Patent Application
20080311005
This application claims priority to Korean Patent Application No. 10-2007-0058572, filed on Jun. 14, 2007, and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which in its entirety are herein incorporated by reference.
1. Field of the Invention
The present invention relates to an apparatus for focusing a particle in a sheath flow and a method of manufacturing the same.
2. Description of the Related Art
Sheath flow is a particular type of laminar flow in which one layer of fluid, or a particle, is surrounded by another layer of fluid on more than one side. The process of confining a particle stream in a fluid is referred to as a ‘sheath flow’ configuration. For example, in sheath flow, a sheath fluid may envelop and pinch a sample fluid containing a number of particles. The flow of the sheath fluid containing particles suspended therein may be narrowed almost to the outer diameter of particles in the center of the sheath fluid. The resulting sheath flow flows in a laminar state within an orifice or channel so that the particles are lined and accurately pass through the orifice or channel in a single file row.
Sheath flow is used in many applications where it is preferable to protect particles or fluids by a layer of sheath fluid, for example, in applications where it is necessary to protect particles from air. For example, in particle sorting systems, flow cytometers and other systems for analyzing a sample, particles to be sorted or analyzed are usually supplied to a measurement position in a central fluid current, which is surrounded by a particle free liquid sheath.
Sheath flow is useful because particles can be positioned with respect to sensors or other components and particles in the center fluid, which is surrounded by the sheath fluid, can be prevented from touching the sides of the flow channel, thereby preventing clogging of the channel. Sheath flow allows for faster flow velocities and higher throughput of sample material. Faster flow velocity is possible without shredding cells in the center fluid because the sheath fluid protects the cells from shear forces at the walls of the flow channel.
Conventional devices that have been employed to implement sheath flow have relatively complex designs and are relatively difficult to fabricate. For example, U.S. Pat. No. 6,506,609 discloses a method of focusing and detecting particles flowing in a first microchannel, the method comprising: flowing the particles in a sample fluid in the first microchannel; focusing the particles in the first microchannel by introducing a fluid into the first microchannel from only one of one or more second microchannels such that the particles are directed towards a first side of at least opposing first and second sides of the first microchannel; directing an interrogating light beam into the focused sample fluid at a location on the first side of the first microchannel; and detecting the particles in the focused sample fluid using the interrogating light beam. In this case, since a two-dimensional channel structure is used, the manufacturing process is less complex. However, when a sample fluid is viewed from a section of the channel, its shape is long in the up and down directions. Therefore, it is difficult to uniformly control the flow velocity of particles (refer to
In addition, U.S. Pat. No. 6,830,729 discloses a sheath flow assembly including: a sample channel; a first sheath fluid channel positioned on either side of and converging with the sample channel; and upper and lower sheath fluid chambers positioned above and below and converging with the sample channel.
However, disadvantages still exist in that it remains difficult to manufacture and use a sheath flow device. In addition, focusing particles in a sample and detecting the focused particles remain difficult.
The present invention provides an apparatus and a method of manufacturing the apparatus for focusing a particle in a sheath flow, wherein the apparatus can be easily manufactured, has excellent focusing efficiency, and optionally easily detects the focused particle.
In particular, the present invention provides an apparatus for focusing a particle in a sheath flow, the apparatus comprising: a sheath fluid inlet for injecting a sheath fluid; a first flow channel for conveying a fluid, wherein the first flow channel extends from the sheath fluid inlet to the fluid outlet and is formed such that a channel extending from the sheath fluid inlet is divided into two subchannels that extend further and are then merged into one channel; and a second flow channel that extends from the sample fluid inlet to the sample fluid outlet, wherein the second flow channel is in fluid communication with the first flow channel at the merged channel region through the sample fluid outlet so as to introduce a particle in a sample into a sheath fluid conveyed through the first flow channel. The first flow channel may comprises a tapering region whose cross-sectional area decreases in a direction along which fluid flows therethrough at the merged channel region of first flow channel. The tapering region facilitates focusing of the sheath fluid around a particle. The apparatus of the present invention focuses the sheath fluid around a particle away from a side wall of surface of the first flow channel at a focusing region which is a merged channel region of the first flow channel extending downstream of the sample fluid outlet.
The apparatus for focusing a particle in a sheath flow, according to the present invention, may include at least two unit structures, each of the unit structures including the first flow channel, the second flow channel and the fluid outlet in separate, and the sheath fluid in common. That is, the apparatus for focusing a particle in a sheath flow may include one sheath fluid inlet and a plurality of unit structures each including the first flow channel, the second flow channel and the fluid outlet.
The apparatus for focusing a particle in a sheath flow may be manufactured by adhering an upper substrate, on which the sheath fluid inlet, the first flow channel, the second flow channel and the fluid outlet are partially or totally formed, to a lower substrate, which may be a flat substrate or a substrate of the corresponding portion of the sheath fluid inlet, the first flow channel, the second flow channel and/or the fluid outlet which are partially formed on the upper substrate.
In the upper substrate, a channel structure, an inlet or outlet of the apparatus for focusing a particle in a sheath flow may be inwardly engraved by injection molding, stamping, microfabrication, machining or sterolithography, but the present invention is not limited thereto. The upper substrate may be formed of a material selected from the group consisting of polydimethylsiloxane (“PDMS”), polycarbonate, polyethylene, polypropylene, polyacrylate, polystyrene and polytetrafluoroethylene (“PTFE”). In addition, the lower substrate may be made of a light transmissive material, for example, glass, cover glass or polydimethylsiloxane (“PDMS”).
The first flow channel in the apparatus for focusing a particle in a sheath flow, according to the present invention, may further comprise an observation region extending from the end of the tapering region and having a constant cross-sectional area. A light detector may be disposed at an outer side of the first flow channel at an observation region, preferably on the lower substrate. The lower substrate may be a light transmissive and planar substrate, and thus the light detector can be disposed close to the sample and the sheath fluid which focuses the sample in the observation region of the first flow channel. Examples of the light detector may include an objective lens, a photodetector, a light source, and the like.
In the apparatus for focusing a particle in a sheath flow, according to the present invention, the first flow channel may be disposed such that a first direction of a sheath fluid flow from the sheath fluid inlet to a merging point of the two subchannels is different from the a second direction of sheath fluid flow from the merging point to the fluid outlet. The first direction may differ from the second direction from 160° to 200°, from 170° to 190°, from 175° to 185°, substantially 180°, or 180°. Therefore, a merged channel region of the first flow channel consisting of a region from a merging point of the two subchannels to the fluid outlet of the first flow channel is preferably formed within an area between the two subchannels of the first flow channel from a diverging point to a merging point. In an example embodiment of the present apparatus, the first flow channel extends from a sheath fluid inlet and diverges into two subchannels at a diverging point, and then the two subchannels extend in parallel with each other and merge into one channel at a merging point. The merged channel extends from the merging point to the fluid outlet in a different direction from a direction of the first flow channel from the sheath fluid inlet to the merging point. As used herein, the term “direction” refers to a net direction of a sheath fluid flow which flows in the first flow channel from the start point to the end point of the sheath fluid flow. The direction of the first flow channel from the sheath fluid inlet to the merging point may differ from that of the first flow channel from the merging point to the fluid outlet from 160° to 200°, from 170° to 190°, from 175° to 185°, substantially 180°, or 80°. Thus, the apparatus according to exemplary embodiments of the present invention can be positioned within a narrow area so that a plurality of apparatuses according to exemplary embodiments of the present invention can be disposed in a multiplex structure, such as in an array.
The cross-sectional area of the first flow channel or the two subchannels may be substantially constant throughout the length of the channel or may vary to facilitate focusing of the sample within the sheath fluid. It is preferable that the first flow channel has a tapering region in a direction along which fluid flows therethrough at a merged channel region, so that the cross-sectional area of the merged channel decreases in a direction along which fluid flows therethrough.
The sample outlet may or may not be formed at the center of the merged channel region. Preferably, the sample outlet may be formed at a position equidistant from each wall surface of the merged channel region in order to inject a sample fluid into the center core of a sheath fluid injected from the two subchannels to the merged channel region.
The apparatus of the present invention may or may not focus a sheath fluid around a particle at the center of a focusing region of the first flow channel. Preferably the apparatus focuses a sheath fluid around a particle on a portion equally distant from each wall surface of the first flow channel, that is, the center core of the first flow channel.
In addition, the sample fluid inlet may be connected to a sample reservoir through a pump. Examples of the pump may include a syringe pump, a high-performance liquid chromatography (HPLC) pump, a diaphragm pump, a peristaltic pump, an electric power pump, and the like, but are not limited thereto. In addition, the sample fluid can be introduced through a pump, and also introduced by applying a positive pressure to the sample fluid inlet or applying a negative pressure to a fluid outlet.
The present invention also provides a method of manufacturing the apparatus for focusing a particle in a sheath flow, the method including: inwardly engraving a surface of an upper substrate to form a sheath fluid inlet, and a first flow channel extending from the sheath fluid inlet to the fluid outlet, wherein the first flow channel is formed such that a channel extending from the sheath fluid inlet is divided into two subchannels that extend further and are then merged into one channel; adhering a light transmissive lower substrate on the engraved surface of the upper substrate to completely form the sheath fluid inlet, the first flow channel and the fluid outlet; and forming a second flow channel at the merged region of the first flow channel, wherein the second flow channel extends from a sample fluid inlet to a sample fluid outlet and is in fluid communication with the first flow channel at the merged channel region through the sample fluid outlet.
The engraving may be performed by injection molding, stamping, microfabrication, machining, or sterolithography, but is not limited thereto. The upper substrate may be formed of a material selected from the group consisting of polydimethylsiloxane (“PDMS”), polycarbonate, polyethylene, polypropylene, polyacrylate, polystyrene and polytetrafluoroethylene (“PTFE”).
The lower substrate may be a light transmissive substrate, for example, glass, cover glass, or a polydimethylsiloxane (“PDMS”) substrate.
In the engraving of the present method, the first flow channel may comprise a tapering region at the merged channel region whose cross-sectional area decreases in a direction along which fluid flows therethrough. The first flow channel may further comprise an observation region extending downstream from the end of the tapering region and having a constant cross-sectional area.
In the engraving of the present method, the first flow channel may be disposed such that a first direction of a sheath fluid flow from the sheath fluid inlet to a merging point of the two subchannels is different from the a second direction of sheath fluid flow from the merging point to the fluid outlet. The first direction may differ from the second direction from 160° to 200°, from 170° to 190°, from 175° to 185°, substantially 180°, or 180°. Therefore, a merged channel region of the first flow channel consisting of a region from a merging point of the two subchannels to the fluid outlet of the first flow channel is preferably formed within an area between the two subchannels of the first flow channel from a diverging region to a merging region.
In the adhering of the present method, the engraved upper substrate and the lower substrate may be attached by using an adhesive material known in the art, or the two substrates may be attached to each other via the adhesive properties of the substrates. For example, the polydimethylsiloxane (“PDMS”) has an adhesive property and can be attached to a substrate such as glass or cover glass by compressing the substrates against each other. As a result, an adhered structure comprising the first flow channel is formed.
In the forming of the present method, a second flow channel is formed at the merged region of the first flow channel, wherein the second flow channel extends from a sample fluid inlet to a sample fluid outlet and is in fluid communication with the first flow channel at the merged channel region through the sample fluid outlet. The second flow channel may be easily formed at the merged region of the first flow channel by simply making a hole and inserting a tube into the merged channel region of the first flow channel.
The above and other aspects, features, and advantages of the present invention will become more apparent by describing in further detail exemplary embodiments thereof with reference to the attached drawings in which:
The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown.
It will be understood that when an element is referred to as being “on” another element, it can be directly on the other element or intervening elements may be present therebetween. In contrast, when an element is referred to as being “disposed on” or “formed on” another element, the elements are understood to be in at least partial contact with each other, unless otherwise specified.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. The use of the terms “first”, “second”, and the like do not imply any particular order but are included to identify individual elements. It will be further understood that the terms “comprises” and/or “comprising”, or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. In the drawings, like reference numerals in the drawings denote like elements.
The term “microfluidic device” as used herein refers to a system or an apparatus for handling, processing, ejecting and/or analyzing a fluid sample, the device including at least one channel of microscale dimensions.
The terms “channel” and “flow channel” as used herein refer to a pathway formed in or through a medium that allows the movement of fluids, such as liquids and gases. A “microchannel” refers to a channel in the microfluidic device, the microchannel preferably having cross-sectional dimensions in the range of about 1.0-500 μm, more preferably of about 25-250 μm, and most preferably of about 50-150 μm. Those of ordinary skill in the art will be able to determine the appropriate volume and length of the flow channel. The flow channel may have various configurations, such as a linear or non-linear configuration and a U-shaped configuration.
Referring to
Still referring to
Referring to
Particles contained in a sample fluid used in the present invention may be a virus, bacteria, cells, microbeads, nanorods, or single fluorescent molecules, for example, but are not limited thereto.
In
The apparatus of the present invention has a simple structure, and is thus inexpensive. A light detector is disposed close to a sample fluid so that particles in a sample can be detected with high detection intensity. In addition, a plurality of the apparatuses can be positioned within a small area, and the formation of multiplex arrays including the apparatus of the present invention is possible. In addition, since the apparatus of the present invention includes a single sheath fluid inlet, it is easy to control the fluid flow.
According to the exemplary method of the present invention described above, the apparatus according to the present invention can be efficiently manufactured.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2007-0058572 | Jun 2007 | KR | national |
