Microfluidic devices and methods of use thereof

Abstract

The present invention provides novel microfluidic substrates and methods that are useful for performing biological, chemical and diagnostic assays. The substrates can include a plurality of electrically addressable, channel bearing fluidic modules integrally arranged such that a continuous channel is provided for flow of immiscible fluids.

1 . A method of pairing sample fluids to form a droplet or nanoreactor comprising: 
 a) providing a microfluidic substrate comprising at least two inlet channels adapted to carry at least two dispersed phase sample fluids and at least one main channel adapted to carry at least one continuous phase fluid;    b) flowing a first sample fluid through a first inlet channel which is in fluid communication with said main channel at a junction, wherein said junction comprises a first fluidic nozzle designed for flow focusing such that said first sample fluid forms a plurality of highly uniform, monodisperse droplets of a first size in said continuous phase;    c) flowing a second sample fluid through a second inlet channel which is in fluid communication with said main channel at a junction, wherein said junction comprises a second fluidic nozzle designed for flow focusing such that said second sample fluid forms a plurality of highly uniform, monodisperse droplets of a second size in said continuous phase, wherein the size of the droplets of the second sample fluid are smaller than the size of the droplets of the first sample fluid;    d) providing a flow and droplet formation rate of the first and second sample fluids wherein the droplets are interdigitized such that a first sample fluid droplet is followed by and paired with a second sample fluid droplet;    e) providing channel dimensions such that the paired first sample fluid and the second sample fluid droplet are brought into proximity;    f) coalescing the paired first and second sample droplets as the paired droplets pass through an electric field, thereby producing a droplet or nanoreactor.    
     
     
         2 . The method of  claim 1 , wherein said inlet and main channels are coated with an anti-wetting or blocking agent for the dispersed phase.  
     
     
         3 . The method of  claim 2 , wherein said channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfluoropolymer, BSA, PEG-silane or fluorosilane.  
     
     
         4 . The method of  claim 3 , wherein the channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound.  
     
     
         5 . The method of  claim 1 , wherein said plurality of droplets comprises a biological/chemical material.  
     
     
         6 . The method of  claim 5 , wherein said biological/chemical material is selected from the group consisting of tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals.  
     
     
         7 . The method of  claim 5 , wherein said biological/chemical material comprises a label.  
     
     
         8 . The method of  claim 7 , wherein each biological/chemical material is labeled with a unique label.  
     
     
         9 . The method of  claim 8 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         10 . The method of  claim 7 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.  
     
     
         11 . The method of  claim 1 , wherein said continuous phase is a non-polar solvent.  
     
     
         12 . The method of  claim 1 , wherein said continuous phase is a fluorocarbon oil.  
     
     
         13 . The method of  claim 1 , wherein said continuous phase further comprises one or more additives.  
     
     
         14 . The method of  claim 13 , wherein said additive is a fluorosurfactant.  
     
     
         15 . The method of  claim 14 , wherein said fluorosurfactant is a perfluorinated polyether.  
     
     
         16 . The method of  claim 1 , wherein the flow of said dispersed phase fluid and said continuous phase fluid is pressure driven.  
     
     
         17 . The method of  claim 1 , wherein said coalescing step occurs within a coalescence module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         18 . The method of  claim 17 , wherein the coalescence module comprises one or more electrodes that generate an electric field.  
     
     
         19 . The method of  claim 1 , wherein said channel dimensions comprise an expanded portion of the main channel between the electrodes to bring successive droplets into proximity.  
     
     
         20 . The method of  claim 1 , wherein said channel dimensions comprise a narrowed portion of the main channel to center droplets within the main channel followed by a expanded portion of the main channel between the electrodes to bring successive droplets into proximity.  
     
     
         21 . The method of  claim 1 , wherein the droplets have no charge.  
     
     
         22 . The method of  claim 1 , further comprising interrogating the droplet or nanoreactor for at least one predetermined characteristic within a detection module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         23 . The method of  claim 22 , wherein said detection module comprising a detection apparatus for evaluating the contents or characteristics of the droplet or nanoreactor.  
     
     
         24 . The method of  claim 23 , wherein said detection apparatus comprises an optical or electrical detector.  
     
     
         25 . The method of  claim 1 , further comprising sorting the droplet or nanoreactor into or away from a collection module within a sorting module in fluid communication with the main channel on the microfluidic substrate in response to the contents or characterization of the droplet or nanoreactor evaluated in the detection module.  
     
     
         26 . The method of  claim 25 , wherein said sorting module comprises a sorting apparatus adapted to direct the droplet or nanoreactor into or away from the collection module.  
     
     
         27 . The method of  claim 26 , wherein the sorting apparatus comprises one or more electrodes that generate an electric field.  
     
     
         28 . The method of  claim 25 , wherein the droplets or nanoreactors have no charge.  
     
     
         29 . A method of forming a droplet emulsion library of a sample fluid comprising: 
 a) providing at least one first channel adapted to carry at least one dispersed phase sample fluid and at least one second channel adapted to carry at least one continuous phase fluid;    b) flowing said sample fluid through said first channel which is in fluid communication with said second channel at a junction, wherein said junction comprises a fluidic nozzle such that said sample fluid forms a plurality of highly uniform, monodisperse droplets of a predetermined size in said continuous phase, wherein the fluidic nozzle is isolated from the dispersed phase fluid, has a three dimensional design to permit flow focusing and eliminates surface wetting.    
     
     
         30 . A method of forming a droplet emulsion library of a sample fluid comprising: 
 a) providing a microfluidic substrate comprising at least one first channel adapted to carry at least one dispersed phase sample fluid and at least one second channel adapted to carry at least one continuous phase fluid;    b) flowing said sample fluid through said first channel which is in fluid communication with said second channel at a junction, wherein said junction comprises a fluidic nozzle such that said sample fluid forms a plurality of highly uniform, monodisperse droplets of a predetermined size in said continuous phase.    
     
     
         31 . A method of forming a droplet emulsion library of a sample fluid comprising: 
 a) providing a microfluidic substrate comprising at least one first channel adapted to carry at least one dispersed phase sample fluid and at least one channel adapted to carry at least one continuous phase fluid;    b) providing a means for storing said sample fluid wherein said storage means is in fluid communication with said first channel and provides means for introducing said sample fluid to said inlet channel, wherein the storage means contains an immiscible phase fluid with a density less than that of the sample fluid;    c) introducing the sample fluid into the storage means wherein said sample fluid flows through the less dense immiscible fluid such that the sample fluid settles at the bottom of the storage means and is subsequently introduced into the first channel;    d) flowing said sample fluid through said first channel which is in fluid communication with said second channel at a junction, wherein said junction comprises a fluidic nozzle such that said sample fluid forms a plurality of highly uniform, monodisperse droplets of a predetermined size in said continuous phase.    
     
     
         32 . A method of forming a droplet emulsion library of a sample fluid comprising: 
 a) providing a microfluidic substrate comprising at least one first channel adapted to carry at least one dispersed phase sample fluid and at least one channel adapted to carry at least one continuous phase fluid;    b) providing a means for storing said sample fluid wherein said storage means is in fluid communication with said first channel and provides means for introducing said sample fluid to said inlet channel, wherein the storage means contains an immiscible phase fluid with a density greater than that of the sample fluid;    c) inserting a sample fluid introduction apparatus into the storage means wherein said sample fluid is forced through the more dense immiscible fluid by the introduction apparatus such that the sample fluid is subsequently introduced into the first channel;    d) flowing said sample fluid through said first channel which is in fluid communication with said second channel at a junction, wherein said junction comprises a fluidic nozzle such that said sample fluid forms a plurality of highly uniform, monodisperse droplets of a predetermined size in said continuous phase.    
     
     
         33 . The method of claims  29 - 32 , wherein said channels are coated with an anti-wetting or blocking agent for the dispersed phase.  
     
     
         34 . The method of  claim 33 , wherein said channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfluoropolymer, BSA, PEG-silane or fluorosilane.  
     
     
         35 . The method of  claim 34 , wherein the channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound.  
     
     
         36 . The method of claims  29 - 32 , wherein said plurality of droplets comprises a biological/chemical material.  
     
     
         37 . The method of  claim 36 , wherein said biological/chemical material is selected from the group consisting of tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals.  
     
     
         38 . The method of  claim 36 , wherein said biological/chemical material comprises a label.  
     
     
         39 . The method of  claim 38 , wherein each biological/chemical material is labeled with a unique label.  
     
     
         40 . The method of  claim 39 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         41 . The method of  claim 38 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.  
     
     
         42 . The method of claims  29 - 32 , wherein said continuous phase is a non-polar solvent.  
     
     
         43 . The method of claims  29 - 32 , wherein said continuous phase is a fluorocarbon oil.  
     
     
         44 . The method of claims  29 - 32 , wherein said continuous phase further comprises one or more additives.  
     
     
         45 . The method of  claim 44 , wherein said additive is a fluorosurfactant.  
     
     
         46 . The method of  claim 45 , wherein said fluorosurfactant is a perfluorinated polyether.  
     
     
         47 . The method of claims  29 - 32 , wherein the flow of said dispersed phase fluid and said continuous phase fluid is pressure driven.  
     
     
         48 . The method of  claim 29 , wherein the nozzle is formed from small bore tubing or from the tip of a molded ferrule.  
     
     
         49 . The method of  claim 31  and  32 , wherein said storage means is a well or reservoir.  
     
     
         50 . The method of  claim 32 , wherein the fluid introduction apparatus is a sample tip loading pump.  
     
     
         51 . A method of forming a uniformed sized droplet emulsion library comprising: 
 a) providing a means for separating droplets of similar sizes, wherein said means comprises a periodic array of geometric parameters defining an obstacle matrix;    b) introducing at least one sample fluid containing various sized droplets to said separating means;    c) subjecting said sample fluid to laminar flow through the microscale obstacles within the separating means, wherein said sample fluids do not mix; and    d) separating and isolating uniformed sized droplets from within said sample fluid by deterministic lateral displacement.    
     
     
         52 . The method of  claim 51 , wherein the means is selected from the group consisting of a microfluidic channel, microfluidic lateral diffusion device, tube, syringe, column and capillary.  
     
     
         53 . A method for solidifying a droplet or nanoreactor comprising: 
 a) providing a microfluidic substrate comprising at least one inlet channel adapted to carry at least one dispersed phase sample fluid and at least one main channel adapted to carry at least one continuous phase fluid;    b) incorporating at least one solidifying agent within a sample fluid;    c) flowing the sample fluid through a first inlet channel which is in fluid communication with said main channel at a junction, such that said first sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    d) providing a means which activates the solidifying agent such that the droplet or nanoreactor forms a matrix.    
     
     
         54 . A method for solidifying a droplet or nanoreactor comprising: 
 a) providing a microfluidic substrate comprising at least two inlet channels adapted to carry at least two dispersed phase sample fluids and at least one main channel adapted to carry at least one continuous phase fluid;    b) incorporating at least one solidifying agent within at least one sample fluid;    c) flowing a first sample fluid through a first inlet channel which is in fluid communication with said main channel at a junction, such that said first sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    d) flowing a second sample fluid through a second inlet channel which is in fluid communication with said main channel at a junction, such that said second sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    e) coalescing at least one droplet formed in step (c) with at least one droplet formed in step (d) as the droplets pass through an electric field, thereby producing a droplet or nanoreactor; and    f) providing a means which activates the solidifying agent such that the droplet or nanoreactor forms a matrix.    
     
     
         55 . The method of  claim 54 , wherein said inlet and main channels are coated with an anti-wetting or blocking agent for the dispersed phase.  
     
     
         56 . The method of  claim 55 , wherein said channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfluoropolymer, BSA, PEG-silane or fluorosilane.  
     
     
         57 . The method of  claim 56 , wherein the channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound.  
     
     
         58 . The method of  claim 54 , wherein said plurality of droplets comprises a biological/chemical material.  
     
     
         59 . The method of  claim 58 , wherein said biological/chemical material is selected from the group consisting of tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals.  
     
     
         60 . The method of  claim 58 , wherein said biological/chemical material comprises a label.  
     
     
         61 . The method of  claim 60 , wherein each biological/chemical material is labeled with a unique label.  
     
     
         62 . The method of  claim 61 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         63 . The method of  claim 60 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.  
     
     
         64 . The method of  claim 54 , wherein said continuous phase is a non-polar solvent.  
     
     
         65 . The method of  claim 54 , wherein said continuous phase is a fluorocarbon oil.  
     
     
         66 . The method of  claim 54 , wherein said continuous phase further comprises one or more additives.  
     
     
         67 . The method of  claim 66 , wherein said additive is a fluorosurfactant.  
     
     
         68 . The method of  claim 67 , wherein said fluorosurfactant is a perfluorinated polyether.  
     
     
         69 . The method of  claim 54 , wherein the flow of said dispersed phase fluid and said continuous phase fluid is pressure driven.  
     
     
         70 . The method of  claim 54 , wherein said coalescing step occurs within a coalescence module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         71 . The method of  claim 70 , wherein the coalescence module comprises one or more electrodes that generate an electric field.  
     
     
         72 . The method of  claim 70 , wherein said coalescence module comprises an expanded portion of the main channel between the electrodes to bring successive droplets into proximity, whereby the paired droplets are coalesced within the electric field.  
     
     
         73 . The method of  claim 70 , wherein said coalescence module comprises a narrowed portion of the main channel to center droplets within the main channel followed by a expanded portion of the main channel between the electrodes to bring successive droplets into proximity, whereby the paired droplets are coalesced within the electric field.  
     
     
         74 . The method of  claim 54 , wherein the droplets have no charge.  
     
     
         75 . The method of  claim 54 , further comprising interrogating the droplet or nanoreactor for at least one predetermined characteristic within a detection module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         76 . The method of  claim 75 , wherein said detection module comprising a detection apparatus for evaluating the contents or characteristics of the droplet or nanoreactor.  
     
     
         77 . The method of  claim 76 , wherein said detection apparatus comprises an optical or electrical detector.  
     
     
         78 . The method of  claim 54 , further comprising sorting the droplet or nanoreactor into or away from a collection module within a sorting module in fluid communication with the main channel on the microfluidic substrate in response to the contents or characterization of the droplet or nanoreactor evaluated in the detection module.  
     
     
         79 . The method of  claim 78 , wherein said sorting module comprises a sorting apparatus adapted to direct the droplet or nanoreactor into or away from the collection module.  
     
     
         80 . The method of  claim 79 , wherein the sorting apparatus comprises one or more electrodes that generate an electric field.  
     
     
         81 . The method of  claim 78 , wherein the droplets or nanoreactors have no charge.  
     
     
         82 . The method of  claim 54 , wherein the solidifying agent is a low temperature agarose or a polymerizing solution.  
     
     
         83 . The method of  claim 54 , wherein the means for activating the solidifying agent is a physical or chemical means.  
     
     
         84 . A method for introducing sample fluid to a microfluidic substrate comprising: 
 a) providing a microfluidic substrate comprising at least one inlet channel adapted to carry at least one dispersed phase sample fluid and at least one main channel adapted to carry at least one continuous phase fluid;    b) providing a means for storing said sample fluid, wherein said storage means is in fluid communication with said inlet channel and provides a means for introducing said sample fluid into said inlet channel;    c) combining the sample fluid with at least one immiscible phase fluid within the storage means, wherein the immiscible phase fluid has a density different from that of the sample fluid such that the fluids separate into distinct layers;    d) providing a force such that the immiscible phase fluid forces the sample fluid completely into the inlet channel of the microfluidic substrate.    
     
     
         85 . A method for introducing sample fluid to a microfluidic substrate comprising: 
 a) providing a microfluidic substrate comprising at least one inlet channel adapted to carry at least one dispersed phase sample fluid and at least one main channel adapted to carry at least one continuous phase fluid;    b) providing a means for storing said sample fluid, wherein said storage means is in fluid communication with said inlet channel and provides a means for introducing said sample fluid into said inlet channel;    c) combining the sample fluid with at least two immiscible phase fluids within the storage means, wherein a first immiscible phase fluid has a density greater than that of the sample fluid and a second immiscible phase fluid has a density less than that of the sample fluid such that the fluids separate into distinct layers with the sample fluid layer residing between the two immiscible phase layers;    d) providing a force such that the more dense immiscible phase fluid forces the less dense immiscible phase fluid and the sample fluid completely into the inlet channel of the microfluidic substrate.    
     
     
         86 . The method of claims  84  and  85 , wherein said inlet and main channels are coated with an anti-wetting or blocking agent for the dispersed phase.  
     
     
         87 . The method of  claim 86 , wherein said channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfluoropolymer, BSA, PEG-silane or fluorosilane.  
     
     
         88 . The method of  claim 87 , wherein the channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound.  
     
     
         89 . The method of claims  84  and  85 , wherein said plurality of sample fluid comprises a biological/chemical material.  
     
     
         90 . The method of  claim 89 , wherein said biological/chemical material is selected from the group consisting of tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals.  
     
     
         91 . The method of  claim 89 , wherein said biological/chemical material comprises a label.  
     
     
         92 . The method of  claim 89 , wherein each biological/chemical material is labeled with a unique label.  
     
     
         93 . The method of  claim 91 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         94 . The method of  claim 91 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.  
     
     
         95 . The method of claims  84  and  85 , wherein said continuous phase is a non-polar solvent.  
     
     
         96 . The method of claims  84  and  85 , wherein said continuous phase is a fluorocarbon oil.  
     
     
         97 . The method of claims  84  and  85 , wherein said continuous phase further comprises one or more additives.  
     
     
         98 . The method of  claim 97 , wherein said additive is a fluorosurfactant.  
     
     
         99 . The method of  claim 98 , wherein said fluorosurfactant is a perfluorinated polyether.  
     
     
         100 . The method of claims  84  and  85 , wherein the flow of said dispersed phase fluid and said continuous phase fluid is pressure driven.  
     
     
         101 . The method of claims  84  and  85 , wherein said storage means is a well or reservoir.  
     
     
         102 . The method of claims  84  and  85 , wherein said immiscible fluid is biologically and/or chemically inert.  
     
     
         103 . A method of extracting biological or chemical material from within a droplet or nanoreactor comprising: 
 a) providing one or more droplets or nanoreactors formed within a microfluidic substrate;    b) combining the droplets or nanoreactors with a first immiscible fluid with a density greater than that of the droplets or nanoreactors such that said droplets or nanoreactors and immiscible fluid form separate layers;    c) removing the droplet or nanoreactor layer formed in step (b);    d) combining and mixing the droplet or nanoreactor layer with a second immiscible fluid comprising a destabilizing surfactant such that the droplets or nanoreactors and immiscible fluid form separate layers; and    e) removing the resulting aqueous layer formed step (d), thereby extracting the biological or chemical material from within the droplet or nanoreactor.    
     
     
         104 . The method of  claim 103 , wherein said immiscible fluid is biologically and/or chemically inert.  
     
     
         105 . The method of  claim 103 , wherein said destabilizing surfactant is a perfluorinated alcohol.  
     
     
         106 . The method of  claim 103 , wherein the droplets or nanoreactors reside on top of the immiscible fluids within said separate layers.  
     
     
         107 . The method of  claim 103 , wherein said biological/chemical material comprises a label.  
     
     
         108 . The method of  claim 107 , wherein each biological/chemical material is labeled with a unique label.  
     
     
         109 . The method of  claim 107 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         110 . The method of  claim 107 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.  
     
     
         111 . A method of amplifying DNA comprising: 
 a) providing a microfluidic substrate comprising at least one inlet channel adapted to carry at least one dispersed phase sample fluid and at least one main channel adapted to carry at least one continuous phase fluid, wherein the main channel comprises a serpentine line with heating and cooling regions;    b) flowing a sample fluid comprising one more target DNA molecules to be amplified, PCR primer pair sets, dNTPs, enzymes and buffer components effective to permit PCR amplification through an inlet channel which is in fluid communication with said main channel at a junction, such that said sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase, wherein said junction is heated to 95° C. to provide a hot start, wherein a portion of at least one of the PCR primer pair sets is attached to a semi-solid substrate;    c) reacting the contents of the droplets for at least twenty heating and cooling cycles to permit the PCR amplification of the target DNA molecules,    such that the amplified target DNA molecules are attached to a semi-solid substrate.    
     
     
         112 . A method of amplifying DNA comprising: 
 a) providing a microfluidic substrate comprising at least two inlet channels adapted to carry at least two dispersed phase sample fluids and at least one main channel adapted to carry at least one continuous phase fluid, wherein the main channel comprises a serpentine line with heating and cooling regions;    b) flowing a first sample fluid comprising one more target DNA molecules to be amplified through a first inlet channel which is in fluid communication with said main channel at a junction, such that said first sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    c) flowing a second sample fluid comprising PCR primer pair sets, dNTPs, enzymes and buffer components effective to permit PCR amplification through a second inlet channel which is in fluid communication with said main channel at a junction, such that said second sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase, wherein said junction is heated to 95° C. to provide a hot start and wherein a portion of at least one of the PCR primer pair sets is attached to a semi-solid substrate;    d) coalescing the droplets comprising the DNA molecules from step (b) with the droplets comprising the PCR primer pair sets, dNTPs, enzymes and buffer components from step (c) within an electric field;    e) reacting the contents of the droplets for at least twenty heating and cooling cycles to permit the PCR amplification of the target DNA molecules,    such that the amplified target DNA molecules are attached to a semi-solid substrate.    
     
     
         113 . A method of amplifying DNA comprising: 
 a) providing a microfluidic substrate comprising at least one inlet channel adapted to carry at least one dispersed phase sample fluid and at least one main channel adapted to carry at least one continuous phase fluid, wherein the main channel comprises a serpentine line with heating and cooling regions;    b) flowing a sample fluid comprising one more target DNA molecules to be sequenced, primer pair sets, dNTPs, enzymes and buffer components effective to permit isothermal amplification through an inlet channel which is in fluid communication with said main channel at a junction, such that said sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase, wherein a portion of at least one of the primer pair sets is attached to a semi-solid substrate;    c) reacting the contents of the droplets to permit the isothermal amplification of the target DNA molecules,    such that the amplified target DNA molecules are attached to a semi-solid substrate.    
     
     
         114 . A method of amplifying DNA comprising: 
 a) providing a microfluidic substrate comprising at least two inlet channels adapted to carry at least two dispersed phase sample fluids and at least one main channel adapted to carry at least one continuous phase fluid, wherein the main channel comprises a serpentine line with heating and cooling regions;    b) flowing a first sample fluid comprising one more target DNA molecules to be amplified through a first inlet channel which is in fluid communication with said main channel at a junction, such that said first sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    c) flowing a second sample fluid comprising primer pair sets, dNTPs, enzymes and buffer components effective to permit isothermal amplification through a second inlet channel which is in fluid communication with said main channel at a junction, such that said second sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase, wherein a portion of at least one of the primer pair sets is attached to a semi-solid substrate;    d) coalescing the droplets comprising the DNA molecules from step (b) with the droplets comprising the primer pair sets, dNTPs, enzymes and buffer components from step (c) within an electric field;    e) reacting the contents of the droplets for at least twenty heating and cooling cycles to permit the isothermal amplification of the target DNA molecules,    such that the amplified target DNA molecules are attached to a semi-solid substrate.    
     
     
         115 . A method of sequencing DNA comprising: 
 a) providing a microfluidic substrate comprising at least two inlet channels adapted to carry at least two dispersed phase sample fluids and at least one main channel adapted to carry at least one continuous phase fluid, wherein the main channel comprises a serpentine line with heating and cooling regions;    b) flowing a first sample fluid comprising one more target DNA molecules to be sequenced, PCR primer pair sets, dNTPs, enzymes and buffer components effective to permit PCR amplification through a first inlet channel which is in fluid communication with said main channel at a junction, such that said first sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase, wherein said junction is heated to 95° C. to provide a hot start;    c) reacting the contents of the droplets for at least twenty heating and cooling cycles to permit the PCR amplification of the target DNA molecules;    d) flowing a second sample fluid comprising shrimp alkaline phosphatase and exonuclease I through a second inlet channel which is in fluid communication with said main channel at a junction, such that said second sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    e) coalescing the droplets comprising the amplified DNA molecules from step (c) with the droplets comprising the shrimp alkaline phosphatase and exonuclease I from step (d) within an electric field and reacting the contents of the combined droplets at 37° C.;    f) inactivating the enzymes within the droplets to terminate the reaction from step (e) by heating the reacted droplets to 95° C.;    g) flowing a third sample fluid comprising universal sequencing primers, labeled ddNTPs and buffer effective to permit nucleotide sequencing through a third inlet channel which is in fluid communication with said main channel at a junction, such that said third sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    h) coalescing the droplets comprising the amplified DNA molecules from step (f) with the droplets comprising the universal sequencing primers, labeled ddNTPs and sequencing buffer from step (g) within an electric field;    i) reacting the contents of the coalesced droplets for at least twenty heating and cooling cycles to permit the sequencing reaction to proceed; and    j) analyzing the sequencing reaction to determine the nucleic acid sequence of the target DNA.    
     
     
         116 . A method of sequencing DNA comprising: 
 a) providing a microfluidic substrate comprising at least two inlet channels adapted to carry at least two dispersed phase sample fluids and at least one main channel adapted to carry at least one continuous phase fluid, wherein the main channel comprises a serpentine line with heating and cooling regions;    b) flowing a first sample fluid comprising one more target DNA molecules to be sequenced, primer pair sets, dNTPs, enzymes and buffer components effective to permit isothermal amplification through a first inlet channel which is in fluid communication with said main channel at a junction, such that said first sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    c) reacting the contents of the droplets to permit the isothermal amplification of the target DNA molecules;    d) flowing a second sample fluid comprising shrimp alkaline phosphatase and exonuclease I through a second inlet channel which is in fluid communication with said main channel at a junction, such that said second sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    e) coalescing the droplets comprising the amplified DNA molecules from step (c) with the droplets comprising the shrimp alkaline phosphatase and exonuclease I from step (d) within an electric field and reacting the contents of the combined droplets at 37° C.;    f) inactivating the enzymes within the droplets to terminate the reaction from step (e) by heating the reacted droplets to 95° C.;    g) flowing a third sample fluid comprising universal sequencing primers, labeled ddNTPs and buffer effective to permit nucleotide sequencing through a third inlet channel which is in fluid communication with said main channel at a junction, such that said third sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    h) coalescing the droplets comprising the amplified DNA molecules from step (f) with the droplets comprising the universal sequencing primers, labeled ddNTPs and sequencing buffer from step (g) within an electric field;    i) reacting the contents of the coalesced droplets for at least twenty heating and cooling cycles to permit the sequencing reaction to proceed; and    j) analyzing the sequencing reaction to determine the nucleic acid sequence of the target DNA.    
     
     
         117 . The method of claims  111 - 116 , wherein said inlet and main channels are coated with an anti-wetting or blocking agent for the dispersed phase.  
     
     
         118 . The method of  claim 117 , wherein said channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfluoropolymer, BSA, PEG-silane or fluorosilane.  
     
     
         119 . The method of  claim 118 , wherein the channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound.  
     
     
         120 . The method of claims  111 - 116 , wherein each ddNTP is labeled with a unique label.  
     
     
         121 . The method of claims  111 - 116 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         122 . The method of  claim 121 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.  
     
     
         123 . The method of claims  111 - 116 , wherein said continuous phase is a non-polar solvent.  
     
     
         124 . The method of claims  111 - 116 , wherein said continuous phase is a fluorocarbon oil.  
     
     
         125 . The method of claims  111 - 116 , wherein said continuous phase further comprises one or more additives.  
     
     
         126 . The method of  claim 125 , wherein said additive is a fluorosurfactant.  
     
     
         127 . The method of  claim 126 , wherein said fluorosurfactant is a perfluorinated polyether.  
     
     
         128 . The methods of claims  115  and  116 , wherein a portion of at least one of the primer pair sets is attached to a semi-solid substrate.  
     
     
         129 . The method of claims  111 - 116 , wherein the flow of said dispersed phase fluid and said continuous phase fluid is pressure driven.  
     
     
         130 . The method of claims  102  and  114 - 116 , wherein at least one coalescing step occurs within a coalescence module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         131 . The method of  claim 130 , wherein the coalescence module comprises one or more electrodes that generate an electric field.  
     
     
         132 . The method of  claim 130  wherein said coalescence module comprises an expanded portion of the main channel between the electrodes to bring successive droplets into proximity.  
     
     
         133 . The method of  claim 130  wherein said coalescence module comprises a narrowed portion of the main channel to center droplets within the main channel followed by an expanded portion of the main channel between the electrodes to bring successive droplets into proximity.  
     
     
         134 . The method of claims  112  and  114 - 116 , wherein the droplets have no charge.  
     
     
         135 . The method of claims  115  and  116 , wherein analysis of the sequence reaction within the droplets occurs within a detection module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         136 . The method of  claim 135 , wherein said detection module comprising a detection apparatus for analyzing the sequence reactor within the droplets.  
     
     
         137 . The method of  claim 136 , wherein said detection apparatus comprises an optical or electrical detector.  
     
     
         138 . The method of claims  111 - 116 , wherein the junction between the inlet channel and main channel comprises a fluidic nozzle for flow focusing.  
     
     
         139 . A method of detecting a single nucleotide polymorphism (SNP) comprising: 
 a) providing a microfluidic substrate comprising at least one inlet channel adapted to carry at least one dispersed phase sample fluid and at least one main channel adapted to carry at least one continuous phase fluid, wherein the main channel comprises a serpentine line with heating and cooling regions;    b) flowing a sample fluid comprising one more target DNA molecules to be analyzed, labeled PCR primer pair sets, dNTPs, enzymes and buffer components effective to permit PCR amplification through an inlet channel which is in fluid communication with said main channel at a junction, such that said sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase, wherein said junction is heated to 95° C. to provide a hot start;    c) reacting the contents of the droplets for at least twenty heating and cooling cycles to permit the PCR amplification of the target DNA molecules; and    d) analyzing the reaction to determine the presence of absence of a SNP.    
     
     
         140 . The method of  claim 139 , wherein said inlet and main channels are coated with an anti-wetting or blocking agent for the dispersed phase.  
     
     
         141 . The method of  claim 140 , wherein said channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfluoropolymer, BSA, PEG-silane or fluorosilane.  
     
     
         142 . The method of  claim 141 , wherein the channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound.  
     
     
         143 . The method of  claim 139 , wherein each primer is labeled with a unique label.  
     
     
         144 . The method of  claim 143 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         145 . The method of  claim 139 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.  
     
     
         146 . The method of  claim 139 , wherein said continuous phase is a non-polar solvent.  
     
     
         147 . The method of  claim 139 , wherein said continuous phase is a fluorocarbon oil.  
     
     
         148 . The method of  claim 139 , wherein said continuous phase further comprises one or more additives.  
     
     
         149 . The method of  claim 148 , wherein said additive is a fluorosurfactant.  
     
     
         150 . The method of  claim 149 , wherein said fluorosurfactant is a perfluorinated polyether.  
     
     
         151 . The methods of  claim 139 , wherein a portion of at least one of the primer pair sets is attached to a semi-solid substrate.  
     
     
         152 . The method of  claim 139 , wherein the flow of said dispersed phase fluid and said continuous phase fluid is pressure driven.  
     
     
         153 . The method of  claim 139 , wherein SNP analysis within the droplets occurs within a detection module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         154 . The method of  claim 153 , wherein said detection module comprising a detection apparatus for analyzing the sequence reactor within the droplets.  
     
     
         155 . The method of  claim 154 , wherein said detection apparatus comprises an optical or electrical detector.  
     
     
         156 . The method of  claim 139 , wherein the junction between the inlet channel and main channel comprises a fluidic nozzle for flow focusing.  
     
     
         157 . A method of forming enzyme emulsions comprising: 
 a) providing a microfluidic substrate comprising at least one inlet channel adapted to carry at least one dispersed phase fluid and at least one main channel adapted to carry at least one continuous phase fluid;    b) flowing a sample fluid comprising at least one cell through an inlet channel which is in fluid communication with said main channel at a junction, such that said sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase, wherein said cells comprises at least one enzyme which can be secreted by said cells;    c) incubating the cells within the plurality of droplets such that the cells secrete at least one enzyme into the droplet,    wherein the cells within the droplets can be incubated within or outside of the microfluidic substrate.    
     
     
         158 . A method of detecting enzyme activity comprising: 
 a) providing a microfluidic substrate comprising at least one inlet channel adapted to carry at least one dispersed phase sample fluid and at least one main channel adapted to carry at least one continuous phase fluid;    b) flowing a sample fluid comprising at least one cell and at least one labeled enzyme substrate through a inlet channel which is in fluid communication with said main channel at a junction, such that said sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase wherein said cells comprises at least one enzyme which can be secreted by said cells;    c) collecting the droplets and incubating them on the microfluidic substrate or off the microfluidic substrate at a temperature and duration appropriate to permit the enzyme/substrate reaction to occur;    d) reintroducing the droplets onto the microfluidic substrate if said droplets removed from the microfluidic substrate in step (c);    e) analyzing the contents of the coalesced droplets using the detection module to detect the presence or absence of an enzyme/substrate reaction; and,    f) selecting the droplets which contain the presence of an enzyme/substrate reaction.    
     
     
         159 . A method of detecting enzyme activity comprising: 
 a) providing a microfluidic substrate comprising at least two inlet channels adapted to carry at least two dispersed phase sample fluids and at least one main channel adapted to carry at least one continuous phase fluid, wherein the main channel comprises a serpentine line with heating and cooling regions;    b) flowing a first sample fluid comprising at least one cell through a first inlet channel which is in fluid communication with said main channel at a junction, such that said first sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase, wherein said cells comprises at least one enzyme which can be secreted by said cells;    c) flowing a second sample fluid comprising at least one labeled enzyme substrate through a second inlet channel which is in fluid communication with said main channel at a junction, such that said second sample fluid forms a plurality of highly uniform, monodisperse droplets in said continuous phase;    d) coalescing the droplets comprising the secreted enzymes from step (b) with the droplets comprising the labeled substrate from step (c) within an electric field;    e) analyzing the contents of the coalesced droplets using a detection module to detect the presence or absence of an enzyme/substrate reaction; and,    f) selecting the coalesced droplets which contain the presence of an enzyme/substrate reaction.    
     
     
         160 . The method of  claim 159 , further comprising incubating the cells within the plurality of droplets from step (b) such that the cells secrete at least one enzyme into the droplet, prior to flowing the second sample fluid in step (c).  
     
     
         161 . The method of  claim 160 , wherein the cells are incubated within the plurality of droplets within the microfluidic substrate.  
     
     
         162 . The method of  claim 160 , wherein the cells are incubated within the plurality of droplets outside the microfluidic substrate.  
     
     
         163 . The method of  claim 159 , further comprising lysing the cells within the plurality of droplets from step (b) such that the cells release at least one enzyme into the plurality droplet, prior to flowing the second sample fluid in step (c).  
     
     
         164 . The method of  claim 159 , further comprising selecting the coalesced droplets which contain the enzyme with the highest level of activity for the labeled substrate.  
     
     
         165 . The method of  claim 159 , wherein said inlet and main channels are coated with an anti-wetting or blocking agent for the dispersed phase.  
     
     
         166 . The method of  claim 165 , wherein said channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfluoropolymer, BSA, PEG-silane or fluorosilane.  
     
     
         167 . The method of  claim 166 , wherein the channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound.  
     
     
         168 . The method of  claim 159 , wherein each enzyme substrate is labeled with a unique label.  
     
     
         169 . The method of  claim 159 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         170 . The method of  claim 159 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.  
     
     
         171 . The method of  claim 159 , wherein said continuous phase is a non-polar solvent.  
     
     
         172 . The method of  claim 159 , wherein said continuous phase is a fluorocarbon oil.  
     
     
         173 . The method of  claim 159 , wherein said continuous phase further comprises one or more additives.  
     
     
         174 . The method of  claim 173 , wherein said additive is a fluorosurfactant.  
     
     
         175 . The method of  claim 174 , wherein said fluorosurfactant is a perfluorinated polyether.  
     
     
         176 . The method of  claim 159 , wherein the flow of said dispersed phase fluid and said continuous phase fluid is pressure driven.  
     
     
         177 . The method of  claim 159 , wherein said coalescing step occurs within a coalescence module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         178 . The method of  claim 177 , wherein the coalescence module comprises one or more electrodes that generate an electric field.  
     
     
         179 . The method of  claim 177  wherein said coalescence module comprises an expanded portion of the main channel between the electrodes to bring successive droplets into proximity.  
     
     
         180 . The method of  claim 177  wherein said coalescence module comprises a narrowed portion of the main channel to center droplets within the main channel followed by a expanded portion of the main channel between the electrodes to bring successive droplets into proximity.  
     
     
         181 . The method of  claim 159 , wherein the droplets have no charge.  
     
     
         182 . The method of  claim 159 , wherein analysis of the enzyme/substrate reaction within the droplets occurs within a detection module in fluid communication with the main channel on the microfluidic substrate.  
     
     
         183 . The method of  claim 182 , wherein said detection module comprising a detection apparatus for analyzing the sequence reactor within the droplets.  
     
     
         184 . The method of  claim 183 , wherein said detection apparatus comprises an optical or electrical detector.  
     
     
         185 . The method of  claim 159 , wherein the junction between the inlet channel and main channel comprises a fluidic nozzle for flow focusing.  
     
     
         186 . A method of detecting an aqueous solution comprising introducing a label into the aqueous solution such that said label alters a physical property of said aqueous solution thereby permitting the detection of aqueous solution comprising said label.  
     
     
         187 . A method of tracking an aqueous solution comprising introducing a label into the aqueous solution such that said label alters a physical property of said aqueous solution thereby permitting the tracking of aqueous solution comprising said label.  
     
     
         188 . The method of claims  186  and  187 , wherein said label is a change in viscosity, a change in opacity, a change in volume, a change in density, a change in pH, a change in temperature, a change in dielectric constant, a change in conductivity, a change in the amount of beads present in the solution, a change in the amount of flocculent in the solution, a change in the amount of a selected solvent within the solution or the change in the amount of any measurable entity within the solution, or combinations thereof.  
     
     
         189 . The method of claims  186  and  187 , wherein said label is an inducible label.  
     
     
         190 . The method of  claim 189 , wherein said inducible label is detectable when combined with at least one additional solution wherein said solution contains at least one entity to which can alter a property of the label such that the aqueous solution comprising said label is detectable.  
     
     
         191 . The method of claims  186  and  187 , wherein said labeled aqueous solution is a labeled aqueous droplet.  
     
     
         192 . The method of  claim 191 , wherein said labeled aqueous droplet can be detected within a microfluidic substrate.  
     
     
         193 . The method of claims  186  and  187 , wherein said labels alter the light scattering properties of the aqueous solution.  
     
     
         194 . The method of claims  186  and  187 , wherein said aqueous solution further comprises a biological/chemical material.  
     
     
         195 . The method of  claim 194 , wherein said biological/chemical material is selected from the group consisting of tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals.  
     
     
         196 . The method of  claim 194 , wherein said biological/chemical material comprises a label.  
     
     
         197 . The method of  claim 196 , wherein each biological/chemical material is labeled with a unique label.  
     
     
         198 . The method of  claim 196 , wherein said label is a protein, a DNA tag, a dye, a quantum dot or a radio frequency identification tag.  
     
     
         199 . The method of  claim 196 , wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.