Abstract:
A device for analyzing sample solutions such as whole blood based on coagulation and agglutination which requires no external power source or moving parts to perform the analysis. Single disposable cartridges for performing blood typing assays can be constructed using this technology.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This patent application takes priority from U.S. Provisional Application Serial No. 60/189,163, filed Mar. 14, 2000, which application is incorporated herein in its entirety by reference.: 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates generally to devices and methods for analyzing samples in microfluidic cartridges, and, in particular, to a device for analyzing sample solutions such as whole blood based on coagulation and agglutination which requires no external power source or moving parts. 
     2. Description of the Related Art 
     Microfluidic devices have recently become popular for performing analytical testing. Using tools developed by the semiconductor industry to miniaturize electronics, it has become possible to fabricate intricate fluid systems which can be inexpensively mass produced. Systems have been developed to perform a variety of analytical techniques for the acquisition of information for the medical field. 
     In microfluidic channels, fluids usually exhibit laminar behavior. U.S. Pat. No. 5,716,852, which patent is herein incorporated by reference in its entirety, is an example of such a device. This patent teaches a microfluidic system for detecting the presence of analyte particles in a sample stream using a laminar flow channel having at least two input channels 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 as a T-Sensor, allows the movement of different fluidic layers next to each other within a channel without mixing other than by diffusion. A sample stream, such as whole blood, and a receptor stream, such as an indicator solution, and a reference stream, which is a known analyte standard, are introduced into a common microfluidic channel within the T-Sensor, and the streams flow next to each other until they exit the channel. Smaller particles, such as ions or small proteins, diffuse rapidly across the fluid boundaries, whereas larger molecules diffuse more slowly. Large particles, such as blood cells, show no significant diffusion within the time the two flow streams are in contact. 
     Two interface zones are formed within the microfluidic channel between the fluid layers. The ratio of a detectable property, such as fluorescence intensity, of the two interface zones is a function of the concentration of the analyte, and is largely free from cross-sensitivities to other sample components and instrument parameters. 
     Usually, microfluidic systems require some type of external fluidic driver to function, such as piezoelectric pumps, micro-syringe pumps, electroosmotic pumps, and the like. In U.S. patent application Ser. No. 09/415,404, which application is assigned to the assignee of the present invention and is hereby incorporated by reference, microfluidic systems are described which are totally driven by inherently available internal forces such as gravity, capillary action, absorption by porous material, chemically induced pressures or vacuums, or by vacuum or pressure generated by simple manual action upon a power source located within the cartridge. Such devices are extremely simple and inexpensive to manufacture and do not require electricity or any other external power source for operation. Such devices can be manufactured entirely out of a simple material such as plastic, using standard processes like injection molding or laminations. In addition, microfluidic devices of this type are very simple to operate. 
     microfluidic devices of this type described can be used to qualitatively or semi-quantitatively determine analyte concentrations, to separate components from particulate-laden samples such as whole blood, or to manufacture small quantities of chemicals. 
     A practical use of these microfluidic devices could be in the determination of several parameters directly in whole blood. A color change in the diffusion zone of a T-Sensor detection channel can provide qualitative information about the presence of the analyte. This method can be made semi-quantitative by providing a comparator color chart with which to compare the color of the diffusion zone, similar to using a paper test strip, but with greate control and reproducibility. 
     It would be desirable, in many situations, to produce a device for analyzing samples in microfluidic channels based on coagulation or agglutination as a function of contact between sample analyte particles and reagent particles. An example of such an assay would be the determination of a person&#39;s blood group by bringing a drop of blood into contact with one or more antisera on a disposable microfluidic cartridge, and visually observing the flow behavior of these two solutions as they flow adjacent to each other, or mixed through sedimentation as they flow with each other through microfluidic channels. If a reaction occurs, the flow will either slow down, stop, or show another observable change that can be attributed to coagulation or agglutination. 
     The accuracy of the device can be enhanced by the addition of a readout system which may consist of an absorbance, fluorescence, chemiluminescence, light scatter, or turbidity detector placed such that the detector can observe an optically observable change caused by the presence or absence of a sample analyte or particle in the detection channel. Alternatively, electrodes can be placed within the device to observe electrochemically observable changes caused by the presence or absence of a sample analyte or particle within the detection channel. 
     SUMMARY OF THE INVENTION 
     Accordingly, it is an object of the present invention to provide a microfluidic device which is capable of performing diagnostic assays without the use of an external power source. 
     It is a further object of the present invention to provide a disposable cartridge for analyzing fluid samples which is inexpensive to produce and simple to operate. 
     It is another object of the present invention to provide a microfluidic analysis cartridge in which a visual analysis can be made of the sample reaction. 
     These and other objects are accomplished in the present invention by a simple cartridge device containing microfluidic channels which perform a variety of analytical techniques based on coagulation or agglutination without the use of external driving forces applied to the cartridge. Single disposable cartridges for performing blood typing assays can be constructed using this technology. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a plan view of a microfluidic cartridge used for performing blood typing according to the present invention; 
     FIG. 2 is a plan view depicting an alternative embodiment of a microfluidic cartridge for performing blood typing according to the present invention; 
     FIG. 3 is a side view of the cartridge of FIG. 2; 
     FIGS. 4A-C show a series of microfluidic cartridges according to FIG. 2 within which a diagnostic test for blood typing has been performed; 
     FIGS. 5A and B are additional views of FIGS. 4C and 4B, respectively, at the conclusion of the diagnostic test; 
     FIG. 6 is a plan view of another alternative embodiment of the microfluidic cartridge of FIG. 2; 
     FIG. 7 is a plan view of another embodiment of the microfluidic cartridge of FIG. 2; and 
     FIG. 8 is a view of a device holding microfluidic cartridges constructed according to the present invention at a constant angle. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The pressure required to drive a blood sample through a microfluidic channel at a specified volume flow rate is determined by the equation: 
     
       
         
           Hc=RQ/ρg 
         
       
     
     where Hc is the head pressure, R is the fluid resistance within the channel, Q is the volume flow rate, ρ is the density of the liquid, and g is the acceleration of gravity. 
     The fluid resistance R can be calculated using the equation: 
     
       
           R= 128μ L/ 4 AF   AR   D   H   
       
     
     where μ is the dynamic viscosity of the fluid, L is the length of the channel, F AR  is the aspect ratio (ratio of length vs. width) of the channel, D H  is the hydraulic diameter of the channel, and A is the cross-sectional flow area of the channel. The characteristic dimension of a cross-sectional flow area A of a channel is the hydraulic diameter D H . For a circular pipe, D H  is the pipe diameter; for a rectangular channel, D H  is four times the area divided by the wetted perimeter, or: 
     
       
           D   H =2/(1 w+ 1/ h ) 
       
     
     where h and w are the channel cross-sectional dimensions. In the present invention, microfluidic channels are fluid passages or chambers which have at least one internal cross-sectional dimension that is less than 500 μm, and typically between about 0.1 μm and 250 μm. 
     The aspect ratio F AR  represents the modification of resistance to flow in the rectangular channel due to the aspect ratio of the cross-sectional flow area. For example, two channels with the same flow area have markedly different resistance to flow if one has a square cross section and the other is very thin but wide. To allow the use of a single formula for resistance, F AR =1 for a circular pipe. A formula for approximating the aspect ratio within 2% for a rectangular channel has been developed: 
     
       
           F   AR =2/3 +11 h (2- h/w )/24 w   
       
     
     where h is less than w. 
     As an example, using these formulas to determine the pressure head H c  required to drive blood (which has a viscosity of 3.6 times the viscosity of water), and using the following parameters: 
     Q=0.2 μl/sec 
     h=250 μm 
     w=1000 μm 
     L=200 mm 
     g=9.81 m/s 2    
     p=1000 kg/m3 
     μ3.6×10 −3  Pa s 
     then F AR =0.867, D H =400 μm, R=6.642 ×10 11  Pa s/m 3 , and the pressure head Hc required to drive blood through this microfluidic channel is calculated to be 13.5 mm. 
     Referring now to FIG. 1, there is shown a cartridge generally indicated at  10  containing the elements of the present invention. Cartridge  10  is preferably constructed from a single material, such as a transparent plastic, using a method such as injection molding or laminations, and is approximately the size and thickness of a typical credit card. Located within cartridge  10  are a series of microfluidic channels  12 ,  14 ,  16 . Each of channels  12 ,  14 ,  16  are individually connected at one end to a circular inlet port  18 ,  20 ,  22  respectively, each of which couples channels  12 ,  14 ,  16  to atmosphere outside cartridge  10 . The opposite ends of channels  12 ,  14 ,  16  all terminate in a circular chamber  24  under a flexible membrane  26  within cartridge  10 , which preferably comprises an aspiration bubble pump. Chamber  24  may also contain a vent  28  which couples its interior to the outside of cartridge  10 . 
     The operation of cartridge  10  can now be described. A sample, such as whole blood, is divided into three parts, to which different reagents are mixed. In the present embodiment, the blood is combined with a physiologic saline, Anti-A antisera, and Anti-B antisera and a drop of each is place on inlet ports  18 ,  20 ,  22  separately. Alternatively; a drop of blood from the sample is placed on ports  18 ,  20 ,  22 , followed by a drop of different reagent for performing the assay, then mixed in the port by conventional means, such as a pipette. 
     The mixture is drawn into channels  12 ,  14 ,  16  via ports  18 ,  20 ,  22  respectively by capillary action, as the channels are sized to create capillary force action and draw the mixtures toward chamber  24 . A reaction of the sample and reagent, such as coagulation, agglutination, or a change in viscosity, is observed within channels  12 ,  14 ,  16  as the fluids travel toward chamber  24 . 
     Chamber  24  can be used for waste storage of the fluids after the assay is complete, and aspiration pump  26  can also assist in driving the fluids through the system. 
     FIG. 2 is directed to an alternative embodiment of the present invention. A microfluidic cartridge  10   a , manufactured in a similar manner to cartridge  10  of FIG. 1, contains a pair of inlet ports  30 ,  32 , which connect to a reaction channel  34  via inlet channels  36 ,  38  respectively. Inlets  36 ,  38  are arranged such that they connect to channel  34  with the one above the other, such that laminar flow in channel  34  is created as shown in FIG. 3. A pair of storage chambers  40 ,  42  are positioned at the end of channel  34  which act as waste storage receptacles. 
     The driving force necessary to perform assays within cartridge  10   a  is provided by gravity. This force can be enhanced by spinning the cartridge in a centrifuge. As an example, an assay to determine blood type of a specimen sample can be performed as follows: a droplet  50  of whole blood to be typed is placed on inlet port  32 , while a suitable reagent solution droplet  52  is placed upon inlet port  30 . Cartridge  10   a  is then positioned at an angle to the vertical plane, allowing fluids  50 ,  52  to flow into channel  34 . As blood drop  50  flows through inlet  38  into channel  34 , it flows in the upper section of channel  34 , while reagent droplet  52  flows through inlet  36  and enters channel  34  flowing in the lower section of channel  34 , with the two fluids exhibiting laminar flow, as can be clearly seen in FIG.  3 . 
     FIG. 8 shows a device  53  which holds the cartridges at a constant angle during the assay. The angle at which the cartridge is held may be varied from vertical to horizontal. The speed of the reaction varies according to the angle. As red blood cells settle under normal gravity at the rate of 1 μm/sec., they will, after some time, settle from fluid  50  across the flow boundary into fluid  52 , and begin to react with the antiserum in the reagent solution. 
     In the instances where the antisera in the reagent solution react with the whole blood in the specimen sample, agglutination will occur, causing a visually observable reaction which indicates the blood type of the sample. A series of channels  55  with graduated width dimensions allow agglutinated particles to travel along according to size. 
     FIGS. 4A-C show a blood typing assay performed on a series of cartridges of the design taught in FIG.  2 . Referring now to these figures, cartridges  10   b ,  10   c ,  10   d  show a blood typing experiment in which a blood sample listed as A-positive from the supplier is assayed. Cartridge  10   b  has whole blood placed in inlet  30  and a physiologic saline solution in inlet  32 , cartridge  10   c  has blood from the same source placed in inlet  30  and Anti-A antisera placed in inlet  32 , while cartridge  10  had a blood sample from the same source placed in inlet  30  and Anti-B antisera placed in inlet  32 . 
     As each of the samples traveled through channel  34 , driven by hydrostatic pressure, the fluids in cartridges  10   b  and  10   d  did not indicate a positive reaction, while the fluid within channel  34  of cartridge  10   c  is showing signs of agglutination, which can be visually detected within channel  34 , indicating a positive reaction for A-positive blood. Views of the completed tests performed within cartridges  10   b  and  10   c  can be more clearly seen in FIGS. 5A-B. 
     An alternative embodiment having a blood typing device integrated into a single cartridge is shown in FIG.  6 . Referring now to FIG. 6, a cartridge  10   e  contains a first chamber  60  which is coupled to a port  62 , and is also connected to a series of microfluidic channels  64 ,  66 ,  68 ,  69 . Channel  64  terminates in a chamber  70 , channel  66  terminates in a chamber  72 , while channel  68  terminates in a chamber  74 . Each of chambers  70 ,  72 ,  74  are connected to another chamber  76  via passageways  78 ,  80 ,  82  respectively. Passageways  78 ,  80 ,  82  each have a section containing a fine grating  78   a ,  80   a ,  82   a  respectively. Chamber  76  is also coupled to atmosphere outside of cartridge  10   e  via a port  84 . Channel  69  couples chamber  60  to another chamber  90 , which is coupled to the exterior of cartridge  10   e  by a port  92 . 
     To perform a blood typing assay with this device, a diluent  94  is pre-inserted into chamber  60 , while chambers  70 ,  72 ,  74  are pre-filled with reagents  96 ,  98 ,  100  for detection blood types A, B and O respectively. After these preliminary steps have been taken, ports  62 ,  84 , and  92  are sealed, preferably by covering with tape. 
     The analysis begins by removing the seal from port  62 , and inserting a quantity of blood of an unknown type into port  62  with a syringe or pipette dropper, which sample enters chamber  60  containing diluent  94 . Port  62  is then resealed, and cartridge  10   e  is shaken, allowing the blood cells to mix with diluent  94 . The cells are then allowed to sediment, positioning cartridge  10   e  in the orientation shown in FIG.  6 . After sedimentation, ports  62  and  92  are unsealed, which allows excess diluent  94  to travel via channel  69  into chamber  90 . Next, port  84  is unsealed, allowing the diluted blood sample to flow into chambers  70 ,  72 ,  74  via channels  64 ,  66 ,  68  respectively, where it can mix with reagents  96 ,  98 ,  100 . Cartridge  10   e  is then shaken briefly, and placed in a temperature-controlled environment in the orientation shown in FIG. 6 for ten minutes. 
     After the specified time period has elapsed, cartridge is taken from the controlled environment, and rotated 90° in the direction shown by arrow A, placing chamber  76  at the lowermost position in cartridge  10   e . This allows the mixed solutions in chambers  70 ,  72 ,  74  to flow toward chamber  76  via passageways  78 ,  80 ,  82  respectively. 
     As the solutions reach fine gratings  78   a ,  80   a ,  82   a , the cells in the chamber which contained the reagent of the unknown blood type will begin to agglutinate, causing a blockage within that particular channel, causing a visual representation of the particular blood type, as the chamber relative to that blood type has not emptied, due to clogging. Cartridge  10   e  can now be safely discarded, with ports  62 ,  84 ,  92  resealed with tape or the like to retain all fluids within the cartridge. This cartridge design is desirable, as it allows the washing of the blood cells to be analyzed prior to their contact with the antisera. 
     An alternative embodiment of a blood typing device (similar to that shown in FIG. 6) can be seen in FIG.  7 . Referring now to FIG. 7, a cartridge  10   f  contains a first chamber  110  which is coupled to the exterior of the cartridge by a port  112 . Chamber  110  is connected to a chamber  114  via a microfluidic channel  116 . Chamber  114  contains a port  118  which couples chamber  114  to the exterior of cartridge  10   f . Port  118  is initially blocked by a plug  120 . 
     Chamber  110  is also connected to a chamber  122  by a channel  124 . Chamber  110  is connected to a chamber  126  by a channel  128 , while chamber  128  is connected to a chamber  130  via a series of parallel channels  132 . Finally, chamber  130  is coupled to the exterior of cartridge  10   f  through a port  134 , which is initially blocked by a plug  136 . 
     To perform an assay using cartridge  10   f , plug  136  is removed from port  134 , and an antisera for a particular blood type is added to cartridge  10   f  through port  112 . This fluid, preferably in the amount of 100 μl, flows through chamber  110  and channel  124  into chamber  122 . Plug  136  is then replaced into port  134 . 
     Next, a blood wash reagent is placed into chamber  110  via port  112 , followed by a sample of blood of unknown type. These fluids are mixed within chamber  110  by shaking, then allowed to settle. 
     After the mixture in chamber  110  has settled, plug  120  is removed from port  118  in chamber  114 , and cartridge  10   f  is carefully tilted such that the supernatant contained within chamber  110  can be removed from cartridge  10   f  through port  118 . When the process is completed, plug  136  is removed from port  134 , which allows the washed cells contained within chamber  110  to flow through channel  124  into chamber  122 , which already contains antisera solution. The fluids are now mixed with chamber  122  by shaking, and cartridge  10   f  is then incubated for a period of time. 
     After incubation, cartridge  10   f  is rotated 90° in the direction shown by arrow B, causing the contents of chamber  122  to flow through channel  128  into chamber  126 . If the unknown blood sample reacts with the antisera inserted into cartridge  10   f , agglutination will clog channel  132 , and chamber  130  will remain empty. If the antisera do not react with the blood sample, chamber will contain fluid from chamber  122 . 
     While the present invention has been shown and described in terms of several preferred embodiments thereof, it will be understood that this invention is not limited to an particular embodiment and that many changes and modifications may be made without deporting from the true spirit and scope of the invention as defined in the appended claims.