CN111819153B - Microfluidic chip devices for photomechanical measurements and cell imaging using microfluidic chip configuration and dynamics - Google Patents

Abstract

A microfluidic chip configuration in which injection is performed in a vertical upward direction with fluid reservoirs located below the chip to minimize particle settling at and before the analytical portion of the chip channels. Inputs and fluids flow upward through the bottom of the chip, using a manifold in one aspect, which avoids orthogonal redirection of fluid dynamics. The contents of the vial are located below the chip and are pumped vertically directly 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, where it makes a short horizontal bend. Fluid is pumped upward to the horizontal analytical portion, which is the highest channel/fluidics point in the chip and is therefore close to the top of the chip, which allows for clearer imaging. The laser can also suspend cells or particles in this channel during analysis.

Description

Microfluidic chip apparatus for optical power measurement and cell imaging using microfluidic chip configuration and dynamics

Technical Field

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

The present invention is directed to a microfluidic chip in which injection occurs in a vertically upward direction with a fluid vial below the chip to minimize particle settling at and before the analysis portion of the chip channel.

The design of the existing microfluidic chip is changed to realize the invention. For example, to vertically align the vial with the chip, a chip interface different from the prior art needs to be established. In particular, the present invention allows for the input and fluid to pass up the bottom of the chip, using a manifold in one aspect, which avoids horizontal reorientation of the fluid/fluid dynamics, rather than having the input tube engage the chip in an orthogonal fashion via a port (typical in microfluidic lab-on-a-chip systems), which is attached to the largest face of the chip, then pumps the fluid first across the chip, and then pumps the fluid over the chip.

According to the invention, the contents of the vial are located below the chip and are pumped directly vertically upwards into the first channel of the chip. The long channels extend from the bottom of the chip to near the top of the chip. The channel then turns a short horizontal bend, but the new channel is too short to nearly cancel out any effect of cell sedimentation due to gravity and zero flow velocity at the wall. The fluid is then pumped up to the analysis section, contrary to the prior art. Thus, the horizontal analysis section is the highest channel/flow control point in the chip, so the horizontal analysis section is close to the top of the chip, which makes the chip material (e.g. glass) between the microscope/camera and the sample less than in the prior art, and the imaging is clearer. During analysis, the laser also suspends the cells in the channel, preventing them from settling.

Background

Description of related Art

According to the prior art, a microfluidic chip vial containing cells or particles to be separated and/or analyzed is placed sideways and pumped horizontally into channels in the microfluidic chip. First, the contents of the vial (e.g., particles or cells) are pumped vertically upward, then travel downward turning a U-bend, and then pumped horizontally into the chip (see, e.g., U.S. patent 9594071).

The horizontal connection to the chip, plus the dead volume in the connection (empty space in the fluidic connection, unavoidable to some extent), causes significant additional sedimentation under the force of gravity. This arrangement also necessitates a relatively large diameter channel for connection, creating a relatively low velocity region in addition to dead volume, further exacerbating the particle settling problem. The chip according to the invention eliminates not only the need for a large horizontal input channel, but also the need for a relatively abrupt change in the relatively thin upward flow from the large horizontal input channel into the first vertical chip conduit. This arrangement eliminates horizontal sedimentation and unnecessary directional changes that result in sedimentation. The problem of sedimentation in dead volumes is also solved by letting the cells enter the bottom edge of the chip by orienting them perpendicular to gravity so that the cells or particles cannot settle at the bottom of the horizontal channel, instead they are continually directed by flow upwards. This is not intuitively known and a lot of experiments need to be performed to understand the problem before designing the solution currently presented. In contrast to the present invention, the currently available microfluidic devices include custom or commercially available connections on polished surfaces and larger area glasses, which typically force any particles, such as cells, contained in the sample stream to turn and travel horizontally immediately after entering the chip.

Furthermore, in the prior art, cells or particles are moved horizontally on the microfluidic chip several times before reaching the analysis channel, which results in sedimentation. When the contents of the vial enter the chip and the channels in the chip, the contents are pumped horizontally with respect to the vertical intra-chip channels. The channel then flows upward turning a long horizontal bend, where the cells tend to settle at the bottom of the channel due to gravity, while the cells tend to have a lower velocity at the walls due to laminar flow conditions. Essentially, the flow is greatest in the middle of the channel, down to zero at or near the channel wall, due to the parabolic velocity profile. After the first horizontal intra-chip channel, the fluid turns down before the analysis channel where particle imaging or separation takes place. With 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 forced down and eventually leave the bottom of the chip.

Furthermore, due to the limitations in the prior art, multiple horizontal movements are required, causing the cells to settle at multiple places in the channel. This in turn leads to reduced image quality because of the need to image with additional material at the chip edge. The prior art channels of the chip have to be pumped vertically upwards, then horizontally and then zigzag again to obtain sufficient cell or particle suspension, then downwards and out of the chip. The present invention avoids zigzagged channels.

There is also prior art regarding 3D image rendering of cells or particles in a fluid. For example, m. Ha Baza, m.kerr Shi Baom, c.root-Ma Ershi nm, g.dadieman, i.ba-in, r.colennostan, c. Du Shener, n.t. Sha Kede teach in tip science (ADVANCED SCIENCE) (2017,4, 1600205) to capture cells, spin the cells at high speed, and determine the refractive index profile within the cells using interferometry. Interferometry is also used to analyze cells in microfluidic channels (see, e.g., Y. Song et al, applied Physics comment (PHYSICAL REVIEW APPLIED), 27, 2014, 2, and 1:014002). However, the present invention requires that multiple images be taken of cells or particles as they travel in a fluid stream and pass through the focal plane of the imaging device(s), thereby eliminating the need to capture cells in order to acquire 3D images. Other techniques, such as using a mechanical displacement platform to move cells or particles (e.g., optical Express by n.lu et al, 29 th year, 2008; 16 (20): 16240-6), do not use the brightfield imaging described herein nor utilize fluid flow to provide cell positioning relative to the image focal plane.

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

Disclosure of Invention

The present invention is directed to a microfluidic chip in which injection is performed and sample vials are located below the chip in order to minimize particle settling. Thus, the contents of the vial are located below the chip and pumped directly vertically upward into the channels of the chip. The long channels extend from the bottom of the chip to near the top of the chip. The channel then turns a short horizontal bend, but the new channel is too short, making it insignificant to any effect of cell sedimentation caused by zero flow velocity at the channel walls. The sample is then pumped up to the analysis section, contrary to the prior art. Thus, 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 glass between the microscope/camera less than in the prior art, and the imaging is clearer. The distance between the analysis channel and the top of the chip may be between 100 micrometers and 2mm, but may also be as long as 100mm, such as from 100 micrometers to 200 micrometers, from 200 micrometers to 300 micrometers, from 300 micrometers to 400 micrometers, etc. In one embodiment, after passing through the chip analysis portion, the sample (e.g., fluid, cells, and/or particles) is pumped down to the bottom of the chip, forced to move outward.

The present invention is further directed to a microfluidic chip in which horizontal movement is minimized, particularly when fluid enters the chip channels. The prior art chip contains about 13mm horizontal channels (non-analysis section) where about 2mm is the injection port of much larger diameter, exacerbating settling caused by low speed. The chip described in the present invention has a (non-analytical) horizontal channel of about 0.2 to 3.0mm in the preferred embodiment, although the length of the horizontal channel may be between 0.01 to 100.0mm, such as from 0.01mm to 0.02mm, from 0.02mm to 0.03mm, from 0.03mm to 0.04mm, and so on. Depending on the channel system of the invention, this is ordered in orders of magnitude different from the prior art, which improves the flow and eliminates cell/particle sedimentation.

Another aspect of the invention is directed to a microfluidic chip in which imaging and analysis is performed based on at or near the corners of the chip, as this allows for less glass, shorter distances between the camera and the analysis channel, to improve imaging from multiple viewpoints. This also enables the use of higher numerical objectives to improve fine imaging by increasing by orders of magnitude. Such design improvements reduce image distortion caused by glass (or other substances that make up the chip, such as plastic or any transparent or translucent material) and distance (e.g., due to imperfections in the glass). The distance between the imaging device and the analysis channel may be between 100 micrometers and 2mm, but may also be as long as 100mm, such as from 100 micrometers to 200 micrometers, from 200 micrometers to 300 micrometers, from 300 micrometers to 400 micrometers, etc.

In addition, the present invention is directed to a microfluidic sorting chip that separates downstream from the analysis channel, allowing for the simultaneous or sequential separate use of pressure and/or laser (or other optical power) to activate the sorting function. In one aspect, for classification functions, flow will continue from the analysis channel. For example, particles will be directed into a vertical channel and then to a horizontal sorting channel. In one embodiment, optical power and/or pressure is applied in the direction of flow of the sorting channel to push particles through the channel. Particles that are not directly acted upon by the optical forces will be diverted to alternative channels due to, for example, gravity, electrodynamic forces, magnetic forces, laminar flow lines, reduced flow rates, orthogonal optical forces, or vacuum (vacuum applied to draw the particles into the alternative channels). In another aspect related to the sorted analysis channels, the invention allows guiding the optical power from the back side of the chip (laser or optical power is directed in the same flow direction) and in some aspects allows the main laser to be resolved. In some embodiments, the optical power and/or pressure may be applied in opposite directions of the movement of the substance through the channel, such as counter-current, or may be applied in the same direction of the movement of the substance in the channel, such as co-current. Cell or particle sorting can be performed on a single device or on separate chips. For example, in fig. 1B, the fifth channel prior to the outlet tube 145 contains one or more branches to achieve single or multiple sorting zones.

In another aspect of the invention, the manifold is connected to the vials in a manner such that the tubing passes through the manifold and connects to the vials or other containers on the other side that is in contact with the substance (e.g., fluid) in the vials. The manifold allows the vials to be connected to the microfluidic chip but to be stored below the chip, and/or the manifold allows the contents of the vials to be injected from the bottom of the chip, alleviating several problems faced by the prior art, such as sedimentation of cells or particles where the vials or tubing from the vials are connected to or in communication with the microfluidic chip.

In another aspect, the invention is directed to a microfluidic chip holder comprising a structure for guiding a light source, the structure comprising an integrated prism cavity that, when fitted with a prism, causes light to be emitted at an angle to the chip, which is a preferred method of illuminating a restricted geometry. In some embodiments, the light source includes, but is not limited to, fiber optic or parallel (collimated) or focused light sources. In particular, the light source is precisely aligned or directed to or into the analysis channel.

In another aspect of the invention, the device includes a second imaging device oriented orthogonal to the first camera and the channel view. The reason for providing the second camera is different. On the one hand, the reason for providing the second imaging device is to aid in analyzing the visual alignment of the laser light or optical power in the channel. On the other hand, using the method according to 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 cell location, size, shape, volume, etc. This additional information about the same cell (or particle) improves accuracy and expands the range of measurement and analysis. In another aspect, the second camera in combination with the first camera enables three-dimensional reconstruction of cells or particles, or groups of cells or particles, imaged by orthogonal cameras and cameras in the stream or cameras positioned towards the sides of the chip. Using the algorithms described herein or other algorithms, including reversing or slowing the flow and taking one or more images for a particular cell or particle, or group of cells or particles, the present invention allows for the analysis and processing of multiple images, thereby allowing for the determination of features/characteristics/quantitative measurements, such as cell volume, cell shape, nuclear site, nuclear volume, organelles or inclusion body sites, etc. 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 serpentine (serpentine) or zig-zag (saw-tooth) channels 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 being injected into the chip, the vertically integrated pumping particles can pass directly up through the channel to the first horizontal channel (referred to herein as the second channel), thereby avoiding a zig-zag channel.

Drawings

The drawings illustrate certain aspects of some embodiments of the invention and are not intended to limit or define the invention. The drawings, together with the description, serve to explain certain principles of the invention.

Fig. 1A is a schematic diagram depicting a global view of a device configuration, including a chip, manifold, and vial. FIG. 1B is a schematic diagram showing some depictions of chip channel orientation and location.

Fig. 2A and 2B contain schematic diagrams depicting microfluidic chip holders according to the invention.

Fig. 3A and 3B are schematic diagrams depicting angles and aspects of a chip holder according to the present invention.

Fig. 4A and 4B are schematic diagrams showing examples of cell paths and imaging configurations associated with a chip. Fig. 4C is a schematic diagram showing an alternative cell path.

Fig. 5 is a schematic diagram showing on-chip multi-planar imaging and how to use it to render 3D images and information.

Fig. 6A and 6B are schematic diagrams showing on-chip multi-planar imaging in a fluid stream and how to use it to render 3D images.

FIG. 7 is a schematic diagram showing the possible ways of capturing cells and/or balancing cells within the analysis portion of the chip, and the possible ways of imaging cells from multiple angles.

Fig. 8 is a schematic diagram showing how the camera and illumination source can be placed in alignment with the laser and flow direction so that the particles are away from the camera.

Fig. 9 is a schematic diagram showing how the camera and illumination source can be placed in alignment with the laser and flow direction to move particles toward the camera.

Detailed Description

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 variations can be made in the practice of the invention without departing from the scope or spirit thereof. 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 incorporating or consisting essentially of these various features may also consist 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 is merely exemplary or explanatory in nature and, thus, variations that do not depart from the gist of the invention are intended to be within the scope of the invention.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments or of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and not of limitation.

Turning now to fig. 1A, fig. 1A shows a global view of an apparatus taught by the present invention. The sample vials 130 are positioned below the microfluidic chip 100 (also commonly referred to as a substrate in the present invention), and the number or size of the vials is not limited. The vial is configured to hold any type of sample that is movable by 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 term fluid or sample may be used to refer broadly to such one or more substances. The vials are directly or indirectly connected, e.g., in operative communication with the underside of the chip using air tubing 110. As further shown in fig. 1A, vials may also be connected by a manifold 120. Fig. 1B shows a preferred embodiment of a microfluidic chip 100 in which substances, fluids, particles and/or cells are injected into one outer surface of the microfluidic chip, such as the edge (e.g., XZ plane in fig. 1B). Fig. 1B shows implantation along one of the minimum distance shared planes (e.g., XZ plane or XY plane). 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, as is typical in the prior art).

Fig. 1A shows that a manifold 120 connects one or more vials 130 to the microfluidic chip 100 so that substances, fluids, particles, and/or cells may 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 electronics, flow sensors and tubing (liquid and air) in a manner that minimizes cell sedimentation, thereby optimizing throughput. The air tubing provides pressure or vacuum, when sealed, providing a closed system within the confines of the manifold device. In one embodiment, the vial(s) are spaced from the manifold by a distance that indicates no seal and the pressure of the interior volume is atmospheric pressure. This allows pumping of substances from or to containers that are open to the atmosphere (pumping of the required vacuum from open containers). The manifold places the electronics, flow sensors and tubing (liquid and air) in a manner that minimizes cell sedimentation, thereby optimizing throughput.

By injecting the contents from the bottom of the chip, the present invention minimizes horizontal movement in the inlet and outlet tubes 140, 145, which results in problems such as settling of particles or cells in the channel(s) 150. In this example, the outlet tube is offset from the inlet tube in the Z and X dimensions (fig. 1B). In some embodiments, the outlet tube is offset, non-offset, straight, aligned, or angled with respect to the inlet tube. This arrangement avoids the need for serpentine or zigzagged vertical channels, as current arrangements address sedimentation problems that occur in the prior art, such as where fluid combined with cells and/or particles is injected or pumped sideways in a horizontal direction to the chip and then must be redirected and hydrodynamically to be pushed upwards. Manifold and injection from the bottom of the chip also allows for placement of additional elements, components, mechanisms or hardware below the chip (see fig. 1A).

The manifold 120 operates on the principle of regulating the air pressure above the contents of the vial and providing the correct geometry for the flow sensor and electronics. A tube, such as a fluid or air tube, passes through the manifold and connects to the vial 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 enclosed area, the system allows for varying parameters such as flow rate and fluid dynamics. The pressurized region is in both the vial(s) 130 and the manifold 120. On the other hand, the vial does not require any air connection, as it is open to the ambient atmosphere. This allows for sampling of a wider variety of containers and sources. In such embodiments, a vacuum is applied to the other vial or vials to create a pressure differential to drive the fluid flow from the open container.

In one embodiment, pressure-based sample injection is employed. The vial is filled with a sample in fluid and sealed with a cap or tube connecting the chip. The vial may be open to the atmosphere prior to attachment to the cap, or may be sealed with a septum or other airtight device. In one aspect, the cap may contain two connectors, one for a fluid such as a gas and the other for a liquid. Optionally, the method embodiment further comprises providing a sample inlet line tip in communication with the sample inlet line, the sample inlet line tip in communication with the first channel.

In another embodiment, vacuum-based sample injection is employed. The vial is filled with the sample in fluid and sealed with a cap or tube connecting the chip. The vial may be open to the atmosphere or may be sealed with a septum prior to attachment to the cap. In one aspect, the cap may contain two connectors, one for a fluid such as a gas and the other for a liquid. Optionally, fluid may be aspirated from the vial open to the atmosphere by applying vacuum pressure to one or more other vials. Optionally, the method embodiment further comprises providing a sample inlet line tip in communication with the sample inlet line, the sample inlet line tip in communication with the first channel.

Fig. 2A-2B and fig. 3A-3B show chip holders 200, 300. The chip holder includes structures that guide a light source 210 (e.g., a fiber light source, a light emitting diode, or a laser) and integrated prism cavities 220, 320 that can be equipped with prisms. The light sources are directed or arrayed to the desired location by an integrated structure or channel 240 within the chip holder. The built-in space of the fiber optic light source allows illumination, such as the illumination cone 250, even in a confined geometry (e.g., on the microfluidic chip 230, 330 or channels in the microfluidic chip). In a preferred aspect, the light source is specifically aligned, or oriented, or focused on the analysis channel 260. In a preferred embodiment, the chip holder includes built-in space for the prisms 220, 320 and the fiber optic light source 210, allowing illumination in a limited geometry. The chip also includes holes or openings 350 in the bottom of the holder to precisely align the fluidic tubing according to the present invention. In one embodiment, the adjustable screw is integral with threaded holes 360 on one or more faces for proper positioning.

Fig. 4A is a preferred embodiment of a microfluidic chip 400 described in the present invention. As shown, the fluid first travels vertically upward through the first channel 410 and then communicates with the second horizontal channel 420. As shown in fig. 4A, another vertical channel 430 brings the fluid closer to the top of the chip, at which point a fourth channel 440 is horizontal and includes an analysis channel. The channels are operably connected to each other to allow the sample to move through the system from one channel to another. In some 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 of both. Pumps and/or vacuum means may be provided to provide positive and/or negative pressure at the opening and/or outlet of the passageway to enable the substance to move through the passageway. The analysis channels are adjacent to one or more outer surfaces of the substrate, such as the face, edge or side of the chip. For example, the analysis channels according to this configuration are close to the top and sides of the chip, thereby improving the imaging and analysis of substances passing through the microfluidic chip. In a preferred embodiment, the distance of the analysis channels to the top and sides of the chip is between about 1mm to about 2 mm. However, the distance of the analysis channel to the top of the chip may be between 0.1mm and 100mm, for example from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, from 0.3mm to 0.4mm, etc. In other words, the analysis channels 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 may be between about 250 micrometers to about 10 mm. However, the length of the analysis channels may be between 100 micrometers and 100mm, for example from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, from 0.3mm to 0.4mm, etc. In other words, the length of the analysis channel may be about 75% or less of the height, width or length of the substrate/chip, e.g., 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 fig. 4B, two imaging devices 450, such as machine vision cameras, are shown. 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 at 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 channel side). In some embodiments, the camera may be positioned at any angle to the one or more streams of matter, such as orthogonal to the one or more streams of matter or at 90 degrees, such as from 0 to 90 degrees, or from 10 to 80 degrees, or from 30 to 60 degrees, etc. In another embodiment, two or more cameras may be used to capture cells or particles in the analysis channel. For example, the camera may be above the chip and oriented orthogonal to the analysis channel. The second camera may be located on the side of the chip and oriented orthogonal to the flow direction or diagonally to the flow direction (e.g. above the channel, below the channel or at an angle to the channel side). As shown in fig. 4B, one or more light sources 460 may be used to illuminate the analysis channel 440, and such light sources may be located below and up the chip, illuminating the sides of the chip, and illuminating in the flow or counter-flow direction, or above the chip and illuminating diagonally down the analysis channel.

Or 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 through, as shown in fig. 8. This would allow the camera 810 to be placed in alignment with the analysis channel. As shown in fig. 8 and 9, there are several embodiments of this case, including placing the optical power laser 830 and the camera 810 at the same or opposite end of the analysis area. An illumination source 860 for a camera may also be required, for example as shown in fig. 8 and 9, 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 described herein, as part of a multi-camera system combined with other viewpoints.

Fig. 4A also shows that a light source 480 (e.g., a laser) may be used to affect cell flow. The laser may be placed in alignment with the cell stream or may be placed opposite the cell stream. The lasers may also be placed and/or oriented orthogonal or diagonal to the cell flow.

Embodiments of the invention include an apparatus for particle analysis. (see, e.g., fig. 4A, 4B, 4C, 5, 6A, 6B, 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 power 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 comprises at least one beam cross section. Embodiments of the present invention include a substrate with a first channel 410, the first channel 410 extending in a vertical direction in the substrate such that a first plane traverses the first channel 410 substantially along its length, whereby a fluid sample is injected into the substrate/chip from the bottom of the chip and forced upward under positive or negative pressure. Embodiments of the present invention include a second channel 420 orthogonal to the first channel, so the second channel 420 is horizontally disposed in the substrate such that a second plane traverses the second channel 420 substantially along its length, and the second plane is disposed orthogonal to the first plane. The 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 vertically upward third channel 430 that brings 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 corners 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 orthogonal to the flow direction in the fourth channel. Embodiments of the invention include a focused particle jet nozzle operatively connected to a first channel. In another aspect of the invention, the second passageway is sized and passes through the nozzle prior to communicating with the third passageway.

Fig. 4C shows another embodiment of a sample path of a microfluidic chip. As shown, the fluid travels in the chip first in a vertically upward direction through a first channel 410, which first channel 410 then communicates directly or indirectly with a second horizontal channel 420, which second horizontal channel 420 is located at the top of the chip in this example and comprises 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 substances passing through the microfluidic chip. In a preferred embodiment, the distance of the analysis channels to the top and sides of the chip is between about 1mm to about 2 mm. However, the distance of the analysis channel to the top of the chip may be between 0.1mm and 100mm, for example from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, from 0.3mm to 0.4mm, etc. In other words, the analysis channels may be arranged within 50%, 33%, 25%, 10% or 5% of the top of the substrate. The length of the horizontal analysis channel according to the present invention may be between about 250 micrometers to about 10 mm. However, the length of the analysis channels may be between 100 micrometers and 100mm, for example from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, from 0.3mm to 0.4mm, etc. In other words, the length of the analysis channel may be about 75% or less of the height, width or length of the substrate/chip, e.g., 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.

Fig. 4C also illustrates that a light source 480 (e.g., a laser) may be used to affect the flow of a substance such as a cell stream. The laser may be placed in alignment with the cell stream or may be placed opposite the cell stream. The lasers may also be placed and/or oriented orthogonal or diagonal to the cell flow.

As shown in fig. 1A-1B and fig. 4A-4C, a fluid stream containing cells or particles is directed vertically through a first channel. One or more substances (fluids, 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 micrometers and 100mm, e.g. from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, etc. The first channel is followed by a second orthogonal/horizontal channel, which in a preferred embodiment is shorter than the first channel. The length of the second channel may be between 250 micrometers 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 may be between 50 micrometers 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 in operable direct or indirect communication to allow one or more substances to move through the plurality of channels. Typical directions of fluid flow are given by the flow arrows in fig. 4A and 4C, but may be reversed.

In a preferred embodiment, the fourth channel comprises an analysis channel, the fourth channel being a channel having a length of between 250 micrometers and 100mm, such as from 0.25mm to 0.5mm, from 0.5mm to 0.75mm, from 0.75mm to 1.0mm, 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, may be between 100 micrometers and 2mm, but may also be as long as 100mm, e.g. from 100 micrometers to 200 micrometers, from 200 micrometers to 300 micrometers, from 300 micrometers to 400 micrometers, 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, which may be between about 100 microns and 2mm from the fourth channel, but may also be as long as 100mm from the fourth channel, such as from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, and so on.

In one embodiment, the laser or other light source has a focusing lens element. Fig. 4A depicts the invention when the laser 480 is operating, emitting a laser beam directing the beam through a focusing lens element into the fourth flow channel 440. The particles are aligned within the laser beam due to the gradient forces attracting the particles to the region of highest laser intensity. The laser scattering force pushes the particles in the direction of laser beam propagation (e.g., left to right in fig. 4A).

In another embodiment, in addition to a camera or photographing device oriented orthogonally to the fourth channel, a second camera or photographing device (see 450) is oriented toward the side of the analysis (or fourth) channel and is aligned orthogonally to the fourth channel or at any angle to the fourth channel; for example, as shown in fig. 4B. Taking one orthogonal view image for each cell allows calculation of multiple cell properties in both directions, such as size and shape, thereby increasing the amount of information that can be acquired for each cell. This will also enable calculation of cell volume properties, including total volume, shape, and provide insight into cells that are not symmetrical along an axis parallel to the flow direction (the Z-axis as shown in fig. 5). Using two or more cameras, a basic three-dimensional model of the cell 550 can be constructed by combining orthogonal images using, for example, existing three-dimensional reconstruction algorithms (e.g., diffraction theory methods or illumination rotation methods). The three-dimensional model and its analysis will enable more accurate analysis of cells, 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.

In fig. 5, portions of analysis channel 540 are shown from two different planes, such as those that may be imaged from an imaging device (see, e.g., fig. 4A-4C). In the first plane 520, a portion of the 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 view angle of another camera. This enables the computation of multicellular attributes, creating a data matrix for each cell for each camera, and creating a three-dimensional presentation of cells or particles 550.

As different portions of the cells pass through the focal planes of the imaging device(s) (e.g., 630 (XZ focal plane) and 640 (YZ focal plane)), different sections of the cells may be imaged as the cells move by, for example, fluid flow 620. This is shown in fig. 6A. In fig. 6A, portions of cells or particles are imaged at multiple points in time and/or space, in different orientations and from different perspectives. In this example, as the cell moves and rotates through the focal plane, it appears to be different sizes in successive images (e.g., four example images from two different cameras for each plane, as shown in fig. 6A). More complex three-dimensional rendering 650 of particles or cells is achieved using a three-dimensional reconstruction algorithm. From this rendering, certain characteristics of the cells or particles, such as cell size, volume, cell nuclei, and other cellular organelle sites and sizes, as well as measurements or summaries of cell morphology, can be inferred. Furthermore, due to changes in biophysical or biochemical properties (including but not limited to refractive index, birefringence, or cell shape or morphology), rotation of the cell may be measured based on a function of torque of optical power.

Fig. 6B depicts multi-plane imaging within a 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 magnification, the working distance of the objective lens is reduced, and therefore it is necessary to bring the lens closer to the cell or particle to be imaged. In one embodiment, a laser or other optical power 670 may be used to affect the flow of cells in the analysis channel. In preferred embodiments, imaging may be performed by moving the laser and/or camera, or adjusting the flow(s) or position using hydrodynamic focusing, to purposefully induce cells or particles into or out of the focal plane of the channel. The laser source distance 660 may be moved using, for example, a piezo actuator or a linear electro-optic bench. This may be done per cell or per population. The hydrodynamic focus of the cells can be changed to affect the initial position and trajectory of the cells. For example, particles may be aligned or oriented in a focal plane within a laser beam due to a gradient force that attracts the particles to the region of highest laser intensity. The laser scattering force pushes the particles in the direction of propagation of the laser beam. See fig. 6B. Moving the laser along the X-axis in this example, imaging of features of different parts of the cell is achieved, as illustrated by disk 680. This may represent, for example, a nucleus, an organelle, an inclusion body or other feature of the cell or particle. The laser pulls the cell to its center due to the gradient force. It is also contemplated that two or more cameras may increase the degree of detail and accuracy.

The embodiment of the invention shown in fig. 7 is a static mode in which the particles or cells 710 are stopped at a specific differential retention site due to the balance of optical forces 730 and fluidic forces 735. The optical power may be applied by, for example, a laser or a parallel light source. As shown and described in fig. 5 and fig. 6A-6B, images may be taken in multiple planes. The flow sensor is used to measure the flow rate of each particle at a given laser power while it is stopped in the flow. Because the optical and fluidic forces are balanced, the fluidic resistance (i.e., resistance from flow rate and channel direction) is equal to the optical force. The properties of each cell can be measured in this manner in turn. While not a high throughput measurement system, embodiments of the invention enable close-up viewing and imaging of captured cells, and also enable dynamic changes in optical power due to biochemical or biological changes in the cells. A reagent stream containing chemicals, biochemicals, cells or other standard biological agents may be introduced into the flow channel to interact with the captured cell(s). These dynamic processes can be monitored quantitatively by measuring changes in optical power during experiments on single or multiple cells.

In one embodiment, the camera or other imaging device is oriented and/or focused along or against the flow in the analysis channel such that the camera or other imaging device is aligned with and parallel to the flow (see, e.g., fig. 8 and 9). Fig. 8 shows a camera 810 aligned with an analysis channel 820 and a laser or parallel light source 830. A dichroic mirror or similar device 840 reflects laser light 835 from the camera elsewhere to prevent damage, but passes light 865 generated by illumination source 860 to effect imaging. The camera is oriented parallel to the fluid flow 870 such that, in one embodiment, the cells or particles 880 move away from the camera. The illumination source is oriented orthogonal to the channel and the laser. The second dichroic mirror 845, which 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 the arrangement transposes the laser and the location of 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 laser light and visible light through the channel.

Fig. 9 shows another embodiment. In this case, the camera or imaging device 910 is oriented such that the cells or particles 980 travel in the fluid flow 970 towards the camera. Thus, the camera and laser 930 are located on the same side of the channel, while the illumination source 960 is located at the opposite end of the channel. Two dichroic mirrors 940 and 945 are used to direct laser light 935 and illumination light 965 into the channel, direct illumination light toward the camera, and divert laser light from the illumination source elsewhere. Another embodiment of the arrangement transposes the position of the laser and 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 toward the camera.

Optionally, embodiments of the present invention further include at least one optical element positioned between the optical power source and the fourth channel and operable to produce a standard TEM00 mode beam, a standard TEM01 mode beam, a standard TEM10 mode beam, a standard Hermite-Gaussian beam mode, a standard lager Gaussian beam mode, a bessel beam, or a standard multimode beam. Optionally, the at least one optical element comprises a standard cylindrical lens, a standard axicon, 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. Alternatively, the power source may comprise a standard circularly polarized beam, a standard linearly polarized beam, or a standard elliptically polarized beam.

Alternatively, the invention comprises an apparatus comprising a microfluidic channel, a laser light source focused into the microfluidic channel by an optical device, an electric field source connected in operation to the microfluidic channel through an electrode; flowing particles in the liquid pass 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 distribution, charge mobility, dielectric constant, and/or deformability. In yet another embodiment, an apparatus includes a microfluidic channel configured to provide a Dielectrophoresis (DEP) field to an interior of the channel through (1) an electrode system or (2) an insulator DEP system, further including 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 in conjunction with a field to act on the particles in the microfluidic channel to capture the particles or change their velocity, wherein the DEP field is linear or nonlinear. Another possible embodiment of the apparatus includes a microfluidic channel including an inlet and a plurality of outlets, and a laser light source focused by the optics to traverse the microfluidic channel at a critical angle that matches a flow rate in the microfluidic channel to generate optical power to the particles while maximizing a residence time of the selected particles in the laser light to separate the particles into the plurality of outlets, wherein the laser light is operable to apply a force to the particles flowing through the microfluidic channel, thereby separating the particles into the plurality of outlets.

Optionally, embodiments of the invention further comprise at least one particle interrogation unit in communication with one or more channels, such as the analysis channel(s), in particular 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 comprises 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 chemiluminescent detector, a standard bioluminescent detector, and/or a standard raman spectral detector.

At least one particle interrogation unit in communication with the fourth channel includes a laser-force based device or apparatus that facilitates identification, selection and classification of cellular disorders. In one aspect, the cell takes advantage of the 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, the near infrared laser beam applies a physical force to the cell, which is then measured. The optical power of the radiation pressure, when balanced with the fluidic resistance on the particles, results in a change in particle velocity that can be used to identify different particles, or a change in particle population based on inherent differences. Flow control and optical power balancing can also be used to change the relative position between particles based on their inherent properties, resulting in physical separation. Another embodiment of the interrogation unit comprises a device for particle analysis and/or separation, for example at least one parallel light source, operable to generate at least one parallel light source beam. The at least one parallel light source beam comprises at least one beam cross section.

Embodiments of the present invention relate to combinations of several of the above design elements discussed in the above monolithic device. Embodiments also include methods of using such devices. Fig. 1A-1B illustrate examples of such unitary devices. The illustrated embodiment of the present invention is a five-layer structure, all five layers being bonded to each other to produce a solid state microfluidic chip, although the chip may be of an opposite construction to the bonding layer. The chips may be fabricated using several standard materials including, but not limited to, fused silica, crown glass, borosilicate glass, soda lime glass, sapphire glass, cyclic Olefin Polymer (COP), poly (dimethyl) siloxane (PDMS), OSTE, polystyrene, polymethyl methacrylate, polycarbonate, other plastics or polymers. The chip allows for sample input, hydrodynamic focusing, optical interrogation, imaging, analysis, sample exit, and transparent optical access to the laser light entry and exit areas. The chips in embodiments may also be 3D printed, molded or otherwise shaped.

Optionally, the at least one particle type comprises a plurality of particle types. Each particle type of the plurality of particle types includes a respective intrinsic property and a respective induced property. Optionally, the intrinsic properties include size, shape, refractive index, morphology, intrinsic fluorescence, and/or aspect ratio. Optionally, the induced property comprises deformation, angular orientation, rotation rate, antibody-labeled fluorescence, aptamer-labeled fluorescence, DNA-labeled fluorescence, staining-labeled fluorescence, differential retention metric, and/or gradient force metric. The method embodiment further includes identifying and separating a plurality of particles according to respective particle types based on at least one of the intrinsic property and the induced property. Optionally, the method embodiment further comprises interrogating or manipulating the sample stream. Optionally, interrogating the sample stream includes determining at least one of an intrinsic property and an induced property of the particle type, and measuring particle velocities of the plurality of particles. The measurement of at least one inherent property is widely applicable, 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 plaque assays or end point dilution assays) for viral quantification, process development and monitoring, sample release assays, adventitious agent testing, clinical diagnosis, biomarker discovery; determining cell productivity for antibodies or proteins for process development and monitoring; determining the efficacy, quality or activation status of cells produced as cell-based therapies, including CAR T and other oncology applications and stem cells; determining the effect of a chemical, bacteria, virus, antibiotic or antiviral drug on a particular cell population; and determining the disease state or likelihood of a study or clinical cell sample. Optionally, the optical power source comprises at least one beam axis and the sample stream comprises a sample stream axis. The step of determining at least one of an intrinsic property and an induced property of the particle type, and the step of measuring particle velocities of the plurality of particles together comprise a shift of the beam axis from the sample flow axis. Optionally, the step of determining at least one of an intrinsic property and an induced property of the particle type, and the step of measuring the particle velocity of the plurality of particles together comprise a calculation of a pitch and a trajectory of one of the plurality of particles that is offset from the sample stream axis to the at least one beam axis.

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 refers to certain features of "comprising," it is understood that the embodiment can alternatively be "consisting of" or "consist essentially of" any one or more of the features described above (consist). Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.

It should be particularly noted that ranges of values are provided in this specification, with each value between the upper and lower limits of the ranges also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is intended that the specification and examples be considered as exemplary or explanatory and that variations that do not depart from the gist of the invention are included within the scope of the invention. Furthermore, all references cited in this disclosure are incorporated herein by reference in their entirety, and are intended to provide an efficient method of supplementing the enabling disclosure of the present disclosure and to provide a background detailing the level of ordinary skill in the art.

Claims (36)

1. A microfluidic device comprising: A substrate comprising a plurality of channels configured to transport one or more substances; and a collimated light source capable of interacting with the one or more substances; wherein the plurality of channels comprises: a first channel oriented vertically upwards in the substrate relative to the direction of gravity, a second channel, operably connected to the first channel and horizontally arranged in the substrate, a third channel operatively connected to the second channel and oriented vertically upward within the substrate relative to the direction of gravity, and a fourth channel, operably connected to the third channel and horizontally arranged in the substrate; wherein the first channel, the second channel, the third channel, and the fourth channel are arranged in a manner to provide 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; wherein the second channel is shorter than the first channel; wherein the one or more substances are injected into the first channel oriented vertically upward relative to gravity within the substrate by pressure or vacuum driving a flow through a bottom horizontal planar surface having an opening leading to the first channel. 2. The microfluidic device according to claim 1 is characterized in that the bottom horizontal plane surface has at least one length from one edge to another edge that is shorter than the length from at least one edge to another edge on the vertical plane surface of the substrate to maintain the direction and volume continuity of the first channel. 3. The microfluidic device according to claim 1 is characterized in that the one or more substances are injected into the first channel arranged vertically 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 substrate, maintaining the direction and volume continuity of the first channel in the vertical direction. 4. The microfluidic device according to claim 1 is characterized in that the first channel includes an opening arranged on the outer surface of the substrate, and the arrangement of the opening provides a path for the one or more substances to vertically enter the substrate and move vertically in the first channel. 5. The microfluidic device according to claim 1 is 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, 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, and 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. 6. The microfluidic device according to claim 1 is characterized in that the one or more substances are injected into the first channel arranged vertically in the substrate through a bottom horizontal plane surface, 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, and the shape, size and orientation of the first channel and the opening are set in a manner to maintain the direction and volume continuity. 7. The microfluidic device according to claim 1 is characterized in that the parallel or focused light source is oriented to propagate along, in the opposite direction, in a direction orthogonal to, or in a diagonal direction of the movement direction of the one or more substances in the fourth channel. 8. The microfluidic device of claim 1, wherein the fourth channel allows for imaging and analysis of particles or cells in multiple focal planes. 9. The microfluidic device of claim 1, wherein the fourth channel allows for imaging and analysis of particles or cells in multiple focal planes during movement of the one or more substances. 10. The microfluidic device of claim 1, wherein the fourth channel allows for imaging and analysis of particles or cells during movement of the one or more substances. 11. The microfluidic device of claim 1 , wherein the fourth channel allows for imaging and analysis of particles or cells from multiple angles and/or orientations during movement of the one or more substances. 12. The microfluidic device of claim 1, wherein the fourth channel allows for imaging and analysis of particles or cells using one or more imaging devices from multiple focal planes, angles, and/or orientations during movement of the one or more substances. 13. The microfluidic device of claim 1, further comprising one or more electrical, optical and/or fluidic forces to move one or more cells or one or more particles in one or more channels. 14. The microfluidic device of claim 1, further comprising one or more electrodynamic forces, electrophoretic forces, and/or dielectrophoretic (DEP) forces to move one or more cells or one or more particles in one or more channels. 15. The microfluidic device of claim 1, further comprising one or more imaging devices, wherein at least one of the imaging devices is movable in a manner that changes a focal plane imaged in the fourth channel. 16. The microfluidic device according to claim 1 is characterized in that one or more cells or one or more particles in the fourth channel can be moved by changes in optical force and/or fluidic force, and the area of the cells imaged in the fourth channel changes with the movement of the particles. 17. The microfluidic device of claim 1, wherein the fourth channel is spaced a distance from two or more outer surfaces of the substrate to allow imaging and analysis of multiple image slices of the cell or particle as the focal plane moves relative to the cell or particle. 18. The microfluidic device of claim 1 , wherein the fourth channel is spaced a distance from two or more outer surfaces of the substrate to allow imaging and analysis of multiple image slices of the moving cells or particles as the moving cells or particles move through the focal plane. 19. The microfluidic device of claim 1, wherein the fourth channel is spaced a distance from two or more outer surfaces of the substrate to allow imaging and analysis of multiple image slices of suspended or static cells or particles. 20. The microfluidic device of claim 1, wherein the one or more substances can be moved by pressure, vacuum, peristalsis, electrokinetic force, electrophoretic force, magnetic force, optical force, or any combination thereof. 21. The microfluidic device of claim 1, further comprising a dichroic mirror for directing light to or away from an imaging device that images and/or analyzes cells or particles in the fourth channel. 22. The microfluidic device according to claim 1, further comprising a dichroic mirror for directing a parallel or focused light source to interact with particles or cells in the fourth channel. 23. The microfluidic device of claim 1, further comprising a dichroic mirror configured to direct parallel or focused light away from an imaging device, a light source, or another portion of the device. 24. The microfluidic device according to claim 1, characterized in that the plurality of channels include a fifth channel, which is arranged horizontally, vertically or diagonally inside the substrate. 25. The microfluidic device of claim 1, wherein the plurality of channels includes a fifth channel that is divided into two or more channels or wells for classifying cells or particles. 26. The microfluidic device according to claim 1, wherein the fourth channel is closer to the top of the substrate than the first channel, the second channel or the third channel. 27. The microfluidic device according to claim 1, wherein the fourth channel is located at a distance of 100 micrometers to 100 mm from the top of the substrate. 28. The microfluidic device according to claim 1, characterized in that the length of the first channel ranges from 0.1 mm to 100.0 mm. 29. The microfluidic device according to claim 1, characterized in that the length of the second channel ranges from 0.1 mm to 100.0 mm. 30. The microfluidic device according to claim 1, characterized in that the length of the third channel ranges from 0.05 mm to 100.0 mm. 31. The microfluidic device according to claim 1, characterized in that the length of the fourth channel ranges from 0.1 mm to 100.0 mm. 32. The microfluidic device according to claim 1, characterized in that the length of the first channel is greater than that of the second channel, the third channel or the fourth channel. 33. The microfluidic device of claim 1, further comprising a cell or particle interrogation unit. 34. The microfluidic device of claim 1, further comprising a cell or particle collection channel. 35. The microfluidic device of claim 1 , further comprising an imaging device selected from at least one of the following: a bright field imager, a light scattering detector, a single wavelength fluorescence detector, a spectral fluorescence detector, a CCD camera, a CMOS camera, a photodiode or a photodiode array, a spectrometer, a photomultiplier tube or a tube array, a chemiluminescence detector, a bioluminescence detector, a standard Raman spectroscopy detection system, surface enhanced Raman spectroscopy (SERS), coherent anti-Stokes Raman spectroscopy (CARS) and/or coherent Stokes Raman spectroscopy (CSRS). 36. The microfluidic device according to claim 1 further includes a channel end for injecting the one or more substances into an opening arranged on the outer surface of the substrate, and the arrangement provides a path for the one or more substances to enter the substrate and move in the first channel, wherein the cross-sectional area of the opening is equal to, greater than or less than the cross-sectional area of the channel end.