Abstract:
A device for performing polymerase chain reaction (PCR) amplification and detection using microfluidic diffusion-based structures. Fluid containing DNA to be amplified is cycled repeatedly across hot and cold zones to enhance the multiplication process. The invention is used in conjunction with other devices to perform both single and multiple target detection.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This patent claims benefit from U.S. Provisional Patent Application Ser. No. 60/206,878, filed May 24, 2000, 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 analytical testing, and, in particular, to a device and method for performing nucleic acid amplification using microfluidic diffusion-based separation processes.  
           [0004]    2. Description of the Related Art  
           [0005]    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. Microfluidic channels are generally defined as a fluid passage which have at least one internal cross-sectional dimension that is less than 500 μm and typically between about 0.1 μm and about 500 μm.  
           [0006]    In microfluidic channels, fluids usually exhibit laminar behavior; that is, they allow the movement of separate fluidic streams next to each other within the channel without mixing, other than diffusion. For example, a sample solution, such as whole blood, and an extraction solution, such as a buffer solution, are introduced into a common microfluidic channel, and flow next to each other until they exit the channel. Smaller particles, such as ions or small parts of DNA, diffuse rapidly across the fluid boundaries, whereas larger particles (e.g., large pieces of DNA or small pieces of DNA attached to a larger particle) diffuse more slowly. Large particles of a diameter of roughly more than 2 μm show no significant diffusion within the time the two flowing streams are in contact.  
           [0007]    The principle of laminar flow has been addressed in a number of patents which have recently issued in the field of microfluidics. U.S. Pat. No. 5,716,852, which is incorporated herein in its entirety, is directed to a device, known as a T-Sensor, having a laminar flow channel and two inlet stream means in fluid communication with the laminar flow channel, which has a depth sufficiently small to allow particles from one stream to diffuse into the other stream. U.S. Pat. No, 5,932,100, which is also incorporated by reference herein in its entirety, is directed to a microfabricated extraction system for extracting desired particles from a sample stream. This device, known as an H-Filter, contains a laminar flow extraction channel and two inlet stream means connected to the extraction channel, with separate outlets at the exit of the extraction channel for a product stream containing the extracted particles and a by-product stream containing the remainder of the sample stream.  
           [0008]    Recently, a number of protocols, test kits, and cartridges have been developed for conducting analyses on biological samples for various diagnostic and monitoring purposes. Immunoassays, agglutination assays, and analyses based on polymerase chain reaction (PCR), various legend-receptor interactions, and differential migration of species in a complex sample have all been used to determine the presence or concentration of various biological compounds or contaminants, or the presence of particular cell types.  
           [0009]    PCR is a method which has been devised for amplifying one or more specific nucleic acid sequences or a mixture thereof using primers, nucleotide triphosphates, and an agent for polymerization, such as DNA polymerase. The extension produced of one primer, when hybridized to the other, becomes a template for the production of the desired specific nucleic acid sequence, and vice versa. The process is repeated as often as necessary to produce the desired amounts of the sequence.  
           [0010]    The basic process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof is described in U.S. Pat. No. 4,683,202, in which a strand of DNA is copied using a polymerase. The process comprises treating complimentary strands of nucleic acid with two primers, for each specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complimentary to each nucleic acid strand, wherein the primers are selected so as to be substantially complimentary to different strands of each specific sequence such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer. The primer extension products are then separated from the templates on which they were synthesized to produce single-stranded molecules. Finally, the single-stranded molecules that are generated are treated with the primer generated under conditions such that a primer extension product is synthesized using each of the single strands as a template. This process is repeated until the desired level of sequence amplification is obtained.  
           [0011]    U.S. Pat. No. 4,683,202, which issued Jul. 28, 1987, is directed to the PCR process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof. In an example disclosed therein, a solution was prepared which was heated to 100° C. for four minutes and allowed to cool to room temperature for two minutes, whereupon DNA polymerase was added and the cycle of heating, cooling, adding polymerase, and reacting was repeated many times. U.S. Pat. No. 5,939,291 is directed to an isothermal method of nucleic acid amplification which incorporates nonthermal means for denaturing the target nucleic acid or resultant amplification products, which enables the avoidance of the use of a thermal cycler component of any amplification equipment. The process can also be used in the context of a microfluidic device.  
           [0012]    Other devices which are directed to microfluidic or microscale devices are: U.S. Pat. No. 5,916,776, which generates copies of a first strand of nucleic acid to generate copies of a second strand, and moves the copies of the second strand to a second location; U.S. Pat. No, 6,057,149, which employs silicon-based microscale microdroplet transport channels wherein the discrete droplets are differentially heated and propelled through stated channels; and U.S. Pat. No. 6,117,634, in which novel sequencing reactions using double-stranded templates are contemplated to take place in microfabricated reaction chambers. U.S. Pat. No. 5,333,675 teaches a device designed for performing automated amplification of nucleic acid sequences and assays using heating and cooling steps.  
           [0013]    U.S. Pat. No. 5,955,029 is directed to devices for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide polymerization reaction. The device may be used to implement a PCR in the reaction chamber, which is provided with the sample polynucleotide, polymerase, nucleotide triphosphates, primers and other reagents required for the PCR, and contains means to thermally control the temperature of the contents of the reaction chamber to dehydridize double stranded polynucleotide, to anneal the primers, and to polymerize and amplify the polynucleotide. U.S. Pat. No. 5,965,410 discloses a device for controlling process parameters, including fluid temperature, of a system by the application of electric current to the material such that the material can be successively heated and cooled for biological applications such as PCR.  
           [0014]    U.S. Pat. No. 6,210,882 is directed to a method for performing rapid and accurate thermocoupling on a sample for performing PCR within microchannels on a microchip using a non-contact heat source. Positive cooling is accomplished by use of a non-contact cooling source directed at the vessel containing the sample. Cooling, like heating, can be accomplished through any member of steps, with a different temperature of steps, with a different temperature being maintained at each step.  
           [0015]    Methodologies using PCR for diagnostic purposes are well established. PCR amplification has been used for the diagnosis of genetic disorders, and generation of specific sequences of closed double standard DNA for use as probes and to create larger amounts of DNA for sequencing.  
           [0016]    Thus, a need has been created for convenient economical systems for PCR analyses, which could be used in a wide range of potential applications in clinical tests, such as test for paternity, genetic and infectious diseases.  
         SUMMARY OF THE INVENTION  
         [0017]    It is therefore an object of the present invention to provide a device for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide polymerization reaction.  
           [0018]    It is a further object of the present invention to provide a compact, single use module capable of analyses involving polymerase chain reaction (PCR) that is economical to manufacture and use.  
           [0019]    These and other objects are accomplished with a device which comprises a substrate microfabricated to define a sample inlet port and a mesoscale flow system extending from the inlet port. The mesoscale flow system includes a polynucleotide polymerization reaction chamber in fluid communication with the inlet port which is provided with reagents required for polymerization and amplification of a preselected polynucleotide. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0020]    [0020]FIG. 1 is a diagram showing non-contact thermal heating of a fluid in a microscale channel;  
         [0021]    [0021]FIG. 2 is a diagram showing the thermocycling in PCR according to the present invention;  
         [0022]    [0022]FIG. 3 is a diagram showing PCR using single target amplification and detection according to the present invention; and  
         [0023]    [0023]FIG. 4 is a diagram showing PCR using multiple target detection according to the present invention. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0024]    [0024]FIG. 1 is a diagram showing a method of heating a fluid plug within a microfluidic channel. A fluid plug  2  is contained within a microfluidic channel which traverses a pair of heat pads  6 ,  8 . Fluid plug  2  can be cycled back and forth within channel  4  until it reaches a desired temperature over heat pads  6 ,  8 .  
         [0025]    Referring now to FIG. 2, there is shown a microfluidic device for performing PCR, generally indicated at  10 . Device  10  includes a microfluidic flow channel  12 , a pair of heat pads  14 ,  16 , and a pair of cooling regions  18 ,  20 . Channel  12  consists of a sinuous S-shaped pathway which traverses across heat pads  14 ,  16  and cooling sections  18 ,  20 . In this arrangement, the contents of channel  10 , which consists of Taq-polymerase, dNTP and two DNA primer sequences which are flowing laminarly within channel  12  alongside the sample containing the DNA to be amplified, can be cycled repeatedly across hot and cold zones which is necessary for the amplification of the described DNA region of interest. Heat pads  14 ,  16  can be manufactured from anything that conducts and/or stores heat, such as metal plates, vices, or hot water. Joule heating or radiation heating may also be used. Typical temperature for pads  14 , 16  generally can be around 95° C., and around 45 to 50° C. for cooling regions  18 ,  20 .  
         [0026]    One embodiment of PCR involving single-target amplification and detection is shown in FIG. 3. Referring now to FIG. 3, a PCR amplification system, generally designated at  30 , contains a main channel  32  and an intersecting channel  34 . A first port  36  is coupled to the inlet of channel  32 , while a second port  38  is coupled to the inlet of channel  34 . Main channel  32  is connected to a mixing structure  40 , which mixer is preferably of the type described in U.S. patent application Ser. No. ______, which application is hereby incorporated by reference in its entirety. However, any mixing structure which provides sufficient mixing may be used. The output of mixer  40  is coupled to PCR thermocycler  10 , which is shown and discussed in detail with respect to FIG. 2.  
         [0027]    Main channel  32  exits thermocycler  10  and is intersected by a second intersecting channel  42  having an input port  43 . Downstream from channel  42 , channel  32  terminates in an exit channel  44 . Exit channel  44  contains a waste section  46  having a port  48 , and a sample section  50 . Section  50  is coupled to a detection means  52 . The output of detection means  52  is coupled to an output port  54  via section  50 .  
         [0028]    The structure formed by channel  42 , main channel  32 , channel  46  and channel  50  operates in the same manner as the absorption enhanced differential extractor device which is described in detail in U.S. Pat. No. 5,971,158, which patent is hereby incorporated by reference in its entirety. This device, which is commonly referred to as an “absorption-enhanced Hfilter”, is useful for extracting desired particles from a sample stream containing the desired particles. A sequestering material within the extraction channel captures the desired particles in the extraction stream.  
         [0029]    In operation, a sample containing DNA is loaded into port  36 , while a sample containing Taq polymerase, a primer  1 , and a primer  2  is loaded into port  38 . Primer  1  preferably consists of large particles or may be attached to larger molecules or particles, while Primer  2  preferably consists of labeled particles. These two substances travel through channel  32  in a laminar fashion where diffusion takes place, as previously discussed, until the streams reach mixer  40 , where the substances are combined to form an essentially homogeneous mixture. This mixture flows from mixer  40  to thermocycler  10 , where conventional PCR amplification is performed in the mixture using the structure shown in FIG. 2. In the present embodiment, the last PCR cycle is ended at the low temperature as DNA is attached to the primers. The output stream of thermocycler  10  flows in main channel  32  and contains multiple copies of DNA attached to labeled primer molecules, as well as excess primer  1  and primer  2 .  
         [0030]    An extraction solution containing primer absorbing particles is loaded into port  43  and flows through channel  42  to main channel  32  where it contacts and flows next to the output stream from thermocycler  10 , without mixing other than diffusion. In this embodiment, the absorbing particles in the solution from channel  42  remove fast-diffusing labeled primer molecules from equilibrium. The length of channel  32  between thermocycler  10  and channel  44  is chosen such that essentially all labeled primer molecules have diffused across the laminar flow boundary between the fluids.  
         [0031]    As the contents of channel  32  reach channel  44 , the extraction solution from channel  42  now contains a waste product containing primer absorbing particles, primer  1  molecules, and other small molecules as a result of diffusion. This stream exits channel  32  by way of section  46  of channel  44 , and flows into exit port  48 , while the stream which contains particles of interest exits channel  32  by way of section  50  of channel  44 , and flows to detection means  52 . In the present embodiment, detection means  52  is preferably a fluorescent detector. The stream now contains multiple copies of the desired DNA, and exits device  30  via port  54 .  
         [0032]    An embodiment showing multiple target detection is shown in FIG. 4. Referring now to FIG. 4, a PCR amplification system, generally designated at  60 , contains a main channel  62  and an intersecting channel  64 . A first port  66  is located at the end of channel  64  opposite to its intersection with channel  62 , while a port  68  is located at the end of channel  62  opposite its intersection with channel  64 . Main channel  62  is connected to a mixing structure  70 , which mixer is preferably of the type shown in FIG. 2 and also described in U.S. patent application Ser. No. ______, but may consist of any suitable mixing device. Mixer  70  receives the contents of channels  62  and  64  which flow in a laminar fashion, and provides an essentially homogeneous mixture to PCR thermocycler  10 , which has previously been described with respect to FIGS. 2 and 3.  
         [0033]    Channel  62  exits thermocycler  10  and is intersected by a channel  72  which extends from an input port  74 . Channel  62  continues downstream where it terminates at a crossing channel  76 . Channel  76  is comprised of a waste section  78  which terminates in an exit port  80 . Channel  76  is connected at its other end to a mixing/heating structure  82 , while a channel  84  which terminates at a port  86  is also coupled to mixer  82 . Channel  76  exits mixer  82  where it is coupled to an intersecting channel  88  coupled to a port  90 . Channel  76  continues along past channel  88  where it intersects a waste channel  92  coupled to a waste port  94 . Channel  76  finally terminates at a detecting device  96 .  
         [0034]    In operation, multiple target amplification and detection is performed by loading a sample containing DNA into port  68 . A mixture of Taq polymerase, primer  1  and primer  2  is loaded into port  66 . These primers in this mixture are intended for multiple targets, and are roughly the same size, with none of the particles very large. The mixture loaded into port  66  flows within channel  62  where it flows laminarly with the sample containing DNA which was loaded into port  68 . The contents of channel  62  enter mixing structure  70 , and exit mixture  70  as an essentially homogeneous fluid.  
         [0035]    The mixed fluid enters PCR thermocycler  10  where DNA amplification occurs using the PCR method. The last PCR cycle performed by thermocycler  10  is ended at high temperature as the DNA is detached from the primers within the fluid mixture. The flow stream exiting thermocycler  10  now contains multiple copies of DNA detached from primer molecules, as well as excess primer  1  and primer  2  for multiple targets.  
         [0036]    An extraction solution containing primer absorbing particles for primers  1  and  2  for each targeted DNA piece is loaded into port  74 , where it flows through channel  72  into main channel  62 , where it contacts with the flow stream exiting thermocycler  10  in a laminar fashion. The combined fluid stream flows through channel  62 , where the primer absorbing particles remove fast-diffusing primer molecules from equilibrium. After sufficient time and travel within channel  62 , almost all primer molecules are removed from system  60  by passing through waste channel  78  into waste port  80 . Waste port  80  contains primer absorbing molecules, primers  1  and  2  for multiple targets and other small molecules, all of which have diffused across channel  62 . The remaining fluid from channel  62  passes into crossing channel  76 , where it enters mixing/heating structure  82 . Also flowing into structure  82  is a fluorescent labeled primer  1  for each of the targeted DNA sequences, which are loaded into port  86 . Structure  82  both mixes the two fluids and heats the solution to annealing temperature, which is approximately 96° C. This process opens up the strands of DNA within structure  82  and are passed along within channel  76 .  
         [0037]    An extraction solution containing primer-absorbing particles is loaded into port  90 , and flows within channel  88  to channel  76 , where it flows laminarly adjacent to fluid exiting structure  82 . As the flow reaches waste channel  92 , waste containing primer absorbing particles, primers  1  and other small molecules which have diffused across the laminar boundary exits channel  92  and flows into port  94 , while the remaining flow within channel  76  which now contains multiple copies of DNA of multiple targets attached to labeled primers  1 .  
         [0038]    The flow from channel  76  enters fluorescent detector structure  96 , where primers  2  for multiple targets are immobilized on the bottom of structure  96 , while the various DNA targets, each labeled with a fluorescent primer  1 , attach to a specific site on structure  96  and can therefore be identified and quantified.  
         [0039]    The structure of device  60  after thermocycler  10  operates in the same manner as two absorption enhanced differential extractor devices, which were discussed previously, which are operating in series.  
         [0040]    The PCR assays performed using the present invention can be used in a wide range of applications such as the generation of specific sequences of cloned double-stranded DNA for use as probes, the generation of probes is specific for uncloned genes by selective amplification of particular segments of cDNA, the generation of libraries of cDNA for sequencing, and the analysis of mutations.  
         [0041]    While the present invention has been shown and described in terms of several embodiments thereof, it will be understood that this invention is not limited to these particular embodiments and that many changes and modifications may be made without departing from the true spirit and scope of the invention as defined in the appended claims.