate at least one collimated light source beam. At least one collimated light source beam includes at least one beam cross section.

One embodiment of the invention includes a combination of several of the above-described design elements within a unitary device. Embodiments also include methods of using such devices. An example of such an integrated device is illustrated in Figure 1. The illustrated embodiment of the present invention is a five-layer structure with all five layers bonded together to create a solid microfluidic chip, but the chip may be a single structure with only bonded layers. Chips are made of fused silica, crown glass, boron silicate glass, soda lime glass, sapphire glass, cyclic olefin polymer (COP), poly(dimethyl)siloxane (PDMS), OSTE, polystyrene, poly(methyl)methacrylate, and polycarbonate. , can be constructed using a number of standard materials, including but not limited to other plastics or polymers. The chip allows clear optical access for sample introduction, hydrodynamic focusing, optical interrogation, imaging, analysis, sample ejection and laser light entering and exiting the regions. Chips in embodiments may also be 3D printed, molded, or otherwise shaped.

Optionally, at least one particle type comprises a plurality of particle types. Each particle type of the plurality of particle types includes its own unique properties and its own induced properties. Optionally, intrinsic properties include size, shape, refractive index, shape, intrinsic fluorescence, and/or aspect ratio. Optionally, the induced properties include strain, angular orientation, rotation, rotation rate, antibody label fluorescence, aptamer label fluorescence, DNA label fluorescence, stain label fluorescence, and differential retention. Includes metric and/or hardness force metric. The method embodiment further includes identifying and separating the plurality of particles according to their respective particle types based on at least one of the intrinsic properties and the derived properties. Optionally, the method embodiment further includes querying or manipulating the sample flow. Optionally, querying the sample flow includes determining at least one of intrinsic properties and derived properties of the particle types, and measuring particle velocities of the plurality of particles. Measurement of at least one of the intrinsic properties can be used for the purposes of viral quantification, process development and monitoring, sample release assays, adventitious agent testing, clinical diagnosis, and biomarker discovery. Determining the viral infectivity (number of functionally infectious virus particles present in a specific cell population similar to a plaque assay or end point dilution assay) of a cell sample, developing a process and Determining the productivity of cells on antibodies or proteins for monitoring, CAR T and other oncology applications, and cells generated as cell-based therapies, including stem cells. determining the potency, quality, or activation state of cells; determining the effect of chemicals, bacteria, viruses, antimicrobial, or antivirals on specific cell populations; and determining the disease status or potential of research or clinical cell samples. It can be used for a range of applications, including but not limited to determining gender. Optionally, the source of optical force comprises at least one beam axis and the sample flow comprises a sample flow axis. Determining at least one of the intrinsic properties and derived properties of the particle types and measuring the particle velocity of the plurality of particles together include offsetting the beam axis from the sample flow axis. Optionally, determining at least one of the intrinsic properties and derived properties of the particle types, and measuring the particle velocity of the plurality of particles together comprises: It includes calculating the tilt and trajectory of the particle.

Those skilled in the art will recognize that the disclosed features may be used alone or in any combination, or may be omitted based on the requirements and specifications of a given application or design. It should be understood that when referring to an embodiment “comprising” certain features, the embodiments may alternatively “consist of” or “consist essentially of” any one or more of the features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.

In particular, it is noted that where a range of values is provided herein, 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 or excluded within the range. The singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. The specification and examples are to be regarded as illustrative or explanatory in nature, and changes that do not depart from the essence of the invention are intended to be within the scope of the invention. Additionally, all references cited in this disclosure are each individually incorporated by reference herein in their entirety, thereby providing an efficient manner of supplementing the possible disclosure of the present invention as well as maintaining the level of ordinary skill in the art. It is intended to provide a detailed explanatory background.

Claims (17)

1. A method of evaluating biological particles for use in cell-based therapies, said method comprising the use of a device, wherein said device:
A substrate comprising a plurality of channels configured to transport one or more biological particles, the plurality of channels comprising:
A first channel disposed vertically within the substrate,
a second channel operably communicating with the first channel and disposed horizontally within the substrate;
a third channel operably communicating with the second channel and disposed vertically within the substrate, and
a fourth channel operably communicating with the third channel and disposed horizontally within the substrate;
The first, second, third and fourth channels provide a path for movement of the one or more biological particles through the substrate from the first channel to the second channel to the third channel to the fourth channel. It is arranged in a way that provides; and,
The biological particle is disposed vertically within the substrate through a lower horizontal planar surface having an opening to the first channel to maintain directional and volumetric continuity with the first channel. Including injection into 1 channel,
wherein the biological particles include CAR T cells, CAR-M cells, TILs, NK cells, other engineered cells or effector cells, or stem cells,
A method of evaluating biological particles for use in cell-based therapy, wherein the evaluation includes optical force-based measurements. The method of claim 1, wherein the optical force-based measurement yields information about the size, shape, refractive index, morphology, internal cellular structural change, cell aggregation, or combinations thereof of the biological particle. What to do, how to do it. According to paragraph 1,
i) determining the number and total number of functionally infectious virus particles present in the biological particle,
ii) Quantification of virus,
iii) process development;
iv) process monitoring;
v) sample release assay,
vi) adventitious agent testing,
vii) Sterility testing,
viii) clinical diagnosis;
ix) Biomarker discovery;
x) Determining the cell killing ability of CAR T cells
xi) measuring transfection efficiency,
xii) measuring virus infectivity,
xiii) measuring differentiation of stem cells,
xiv) measuring the differentiation capacity of stem cells;
xv) measuring viral transduction efficiency,
xvi) determining the productivity of cells from said biological particles in terms of antibodies, proteins, or cell phenotypes for process development and monitoring;
xvii) determining the potency, quality, or activation state of said biological particle;
xviii) determining the effect of a chemical, bacterial, viral, antimicrobial, or antiviral agent on said biological particle;
xix) determining the disease state of said biological particle,
xx) measuring cell death, viability, apoptosis, cytotoxicity, or other cellular health-related parameters, or
xxi) For combinations thereof,
The method further comprising using the optical force-based measurement. The method of claim 1 , further comprising separating the biological particles based on size, shape, refractive index, morphology, charge, charge distribution, charge mobility, permittivity, deformability, or combinations thereof. The method of claim 1 further comprising imaging the biological particle. The method of claim 5 , further comprising using the imaging to determine volume, shape, nuclear location, nuclear volume, organelle location, inclusion body location, or combinations thereof of the biological particle. 6. The method of claim 5, wherein said imaging:
i) within the fourth channel of the device,
ii) comprising capturing images of said biological particles in multiple focal planes;
iii) comprising capturing three-dimensional images of said biological particles,
iv) comprising capturing images of the biological particle during its movement,
v) comprising capturing images of said biological particle from multiple angles and/or orientations,
vi) involving the use of multiple imaging devices;
vii) comprising analysis of multiple image slices of the biological particle according to movement of the plane of focus relative to the biological particle,
viii) moving the imaging device to change the focal plane imaged in the fourth channel;
ix) comprising analysis of multiple image slices of the biological particle according to its movement through the focal plane,
x) comprising analysis of multiple image slices of suspended or static biological particles from among said biological particles,
xi) directing light away from the imaging device during image collection in the fourth channel, or
xii) A method that is a combination thereof. 6. The method of claim 5, further comprising moving the biological particle in the fourth channel using optical force, fluidic force, or a combination thereof, wherein the area within the fourth channel where the biological particle is imaged wherein the biological particle is altered during movement. The method of claim 5, wherein the imaging is performed using a bright field imager, a light scattering detector, a single wavelength fluorescence detector, a spectroscopic fluorescence detector, a CCD camera, a CMOS camera, a photodiode, a photodiode array (PDA), a spectrometer, a light multiplier tube or tube. Array, photodiode array, chemiluminescence detector, bioluminescence detector, standard Raman spectroscopic detection system, surface enhanced Raman spectrometer (SERS), coherent van Stokes Raman spectrometer (CARS), coherent Stokes Raman spectrometer (CSRS) , or a combination thereof. The method of claim 1 , further comprising moving the biological particles in one or more of the plurality of channels using electrical forces, optical forces, fluid forces, electrophoretic forces, dielectrophoretic forces, or a combination thereof. 2. The method of claim 1, wherein the lower horizontal planar surface has at least one length from one edge to another edge that is shorter compared to at least one length from one edge to another edge on the vertical planar surface of the substrate. What to have, how to have. The method of claim 1 , wherein the device further comprises a collimated or focused light source oriented to interact with the biological particle in the fourth channel. According to paragraph 1,
i) the fourth channel is located closer to the top of the substrate than the first channel, the second channel, or the third channel,
ii) the fourth channel is located 100㎛ to 100mm from the top of the substrate,
iii) the first channel has a length ranging from 0.1 mm to 100.0 mm,
iv) the second channel has a length ranging from 0.1 mm to 100.0 mm,
v) the third channel has a length ranging from 0.05 mm to 100.0 mm,
vi) the fourth channel has a length ranging from 0.1 mm to 100.0 mm,
vii) the first channel is greater in length than the second channel, the third channel or the fourth channel, or
viii) a method that is a combination thereof. The method of claim 1 , wherein the device further comprises a cell or particle interrogation unit, a cell or particle collection channel, or a combination thereof. The method of claim 1, wherein the lower horizontal planar surface has a surface area that is less than or equal to the surface area on the vertical planar surface of the chip. The method of claim 1, wherein the second channel and the fourth channel are shorter than the first channel. The method of claim 1, wherein said injection includes providing a sample inlet line tip in communication with a sample inlet line that communicates with said first channel.