Patent Publication Number: US-2009226971-A1

Title: Portable Rapid Microfluidic Thermal Cycler for Extremely Fast Nucleic Acid Amplification

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61/022,692 filed on Jan. 22, 2008 entitled “portable rapid microfluidic thermal cycler for extremely fast nucleic acid amplification,” the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Related inventions are disclosed and claimed in U.S. patent application Ser. No. ______ titled Rapid Microfluidic Thermal Cycler for Nucleic Acid Amplification filed on the same as this application. The disclosure of U.S. patent application Ser. No. ______ titled Rapid Microfluidic Thermal Cycler for Nucleic Acid Amplification is hereby incorporated by reference. 
    
    
     STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT 
     The United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC for the operation of Lawrence Livermore National Laboratory. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of Endeavor 
     The present invention relates to thermal cycling and more particularly to a portable rapid microfluidic thermal cycler. 
     2. State of Technology 
     United States Published Patent No. 2005/0252773 for a thermal reaction device and method for using the same includes the following state of technology information:
         “Devices with the ability to conduct nucleic acid amplifications would have diverse utilities. For example, such devices could be used as an analytical tool to determine whether a particular target nucleic acid of interest is present or absent in a sample. Thus, the devices could be utilized to test for the presence of particular pathogens (e.g., viruses, bacteria or fungi), and for identification purposes (e.g., paternity and forensic applications). Such devices could also be utilized to detect or characterize specific nucleic acids previously correlated with particular diseases or genetic disorders. When used as analytical tools, the devices could also be utilized to conduct genotyping analyses and gene expression analyses (e.g., differential gene expression studies). Alternatively, the devices can be used in a preparative fashion to amplify sufficient nucleic acid for further analysis such as sequencing of amplified product, cell-typing, DNA fingerprinting and the like. Amplified products can also be used in various genetic engineering applications, such as insertion into a vector that can then be used to transform cells for the production of a desired protein product.”       

     United States Published Patent No. 2008/0166793 by Neil Reginald Beer for sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture provides the following state of technology information:
         “A complex environmental or clinical sample  201  is prepared using known physical (ultracentrifugation, filtering, diffusion separation, electrophoresis, cytometry etc.), chemical (pH), and biological (selective enzymatic degradation) techniques to extract and separate target nucleic acids or intact individual particles  205  (e.g., virus particles) from background (i.e., intra- and extra-cellular RNA/DNA from host cells, pollen, dust, etc.). This sample, containing relatively purified nucleic acid or particles containing nucleic acids (e.g., viruses), can be split into multiple parallel channels and mixed with appropriate reagents required for reverse transcription and subsequent PCR (primers/probes/dNTPs/enzymes/buffer). Each of these mixes are then introduced into the system in such a way that statistically no more than a single RNA/DNA is present in any given microreactor. For example, a sample containing 106 target RNA/DNA would require millions of microreators to ensure single RNA/DNA distribution.   An amplifier  207  provides Nucleic Acid Amplification. This may be accomplished by the Polymerase Chain Reaction (PCR) process, an exponential process whereby the amount of target DNA is doubled through each reaction cycle utilizing a polymerase enzyme, excess nucleic acid bases, primers, catalysts (MgCl2), etc. The reaction is powered by cycling the temperature from an annealing temperature whereby the primers bind to single-stranded DNA (ssDNA) through an extension temperature whereby the polymerase extends from the primer, adding nucleic acid bases until the complement strand is complete, to the melt temperature whereby the newly-created double-stranded DNA (dsDNA) is denatured into 2 separate strands. Returning the reaction mixture to the annealing temperature causes the primers to attach to the exposed strands, and the next cycle begins.   The heat addition and subtraction powering the PCR chemistry on the amplifier device  207  is described by the relation:       

         Q=hA ( T   wall   −T   ∞ )         The amplifier  207  amplifies the organisms  206 . The-nucleic acids  208  have been released from the organisms  206  and the nucleic acids  208  are amplified using the amplifier  207 . For example, the amplifier  207  can be a thermocycler. The nucleic acids  208  can be amplified in-line before arraying them. As amplification occurs, detection of fluorescence-labeled TaqMan type probes occurs if desired. Following amplification, the system does not need decontamination due to the isolation of the chemical reactants,”       
     U.S. Pat. No. 3,635,037 for a Peltier-effect heat pump provides the following state of technology information:
         “The Peltier-effect has been used heretofore in heat pumps for the heating or cooling of areas and substances in which fluid-refrigeration cycles are disadvantageous. For example, for small lightweight refrigerators, compressors, evaporators and associated components of a vapor/liquid refrigerating cycle may be inconvenient and it has, therefore, been proposed to use the heat pump action of a Peltier pile. The Peltier effect may be described as a thermoelectric phenomenon whereby heat is generated or abstracted at the junction of dissimilar metals or other conductors upon application of an electric current. For the most part, a large number of junctions is required for a pronounced thermal effect and, consequently, the Peltier junctions form a pile or battery to which a source of electrical energy may be connected. The Peltier conductors and their junctions may lie in parallel or in series-parallel configurations and may have substantially any shape. For example, a Peltier battery or pile may be elongated or may form a planar or three-dimensional (cubic or cylindrical) array. When the Peltier effect is used in a heat pump, the Peltier battery or pile is associated with a heat sink or heat exchange jacket to which heat transfer is promoted, the heat exchanger being provided with ribs, channels or the like to facilitate heat transfer to or from the Peltier pile over a large surface area of high thermal conductivity. A jacket of aluminum or other metal of high thermal conductivity may serve for this purpose.”       

     SUMMARY 
     Features and advantages of the present invention will become apparent from the following description. Applicants are providing this description, which includes drawings and examples of specific embodiments, to give a broad representation of the invention. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this description and by practice of the invention. The scope of the invention is not intended to be limited to the particular forms disclosed and the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims. 
     The present invention provides a system for extremely fast continuous flow or batch PCR amplification of target nucleic acids in a compact, portable microfluidic-compatible platform. The present invention also provides a system for extremely fast thermal cycling, precise thermal control, and low power consumption due to innovative heat transfer characteristics. In addition, present invention also provides a method for thermally calibrating the system to ensure the proper heating and cooling set points are reached during the extremely rapid cycling. 
     In one embodiment the present invention provides a portable apparatus for thermal cycling a material to be thermal cycled, including a portable microfluidic-compatible platform, a microfluidic heat exchanger carried by the portable microfluidic-compatible platform; a porous medium in the microfluidic heat exchanger; a microfluidic thermal cycling chamber containing the material to be thermal cycled, the microfluidic thermal cycling chamber operatively connected to the microfluidic heat exchanger; a working fluid at first temperature, a first system for transmitting the working fluid at first temperature to the microfluidic heat exchanger; a working fluid at a second temperature, a second system for transmitting the working fluid at second temperature to the microfluidic heat exchanger; a pump for flowing the working fluid at the first temperature from the first system to the microfluidic heat exchanger and through the porous medium; and flowing the working fluid at the second temperature from the second system to the heat exchanger and through the porous medium. 
     In one embodiment the first system for transmitting the working fluid at first temperature to the microfluidic heat exchanger is a first container for containing the working fluid at first temperature and the second system for transmitting the working fluid at second temperature to the microfluidic heat exchanger is a second container for containing the working fluid at second temperature. In another embodiment the first system for transmitting the working fluid at first temperature to the microfluidic heat exchanger and the second system for transmitting the working fluid at second temperature to the microfluidic heat exchanger comprises a single container and separate line with a heater or cooler that are connected to provide the working fluid at first temperature to the microfluidic heat exchanger and to provide the working fluid at second temperature to the microfluidic heat exchanger. 
     In one embodiment the present invention provides a portable apparatus for thermal cycling a material to be thermal cycled. The apparatus includes a portable microfluidic-compatible platform, a microfluidic heat exchanger carried by the portable microfluidic-compatible platform; a porous medium in the microfluidic heat exchanger; a microfluidic thermal cycling chamber containing the material to be thermal cycled, the microfluidic thermal cycling chamber operatively connected to the microfluidic heat exchanger; a working fluid at first temperature, a first container for containing the working fluid at first temperature, a working fluid at a second temperature, a second container for containing the working fluid at second temperature, a pump for flowing the working fluid at the first temperature from the first container to the microfluidic heat exchanger and through the porous medium; and flowing the working fluid at the second temperature from the second container to the heat exchanger and through the porous medium. In another embodiment the present invention provides a method of thermal cycling a material to be thermal cycled between a number of different temperatures. The method includes the steps of providing a portable microfluidic-compatible platform, providing a microfluidic heat exchanger on the portable microfluidic-compatible platform, the microfluidic heat exchanger operatively positioned with respect to the material to be thermal cycled providing working fluid at a first temperature, flowing the working fluid at the first temperature to the microfluidic heat exchanger to hold the material to be thermal cycled at the first temperature, providing working fluid at a second temperature, and flowing the working fluid at the first temperature to the heat exchanger to hold the material to be thermal cycled at the second temperature. 
     The present invention has use in a number of applications. For example, the present invention has use in biowarfare detection applications. The present invention has use in identifying, detecting, and monitoring bio-threat agents that contain nucleic acid signatures, such as spores, bacteria, etc. The present invention has use in biomedical applications. The present invention has use in tracking, identifying, and monitoring outbreaks of infectious disease. The present invention has use in automated processing, amplification, and detection of host or microbial DNA in biological fluids for medical purposes. The present invention has use in genomic analysis, genomic testing, cancer detection, genetic fingerprinting. The present invention has use in forensic applications. The present invention has use in automated processing, amplification, and detection DNA in biological fluids for forensic purposes. The present invention has use in food and beverage safety. The present invention has use in automated food testing for bacterial or viral contamination. The present invention has use in environmental monitoring and remediation monitoring. 
     The invention is susceptible to modifications and alternative forms. Specific embodiments are shown by way of example. It is to be understood that the invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings, which are incorporated into and constitute a part of the specification, illustrate specific embodiments of the invention and, together with the general description of the invention given above, and the detailed description of the specific embodiments, serve to explain the principles of the invention. 
         FIG. 1  illustrates one embodiment of the present invention. 
         FIG. 2  illustrates another embodiment of the present invention. 
         FIG. 3  illustrates yet another embodiment of the present invention. 
         FIG. 4  illustrates an embodiment of the present invention utilizing a glass micro array. 
         FIG. 5  illustrates an embodiment of the present invention utilizing microreactors. 
         FIG. 6  illustrates another embodiment of the present invention. 
         FIG. 7  illustrates yet another embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS 
     Referring to the drawings, to the following detailed description, and to incorporated materials, detailed information about the invention is provided including the description of specific embodiments. The detailed description serves to explain the principles of the invention. The invention is susceptible to modifications and alternative forms. The invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims. 
     Referring now to the drawings and in particular to  FIG. 1 , one embodiment of a system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral  100 . The system  100  provides extremely fast continuous flow or batch PCR amplification of target nucleic acids in a compact, portable microfluidic-compatible platform  120 . Some of the technical challenges that were met in producing the system were (1) realizing a high throughput, field portable, real time PCR instrument that can run 10 assays in 1 minute, (2) a porous media heat exchanger coupled to an on-chip PCR device to optimize PCR (˜3 sec per cycle), and (3) field portable fluid reservoirs, valving, power supply, and pumps integrated with a real-time detector. 
     The system  100  provides thermal cycling a material  115  (DNA Sample) to be thermal cycled between a temperature T 1  and T 2  using a microfluidic heat exchanger  101  operatively positioned with respect to the material  115  to be thermal cycled. A working fluid  102  at T 1  is provided and the working fluid  102  at T 1  is flowed to the microfluidic heat exchanger  101 . A working fluid  104  at T 2  is provided and the working fluid  104  at T 2  is flowed to the heat exchanger  101 . The steps of flowing the working fluid at T 1  and at T 2  to the microfluidic heat exchanger  101  are repeated for a predetermined number of times. A porous medium  113  is located in the microfluidic heat exchanger  101 . The working fluids at T 1  and T 2  flow through the porous medium  113  during the steps of flowing the working fluid at T 1  and T2 through the microfluidic heat exchanger  101 . The system  100  is contained in a compact, portable microfluidic-compatible platform  120 . 
     The material  115  to be thermal cycled is contained on a chip  118  (microarray  118 ) containing the DNA. Examples of microarrays are shown in U.S. Pat. No. 7,354,389 for a microarray detector and methods which states, “The present invention is directed to an analytic system for detection of a plurality of analytes that are bound to a biochip, wherein an optical detector uses registration markers illuminated by a first light source to determine a focal position for detection of the analytes that are illuminated by a second light source.” U.S. Pat. No. 7,354,389 for a microarray detector and methods is incorporated herein by reference. The DNA sample  115  is contained on the chip  118  containing the DNA sample. A highly conductive plate  116  connects the chip  118  to the heat exchanger  101 . Conductive grease  117  is used to provide thermal conductivity between the chip  118  and the heat exchanger  101 . Instead of conductive grease  117  between the chip  118  and the heat exchanger  101  other forms of connection may be used. For example, press-fit contact or thermally-conductive tape may be used between the chip  118  and the heat exchanger  101 . 
     The steps of repeatedly flowing the working fluid at T 1  and at T 2  to the microfluidic heat exchanger  101  provide PCR fast and efficient nucleic acid analysis. The microfluidic polymerase chain reaction (PCR) thermal cycling method  100  is capable of extremely fast cycles, and a resulting extremely fast detection time for even long amplicons (amplified nucleic acids). The method  100  allows either singly or in combination: reagent and analyte mixing; cell, virion, or capsid lysing to release the target DNA if necessary; nucleic acid amplification through the polymerase chain reaction (PCR), and nucleic acid detection and characterization through optical or other means. An advantage of this system lies in its complete integration on a microfluidic platform and its extremely fast thermocycling. 
     The system  100  includes the following structural components: microfluidic heat exchanger  101 , microfluidic heat exchanger housing  112 , porous medium  113 , micropump  110 , lines  111 , chamber  103 , working fluid  102  at T 1 , chamber  105 , working fluid  104  at T 2 , lines  106  and  108 , multi position valve  107 , line  109 , highly conductive plate  116 , thermal grease  117 , chip containing DNA sample  118 , and DNA sample  115 . 
     The structural components of the system  100  having been described, the operation of the system  100  will be explained. The valve  107  is actuated to provide flow of working fluid  102  at T 1  from chamber  103  to the microfluidic heat exchanger  101 , Micro pump  110  is actuated driving working fluid  102  at T 1  from chamber  103  to the microfluidic heat exchanger  101 . The working fluid  102  at T 1  passes through the porous medium  113  in the microfluidic heat exchanger  101  raising the temperature of the material to be thermalcycled  115  to temperature T 1 . The porous medium  113  in the microfluidic heat exchanger  101  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     Next the valve  107  is actuated to provide flow of working fluid  104  at T 2  from chamber  105  to the microfluidic heat exchanger  101 . Micro pump  110  is actuated driving working fluid  104  at T 2  from chamber  105  to the microfluidic heat exchanger  101 . The working fluid  104  at T 2  passes through the porous medium  113  in the microfluidic heat exchanger  101  lowering the temperature of the material to be thermalcycled  115  to temperature T 2 . The steps of flowing the working fluid at T 1  and at T 2  to the microfluidic heat exchanger  101  are repeated for a predetermined number of times to provide the desired PCR. The porous medium  113  in the microfluidic heat exchanger  101  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     The heat exchanger  101  of the system  100  utilizes inlet and exit channels where heating/cooling fluid  102  and  104  is passing through, an enclosure, and a layer of conductive plate attached to a PCR micro-chip. The enclosure is filled with a conductive porous medium  113  of uniform porosity and permeability. In another embodiment the enclosure is filled with a conductive porous medium  113  with a gradient porosity. The nominal permeability and porosity of the porous matrix are taken as 3.74×10 −10  m 2  and 0.45, respectively. The porous medium  113  is saturated with heating/cooling fluid  102 ,  104  coming through an inlet channel. The inlet channel will be connected to hot and cold supply tanks  103  and  105 . A switching valve  107  is used to switch between hot  102  and cold tanks  105  for heating and cooling cycles. All lateral walls and top of the porous medium are insulated to minimize losses. The micropump  110  is positioned to drive the working fluids  102  and  105  directly into the microfluidic heat exchanger  101 . By positioning the micropump  110  outside the hot and cold supply tanks  103  and  105  and lines to the microfluidic heat exchanger  101  it eliminates the time the would be required to bring the micropump  110  up to the new temperature after each change. 
     Referring now to  FIG. 2 , another embodiment of a system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral  200 . The system  200  provides provides extremely fast continuous flow or batch PCR amplification of target nucleic acids in a compact, portable microfluidic-compatible platform  220 . The material  215  to be thermal cycled is contained on a chip  218  (microarray  218 ) containing the DNA. The DNA sample  215  is contained on the chip  218  containing the DNA sample. A highly conductive plate  216  connects the chip  218  to the heat exchanger  201 . Conductive grease is used to provide thermal conductivity between the chip  218  and the heat exchanger  201 . 
     A working fluid  202  at T 1  is provided in “Tank A”  203 . The working fluid is maintained at the temperature T 1  in Tank A ( 203 ) by appropriate heating and cooling equipment. The working fluid  202  at T 1  from Tank A ( 203 ) is flowed to the microfluidic heat exchanger  201 . 
     A working fluid  204  at T 2  is provided in “Tank B”  205 . The working fluid is maintained at the temperature T 2  in Tank B ( 205 ) by appropriate heating and cooling equipment. The working fluid  204  at T 2  from Tank B ( 205 ) is flowed to the heat exchanger  201 . 
     The system  200  includes the following additional structural components: microfluidic heat exchanger housing  212 , porous medium  213 , lines  206 ,  208 ,  209 , &amp;  211 , micropump  210 , multiposition valves  207 , and supply tank  221 . The system  200  is contained in a compact, portable microfluidic-compatible platform  220 . 
     The structural components of the system  200  having been described, the operation of the system  200  will be explained. When used for PCR, the system  200  provides thermal cycling a material  215  to be thermal cycled between a temperature T 1  and T 2  using a microfluidic heat exchanger  201  operatively positioned with respect to the material  215  to be thermal cycled. A working fluid  202  at T 1  is provided in “Tank A”  203 . The working fluid  202  at T 1  from Tank A ( 203 ) is flowed to the microfluidic heat exchanger  201 . A working fluid  204  at T 2  is provided in “Tank B”  205 . The working fluid  204  at T 2  from Tank B ( 205 ) is flowed to the heat exchanger  201 . 
     The multiposition valves  207  are actuated to provide flow of working fluid  202  at T 1  from Tank A ( 203 ) to the microfluidic heat exchanger  201 . Micro pump  210  is actuated driving working fluid  202  at T 1  from Tank A ( 203 ) to the microfluidic heat exchanger  201 . The working fluid  202  at T 1  passes through the porous medium  213  in the microfluidic heat exchanger  201  raising the temperature of the material to be thermalcycled  215  to temperature T 1 . The porous medium  213  in the microfluidic heat exchanger  201  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     Next the valves  207  are actuated to provide flow of working fluid  204  at T 2  from Tank B ( 205 ) to the microfluidic heat exchanger  201 . Micro pump  210  is actuated driving working fluid  204  at T 2  from chamber  205  to the microfluidic heat exchanger  201 . The working fluid  202  at T 2  passes through the porous medium  213  in the microfluidic heat exchanger  201  lowering the temperature of the material to be thermalcycled  215  to temperature T 2 . The porous medium  213  in the microfluidic heat exchanger  201  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     Referring now to  FIG. 3 , another embodiment of a thermal cycling system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral  300 . The system  300  provides thermal cycling of a material  315  between different temperatures using a microfluidic heat exchanger  301  operatively positioned with respect to the material  315 . The material to be thermal cycled  315  illustrated in  FIG. 3  is a DNA sample. The DNA sample  315  is contained on the chip  318  containing the DNA sample. A highly conductive plate  316  connects the chip  318  to the heat exchanger  301 . Conductive grease is used to provide thermal conductivity between the chip  318  and the heat exchanger  301 . 
     A working fluid  302  at T 1  is provided in “Tank A”  303 . The working fluid is maintained at the temperature T 1  in Tank A ( 303 ) by appropriate heating and cooling equipment. The working fluid  302  at T 1  from Tank A ( 303 ) is flowed to the microfluidic heat exchanger  301 . 
     A working fluid  304  at T 2  is provided in “Tank B”  305 . The working fluid is maintained at the temperature T 2  in Tank B ( 305 ) by appropriate heating and cooling equipment. The working fluid  304  at T 2  from Tank B ( 305 ) is flowed to the heat exchanger  301 . 
     A working fluid  319  at T 3  is provided in “Tank C”  320 . The working fluid is maintained at the temperature T 3  in Tank C ( 320 ) by appropriate heating and cooling equipment. The working fluid  319  at T 3  from Tank C ( 320 ) is flowed to the heat exchanger  301 . The system  300  includes the following additional structural components: microfluidic heat exchanger housing  312 , porous medium  313 , lines  306 ,  308 ,  309 , &amp;  311 , micropump  310 , multiposition valves  307 , and supply tank  321 . 
     The structural components of the system  300  having been described, the operation of the system  300  will be explained. The system  300  will be described as a polymerase chain reaction (PCR) system; however, it is to be understood that the system  300  can be used as other thermal cycling systems. 
     When used for PCR, the system  300  provides thermal cycling a material  315  to be thermal cycled between a temperatures T 1  and T 2  and T 3  using a microfluidic heat exchanger  301  operatively positioned with respect to the material  315  to be thermal cycled. The material  315  to be thermal cycled is contained on a chip  318  (microarray  318 ) containing the DNA. 
     A working fluid  302  at T 1  is provided in “Tank A”  303 . The working fluid  302  at T 1  from Tank A ( 303 ) is flowed to the microfluidic heat exchanger  301 . A working fluid  403  at T 2  is provided in “Tank B”  305 . The working fluid  303  at T 2  from Tank B ( 305 ) is flowed to the heat exchanger  301 . A working fluid  319  at T 3  is provided in “Tank C”  320 . The working fluid  319  at T 3  from Tank C ( 320 ) is flowed to the heat exchanger  301 . 
     The multiposition valves  307  are actuated to provide flow of working fluid  302  at T 1  from Tank A ( 303 ) to the microfluidic heat exchanger  301 . Micro pump  310  is actuated driving working fluid  302  at T 1  from Tank A ( 303 ) to the microfluidic heat exchanger  301 . The working fluid  302  at T 1  passes through the porous medium  313  in the microfluidic heat exchanger  301  raising the temperature of the material to be thermalcycled  315  to temperature T 1 . The porous medium  313  in the microfluidic heat exchanger  301  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     Next the valves  307  are actuated to provide flow of working fluid  304  at T 2  from Tank B ( 305 ) to the microfluidic heat exchanger  301 . Micro pump  310  is actuated driving working fluid  304  at T 2  from chamber  305  to the microfluidic heat exchanger  301 . The working fluid  304  at T 2  passes through the porous medium  313  in the microfluidic heat exchanger  301  lowering the temperature of the material to be thermalcycled  315  to temperature T 2 . The porous medium  313  in the microfluidic heat exchanger  301  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     The valves  307  can also be actuated to provide flow of working fluid  319  at T 3  from Tank C ( 320 ) to the microfluidic heat exchanger  301 . Micro pump  310  is actuated driving working fluid  319  at T 3  from Tank C ( 320 ) to the microfluidic heat exchanger  301 . The working fluid  319  at T 3  passes through the porous medium  313  in the microfluidic heat exchanger  301  changing the temperature of the material to be thermalcycled  315  to temperature T 3 . The porous medium  313  in the microfluidic heat exchanger  301  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     The heat exchanger  301  of the system  300  utilizes inlet and exit channels where heating/cooling fluid  302 ,  304 , and  319  pass through the porous media  313 . In one embodiment the porous media  313  has a uniform porosity and permeability. The nominal permeability and porosity of the porous matrix are taken as 3.74×10 −10  m 2  and 0.45, respectively. In other embodiments the porous media  313  has gradient porosity. The system  300  allows the heat exchanger  301  to change the temperature of the material to be thermal cycled  315  between and to a variety of different temperatures. By various combinations of settings of the multiposition valves  307  it is possible to supply working fluid from tanks A, B, and C at a near infinite variety of different temperatures. This provides a full spectrum of heat transfer control by a combination of T 1 , T 2 , and T 3  as well as coolant flow rate. 
     Referring now to  FIG. 4 , another embodiment of a thermal cycling system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral  400 . The system  400  provides thermal cycling a material  415  to be thermal cycled between different temperatures using a microfluidic heat exchanger  401  operatively positioned with respect to the material  415  to be thermal cycled. The system  400  is contained in a compact, portable microfluidic-compatible platform  420 . 
     The material  415  to be thermal cycled is contained on a microarray  416 . Examples of microarrays are shown in U.S. Pat. No. 7,354,389 for a microarray detector and methods which states, “The present invention is directed to an analytic system for detection of a plurality of analytes that are bound to a biochip, wherein an optical detector uses registration markers illuminated by a first light source to determine a focal position for detection of the analytes that are illuminated by a second light source.” U.S. Pat. No. ______ for a microarray detector and methods is incorporated herein by reference. 
     The system  400  includes the following additional structural components: microfluidic heat exchanger housing  412 , porous medium  413 , micropump  410 , lines  411 , chamber  403 , working fluid  402  at T 1 , chamber  405 , working fluid  404  at T 1 , lines  408 , multi-position valve  407 , and lines  409 . The structural components of the system  400  having been described, the operation of the system  400  will be explained. The multi-position valve  407  is actuated to provide flow of working fluid  402  at T 1  from chamber  403  to the microfluidic heat exchanger  401 . Micro pump  410  is actuated driving working fluid  402  at T 1  from chamber  403  to the microfluidic heat exchanger  401 . The working fluid  402  at T 1  passes through the porous medium  413  in the microfluidic heat exchanger  401  raising the temperature of the material to be thermalcycled  415  to temperature T 1 . The porous medium  413  in the microfluidic heat exchanger  401  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     Next the multi-position valve  407  is actuated to provide flow of working fluid  404  at T 2  from chamber  405  to the microfluidic heat exchanger  401 . Micro pump  410  is actuated driving working fluid  404  at T 2  from chamber  405  to the microfluidic heat exchanger  401 . The working fluid  402  at T 2  passes through the porous medium  413  in the microfluidic heat exchanger  401  lowering the temperature of the material to be thermalcycled  415  to temperature T 2 . 
     The heat exchanger  401  of the system  400  utilizes inlet and exit channels where heating/cooling fluid  402  and  404  is passing through, an enclosure, and microarray  416  containing the material to be thermal cycled. The heat exchanger  401  is filled with a conductive porous medium  413  of uniform porosity and permeability. In another embodiment the enclosure is filled with a conductive porous medium  413  with a gradient porosity. The nominal permeability and porosity of the porous matrix are taken as 3.74×10 −10  m 2  and 0.45, respectively. The porous medium  413  is saturated with heating/cooling fluid  402 ,  404  coming through an inlet channel. The inlet channel will be connected to hot and cold supply tanks  403  and  405 . The switching multi-position valve  407  is used to switch between hot  402  and cold tanks  405  for heating and cooling cycles. All lateral walls and top of the porous medium are insulated to minimize losses. The micropump  410  is positioned to drive the working fluids  402  and  405  directly into the microfluidic heat exchanger  401 . By positioning the micropump  410  outside the hot and cold supply tanks  403  and  405  and lines to the microfluidic heat exchanger  401  it eliminates the time the would be required to bring the micropump  410  up to the new temperature after each change. 
     Referring now to the drawings and in particular to  FIG. 5 , one embodiment of a system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral  500 . The system  500  provides extremely fast continuous flow or batch PCR amplification of target nucleic acids in a compact, portable microfluidic-compatible platform  520 . 
     The system  500  provides thermal cycling a material  515  (DNA Sample) to be thermal cycled between a temperature T 1  and T 2  using a microfluidic heat exchanger  501  operatively positioned with respect to the material  515  to be thermal cycled. A working fluid  502  at T 1  is provided and the working fluid  502  at T 1  is flowed to the microfluidic heat exchanger  501 . A working fluid  504  at T 2  is provided and the working fluid  504  at T 2  is flowed to the heat exchanger  501 . The steps of flowing the working fluid at T 1  and at T 2  to the microfluidic heat exchanger  501  are repeated for a predetermined number of times. A porous medium  513  is located in the microfluidic heat exchanger  501 . The working fluids at T 1  and T 2  flow through the porous medium  513  during the steps of flowing the working fluid at T 1  and T2 through the microfluidic heat exchanger  501 . The system  500  is contained in a compact, portable microfluidic-compatible platform  520 . 
     The material  515  to be thermal cycled is contained in droplets or microreactors  518 . Systems for thermal cycling the droplets or microreactors  518  are described and illustrated in United States Published Patent No. 2008/0166793 by Neil Reginald Beer for sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture. The disclosure of United States Published Patent No. 2008/0166793 by Neil Reginald Beer is incorporated herein by reference. The material  515  to be thermal cycled can for example be a DNA sample. The droplets or microreactors  518  are carried through a microchannel  520  in a chip  516  by a fluid  519 . The material  515  (DNA sample) is analyzed by a laser detector system  517 . The droplets or microreactors  518  are thermal cycled by the heat exchanger  501 . the heat exchanger  501  provides microfluidic polymerase chain reaction (PCR) with extremely fast cycles, and a resulting extremely fast detection time for even long amplicons (amplified nucleic acids). The system  500  allows either singly or in combination: reagent and analyte mixing; cell, virion, or capsid lysing to release the target DNA if necessary; nucleic acid amplification through the polymerase chain reaction (PCR), and nucleic acid detection and characterization through optical or other means  517 . An advantage of this system lies in its complete integration on a microfluidic platform and its extremely fast thermocycling. 
     The system  500  includes the following additional structural components: microfluidic heat exchanger housing  512 , porous medium  513 , micropump  510 , lines  508 ,  509 ,  511 , &amp;  514 , and multi position valve  507 . 
     The structural components of the system  500  having been described, the operation of the system  500  will be explained. The valve  507  is actuated to provide flow of working fluid  502  at T 1  from chamber  503  to the microfluidic heat exchanger  501 . Micro pump  510  is actuated driving working fluid  502  at T 1  from chamber  503  to the microfluidic heat exchanger  501 . The working fluid  502  at T 1  passes through the porous medium  513  in the microfluidic heat exchanger  501  raising the temperature of the material to be thermalcycled  515  to temperature T 1 . The porous medium  513  in the microfluidic heat exchanger  501  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     Next the valve  507  is actuated to provide flow of working fluid  504  at T 2  from chamber  505  to the microfluidic heat exchanger  501 . Micro pump  510  is actuated driving working fluid  504  at T 2  from chamber  505  to the microfluidic heat exchanger  501 . The working fluid  502  at T 2  passes through the porous medium  513  in the microfluidic heat exchanger  501  lowering the temperature of the material to be thermalcycled  515  to temperature T 2 . The steps of flowing the working fluid at T 1  and at T 2  to the microfluidic heat exchanger  501  are repeated for a predetermined number of times to provide the desired PCR. The porous medium  513  in the microfluidic heat exchanger  501  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     The heat exchanger  501  of the system  500  utilizes inlet and exit channels where heating/cooling fluid  502  and  504  is passing through, an enclosure, and a layer of conductive plate attached to a PCR micro-chip. The enclosure is filled with a conductive porous medium  513  of uniform porosity and permeability. In another embodiment the enclosure is filled with a conductive porous medium  513  with a gradient porosity. The nominal permeability and porosity of the porous matrix are taken as 3.74×10 −10  m 2  and 0.45, respectively. The porous medium  513  is saturated with heating/cooling fluid  502 ,  504  coming through an inlet channel. The inlet channel will be connected to hot and cold supply tanks  503  and  505 . A switching valve  507  is used to switch between hot  502  and cold tanks  505  for heating and cooling cycles. All lateral walls and top of the porous medium are insulated to minimize losses. The micropump  510  is positioned to drive the working fluids  502  and  505  directly into the microfluidic heat exchanger  501 . By positioning the micropump  510  outside the hot and cold supply tanks  503  and  505  and lines to the microfluidic heat exchanger  501  it eliminates the time the would be required to bring the micropump  510  up to the new temperature after each change. 
     Results 
     Tests and analysis were performed that provided unexpected and superior results and performance of apparatus and methods of the present invention. Some of the results and analysis of apparatus and methods of the present invention are described in the article “rapid microfluidic thermal cycler for polymerase chain reaction nucleic acid amplification,” by Shadi Mahjoob, Kambiz Vafai, and N. Reginald Beer in the  International Journal of Heat and Mass Transfer  51 (2008) 2109-2122. The “Conclusions” section of the article states, “An innovative and comprehensive methodology for rapid thermal cycling utilizing porous inserts was presented for maintaining a uniform temperature within a PCR microchip consisting of all the pertinent layers. An optimized PCR design which is widely used in molecular biology is presented for accommodating rapid transient and steady cyclic thermal management applications. Compared to what is available in the literature, the presented PCR design has a considerably higher heating/cooling temperature ramps and lower required power while resulting in a very uniform temperature distribution at the substrate at each time step. A comprehensive investigation of various pertinent parameters on physical attributes of the PCR system was presented. All pertinent parameters were considered simultaneously leading to an optimized design.” The article “rapid microfluidic thermal cycler for polymerase chain reaction nucleic acid amplification,” by Shadi Mahjoob, Kambiz Vafai, and N. Reginald Beer in the  International Journal of Heat and Mass Transfer  51 (2008) 2109-2122 is incorporated herein in it entirety by this reference. 
     The systems described above can include reprogrammable intermediate steps. The reprogrammable intermediate steps are described as follows and can be used with the systems described in connection with  FIGS. 1-8 : 
     A) With 2 tanks and the variable electronically-controlled valve, a thermal sensor upstream of the valve that is running under automated closed loop control provides the ability to adjust the ratios of the volume of flow from the T 1  and T 2  reservoirs. By adjusting these ratios ANY temperature between (and including) T 1  and T 2  are attainable. So say a thermal setpoint for T 3  is known by the user, they input T 1 , T 2 , &amp; T 3  into their keypad, PC, pendant etc and the machine can thermal cycle between T 1  and T 2  and stop at T 3  if desired. For that matter, there can be multiple different “T 3 ”s as long as they are between T 1  and T 2 . 
     B) This capability would be highly desirable for PCR since most protocols are 3-step, that is they cycle from the annealing (low) temperature (˜50 C) to an extension temperature (˜70 C) which is the temperature that the DNA polymerase enzyme performs optimally, to the high temperature (˜94 C) where the doubles strands separate. The sample is then brought back down to the anneal temp (˜50 C) and the cycle repeats. An example of the complete thermal cycling protocol, including one time reverse transcription (converts RNA to DNA) and enzyme activation (“hot start”) is given in the Experimental section (page 1855) of the publication “On-Chip Single-Copy Real-Time Reverse-Transcription PCR in Isolated Picoliter Droplets,” by N. Reginald Beer, Elizabeth K. Wheeler, Lorenna Lee-Houghton, Nicholas Watkins, Shanavaz Nasarabadi, Nicole Hebert, Patrick Leung, Don W. Arnold, Christopher G. Bailey, and Bill W. Colston in  Analytical Chemistry  Vol. 80, No. 6: Mar. 15, 2008 pages 1854-1858. The publication “On-Chip Single-Copy Real-Time Reverse-Transcription PCR in Isolated Picoliter Droplets,” by N. Reginald Beer, Elizabeth K. Wheeler, Lorenna Lee-Houghton, Nicholas Watkins, Shanavaz Nasarabadi, Nicole Hebert, Patrick Leung, Don W. Arnold, Christopher G. Bailey, and Bill W. Colston in  Analytical Chemistry  Vol. 80, No. 6: Mar. 15, 2008 pages 1854-1858 is incorporated herein by reference. 
     C) This capability also provides the ability for powering small molecule amplification that has multiple temperature steps that repeat in cycles. As time goes on, more and more of these molecular amplifications (not necessarily using DNA) will enter the art. 
     D) This also may be useful in other general chemical or complex synthesis reactions where endothermal and exothermal steps are required, such that an array or multi-well plate attached to this thermal cycler receives new reagents pipetted in (robotically or manually) at different temperatures in the repeating cycle. 
     Referring now to  FIG. 6 , another embodiment of a thermal cycling system constructed in accordance with the present invention is illustrated. The system is designated generally by the reference numeral  600 . The system  600  provides thermal cycling of a material to be thermal cycled between a temperature T 1  and T 2  using a microfluidic heat exchanger  601  operatively positioned with respect to the material  606  to be thermal cycled. The material to be thermal cycled is positioned in contact with the microfluidic heat exchanger  601  as illustrated in the previous figures. 
     A working fluid at T 1  is provided and the working fluid at T 1  is flowed to the microfluidic heat exchanger  601  through the inlet  602 . A working fluid at T 2  is provided and the working fluid at T 2  is flowed to the heat exchanger  601 . The steps of flowing the working fluid at T 1  and at T 2  to the microfluidic heat exchanger  601  are repeated for a predetermined number of times. A porous medium is located in the microfluidic heat exchanger  601 . The working fluids at T 1  and T 2  flow through the porous medium during the steps of flowing the working fluid at T 1  and T 2  through the microfluidic heat exchanger  601 . The porous medium is a porous medium of gradient permeability and porosity. The porous medium is made up of a first porous medium  603 , a second porous medium  604 , and a third porous medium  605 . The first porous medium  603 , second porous medium  604 , and third porous medium  605  have different permeability and porosity. The first porous medium  603 , second porous medium  604 , and third porous medium  605  are arrange to provide a gradient permeability and porosity. 
     The structural components of the system  600  having been described, the operation of the system  600  will be explained. A valve is actuated to provide flow of working fluid at T 1  from a chamber to the microfluidic heat exchanger  601 . A micro pump is actuated driving working fluid at T 1  from chamber to the microfluidic heat exchanger  601 . The working fluid at T 1  passes through the porous medium in the microfluidic heat exchanger  601  raising the temperature of the material to be thermalcycled to temperature T 1 . The porous medium with gradient permeability and porosity  603 ,  604 ,  605  in the microfluidic heat exchanger  601  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     Next a valve is actuated to provide flow of working fluid at T 2  from a chamber to the microfluidic heat exchanger  601 . A micro pump is actuated driving working fluid at T 2  from chamber to the microfluidic heat exchanger  601 . The working fluid at T 2  passes through the porous medium  602  in the microfluidic heat exchanger  601  lowering the temperature of the material to be thermalcycled to temperature T 2 . The steps of flowing the working fluid at T 1  and at T 2  to the microfluidic heat exchanger  601  are repeated for a predetermined number of times to provide the desired PCR. The porous medium with gradient permeability and porosity  603 ,  604 ,  605  in the microfluidic heat exchanger  601  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. 
     The aqueous channel can be used to mix various assay components (i.e., analyte, oligonucleotides, primer, TaqMan probe, etc.) in preparation for amplification and detection. The channel geometry allows for dividing the sample into multiple aliquots for subsequent analysis serially or in parallel with multiple streams. Samples can be diluted in a continuous stream, partitioned into slugs, or emulsified into droplets. Furthermore, the nucleic acids may be in solution or hybridized to magnetic beads depending on the desired assay. The scalability of the architecture allows for multiple different reactions to be tested against aliquots from the same sample. Decontamination of the channels after a series of runs can easily be performed by flushing the channels with dilute solution of sodium hypochlorite, followed by deionized water. 
     The heat exchanger  601  of the system  600  utilizes inlet and exit channels where heating/cooling fluid is passing through, an enclosure, and a layer of conductive plate attached to a PCR micro-chip or microarray. The enclosure is filled with a conductive porous medium of gradient porosity and permeability. The porous medium is saturated with heating/cooling fluid coming through an inlet channel  602 . The inlet channel will be connected to hot and cold supply tanks. A switching valve is used to switch between hot and cold tanks for heating and cooling cycles. All lateral walls and top of the porous medium are insulated to minimize losses. 
     Referring now to the drawings and in particular to  FIG. 7 , another embodiment of a system constructed in accordance with the present invention utilizing a single tank is illustrated. The system is designated generally by the reference numeral  700 . The system  700  provides extremely fast continuous flow or batch PCR amplification of target nucleic acids in a portable compact, portable microfluidic-compatible platform  720 . The system  700  provides a 1-tank version where the single tank  702  is kept at a constant temperature and is fed by a return line(s)  714  and  706  from the heat exchanger  701 . The same return line(s)  714  and  706  however feeds both the tank  702  as well as a separate tank bypass line  705 . The bypass line  705  is essentially a coil with or without heatsinks and fans blowing over it that connects to the variable valve just upstream of the chip input. By placing a thermister or thermocouple  704  upstream of the variable valve  707 , it is possible to send working fluid at T 1  or T 2  or any temperature in-between, and only requires 1 tank and heating system. 
     The material  715  to be thermal cycled is contained on a chip  718  containing the DNA. The DNA sample  715  is contained on the chip  718  containing the DNA sample. A highly conductive plate  716  connects the chip  718  to the heat exchanger  701 . Conductive grease  717  is used to provide thermal conductivity between the chip  718  and the heat exchanger  701 . Instead of conductive grease  717  between the chip  718  and the heat exchanger  701  other forms of connection may be used. For example, press-fit contact or thermally-conductive tape may be used between the chip  718  and the heat exchanger  701 . 
     The system  700  provides thermal cycling a material  715  (DNA Sample) to be thermal cycled between a temperature T 1  and T 2  or any temperature in between using a microfluidic heat exchanger  701  operatively positioned with respect to the material  715  to be thermal cycled. The steps of repeatedly flowing the working fluid at T 1  and at T 2  to the microfluidic heat exchanger  701  provide PCR fast and efficient nucleic acid analysis. The microfluidic polymerase chain reaction (PCR) thermal cycling method  700  is capable of extremely fast cycles, and a resulting extremely fast detection time for even long amplicons (amplified nucleic acids). The method  700  allows either singly or in combination: reagent and analyte mixing; cell, virion, or capsid lysing to release the target DNA if necessary; nucleic acid amplification through the polymerase chain reaction (PCR), and nucleic acid detection and characterization through optical or other means. An advantage of this system lies in its complete integration on a microfluidic platform and its extremely fast thermocycling. 
     The system  700  includes the following structural components: microfluidic heat exchanger  701 , microfluidic heat exchanger housing  712 , porous medium  713 , micropump  710 , lines  705 ,  706 ,  708 ,  709 , and  714 , multi position valve  707 , highly conductive plate  716 , thermal grease  717 , chip containing DNA sample  718 , and DNA sample  715 . 
     The structural components of the system  700  having been described, the operation of the system  700  will be explained. The valve  707  is actuated to provide flow of working fluid at T 1  from tank  702  to the microfluidic heat exchanger  701 . The system  700  provides a 1-tank version where the single tank  702  is kept at a constant temperature and is fed by a return line(s)  714  and  706  from the heat exchanger  701 . The same return line(s)  714  and  706  however feeds both the tank  702  as well as a separate tank bypass line  705 . The bypass line  705  is essentially a coil with or without heatsinks and fans blowing over it that connects to the variable valve just upstream of the chip input. By placing a thermister or thermocouple  704  upstream of the variable valve  707 , it is possible to send working fluid at T 1  or T 2  or any temperature in-between, and only requires 1 tank and heating system. 
     The porous medium  713  in the microfluidic heat exchanger  701  results in substantial surface area enhancement and increased fluid flow-path tortuosity, both of which enhance heat transfer and the resulting heat flux between the working fluid and the porous matrix. The heat exchanger  701  of the system  700  utilizes inlet and exit channels where heating/cooling fluid is passing through, an enclosure, and a layer of conductive plate attached to a PCR micro-chip. The enclosure is filled with a conductive porous medium  713  of uniform or gradient porosity and permeability. The porous medium  713  is saturated with heating/cooling fluid coming through an inlet channel. The switching valve  707  is used to switch between hot and cold for heating and cooling cycles. All lateral walls and top of the porous medium are insulated to minimize losses. The micropump  710  is positioned to drive the working fluids directly into the microfluidic heat exchanger  701 . By positioning the micropump  710  outside the hot and cold supply tanks it eliminates the time that would be required to bring the micropump  710  up to the new temperature after each change. 
     While the invention may be susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, it should be understood that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the following appended claims.