KR20050047540A - Method and apparatus for sorting particles - Google Patents

Abstract

A method and apparatus for sorting particles moving through a closed channel system of capillary size comprises a bubble valve for selectively generating a pressure pulse to separate a particle having a predetermined characteristic from a stream of particles. The particle sorting system may further include a buffer for absorbing the pressure pulse. The particle sorting system may include a plurality of closely coupled sorting modules which are combined to further increase the sorting rate. The particle sorting system may comprise a multi-stage sorting device for serially sorting streams of particles, in order to decrease the error rate.

Description

Particle sorting method and apparatus {METHOD AND APPARATUS FOR SORTING PARTICLES}

The present invention claims priority to U.S. Provisional Application No. 60 / 411,143, filed Sep. 16, 2002 and U.S. Provisional Application No. 60 / 411,058, filed Sep. 16, 2002, the content of which is herein incorporated by reference. To quote. The invention also claims priority to US patent application Ser. No. 10 / 329,008, filed Dec. 23, 2002, which is part of a serial application of US patent application Ser. No. 10 / 179,488, filed Jun. 24, 2002. As part of the continuous applications, the contents are incorporated herein by reference.

The present invention relates to a method and apparatus for sorting particles contained in a suspension in which the inlet flow path of the sorting module can be divided into a plurality of outlet channels. More specifically, the present invention relates to a particle sorting system in which a plurality of sorting modules are interconnected to increase particle throughput.

There is a need for high efficiency screening of particles in biotechnology, particularly in the field of cytology and drug screening. Examples of particles that require filtration include various cells such as platelets, white blood cells, tumor cells, embryonic cells, and the like. Such particles are particularly important in the field of cytology. (Macro) molecular species such as proteins, enzymes and poly-nucleotides. Such particle species are particularly important in the field of drug screening during the development of new drugs.

Particle sorting methods and apparatus are known and most prior art is suspended in a fluid flowing through a channel network where the particles are at least downstream of the branch point and are also subject to the detect-dicide-deflect principle. Work under controlled conditions. Moving particles are first analyzed for certain characteristics such as light absorption, fluorescence brightness, size, and the like. Depending on the result of the detection step, it is determined how the particles are to be further processed downstream. The result of the determination is then applied to deflect the direction of the particular particle towards the branch of the predetermined channel network.

What matters is the throughput of the sorting device, ie how much particles can be sorted per unit time. Typical sorting rates for sorters using a flow of particle suspension in a closed channel range from hundreds of particles per second to thousands of particles per second for a single sorting unit.

Examples of screening devices are described in US Pat. No. 4,175,662, the contents of which are incorporated herein by reference. In U. S. Patent No. 4,175, 662, particles, in this case cells, flow through a straight channel, the straight channel branching into two vertical channels downstream of the branching point (T-branch). The entrapping particles are surrounded by a sheath made of a compatible liquid, so that the particles are confined to the center of the channel. Under normal conditions, the flow ratio through the two branches is adjusted so that the particles automatically flow through one of the branches. In a portion of the channel, particle characteristics are measured by a detector (eg an optical system) (detection step). The detector generates a signal when the detector detects particles having predetermined characteristics in the detection step. Once the particles are detected, the deflector is activated in the deflection step to deflect the particles. In this case, the deflector has a pair of electrodes arranged inside the channel branch through which the particles normally flow through without the deflector operating. When a current pulse is applied, the aqueous solution is electrolyzed to produce and release gas bubbles between the pair of electrodes. As the size of the gas bubbles increases, the flow rate through this branch decreases during the discharge phase. After the current pulse is applied, if bubble growth stops, gas bubbles move along the flow. As a result, the flow through a particular branch temporarily decreases, and the particles change the flow path and flow to another branch.

The apparatus of US Pat. No. 4,175,662 is effective for screening particles. However, there is a serious drawback that gas bubbles are generated that can potentially accumulate at any point in the fluid network. This bubble generation blocks the flow channel, causing a screening error. Another drawback is that the generated gases (mainly coral and hydrogen) and ionic species (mainly OH ⁻ and H ⁺ ) affect particles flowing through branches having a pair of electrodes. In addition, sensitive proteins such as cells and enzymes are very fragile and can be destroyed by contaminants generated with gas bubbles. Another drawback is the complexity of the overall sorting device. In particular, mounting and assembling the microelectrode structure inside a small channel of the system is very complex. As a result, the price of the sorting unit is relatively high.

Another example of a conventional particle sorting apparatus is described in US Pat. No. 3,984,307, the contents of which are incorporated herein by reference. In US Pat. No. 3,984,307, the particles flow through the center of the channel in a state defined by the flow sheath fluid. After passing through the detector, the channel branches into two branches and forms an acute angle between the two branches (eg, a Y-shaped branch). Immediately before the fork, an electrically operated transducer is placed inside the channel to detect specific particles with appropriate predetermined characteristics. The disclosed transducer is a piezoelectric actuator or ultrasonic transducer, which generates pressure waves inside the channel during electrical operation. The generated pressure wave temporarily disturbs the flow inside one branch, deflecting the particles to the other branch.

As with the apparatus described previously, in the apparatus of US Pat. No. 3,984,307, a deflector is installed inside the channel system, resulting in relatively high manufacturing costs. The generated pressure wave is not limited to the branching point and transmits not only the two branching points but also the deflections upstream. This affects the overall flow through the channel. In particular, this is a drawback when connecting this type of sorter in series or in parallel which is conventional in constructing a high efficiency sorting system. Pressure waves generated in one sorter affect the deflection and flow of particles in adjacent sorting units.

Another example of a conventional particle sorting apparatus is described in US Pat. No. 4,756,427, the contents of which are incorporated herein by reference. This selector is similar to the device disclosed in US Pat. No. 4,175,662. However, in the case of this patent, the flow inside the branch is disturbed by temporarily changing the resistance of the branch. The resistance is changed by changing the height of the branch channel with an external actuator. In a preferred embodiment, the external actuator is a piezoelectric disk adhered to the top of the channel, which, in operation, moves the channel downstream.

Although the structure of the sorter described in US Pat. No. 4,756,427 is less complex than the sorter structure described above, there is still a problem in combining multiple sorter modules of the type described above to increase the screening rate. This is due to the generated pressure wave that causes interference with other sorter modules, such as in the sorter disclosed in US Pat. No. 3,984,307.

Another example of a conventional particle sorting apparatus is described in US Pat. No. 5,837,200, the contents of which are incorporated herein by reference. U. S. Patent No. 5,837, 200 discloses a sorting device that uses a magnetic deflection module to classify or select particles based on magnetic properties. U. S. Patent 5,837, 200 also describes treating and separating individual particle streams in parallel.

1 is a schematic diagram of a particle sorting system according to an exemplary embodiment of the present invention.

2-4 illustrate the operation of the particle sorting system of FIG.

FIG. 5 shows a particle sorting system showing alternate positions for the actuator chamber and buffer chamber. FIG.

Fig. 6 is a diagram showing another particle sorting system according to another embodiment of the present invention.

Figure 7 illustrates a bubble valve suitable for use in the particle sorting system of the present invention.

8 is a schematic diagram of a particle sorting system according to an exemplary embodiment of the present invention.

9 illustrates an embodiment of a particle sorting system for sorting parallel streams of particles in accordance with the teachings of the present invention.

FIG. 10 is a diagram illustrating one embodiment of a particle sorting system comprised of a binary tree-type structure of a sorting module in accordance with the teachings of the present invention.

11 illustrates another embodiment of a multistage particle sorting system for multistage sorting of parallel particle streams.

12 illustrates a parallel particle sorting system according to another embodiment of the present invention.

Figure 13 illustrates a parallel particle sorting system according to another embodiment of the present invention.

14A and 14B illustrate a particle sorting system according to another embodiment of the present invention having an optical mask for measuring particle size and / or velocity.

Figure 15 illustrates a parallel sorting system with variable channels according to another embodiment of the present invention.

Figure 16 illustrates a variable array structure of a parallel sorting system according to another embodiment of the present invention.

17 illustrates a parallel sorting system according to another embodiment of the present invention.

Figure 18 illustrates a drug screening system for implementing the particle sorting system of the present invention.

The present invention provides a method and apparatus for sorting particles moving through capillary sized closed channel systems. The particle sorting system of the present invention is to provide a sorting module that can be assembled at low cost while providing a means for precisely sorting a large amount of particles per unit time. The particle sorting system may be equipped with a plurality of closely coupled sorting modules mixed to increase the fraction. The particle sorting system may be equipped with a multistage sorting device for sorting the particle stream in series to reduce the error rate.

The particle sorting system implements an improved fluid particle switching method and switching device according to the present invention. The particle sorting system comprises a closed channel system of capillary size for sorting particles. The channel system has a first supply line for introducing a stream of particles and a second supply line for supplying a carrier fluid. The first feed canal forms a nozzle to introduce a stream of particles into the flow of carrier fluid. The first supply pipe and the second supply pipe are in fluid communication with the measurement tube, the measurement tube branches from the first branch point to the first branch channel and the second branch channel. A measuring zone is formed inside the measuring tube, which is connected with a detector to sense the predetermined characteristics of the particles passing through the measuring zone. Two opposing bubble valves are arranged in fluid communication with the measuring tube. Bubble valves are spaced apart from each other. The bubble valve communicates with the measuring tube through a pair of opposite side passages, respectively. The fluid may partially fill this side passage, forming a meniscus therein. The meniscus forms an interface between the carrier fluid and other fluids, such as the gas inside the reservoir of the associated bubble valve. An external actuator is provided to drive the bubble valve. When the actuator is driven, the pressure inside the reservoir of the activated bubble valve increases, deflecting the meniscus and causing a flow disturbance inside the measuring tube to deflect the flow therein.

When a sensor disposed in the measurement area senses a predetermined feature of particles flowing through the measurement area, the sensor generates a signal in response to the detected feature. The external actuator responds to the sensor, causing a pressure wave inside the compression chamber of the first bubble valve to deflect particles having the predetermined characteristics and thus the selected particles flow through the second branch conduit.

In one embodiment, the present invention provides a method for providing a measuring tube having an inlet and a branching point (in which the measuring tube is separated into two branch conduits), and discharging the fluid stream with a suspended particle stream therein. Wherein the particles flow normally through the first conduit of a branch conduit and upstream from the branch point, two opposing side passages for temporarily deflecting the stream inside the conduit. It includes a method of providing. The first side passage of the side passages is hydraulically connected to the compression chamber of the first bubble valve and is operated by an external actuator to change the pressure inside. The second side passage of the side passages is hydraulically connected to the buffer chamber of the second bubble valve for absorbing pressure fluctuations. The method also includes providing a measuring station along the upstream side of the measuring tube of the lateral passage, thereby detecting a predetermined characteristic of the particles inside the stream and generating a signal when the predetermined characteristic is detected. The method also drives an external actuator in response to sensing the predetermined feature to generate a flow disturbance inside the measuring tube between the side passages, deflect the particles having the predetermined feature and flow the selected particles into the second branch conduit.

In another embodiment of the present invention, the particle sorting rate is increased by connecting a plurality of sorting modules in parallel or in series respectively in a binary tree configuration, or the type of sorted particles is increased.

According to one embodiment of the invention, a particle sorting system is provided. The particle sorting system delivers a suspended cell stream to a carrier fluid, the first conduit having an inlet, a first outlet, and a second outlet, a sensor for sensing one of the predetermined characteristics of the particle, the size and velocity of the particle, A side channel communicating with the first conduit, the sealing chamber disposed adjacent the side channel, wherein the carrier fluid forms a meniscus inside the side channel to separate the sealing channel and the carrier fluid; , Including the actuator. An actuator changes the pressure inside the sealing chamber to deflect the meniscus when the sensor senses a predetermined feature. When the meniscus deflects, cells with predetermined characteristics flow to the second outlet, and cells without the predetermined characteristics flow to the first outlet.

The present invention can be expected to have the major value of screening multiple candidate components for high efficiency screening, eg, activity on one or more species of cells. It is particularly valuable for screening synthetic or natural product libraries, for example for active ingredients or biochemical characteristics.

The present invention can be expected to be of value in screening samples for a large number of molecules, such as biochemical characters, with high efficiency. The present invention can also be used to screen samples for the presence of multiple biochemical molecules such as polypeptides, receptor ligands, enzyme substrates, antagonists of enzyme or receptor activity or nucleic acids.

The present invention provides a particle sorting system for sorting particles suspended in a fluid. The particle sorting system increases the sorting efficiency of the particles and lowers the sorting error based on the predetermined characteristics. The present invention will be described below with respect to exemplary embodiments. Those skilled in the art will appreciate that the present invention may be practiced in many different applications and examples and is not particularly limited to the examples for the particular embodiments shown herein.

As used herein, the terms ducts, channels and flow channels refer to passages formed within or through the media that enable the movement of fluids such as liquids and gases. The channels within the microfluidic control system have cross-sectional dimensions ranging from about 1.0 μm to about 500 μm, preferably from about 25 μm to about 250 μm, most preferably from about 50 μm to about 150 μm. Those skilled in the art will be able to determine the proper volume and length of the flow channel. The range is intended to include the above quoted values as upper or lower limits. The flow channel can have any selected shape or arrangement, such as linear or nonlinear configurations and U-shaped configurations.

The term particle refers to a discontinuous unit substance including, but not limited to, cells.

As used herein, the term sensor refers to a device for measuring the characteristics of an object, such as a particle.

As used herein, the term bubble valve refers to a device that generates a pressure wave to control the flow through the channel.

As used herein, the term carrier fluid refers to a sheath of compatible fluid that surrounds particles for delivering one or more particles through a conduit or channel.

1 is a schematic diagram illustrating a particle sorting system 10 in accordance with the teachings of the present invention. According to one application of the invention, the particle sorting system 10 comprises a closed channel system of capillary size for sorting particles. The channel system has a first supply line 12 for introducing a stream of particles 18 and a second supply line 14 for supplying a carrier fluid. The first feed canal 12 forms a nozzle 12a and introduces a stream of particles into the flow of the carrier fluid. The first supply pipe 12 and the second supply pipe 14 are in fluid communication with the measuring tube 16 for transporting particles suspended in the carrier fluid. The measuring tube branches from the first branch point 21 to the first branch channel 22a and the second branch channel 22b. A measuring region 20 is formed inside the measuring tube 16, which is connected with a detector 19 to detect predetermined characteristics of particles passing through the measuring region 20. Two opposing bubble valves 100a and 100b are positioned in connection with the measuring tube, and are arranged in fluid communication with the measuring tube. The valves are spaced apart from each other, but other configurations can be used by those skilled in the art. The bubble valves 100a and 100b communicate with the measuring tube 16 through a pair of opposed side passages 24a and 24b, respectively. The fluid may partially fill these side passages 24a and 24b to form a meniscus 25 therein. The meniscus forms an interface between the carrier fluid and other fluids, such as the gas inside the reservoir of the associated bubble valve 100. An actuator 26 is provided to drive the bubble valve, which temporarily causes a flow disturbance inside the measuring tube to deflect the flow therein when driven by the actuator 26. As shown, the actuator is coupled to bubble valve 100b. The second bubble valve 100a acts as a shock absorber for absorbing the pulse pressure generated by the first bubble valve 100b.

The first side passage 24b is hydraulically connected to the compression chamber 70b in the first bubble valve 100b so that if this pressure inside the compression chamber increases, the flow inside the measuring tube adjacent to the side passage is inward, i.e. It moves almost perpendicular to the normal flow inside the measuring tube. The second side passage 24a disposed opposite the first side passage 24b is hydraulically connected to the buffer chamber 70 in the second bubble valve 100a to absorb the pressure transient. This first side passage 24a cooperates with the second side passage 24a to guide the above-described fluid movement by pressurizing the compression chamber 70b so that this movement is a normal flow of particles through the measuring tube. It has a component orthogonal to.

When the compression chamber 70b is pressurized, a predetermined amount of fluid is temporarily discharged from the first side passage 24b. Due to the elasticity of the second side passage 24a, the temporary flow of the fluid inside the measuring tube is pressurized to the second side passage 24a. By cooperating the interconnected fluid structure and the two side passages, the flow through the measuring tube 16 is compressed by the external actuator 26 in response to the signal output from the detection means 19. As it compresses or decompresses, it temporarily moves back and forth along the side. This temporary fluid displacement has a component perpendicular to the normal flow inside the measuring tube, which can be applied to the deflecting particles having the desired characteristics to separate the deflecting particles from the residual particles contained in the mixture.

As shown, the measuring tube 16 branches into two branches 22a and 22b at the branch point 21. The flow rate in these two branches is adjusted such that the particles normally pass through the second branch of the two branches 22b. The angle between these branches 22a, 22b is between 0 degrees and 180 degrees, preferably between 10 degrees and 45 degrees. However, the angle may even be zero degrees corresponding to two parallel conduits with a straight dividing wall therebetween.

The particles to be screened are preferably fed to a measurement position in the central fluid stream surrounded by a particle free fluid sheath. The process of confining a particle stream is known and is referred to as a "sheath flow" configuration. Generally, the foregoing limitations are made by injecting a suspended particle stream through a narrow discharge nozzle into a particle-free carrier fluid flowing inside the measuring tube 16. By adjusting the flow rate ratio of the suspension and the carrier fluid, not only the distance between particles but also the radial limitation inside the measuring tube can be adjusted. A relatively high flow rate of the carrier fluid produces a more defined particle stream with a longer distance between the particles.

In the suspension introduced by the first feed canal 12, the particles can be classified into two types: normal particles 18a and corresponding particles 18b. Upon detecting a predetermined characteristic of the particle 18b in the measurement area 20, the detector 19 outputs a signal. The external actuator 26 receives a signal generated from the detector 19 in response to detecting a predetermined characteristic to drive the bubble valve 100b of the first actuator, so that the side passage 24a inside the measuring tube 16 is provided. , 24b) creates a flow disturbance. The flow disturbance deflects the particles 18b having predetermined characteristics to flow toward the first branch pipe 22a rather than the second branch pipe 22b. The detector communicates with the actuator 26, and when the detector 19 detects a predetermined characteristic in the particle, the actuator drives the first bubble valve 100b to change the pressure inside the reservoir 70b of the first bubble valve. Causes When the first bubble valve is operated, the meniscus 25b is deflected inside the first bubble valve 100b, and a temporary pressure fluctuation occurs inside the first side passage 24b. The second side passage 24a and the second bubble valve 100a absorb a temporary pressure fluctuation inside the measuring tube 16 induced through the actuator 26. Basically, the reservoir 70a of the second bubble valve 100a is a buffer chamber having an elastic wall or containing a compressive fluid such as a gas. The elastic properties allow fluid to flow from the measuring tube to the second side passage 24a, absorbing pulse pressure and preventing disturbances in the flow of particles not selected in the particle stream.

In the measurement area 20, a suitable sensor is used to inspect individual particles for special features such as size, shape, fluorescence luminance, as well as features that will be apparent to those skilled in the art. Examples of applicable sensors known in the art are various forms of optical detection systems such as microscopes, computer imaging systems and electronic means for measuring the electronic properties of particles. Particularly well known systems in the art include systems for measuring the fluorescence brightness of particles. Such a system includes a light source having a wavelength suitable for inducing fluorescence and a detection system for measuring the luminance of the induced fluorescent light. Such devices are often used in combination with particles labeled with a fluorescent marker (i.e., an attached molecule that, when illuminated with light of a particular first wavelength, generates light of another particular second wavelength (fluorescence)). When light of this second wavelength is detected, the feature is detected and a signal is output.

Another example includes measuring light scattered by particles flowing through the measurement region. Interpretation of the scatter yields information about the size and shape of the particles, which can be adapted to output a signal when detecting certain features.

The actuator 26 for pressurizing the compression chamber of the first bubble valve may comprise an external actuator responsive to a signal from a sensor having a predetermined characteristic in which particles are selected. There are two types of external actuators suitable for increasing pressure. The first kind directly supplies gas pressure to the fluid inside the first side passage 24b. For example, the actuator may comprise a source of compressed gas connected to a switching valve with respect to the fluid column inside the side passage 24b. Actuation of a switching valve connects a passage to the source of compressed gas, thereby deflecting the meniscus inside the fluid. When stopped, the switching valve returns the passage 24b to normal operating pressure.

Alternatively, displacement actuators may be used in combination with closed compression chambers having movable walls. When the displacement actuator displaces the movable wall of the compression chamber inward, the internal pressure increases. When the movable wall returns to its original position, the pressure decreases to the normal operating pressure. An example of a suitable displacement actuator is an electromagnetic actuator, which displaces the plunger when the coil is excited. Another example is the use of piezoelectric materials in the form of cylinders or disk piles, which, when voltage is applied, cause linear displacements. The two types of actuators described above combine the movable walls of the compression chamber 70 to cause pressure variations therein.

2-4 illustrate the switching operation of the switch 40 in the particle sorting system 10 of FIG. In FIG. 2, the detector 19 senses the predetermined characteristics of the particles and generates a signal to drive the actuator 26. When the actuator is driven, the internal pressure of the reservoir 70b of the first bubble valve 100b increases, deflecting the meniscus 25b and temporarily displacing the fluid from the first side passage 24b, as shown by the arrow. Release. Due to the elastic properties of the reservoir of the second bubble valve 100a, the fluid flows to the second side passage 24a when the pressure suddenly increases at this point inside the measuring tube. This fluid movement to the second side passage 24a is indicated by an arrow. As a result, as shown in the figure, the flow through the measuring tube 16 is deflected such that the selected corresponding particles 18b disposed between the first side passage 24b and the second side passage 24a are normal. Move at right angles to the flow direction of the state. The flow resistance to the measuring tube 16, the first branch 22a and the second branch 22b is such that the preferred flow direction to or from the first side passage 24b and the second side passage 24a is determined by the measuring tube ( It is chosen to have a white component that is perpendicular to the steady flow through 16). This purpose is achieved by the first and second branches 22a and 22b, for example, in order for the flow resistance to be selected to be large compared to the flow resistance of the first side passage 24b and the second side passage 24a. Is done.

FIG. 3 shows the particle sorting system 10 during the relief of the first bubble valve reservoir when the particles 18b remain large between the first side passage 24b and the second side passage 24a. . When the actuator 26 stops, the internal pressures of the reservoirs 70a and 70b return to normal pressure. During this relief step, there is a negative pressure difference between the two reservoirs 70a, 70b of the bubble valve, so that the flow through the first side passage 24b and the second side passage 24a is reduced, as shown in the previous figure. And oppose the fluid flow as indicated by the arrow.

4 shows the particle sorting system 10 after the switching process has ended. When the reservoir internal pressure of the bubble valve is equalized, the flow through the measuring tube 16 is normalized. When the particles 18b are displaced in the radial direction, they flow to the first branch 22a and other particles continue to flow to the second branch 22b to separate the particles based on the predetermined characteristics.

The process of detecting and optionally deflecting the particles can be repeated several times per second to screen particles at a high rate. Employing the above-described fluid switching, the switching operation is performed at about thousands of switching operations per second, sorting at a rate of about 1 million selected particles per hour.

According to another embodiment of the present invention, the bubble valve 100a and the buffer bubble valve 100b of the actuator may be arranged in different positions. For example, as shown in FIG. 5, the bubble valve 100a and the first sidewall passage 24a and / or the buffer bubble valve 100b and the second sidewall passage 24b of the actuator are separated from the branch point 21. It may be arranged upstream. The component may be disposed in any suitable position such that the flow resistance between the actuator chamber 70a and the buffer chamber 70b is less than the flow resistance between any one of the latter components and the other pressure source. More specifically, the actuator chamber 70a and the buffer chamber 70b may be arranged such that the flow resistance therebetween is less than the flow resistance between the selected particles and the subsequent particles downstream of the particles. Positioning the component in the manner described above causes the pressure waves generated by the aforementioned method of deflecting a single selected particle to move upstream or downstream to affect the flow of residual particles contained in the stream of particles. Can be prevented. If the difference in flow resistance is large, the fluid switching action due to the associated pressure variation is separated from the flow characteristics of the rest of the system to a high level. In addition, the generated pressure wave applied for sorting can be in-situ attenuated to implement a sorting network having a plurality of switches 40, each of which is hydraulically and pneumatically isolated from the other. .

According to another embodiment, as shown in FIG. 6, the particle sorting system of the present invention employs an appropriate pressure wave generator (instead of a bubble valve) in cooperation with one or more bubble valves acting as buffers, such as valve 100b. Can be. For example, pressure wave generator 260 may act on a fluid that flows directly or through an actuator or stepper motor (such as a piezoelectric column) or a stepper motor to selectively deflect particles when the actuator is driven by a signal. With a plunger). Another suitable pressure wave generator includes an electromagnetic actuator, a thermopneumatic actuator and a thermal wave generator for applying steam to generate steam bubbles in the flowing fluid. The buffer bubble valve 100b is arranged to absorb the pressure wave generated by the pressure wave generator 260 to prevent flow disturbances in other particles of the particle stream. The spring constant of the buffer 100b determines the volume of the buffer chamber 70b, the cross-sectional area of the side passages 24b and / or the stiffness or thickness of the flexible membrane (reference 72 in FIG. 7) forming the buffer chamber 70b. By changing, it can be changed according to specific requirements.

FIG. 7 shows an embodiment of a valve 100 suitable for generating a pressure wave to separate the particle from other particles included in the particle stream and / or a buffer for absorbing the pressure wave in accordance with the teachings of the present invention. . As shown, the valve 100 is formed adjacent to the side passages 24a and 24b formed inside the substrate extending to the measuring tube 16. The side passage 24a has a fluid interface port 17 formed by an opening in the inner wall of the passage. Adjacent to the side passage 24a is a sealed compression chamber 70, which communicates with the side passage through a fluid interface port. The illustrated compression chamber 70 is formed of a seal 71 and a flexible membrane 72. The carrier fluid inside the side passages 24a forms a meniscus 25 at the interface between the side passages and the compression chamber. The actuator 26 compresses the flexible membrane to increase the pressure inside the compression chamber, resulting in a pressure wave in the carrier fluid.

FIG. 8 shows a sorting module 50 having a first feed conduit 54 and a second discharge conduit 56 as well as a suitable feed conduit 52 for providing a stream of particles to be sorted, either of which The sorted particles may be carried in the sorting module 50. The sorting module 50 includes a detector system 19 for detecting particles entering the sorting module 50 through the feed duct 52, which is connected to a switch 40 to provide the necessary switching performance. It is operatively connected to screen particles. The first branch 22b and the second branch 22a of FIG. 1 may be disposed in fluid connection with the discharge pipe 54 and the second discharge pipe 56.

9 illustrates a particle sorting system 500 according to an embodiment of the present invention having a plurality of sorting modules 50 that can be coupled together in a suitable configuration. For example, the modules 50 of this embodiment are combined in parallel. The outlet pipe 54 of the sorting module 50 is coupled to the first composite outlet 58, and the second outlet tube 56 is coupled to the second composite outlet 60. The parallel arrangement of the sorting modules forms a system of complex sorting modules 50 having a total sorting rate of N times the sorting rate of the individual sorting modules 50, where N is the number of sorting modules 50 coupled in parallel. Count

10 shows a particle sorting system 550 according to another embodiment having a first sorting module 50a and a second sorting module 50b coupled in series with it. The second sorting module 50b may be mounted to sort particles having predetermined characteristics that are the same or different than the predetermined characteristics of the particles sorted by the first sorting module 50a. The particle stream enters the first sorting module 50a through the feed conduit 52 and may comprise at least two kinds of particles. Particles of the first type are sorted in the first sorting module 50a and are discharged through the first discharge pipe 54a. The residual particles are discharged from the first sorting module 50a through the second discharge pipe 56a and introduced into the second sorting module 50b through the second supply pipe 52b. From the particle stream, particles having other predetermined characteristics are sorted and discharged through the second discharge pipe 54b. Particles not having any of the two predetermined features are discharged from the second sorting module 50b through the second discharge pipe 56b. Those skilled in the art will appreciate that any suitable type of sorting module 50 can be used and can be combined with one another in a variety of ways depending on the desired result.

Figure 11 shows a hierarchical structure for sorting with high efficiency and low error in accordance with another embodiment of the present invention. The illustrated embodiment shows a two stage particle sorting system 800 for sorting a plurality of parallel particle streams in a first stage, collecting the output of the first stage, and performing a second sorting process on the output of the first stage. )to be. The input stream of particles contained in the suspension 80 from the particle input chamber 88 is divided into N single sorting channels 81a through 81n, each channel being capable of sorting a selected number of particles per second. Each channel 81 has a detection area 84 for inspecting particles and identifying particles having predetermined characteristics, and a switching region for separating particles having predetermined characteristics from other particles included in the stream, as described above. 82). The switching region 82 produces two particle output streams, the selected stream and the rejected stream included in the switching region 82 based on the particle characteristics measured in the detection region 84. The stream selected from each channel is collected in collection area 86 as a stream that is reselected in secondary sorting channel 810. As shown, the secondary screening channel 810 repeats the screening process of detection and screening based on the predetermined characteristics.

Assuming that each single channel screening process produces some degree of error (y) of false choices (y is a probability less than one of the particles chosen by mistake), the hierarchical structure is y ² for the two-stage classification system. Or produce a low error rate of y ⁿ for the n-stage classification system. For example, if the single channel error rate is 1%, and step 2, the error rate was 0.01% or 10 ^{a quarter} of.

Alternatively, the structure may have M primary sets of N selection channels per secondary channel. Assuming the application wants to capture particles present at the input at the ratio z, the single channel selector has a maximum screening rate x of particles per second. System throughput is the particle count of M * N * x per second. The number of particles collected in the B channel per second is N * x * z, where N * z must be less than 1 so that all particles accumulated from the N channel can be sorted into a single secondary channel. In order to increase throughput over N = 1 / z, a group of N primary channels and one secondary channel must be added in parallel. The total throughput is M * N * x by M secondary channels.

12 shows a parallel-serial particle sorting system 160 according to another embodiment of the present invention. The parallel-serial particle sorting system 160 includes a first parallel sorting module 161 and a second parallel sorting module 162. The first sorting module 161 is applied to a plurality of labeled particles, with particles having two markers sorted by the discharge channel 165 and transported through it.

13 illustrates another parallel-serial particle sorting system 170. The first parallel sorting module 171 separates particles with a first marker, collects particles from different channels, and transports particles with a first marker through the first outlet channel 175. All other particles are fed to a second parallel sorter 172 to sort particles with a second marker. Particles with a second marker are collected and transported through the second outlet channel 176. Particles having neither the first marker nor the second marker are transported through the third outlet channel 177.

According to the embodiment of the present invention shown in FIGS. 14A and 14B, the particle sorting system may be equipped with sensors for measuring the velocity, position and / or size of the particles. Measurement of velocity, position and / or size may be performed during screening or at the same time as the classification of particles or at different times. As shown in Figure 11, in a system based on parallel channels, different channels may have different flow resistances and may vary particle or cell velocity in each channel. In a system in which the detection zone 84 is separated from the switching zone by a distance L, the particle velocity in the channel 81 sets the switching time delay T (i.e., the switch actuation delay time relative to the detection moment of the target particle). In order to be known.

In most optical systems for detecting cells or particles, the area in which the cells generate light in the photo detector in the detection area has a size that is significantly larger than the size of the cell diameter. Therefore, when detecting light in the detection area, the cells may exist anywhere in the area, making it difficult to indicate the exact position of the cell. To provide more accurate detection, multiple pixels of the optical detector can be packed across the detection area, but are expensive and require complex support electronics.

According to an exemplary embodiment of the present invention, an optical mask 140 may be added to the detection region to deposit the masking pattern directly on the select chip to provide accurate speed detection. The masking pattern may be deposited such that the edges in the masking pattern are precisely positioned (with precision less than 1 um in current technology) with respect to cell selection actuator region 82. A single optical detector that captures light from the cells in the detection region 84 can see the light when the cells are not masked. The duration of light blocked by one of the "bars", the connected opaque portion of the mask having a known length, provides a velocity measure.

Mask patterns with multiple bars ranging in size from 10um to 30um in 1um steps produce only bars of larger size than cells that minimize the signal generated from the cells. Therefore, this pattern can be used to measure the size of a cell independently of a signal. Such gradient masks also produce certain patterns in optical detectors that can be analyzed to measure several speeds to reduce discrepancies in speed measurements. The pattern of light caused by the mask 140 also allows the detector to identify each edge in the mask 140. If the bars 141 are the same, the light signals for each bar are the same, and the detectors can distinguish one by one sequentially. Therefore, the gradient mask pattern allows a single detector that observes a large area (multiplied by the size of the cell) to measure the speed of the cell and inside the detection area 84 with about 1 um accuracy with respect to the channel structure and actuator position on the chip. The exact position can be measured and the size of the cell can be identified with the precision imposed by the gradient pattern. Gradient mask 140 allows the detector to measure these parameters regardless of the magnification of the optical system or the nature of the optical detector.

Those skilled in the art will recognize other devices for measuring the size, position and / or velocity of particles in the sorting system according to the teachings of the present invention. Suitable devices are readily available and known to those skilled in the art.

According to another embodiment, as shown in Figure 15, the particle sorting system includes an array 8000 of unequal sorting channels. Using a parallel array with a series of non-identical select channels 810a through 810n is more efficient in terms of space for optimal external actuators, use of light output and adaptation. Since the particle velocity can be precisely measured, the channel does not require a fixed delay between the operation of the switch and the detection of the characteristic in order to deflect particles having the detected characteristic. Therefore, certain parameters of the channel such as the distance L between the detector 84 and the switch 82 or the shape of the passageway between the detector 84 and the switch 82 can be changed.

Using a single laser for each wavelength light irradiation oriented perpendicular to the chip, the laser irradiates an area defined by (number of channels) x ((channel width in the detection area) + (internal channel spacing C)) (See Fig. 15). However, the active region capable of absorbing light to generate fluorescence is only a channel (number of channels) x (channel width), leaving a fill factor of (channel width) / (channel width + C). . The fill factor is preferably close to 100% to avoid wasting available input light.

Therefore, minimizing the interchannel spacing in a parallel sorting system is important for light detection area and optical system efficiency. In the usable array structure of the present invention, as shown in FIG. 16, the spacing of the channels in the detection region 84 is similar to the width of the channel, thus bringing light utilization close to about 50%. The channel spacing in the active region 82 may be large as shown in FIG. Placing the actuator 26 along the channel may also be modified to increase the effective radius for the external driver actuator.

Also, the variable array 8000 may have a meander inside the selected channel to balance the flow resistance of all the channels so that the particle velocity is approximately equal, assuming a constant pressure drop across all the channels. The meander may be added upstream or downstream of the illustrated system or in the region between the detector and the actuator. Since the length L between each channel detection region 82I and the actuator 26i is known per structure, measuring the particle velocity at the same time as that determined for that particle provides an improved cell selection system.

Figure 17 illustrates a particle sorting system according to another embodiment of the present invention. The particle sorting system 1700 includes a plurality of sorting modules 1701 that operate in parallel. System 1700 has an input area 1710 for introducing a sample to each sorting module and a detection area 1702 for measuring a predetermined characteristic of each particle sorting channel 1702 in the detection area. The system also includes a switch region 1730 having an actuator in each of the sorting modules for separating particles having predetermined characteristics from particles having no predetermined characteristics. As shown in FIG. 17, the selection channel 1702 distance between each selection channel in the detection region 1720 is smaller than the interchannel distance in the switch region 1730. The close spacing in the detection area can reduce costs when detecting particles using a laser, and larger distance separation in the switch area 1730 can accommodate actuators of various sizes.

Particle sorting system 1700 also includes a secondary sorting module 1740 for repeating the sorting process consisting of detection and sorting based on predetermined characteristics to increase the precision of the sorting process. According to one embodiment, the sorting system comprises an enrichment region 1750 between the array of primary sorting module 1701 and the secondary sorting module 1740 to transfer particles from the primary sorting process to the secondary sorting process. ) May be provided. According to an exemplary embodiment, the rich region 1750 transfers particles by removing excess carrier fluid from the particles before they pass through the secondary sorting module 1740. The rich region 1750 may also include a hydration device that adds secondary sheet fluid to the particles after they are thick. The rich region 1750 can include a membrane inserted into the outlet channel 1703, a rich channel across the outlet channel 1703, and a membrane separating the outlet channel from the rich channel. Excess carrier fluid is removed from the stream of selected particles in the outlet channel 1703 through the membrane into the rich channel before the selected particles pass into the secondary sorting module 1740.

A system suitable for forming a rich region is disclosed in US patent application Ser. No. 10 / 329,018, entitled "Performance of Microfluidic Control Members in a Microfluidic Control System," the contents of which are described herein. It is cited for reference.

According to an exemplary embodiment, the removed carrier fluid may be regenerated and fed back to the inlet of the primary channel. The regeneration channel or other device connects the rich zone to the primary channel to make the carrier fluid available again for subsequent sorting processes. Alternatively, the carrier fluid can be removed from the failed particles and introduced to the primary channel inlet before discarding the failed particles.

The particle sorting system of the present invention is used in various applications in the field of microfluidics. According to one application, the particle sorting system may be realized in a drug sorting system as shown in FIG. As shown in FIG. 18, the drug selection system 140 includes a particle selection module 10 for recognizing target cells and separating target cells from a sample comprising the target cells. The target cell containing sample is introduced into the selection system through an input channel and passed to the particle selection module, which separates the target cell from the rest of the sample and passes the target cell into the mixing and culture region 141. The test compound is introduced into the mixing and culture zone 141 through the test channel 142 and contacted with target cells provided from the particle sorting system 10. Thereafter, the effect of the test compound on the target cell is detected in the detection region 145.

Exemplary drug selection system 140 with particle sorter 10 can be used with various types of markers. The drug screening system may utilize markers that allow for the determination of the activity of a particular enzyme to allow for the search of modulators of the pathway in which the enzyme is present. In addition, drug screening systems allow markers to measure the concentration of any intracellular messenger / signal, and to identify specific cell types, particularly rare (less than a hundred) cells. You can also use markers. Suitable markers include, for example, cell surface markers as in antibody and recombinant display techniques, as well as enzyme substrate markers that can be fluorescent, and cells such as Ca ⁺⁺ binding fluorescent dyes (Fura-3, Indo-1). May include, but are not limited to, signal binding compounds, and bio-luminescent enzyme substrate markers. For enzyme substrate markers that can be fluorescent, the compound enters the cell and is converted into fluorescent material by specific intracellular enzymes. Examples include Bodipy aminoacetaldehyde or BAAA for ALDH enzymes, 4-methylumbelliferyl phosphate for Phosphatases, and dihydrorhodamine 123 for cell redox systems. Can be mentioned. Another example of a fluorescent dye that may be used in the methods of the present invention is an In bio-luminescent enzyme based marker, wherein the compound enters a cell and reacts with a particular intracellular enzyme to generate light directly. Let's do it. Often, this technique can be confined to a reporter gene that observes gene activity. Examples include D-Luciferin and related report genes, as well as DMNPE and related report genes.

According to exemplary applications, any different number of compounds can be selected for their effect on various chemical and biochemical systems. For example, compounds may be selected for effects in other inhibitory key events with respect to biochemical systems that have blocking, slowing or unfavorable effects. For example, test compounds may be selected for their ability to block a system that is at least partly responsible for the development or disease development of certain symptoms of disease, including genetic diseases, cancer, bacterial or viral infections, and the like. Subsequently, the compounds showing favorable results for these screening assays may be further tested to identify agents effective for the treatment of symptoms or diseases of the vagina.

Alternatively, the compounds may be selected for their ability to promote, ameliorate, or otherwise induce a biochemical system having a function that is believed to be advantageous, such as a function that is believed to treat an existing deficiency in a patient.

The present invention comprises rare cells (i.e., less than 0.1%-less than 1% of the input sample) for a variety of applications, such as cell transplantation, cell transplantation and cell transfection, or genetic modification following transplantation or transplantation. Cells). The invention can also be used to isolate tumor cells in a sample for the development of custom treatments.

The present invention can be used to select compounds of primary cells, for example taking 10 ¹¹ cells by plasma exchange and selecting any subpopulation instead of the cell line. The invention can also be used to screen for compounds of incomplete main cells in which less than 100% of the main cells display the correct genes for the screening program. In addition, the present invention can be used to select a compound of cells, for example, at a particular cell-cycle stage, which selects cells only in the replication phase.

The present invention has been described with respect to exemplary embodiments. Since some modifications may be made in the casting without departing from the scope of the invention, it is intended that all matter contained in the foregoing description as shown in the accompanying drawings shall be interpreted as illustrative and not limiting.

The following claims are to be understood as including all expressions of the scope of the invention which may be referred to as belonging there between as a matter of all the general and specific features and language of the invention described herein.

Claims (23)

Substrate, A cell sorting system formed on the substrate for sorting the cells according to predetermined characteristics, A screening system formed on the substrate for screening cells, The screening system includes a mixing region for mixing cells of a predetermined feature with a test compound, and a detection region for detecting the influence of the test compound on cells of the predetermined feature. The microfluidic system of claim 1, further comprising a connecting channel between the cell selection system and the screening system for transferring selected cells from the cell selection system to the screening system. The method of claim 1, wherein the cell selection system, A first duct for conveying the flow of suspended cells in the carrier fluid, said first duct having an inlet, a first outlet and a second outlet, A sensor for detecting one of the particle size and velocity and a predetermined characteristic of the particle, A side channel in communication with the first duct, A sealing chamber in which the carrier fluid forms a meniscus in the side channel and is separated from the carrier fluid and disposed adjacent to the side channel; When the sensor senses certain features, it includes an actuator that changes the pressure inside the sealing chamber to deflect the meniscus, A microfluidic system that causes cells of the meniscus not having certain features to flow into the first outlet, and cells of certain features to flow into the second outlet. The microfluidic system of claim 3, wherein the cell sorting system further comprises a buffer for absorbing pressure variations in the first duct. 4. The microfluidic system of claim 3, wherein the actuator comprises a source of compressed gas. 4. The microfluidic system of claim 3, wherein the sealing chamber comprises a movable wall. 6. The microfluidic system of claim 5, wherein the actuator comprises a displacement actuator that moves the movable wall of the sealing chamber to change the pressure in the sealing chamber. 8. The microfluidic system of claim 7, wherein the actuator comprises one of an electromagnetic actuator and a piezoelectric element. A system for screening test compounds, A first duct having an inlet, a first outlet and a second outlet for conveying the sample, a sensor for sensing a predetermined feature of the target particle, a side channel in communication with the first duct, and a carrier fluid in the first duct A sealing chamber that forms a meniscus in the side channel to be separated from the carrier fluid and disposed adjacent to the side channel, and an actuator that deflects the meniscus to change the pressure in the sealing chamber when the sensor senses a predetermined feature. Deflection of the meniscus causes particles that do not have a predetermined feature to flow into the first outlet, target particles of the desired feature into the second outlet, and particle separation to separate the target particles from the sample containing the target particles. System, A mixing and incubation zone in communication with a second outlet containing the target particles and the test compound, for mixing the target particles and the test compound, A screening system comprising a detection region for detecting the effect of a test compound on a target particle. A method for testing the effect of a compound on a target cell, Selecting a set of cells according to a predetermined feature, Transferring cells of a predetermined feature through the connecting channel to the mixing region; Introducing the compound into the mixing zone. Is a method of screening major cells, A selection step of selecting a set of cells to separate subpopulations from the set, Adding the compound to a subpopulation of cells, And analyzing the subpopulations of the cells to determine the effect of the compound on the subpopulations of the cells. Is a method of analyzing the side effects of compounds, Providing a set of primary cells, Adding the compound to the main cell set, Measuring the primary cell to determine activity by the compound, Separating the active primary cell from the active primary cell. The method of claim 10, wherein the predetermined feature is the presence of a predetermined marker. The method of claim 13, wherein the predetermined marker is an antibody, enzyme, substrate, cofactor, fluorescent dye, or reporter gene. A method of screening the activity of a compound to modulate the activity of a cell, wherein the cell is in contact with the test compound and the cells responding to the test compound are monitored using the microfluidic system of claim 1. A method of separating a subpopulation of cells from a cell population using a microfluidic system, Identifying from the population cells having a given phenotype; Separating said cell from cells that do not have a predetermined phenotype. The method of claim 16, wherein the cell population is culture of isolated primary cells. The method of claim 16, wherein the cell population is cell culture. Is a method of separating subpopulations of cells used for cell transplantation, Identifying cells having a given phenotype, Separating the cells using the microfluidic device, Separating the subpopulations of cells used for transplantation. To isolate a subpopulation of genetically altered cells, Identifying a subpopulation of cells in the cell population according to a given phenotype; Separating the cells using the microfluidic device, Isolating a subpopulation of genetically altered cells. The method of claim 20, wherein the genetically altered isolated cells are transplanted into the patient. Is a method of separating subpopulations of cells, Identifying a subpopulation of cells that exhibit cell cycle stage specific markers; Separating the cells using the microfluidic device, Separating the subpopulations of cells at the same stage of the cell cycle. A method of testing the activity of a compound that modulates enzyme activity in vivo, Administering to the cell population a compound that becomes fluorescent when actuated by a cell enzyme, Measuring cell fluorescence to determine the activity of a compound that modulates enzyme activity.