Abstract:
A device for promoting sedimentation within microfluidic channels which uses gravity to separate particles from fluid. Particles such as blood cells or beads are separated from a carrier fluid using gravity combined with various devices such as membranes and sonic energy in different embodiments.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims benefit from U.S. Provisional Patent Application Serial No. 60/281,114, filed Apr. 3, 2001, which application is incorporated herein by reference. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    This invention relates generally to microfluidic devices for performing analytic testing, and, in particular, to devices for rapidly increasing sedimentation within microfluidic channels.  
           [0004]    2. Description of the Related Art  
           [0005]    Microfluidic devices have recently become popular for performing analytic testing. Using tools developed by the semiconductor industry to miniaturize electronics, it has become possible to fabricate intricate fluid systems which can be inexpensively means produced. Systems have been developed to perform a variety of analytical techniques for the acquisition of information for the medical field.  
           [0006]    Microfluidic devices may be constructed in a multi-layer laminated structure where each layer has channels and structures fabricated from a laminate material to form microscale voids or channels where fluids flow. A microscale channel is generally defined as a fluid passage which has at least one internal cross-sectional dimension that is less than 500 μm and typically between about 0.1 μm and about 500 μm. The control and pumping of fluids through these channels is affected by either external pressurized fluid forced into the laminate, or by structures located within the laminate.  
           [0007]    U.S. Pat. No. 5,716,852 teaches a method for analyzing the presence and concentration of small particles in a flow cell using diffusion principles. This patent, the disclosure of which is incorporated herein by reference, discloses a channel cell system for detecting the presence of analyte particles in a sample stream using a laminar flow channel having at least two inlet means which provide an indicator stream and a sample stream, where the laminar flow channel has a depth sufficiently small to allow laminar flow of the streams and length sufficient to allow diffusion of particles of the analyte into the indicator stream to form a detection area, and having an outlet out of the channel to form a single mixed stream. This device, which is known at a T-Sensor, may contain an external detecting means for detecting changes in the indicator stream. This detecting means may be provided by any means known in the art, including optical means such as optical spectroscopy, or absorption spectroscopy of fluorescence.  
           [0008]    U.S. Pat. No. 5,932,100, which patent is also incorporated herein by reference, teaches another method for analyzing particles within microfluidic channels using diffusion principles. A mixture of particles suspended in a sample stream enters an extraction channel from one upper arm of a structure, which comprises microchannels in the shape of an “H”. An extraction stream (a dilution stream) enters from the lower arm on the same side of the extraction channel and due to the size of the microfluidic extraction channel, the flow is laminar and the streams do not mix. The sample stream exits as a by-product stream at the upper arm at the end of the extraction channel, while the extraction stream exits as a product stream at the lower arm. While the streams are in parallel laminar flow is in the extraction channel, particles having a greater diffusion coefficient (smaller particles such as albumin, sugars, and small ions) have time to diffuse into the extraction stream, while the larger particles (blood cells) remain in the sample stream. Particles in the exiting extraction stream (now called the product stream) may be analyzed without interference from the larger particles. This microfluidic structure, commonly known as an “H-Filter,” can be used for extracting desired particles from a sample stream containing those particles.  
           [0009]    It is often desirable to remove particles from a liquid for analysis purposes. One method of performing this procedure is to use a centrifuge. Centrifugation is a process by which particles in suspension in a fluid are separated by spinning the fluid, usually in a test tube, such that centrifugal force throws the particles to the periphery of the rotated vessel. Sedimentation is also an important method to separate particles by density. In many cases, the difference in the rate of sedimentation of particles to be separated is very small, as is the rate of separation itself. Frequently, small particles such as blood cells sediment at a rate of only a few micrometers per second. This problem is usually solved by increasing the apparent gravitational force that drives the sedimentation by using a centrifuge.  
           [0010]    In microfluidic structures, sedimentation structures can be used to achieve sedimentation without the use of centrifuges. The rate of sedimentation of a few micrometers/second provides a sufficient speed in channels that have dimensions in the order of hundreds of micrometers. For example, blood cells will settle in a channel of 100 micrometer depth in about 100 seconds at standard gravity.  
         SUMMARY OF THE INVENTION  
         [0011]    It is therefore an object of the present invention to provide a device which will allow sedimentation in microfluidic channels.  
           [0012]    It is a further object of the present invention to provide a device using microfluidic channels to separate blood cells from plasma.  
           [0013]    It is still a further object of the present invention to provide a microfluidic sedimentation device which is simple and easy to use.  
           [0014]    These and other objects of the present invention will be more readily apparent in the description and drawings which follow. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0015]    [0015]FIG. 1 is a side view of a sedimentation device according to the present invention;  
         [0016]    [0016]FIG. 2 is a side view of an alternative embodiment of the device of FIG. 1;  
         [0017]    [0017]FIG. 3 is another embodiment of a sedimentation device according to the present invention;  
         [0018]    [0018]FIG. 4 is a side view of a microfluidic channel having a second channel passing beneath for use in the present invention;  
         [0019]    [0019]FIG. 5 is a bottom view of the device of FIG. 4;  
         [0020]    [0020]FIG. 6 is another embodiment for carrying out the present invention;  
         [0021]    FIGS.  7 A-I show several different embodiments of a device for capturing beads for use in the present invention; and  
         [0022]    [0022]FIG. 8 is a plan view of an analysis card according to the principles of the present invention. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0023]    [0023]FIG. 1 shows a microfluidic device used for promoting sedimentation. Referring now to FIG. 1, a microfluidic channel  10  is filled with whole blood. An audio speaker  12  is positioned below channel  10 . Speaker  10  is then activated, subjecting channel  10  to sonic energy, vibrating blood cells  14  within the whole blood sample. This vibration is sufficient to exceed the minimum shear stress in the fluid surrounding cells  14 , allowing motion of cells  14  in response to gravity. After sufficient time for sedimentation, a pusher fluid is used to flush the plasma from above and around settled cells  14  by passing it through channel  10  in the direction of arrows A. This technique is insensitive to channel geometry except for a requirement that the height of channel  10  be small enough that sedimentation occurs rapidly. Although this device is shown as a sedimentation device for blood cells, it could also be used to isolate beads within a channel to be used for analysis purposes.  
         [0024]    An alternative structure for the device of FIG. 1 is shown in FIG. 2. In this embodiment, channel  10  is saturated at an angle above the horizontal plane. Whole blood is loaded into channel  10  and speaker  12  activated to subject the sample to sonic energy. Blood cells  14  settle along the bottom of channel  10 , and as channel  10  is angled, cells  14  tend to move along the bottom surface of channel  10  in the direction of arrows B. As a pusher fluid is injected into channel  10 , plasma from the blood sample travels in the direction of arrows C, which is in the opposite direction of the movement of cells  14 . The speed of sedimentation can be varied by varying the angle of inclination of channel  10 .  
         [0025]    Another embodiment which can be used for promoting sedimentation is shown in FIG. 3. A sample fluid containing particles  28  which are denser than the sample fluid is inputted into a microfluidic channel  30 . Channel  30  contains a recessed well section  32  on the bottom surface of channel  30 . As particles  28  flow along within channel  30 , they drop down into section  32  of channel  30 , as they are denser than the fluid. As a result, a particle-free sample passes out of channel  30  as shown at arrow D.  
         [0026]    Another embodiment of the principles of this invention is shown in FIGS. 4 and 5. Referring now to FIG. 4, a main microfluidic channel  40  is shown having a circuitous or S-shaped channel  42  coupled to the bottom surface and is open to channel  40  in periodic locations along channel  40  as can be clearly seen in FIG. 5. In addition, a filter or membrane  44  is situated on the bottom surface of channel  40 . A diluted fluid containing particles  46  flows into channel  40  at  48 . Particles  46  tend to move slightly away from the walls of channel  40  to avoid the shear gradient that is present in that area. Membrane  44 , which is fluid permeable, excludes particles  46  from entering into channel  42 ; however, when channel  42  is held at a lower absolute pressure than main channel  40 , a small portion of the fluid will flow through membrane  44  into channel  42  at each intersection. This clear fluid flowing within channel  42  may be collected at the end of channel  42 , while the particle  46  suspension within channel  40  becomes more concentrated as it moves through main channel  40 . This structure may be used for the extraction of undiluted plasma from whole blood.  
         [0027]    Another structure which may be used to separate plasma from whole blood is shown in FIG. 6. Referring now to FIG. 6, a microfluidic channel  60  is shown. The inner walls  62  of channel  60  contain a chemical that initiates aggregation of blood cells into dense formations called rouleaux. A sample of whole blood flows into channel  60  at  64 , and blood cells  66  react with the chemical on walls  62  and begin to aggregate. After a sufficient amount of time has passed, a pusher fluid enters channel  60  at  64 , and flows through aggregated cells  66  to flush the plasma from between the rouleaux and out of channel  60  at  68 .  
         [0028]    FIGS.  7 A-I represent different embodiments in which beads may be trapped within a microfluidic channel to assist in analyzing a particular fluid. Referring now to FIG. 7A, there is shown a microfluidic channel  80  through which a plurality of beads  82  are transmitted. Beads  82  are preferably functionalized with antibodies such that the beads will fluoresce upon contact with a specific substance. A membrane or filter  84  is located within channel  80  such that beads will not pass through channel  80 , but a fluid can flow across beads  82  for analysis purposes and flow out through opening  84 . Other means for capturing beads  82  are also shown in the figures; channel  80  may have a narrow section  90  which will restrict passage of beads  82  (FIG. 7B); beads  82  may be denser that the fluid flowing in channel such that they will settle on the bottom surface  92  of channel  80  due to gravity (FIG. 7C); beads  80  may have magnetic properties such that their travel within channel  80  is stopped using a magnet  94  located outside channel  80  (FIG. 7D); channel  80  may have an inlet  96  in which beads  82  are inserted into a wide section  98  of channel  80  whereas beads  82  cannot pass into channel  80  from section  98  (FIG. 7E); beads  82  may be less dense than the fluid flowing in channel  80  such that they would settle into a section  100  on the upper surface of channel  80  and remain in section  100  (FIG. 7F); channel  80  may have a section  102  which is above the level of channel  80  wherein beads  82  which are less dense than the fluid in channel  80  such that they will be trapped in section  102  (FIG. 7G); channel  80  may have a recessed section  104  wherein beads  82  which are more dense than the fluid will settle in section  104  (FIG. 7H); and channel  80  may have a downwardly depending section  106  such that beads  82  which are more dense than the fluid remain in section  106  (FIG. 7I). In all of these embodiments, beads  82  will react of a specific substance within the fluid such that they will fluoresce to indicate a particular concentration of that substance.  
         [0029]    [0029]FIG. 8 shows a laminate analysis card  120  which also embodies the principles of the present invention. Card  120  has a first input  122  into which a solution of beads that are functionalized with antibodies is injected, a second input  124  into which a sample such as whole blood is injected, and a third input  126  into which a wash solution is injected. Input  122  is coupled through a channel  128  to a junction  130 , input  124  is coupled to junction  130  through a channel  132 , and input  126  is coupled to junction  130  through a channel  134 .  
         [0030]    Junction  130  is connected to a channel  140  having a series of recessed well-like structures  141  similar to well  32  shown in FIG. 3. The output of channel  140  is coupled to a reservoir  142  through a channel  144 .  
         [0031]    The operation of analysis card  120  is as follows: a bead solution is injected into input  122 , a whole blood sample into inlet  124 , and a wash solution into inlet  126 . Bead solution is first pumped into channel  140  through a valve  150 , and the beads in the solution settle into well structures  141 . Then the blood sample is pumped into channel  140  through a valve  152 , where the blood analytes interact with the antibodies on the beads in wells  141 . Finally, the wash solution is pumped through a valve  154  through channel  140  to wash the blood away. The beads in wells  141  will change color or fluoresce to indicate the presence or concentration of the desired substance in the blood.  
         [0032]    While the present invention has been shown and described in terms of a preferred embodiment thereof, it will be understood that this invention is not limited to this particular embodiment and that changes and medications may be made without departing from the true spirit and scope of the invention as defined in the appended claims.