Separation of particles based on size is one of the essential components in biochemical analysis, environmental assays, and industrial and biomedical applications. Filtration is one of the most frequently used techniques to separate particles. A mechanical filter can be used to remove, filter, or collect particles. This filtering and collection of particles can be used for sampling of particles, chemical detection, and/or biological cell analysis.
Existing filtration methods are performed in a batch or a continuous manner. However, when the particle size is much smaller or when the difference in particle size is smaller, separation becomes difficult. Pore clogging or membrane fouling may be an issue.
Separation of specific cells from a mixed cell population is important in medicine for biological and immunological measurements, and for use in cell therapy (e.g. transfusion medicine). For example, in the medical field, it is often necessary to filter blood. Human blood cell separation is the first challenging step towards total blood count and the subsequent disease diagnosis, prognosis and management. Normal erythrocytes vary in dimension from 5 μm to 8 μm. Leukocytes have an average diameter of between 7 μm to 20 μm.
Several techniques are available for separation of blood elements. Most current approaches involve centrifugation (e.g. distinguishing the cells based on density) or surface characteristics. Such procedures are typically not able to separate all of the white blood cells from the platelets and the forces involved in separation of the cells can damage the final product. Cell labeling-based separation techniques are expensive, inconvenient and in most cases, labeled cells cannot be infused in patients and the harsh washing conditions necessary to remove the label can damage the cells. Passive matrix-based separation techniques are not sufficiently selective or adaptive for separation of specific cell types. Similarly, column chromatography and magnetic bead adsorption techniques cannot separate cell subtypes quickly and cheaply.
Therefore, there is a need to develop devices and methods for continuous separation of particles of different sizes, in particular, the separation of various cells and particles that exist in blood. The present invention satisfies these and other needs.
The present invention provides a streamline-based microfluidic device and a method of using the device for continuous separation of particles and cells.
According to the present invention, a microfluidic device is provided, which includes components linked in fluid communication. The components include one or more sample inlet ports, one or more microchannels, and one or more outlet ports. The device is capable of sorting particles (such as cells) according to their characteristics, such as particle size and shape. The various components are compatible with various microscale systems. Moreover, the design is modular, which permits the addition of other elements (e.g. detectors, cell collection chambers, and the like.)
According to one aspect of the present invention, a microfluidic device for streamline separation of particles is provided. The device includes one or more inlets, one or more outlets, a main microchannel and a plurality of side microchannels. The microchannels are disposed on a substrate. The main channel and the side channels are in fluid communication. The main channel contains a plurality of geometric stagnation points, one or more inlets and one or more outlets. In one embodiment, each stagnation point has a predetermined geometric design, for example, each of the stagnation points has a predetermined distance from the edge of each of the side microchannels. In another embodiment, one or more of the side microchannels are substantially perpendicular to the main microchannel.
According to another aspect of the present invention, a method for streamline separation of particles using the microfluidic device of the present invention is provided. The method includes administering a fluid containing a plurality of particles through the main microchannel, optionally applying a positive or a negative pressure to the main microchannel to separate each particle, and collecting the plurality of particles from each of the side microchannels. In one embodiment, the particles to be separated include red blood cells and white blood cells.
Reference to the remaining portions of the specification, including the drawings and claims, will realize other features and advantages of the present invention. Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with reference to the accompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements.
The present invention provides a novel streamline-based microfluidic device and a method of using such a device for continuous separation of particles of various sizes. The device is especially useful for the separation of erythrocytes from leukocytes in the blood. In certain instances, the present invention provides a system, which does not require moving parts, and thus it is desirable for integration with other kinds of micro unit operation for other treatment, analysis and utilization. The present invention demonstrates a high separation efficiency, an ease of operation, capability of simultaneous particle concentration and size dependent separation and elimination of channel clogging. Advantageously, the microfluidic device can have a tailored design with a tunable capability to allow easy and accurate manipulation of particles of various sizes found in biological, ecological and industrial environments, especially in the separation of particles and cells in the blood to obtain accurate blood counts for diagnosis and treatment.
As used herein, the term “microchannel” or “channel” refers to a micrometer dimension pathway through a medium that allows for movement of liquids and gases. Channels can connect with other components in fluid communication.
As used herein, the term “streamline” refers to the path of a particle that is flowing steadily and without turbulence in a fluid past an object, or the flow of a fluid past an object such that the velocity at any fixed point in the fluid is constant or varies in a regular manner. For example, streamline flow means a flow of a gas or liquid in which the velocity at any point is relatively steady.
As used herein, the term “stagnation point” refers to a point in a flow where the velocity is zero, where any streamline touches a solid surface at an angle.
As used herein, the term “edge distance of side channel” refers to the height between the downstream edge and the upstream edge of each of the side microchannels.
As used herein, the term “particle(s)” refers to particles found, for example, in vitro or in vivo including, but not limiting to, aersols, cells, bacteria, microorganisms, fiberins and particulates.
As used herein, the term “side channel length” or “side microchannel length” refers to the length of the side microchannels as shown in
Simulation results have shown that that the minimal separation lane width D is a function of both the side channel edge distance Δλ and the side microchannel length L (
According to one aspect of the present invention, the separation lane width D can be controlled by the local geometry of the separation region and the flow resistance of the side microchannels. Table 1 shows the results of simulation software (FEMLAB) using the Navier Stokes equation.
In certain aspect, the present invention provides a device with tailored designs, where the separation lane width and selection of side microchannels can be precisely controlled through the geometric design of the stagnation points and the variation of the side channel lengths. The device with such designs enables accurate and efficient separation of particles having various dimensions.
Many materials can be used as substrates for the construction of the microfluidic device. The materials include, but are not limited to, polysiloxane, paraylene, glass, silicon, polyacrylate, polyethylene, polypropylene, polystyrene, polycarbonate and the like. Preferably, polydimethysiloxane (PDMS) is used as a substrate for the fabrication of the device. PDMS is a preferred substrate material because of its optical transparency, ease of molding, elastomer character, controlled surface chemistry of oxidized PDMS using conventional siloxane chemistry; and compatibility with cell culture (e.g. non-toxic and gas permeable). Soft lithographic rapid prototyping can be employed to fabricate the desired microfluidic microchannel systems. Soft lithography is an alternative to silicon-based micromachining that uses replica molding of nontraditional elastomeric materials to fabricate microfluidic channels. The softness of the materials used allows the device areas to be reduced by more than two orders of magnitude compared with silicon-based device. The devices can be fabricated with deep reactive ion etching (DRIE) of silicon molds. An example of DRIE of single crystal silicon has been demonstrated by the BOSCH process, See, Ayon, A. A., and et al “Characterization of a time multiplexed inductively coupled plasma etcher,” J. Electrochem. Soc., 1999, 146, 339-349 incorporated herein by reference.
In other embodiments, the present invention contemplates fabricating devices using glass or silicon substrates. Silicon has well-known fabrication characteristics and associated photographic reproduction techniques. The principal modern method for fabricating semiconductor integrated circuits is the so-called planar process. The planar process relies on the unique characteristics of silicon and comprises a sequence of manufacturing steps involving deposition, oxidation, photolithography, diffusion and/or ion implantation, and metallization, to fabricate a “layered” integrated circuit device in a silicon substrate, see, e.g., U.S. Pat. No. 5,091,328, hereby incorporated by reference.
A skilled artisan will appreciate that the present invention is not limited to the arrangements of the microchannels shown in
The main microchannel and the side microchannels can have a variety of shapes. The cross-section geometries of the microchannels include, but are not limited to, a circle, an oval, a symmetric polygon and an unsymmetric polygon. The shapes and the sizes of the cross-section can vary along the microchannels. In one embodiment, the number of sides of the polygonal cross-section can vary from 3 to about 29. One example is a four-sided polygon such as a square or rectangle. Each of the side microchannels can have the same or different dimensions. Depending on the types, numbers and density of the particles to be separated, a device can have any desirable numbers of side microchannels to meet the operation requirements, for example, from 1 to 10,000; from 1 to 1,000; or from 1 to 100.
The present invention is not limited by precise dimensions of the microchannels employed in the separating devices. Illustrative ranges for microchannels are as follows: the microchannels can be between 0.35 μm and 200 μm in depth (preferably 20 μm) and between 2 μm and 1000 μm in width (preferably between 10 μm and 500 μm). The microchannels can be fabricated into any desirable length and width. The main channel can have a length from about 100 μm to about 500 μm, 200 μm to 700 μm, 500 μm to 1000 μm, 800 um to 1200 μm, 1000 μm to 1500 μm, or 1400 um to 2000 μm, (preferably 1,000 μm). The side microchannels can be evenly or unevenly spaced. In one embodiment, the side microchannels are evenly spaced. The distance between the adjacent side microchannels can be in the range of 1 μm to 5 μm, 2 μm to 6 μm, 5 μm to 10 μm, 8 μm to 12 μm, 10 μm to 15 μm, 15 μm to 20 μm or 18 μm to 25 μm, preferably between about 5 μm and about 25 μm, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μm. In one embodiment, the device has a plurality of evenly spaced microchannels. In another embodiment, the distance between each of the adjacent microchannels is 20 μm. It is specifically contemplated that the present invention can employ both channels of uniform dimensions, and channels of different dimensions. For example, the present invention contemplates channels that are uniform and channels that are non-uniform. With regard to the latter, the beginning of the channel may be wider (e.g. have a greater radius) than the middle or end of the channel. In one embodiment, a “v” design is employed, whereby a channel gradually narrows (e.g. the radius gradually decreases) from the beginning to the end, along the length of the channel. In other aspects, the present invention also contemplates side microchannels wherein the channel gradually widens (e.g. the radius of the channel gradually increases) from the beginning of the channel to the end of the channel.
While
The fluid used for separation can be, for example, a gas or a liquid. Suitable liquids include, but are not limited to, water and organic solvents. Suitable organic solvents include, but are not limited to, polar solvents, such as an alcohol, a ketone, an amide, such as dimethylormamide and dimethylacetamide; dimethysulfoxide, tetrahydrofuran, an ether and a chlorinated hydrocarbon solvent, such as chloroform, dichloromethane, dichloroethane, carbon tetrachloride, tetrachloroethylene and chlorobenzene; less polar solvents, such as an aromatic solvent, for example, benzene, toluene, xylene or an hydrocarbon solvent, for example, C4-C8 alkanes, such as butanes, pentanes, hexanes, heptanes and octanes; and combinations thereof. Suitable gases include, but are not limited to, air, CO, CO2, H2, N2, O2, methane, ethane, propane and a noble gas. The particles to be separated include, but are not limited to, beads, aerosols, cells, bacteria, fibrins and particulates present in a biological environment.
The stagnation points disposed within the main microchannel can be formed by, for example, the downstream edge of each side microchannels. The downstream edge can be either above or below the upstream edge. The stagnation points have a geometric design including a size, a shape and a distance. In one embodiment, the stagnation point is a sharp edge, corner, tip or point with a diameter from about 1 nm to about 1 μm. Alternatively, the stagnation points can be a smooth, or a rough surface and the surface can be either concave or convex. In another embodiment, the stagnation point can be modified with a layer of material other than the substrate material. For example, the stagnation point can be modified by depositing a layer of polymer, glass, ceramic material or metal. Suitable polymers for coating include, but are not limited to, polyester, polyacrylate, polyimide, parylene and polycarbonate. Suitable metals for coating include, but are not limited to, Au, Pt, Ag, Pd, Cu, Ir, Zn, Ni, Fe, Ru, Rh and Si. The distance between the upstream edge and the downstream edge can vary from about 0 μm to about 100 μm, preferably, from 0 μm to about 20 μm. The upstream edge can obtrude or withdraw from the downstream edge. In one embodiment, the upstream edge obtrudes, such as from the downstream edge. Alternatively, the upstream edge withdraws from the downstream edge.
Turning back to
Optionally, pressure can be applied to the fluid or the microchannels. A pressure generating means can include, but is not limited to, a pump such as, a syringe pump, a peristaltic pump, an electrokinetic pump, a bubble pump, air pressure driven pump and a gravity driven pump. In one embodiment, the pressure applied to the fluid is generated by gravity. In another embodiment, the pressure applied to the fluid is generated by an electrokinetic means, for example, electroosmosis means, or a ratchet pump. In yet another embodiment, fluid pressure is generated using pneumatic or magneto hydrodynamic pumps. In still another embodiment, the pressure applied to the fluid is generated by a mechanical device. One example of a mechanical pressure generating device is a screw-type pumping device or a peristaltic pump.
The side microchannels can also be arranged into groups to facilitate the separation and collection. In certain aspects, each group can have, for example, from 2 to 20 side microchannels or more. The side microchannels in each group can have the same or different designs including variation in sizes and/or shapes. In one embodiment, each group contains microchannels of substantially the same dimensions and shapes. For example, each of the side microchannels in the group can be substantially parallel to one another.
The device of the present invention is not limited for the separation of in vitro particles, but can also be used to separate particles in a biological environment, such as cells, fibrins, bacteria, microorganisms, particulates and the like.
The present invention also provides a method for streamline separation of particles, The method utilizes the device of the present invention. In one aspect, the method includes contacting the main microchannel with a fluid containing a plurality of particles, optionally applying a positive or a negative pressure to the fluid or the main microchannel, separating each of the particles and collecting each of the separated particles from the side microchannels. The pressure can be applied either directly to the fluid or through a media. The particles to be separated can be synthetic particles, such as polymer beads or particles present in the biological fluid, such as blood cells, cancer cells, bacteria cells, fibrins, particulates and stem cells.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This application claims priority to U.S. Provisional Patent Application No. 60/673,572, filed Apr. 21, 2005, which is hereby incorporated by reference in its entirety for all purposes.
A portion of the present invention was made under federally sponsored research and development under NASA through the National Space Biomedical Research Institute (NSBRI). The co-operative agreement number is NCC 9-58-317. The Government may have rights in certain aspects of this invention.
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Number | Date | Country | |
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60673572 | Apr 2005 | US |