METHODS AND SYSTEMS FOR DETECTING BIOLOGICAL COMPONENTS

• US 20150232942A1
• Filed: 08/12/2013
• Published: 08/20/2015
• Est. Priority Date: 08/13/2012
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Abate, Adam R., Eastbum, Dennis Jay, Sciambi, Adam R.
• Granted Patent: US 10,161,007 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 25/4335 Mixers with a converging-diverging cross-section
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C12Q 2600/118 : Prognosis of disease development
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays

Abstract

Methods for the detection of components from biological samples are provided. In certain aspects, the methods may be used to detect and/or quantify specific components in a biological sample, such as tumor cells (e.g., circulating tumor cells). Systems and devices for practicing the subject methods are also provided.

Claims

1. A method for the detection of cells, the method comprising:

encapsulating in a microdroplet a cell obtained from a biological sample from a subject, wherein at least one cell is present in the microdroplet;

incubating the microdroplet under conditions effective for cell lysis;

introducing polymerase chain reaction (PCR) reagents, a detection component, and a plurality of PCR primers into the microdroplet and incubating the microdroplet under conditions allowing for PCR amplification to produce PCR amplification products, wherein the plurality of PCR primers comprise one or more primers that each hybridize to one or more oligonucleotides; and

detecting the presence or absence of the PCR amplification products by detection of the detection component, wherein detection of the detection component indicates the presence of PCR amplification products;

wherein one or more steps are performed under microfluidic control.

2. The method according to claim 1, wherein incubating the microdroplet under conditions effective for cell lysis comprises introducing a lysing agent into the microdroplet.

3. The method according to claim 1 or 2, wherein the one or more oligonucleotides are oncogenes.

4. The method according to any of claims 1-3, wherein the biological sample is blood and the method comprises determining the number of circulating tumor cells (CTCs) present in the sample of the subject'"'"'s blood based at least in part on the number of microdroplets in which PCR amplification products were detected.

5. The method according to any of claims 1-4, wherein all steps are performed under microfluidic control.

6. The method according to claim 5, wherein all steps are performed on the same microfluidic device.

7. The method according to any of the above claims, wherein the plurality of PCR primers comprises 10 or more primers.

8. The method according to any of the above claims, wherein the plurality of PCR primers comprises 20 to 100 primers.

9. The method according to any of the above claims, wherein the plurality of PCR primers comprises primers for 10 or more oncogenes.

10. The method according to any of the above claims, wherein incubating the microdroplet under conditions allowing for PCR amplification is performed on the same microfluidic device used to encapsulate the cells and lyse the cells.

11. The method according to any of the above claims, wherein the PCR reagents and PCR primers are added at the same time as the lysing agent.

12. The method according to any of the above claims, wherein the PCR reagents are added in two steps or more.

13. The method according to any of the above claims, further comprising introducing a probe into the microdroplet.

14. The method according to claim 13, wherein the probe is introduced prior to incubating the microdroplet under conditions allowing for PCR amplification.

15. The method according to claim 13 or 14, wherein the probe is a TaqMan®

probe.

16. The method according to any of the above claims, wherein a reagent is added to the microdroplet by merging the microdroplet with a second microdroplet comprising the reagent.

17. The method according to any of the above claims, wherein a reagent is added to the microdroplet using either droplet coalescence or picoinjection.

18. The method according to any of the above claims, wherein a reagent is added to the microdroplet by a method comprising:

d) emulsifying the reagent into a stream of droplets, wherein the droplets are smaller than the size of the microdroplet;

e) flowing the droplets together with the microdroplet; and

f) merging a droplet with the microdroplet.

19. The method according to claim 18, wherein the diameter of the droplets is 25% or less than that of the diameter of the microdroplet, and a plurality of droplets are merged with the microdroplet.

20. The method according to claim 18 or 19, wherein the merging comprises applying an electric field.

21. The method according to any of the above claims, wherein a reagent is added to the microdroplet by a method comprising:

d) jetting the reagent into a fluid jet;

e) flowing the fluid jet alongside the microdroplet; and

f) merging a droplet with the microdroplet.

22. The method according to claim 21, wherein merging comprises applying an electric field.

23. The method according to claim 21 or 22, wherein jetting the reagent comprises adding a viscosity-increasing agent or surfactant.

24. The method according to any of the above claims, wherein a reagent is added to the microdroplet by a method comprising using a fluid injected into the microdroplet as an electrode.

25. The method according to any of the above claims, wherein the detection component is detected based on a change in fluorescence.

26. The method according to claim 25, where in the change in fluorescence is due to fluorescence resonance energy transfer (FRET).

27. The method according to claim 25, where in the change in fluorescence is due to fluorescence polarization.

28. The method according to claim 25 or 27, wherein the detection component is an intercalating stain.

29. The method according to any of the above claims, wherein detecting the presence or absence of the PCR amplification products comprises repeatedly imaging the microdroplet.

30. The method according to claim 29, wherein the microdroplet is repeatedly imaged while the microdroplet is subjected to conditions allowing for PCR amplification to produce the PCR amplification products.

31. The method according to any of the above claims, wherein incubating the microdroplet under conditions allowing for PCR amplification and detecting the presence or absence of the PCR amplification products are performed on a Megadroplet Array.

32. The method according to any of the above claims, comprising sorting a microdroplet.

33. The method according to claim 32, wherein the sorting comprises using membrane valves, bifurcating channels, surface acoustic waves, or dielectrophoresis.

34. The method according to claim 32 or 33, wherein the microdroplet is sorted based on a property comprising size, viscosity, mass, buoyancy, surface tension, electrical conductivity, charge, or magnetism.

35. The method according to any of claims 32-34, comprising sorting based at least in part based upon the detection of the presence or absence of PCR amplification products.

36. The method according to any of claims 32-35, wherein the microdroplet is sorted prior to the introduction of a PCR reagent.

37. The method according to any of claims 32-36, wherein the microdroplet is sorted prior to the introduction of a lysing agent.

38. The method according to any of the above claims, further comprising:

injecting a diluent into the microdroplet; and

flowing the microdroplet through a microfluidic channel on which an electric field is being applied, under conditions in which the microdroplet is split.

39. The method according to any of the above claims, wherein the subject is mammalian.

40. The method according to any of the above claims, wherein the subject is human.

41. The method according to any of the above claims, wherein the subject has been diagnosed with cancer.

42. The method according to any of the above claims, wherein the biological sample is a blood sample.

43. The method of claim 42, wherein the blood sample is whole blood.

44. The method of claim 42 or 43, comprising fractionating the blood sample.

45. The method of any one of claims 42-44, comprising drawing 30 mL or less of the subject'"'"'s blood.

46. The method of claim 45, wherein the blood sample is 15 mL or less.

47. The method of any one of the above claims, comprising fixing and/or permeabilizing the cell.

48. The method of any one of the above claims, comprising introducing a plurality of different detection components, and detecting the presence or absence of the PCR amplification products by detection of the plurality of detection components, wherein detection of the detection components indicates the presence of PCR amplification products.

49. The method of any one of the above claims, comprising contacting the cell or a component thereof with a detectably labeled antibody.

136. The method of any one of claims 1-20, wherein a reagent is added to the microdroplet by:

contacting the microdroplet with oil so that the oil encapsulates the microdroplet to form a double emulsion;

contacting the double emulsion with a drop containing the reagent so that the drop containing the reagent encapsulates the double emulsion to form a triple emulsion;

applying an electrical field to the triple emulsion so that the fluid interfaces of the triple emulsion are ruptured and allow the microdroplet and reagent to mix.

137. The method of claim 136, wherein the electric field is applied by one or more liquid electrodes.

50. A method for the detection of tumor cells, the method comprising:

encapsulating a plurality of cells in a plurality of microdroplets under conditions in which a majority of microdroplets comprise zero or one cell, wherein the plurality of cells are obtained from a subject'"'"'s blood sample suspected of containing circulating tumor cells (CTCs);

enriching the plurality of microdroplets for microdroplets containing one cell;

introducing a lysing agent into the plurality of microdroplets and incubating under conditions effective for cell lysis;

introducing polymerase chain reaction (PCR) reagents, a detection component, and a plurality of PCR primers into the plurality of microdroplets and incubating the plurality of microdroplets under conditions allowing for PCR amplification to produce PCR amplification products, wherein the plurality of PCR primers comprise one or more primers that each hybridize to one or more oncogenes;

detecting the presence or absence of the PCR amplification products by detection of the detection component, wherein detection of the detection component indicates the presence of the PCR amplification products; and

determining the number of CTCs present in a sample of the subject'"'"'s blood based at least in part on the number of microdroplets in which the PCR amplification products were detected;

wherein one or more steps are performed under microfluidic control.

51. The method according to claim 50, wherein all steps are all performed under microfluidic control.

52. The method according to claim 50 or 51, wherein all steps are performed on the same microfluidic device.

53. The method according to any of claims 50-52, wherein the plurality of PCR primers comprises 10 or more primers.

54. The method according to any of claims 50-53, wherein the plurality of PCR primers comprises primers for 10 or more oncogenes.

55. The method according to any of claims 50-54, wherein the plurality of PCR primers comprises a plurality of probes.

56. The method according to claim 55, wherein the probes comprise TaqMan®

probes.

57. The method according to any of claims 50-56, wherein the PCR reagents are added in two steps or more.

58. The method according to any of claims 50-57, further comprising introducing a probe into the microdroplet.

59. The method according to any of claims 50-58, wherein a reagent is added to the plurality of microdroplets by merging a microdroplet with a second microdroplet comprising the reagent.

60. The method according to any of claims 50-59, wherein a reagent is added to the plurality of microdroplets using either droplet coalescence or picoinjection.

61. The method according to any of claims 50-60, wherein a reagent is added to the plurality of microdroplets by a method comprising:

d) emulsifying the reagent into a stream of droplets, wherein the droplets are smaller than the size of a microdroplet;

e) flowing the droplets together with the microdroplet; and

f) merging a droplet with the microdroplet.

62. The method according to claim 58, wherein the merging comprises applying an electric field.

63. The method according to any of claims 50-62, wherein a reagent is added to the plurality of microdroplets by a method comprising:

d) jetting the reagent into a fluid jet;

e) flowing the fluid jet alongside a microdroplet; and

f) merging a droplet with the microdroplet.

64. The method according to any of claims 50-63, wherein a reagent is added to the microdroplet by a method comprising using a fluid injected into the microdroplet as an electrode.

65. The method according to any of claims 50-64, comprising sorting a microdroplet.

66. The method according to claim 65, wherein the plurality of microdroplets is sorted based on a property comprising size, viscosity, mass, buoyancy, surface tension, electrical conductivity, charge, or magnetism.

67. The method according to any of claims 65-66, wherein the plurality of microdroplets is sorted prior to the introduction of a PCR reagent.

68. The method according to any of claims 50-67, wherein detecting the presence or absence of the PCR amplification products comprises repeatedly imaging the plurality of microdroplets.

69. The method according to claim 68, wherein the plurality of microdroplets is repeatedly imaged while the plurality of microdroplets is subjected to conditions allowing for PCR amplification to produce the PCR amplification products.

70. The method according to any of claims 50-69, wherein incubating the plurality of microdroplets under conditions allowing for PCR amplification and detecting the presence or absence of the PCR amplification products are performed on a Megadroplet Array.

71. The method according to any of claims 50-70, wherein the subject is mammalian.

72. The method according to any of claims 50-71, wherein the subject is human.

73. The method according to any of claims 50-72, wherein the subject has been diagnosed with cancer.

74. A method for genotyping of cells, the method comprising:

encapsulating in a microdroplet a cell obtained from a biological sample from a subject, wherein one cell is present in the microdroplet;

introducing a lysing agent into the microdroplet and incubating the microdroplet under conditions effective for cell lysis;

introducing polymerase chain reaction (PCR) reagents and a plurality PCR primers into the microdroplet, and incubating the microdroplet under conditions allowing for PCR amplification to produce PCR amplification products, wherein the plurality of PCR primers comprise one or more primers that each hybridize to one or more oncogenes;

introducing a plurality of probes into the microdroplet, wherein the probes hybridize to one or more mutations of interest and fluoresce at different wavelengths; and

detecting the presence or absence of specific PCR amplification products by detection of fluorescence of a probe, wherein detection of fluorescence indicates the presence of the PCR amplification products;

wherein one or more of steps are performed under microfluidic control.

75. The method according to claim 74, wherein the probes comprise TaqMan®

probes.

76. The method according to claim 74 or 75, wherein detecting the presence or absence of specific PCR amplification products by detection of fluorescence of a probe comprises repeatedly imaging the microdroplet while the microdroplet is subjected to conditions allowing for PCR amplification to produce PCR amplification products.

77. The method according to claim 76, comprising obtaining time-dependent fluorescence information.

78. The method according to any of claims 74-77, wherein a reagent is added to the microdroplet by merging the microdroplet with a second microdroplet comprising the reagent.

79. The method according to any of claims 74-78, wherein a reagent is added to the microdroplet using either droplet coalescence or picoinjection.

80. The method according to any of claims 74-79, wherein a reagent is added to the microdroplet by a method comprising:

d) emulsifying the reagent into a stream of droplets, wherein the droplets are smaller than the size of the microdroplet;

e) flowing the droplets together with the microdroplet; and

f) merging a droplet with the microdroplet.

81. The method according to any of claims 74-80, wherein a reagent is added to the microdroplet by a method comprising:

d) jetting the reagent into a fluid jet;

e) flowing the fluid jet alongside the microdroplet; and

f) merging a droplet with the microdroplet.

82. The method according to any of claims 74-81, wherein a reagent is added to the microdroplet by a method comprising using a fluid injected into the microdroplet as an electrode.

83. The method according to any of claims 74-82, comprising sorting a microdroplet.

84. The method according to claim 83, wherein the microdroplet is sorted based on a property comprising size, viscosity, mass, buoyancy, surface tension, electrical conductivity, charge, or magnetism.

85. The method according to any of claims 74-84, wherein the subject is mammalian.

86. The method according to any of claims 74-85, wherein the subject is human.

87. The method according to any of claims 74-86, wherein the subject has been diagnosed with cancer.

88. A method for the detection of cancer in a subject, the method comprising:

encapsulating in a microdroplet oligonucleotides obtained from a biological sample from the subject, wherein at least one oligonucleotide is present in the microdroplet;

introducing polymerase chain reaction (PCR) reagents, a detection component, and a plurality of PCR primers into the microdroplet and incubating the microdroplet under conditions allowing for PCR amplification to produce PCR amplification products, wherein the plurality of PCR primers comprise one or more primers that each hybridize to one or more oncogenes;

detecting the presence or absence of the PCR amplification products by detection of the detection component, wherein detection of the detection component indicates the presence of the PCR amplification products; and

diagnosing the subject as having cancer or not based at least in part on the presence or absence of the PCR amplification products;

wherein one or more steps are performed under microfluidic control.

89. The method according to claim 88, wherein the plurality of PCR primers comprises 10 or more primers.

90. The method according to any of claims 88-89, wherein the plurality of PCR primers comprises primers for 10 or more oncogenes.

91. The method according to any of claims 88-90, further comprising introducing a probe into the microdroplet.

92. The method according to claim 91, wherein the probe is introduced prior to incubating the microdroplet under conditions allowing for PCR amplification.

93. The method according to claim 91 or 92, wherein the probe is a TaqMan®

probe.

94. The method according to any of claims 88-93, wherein a reagent is added to the microdroplet by merging the microdroplet with a second microdroplet comprising the reagent.

95. The method according to any of claims 88-94, wherein a reagent is added to the microdroplet using either droplet coalescence or picoinjection.

96. The method according to any of claims 88-95, wherein a reagent is added to the microdroplet by a method comprising:

d) emulsifying the reagent into a stream of droplets, wherein the droplets are smaller than the size of the microdroplet;

e) flowing the droplets together with the microdroplet; and

f) merging a droplet with the microdroplet.

97. The method according to any of claims 88-96, wherein a reagent is added to the microdroplet by a method comprising:

d) jetting the reagent into a fluid jet;

e) flowing the fluid jet alongside the microdroplet; and

f) merging a droplet with the microdroplet.

98. The method according to any of claims 88-97, wherein a reagent is added to the microdroplet by a method comprising using a fluid injected into the microdroplet as an electrode.

99. The method according to any of claims 88-98, wherein the detection component is detected based on a change in fluorescence.

100. The method according to any of claims 88-99, wherein detecting the presence or absence of the PCR amplification products comprises repeatedly imaging the microdroplet.

101. The method according to claim 100, wherein the microdroplet is repeatedly imaged while the microdroplet is subjected to conditions allowing for PCR amplification to produce the PCR amplification products.

102. The method according to any of claims 88-101, comprising sorting a microdroplet.

103. The method according to claim 102, wherein the microdroplet is sorted based on a property comprising size, viscosity, mass, buoyancy, surface tension, electrical conductivity, charge, or magnetism.

104. The method according to any of claims 88-103, comprising sorting based at least in part based upon the detection of the presence or absence of PCR amplification products.

105. The method according to any of claims 88-104, further comprising:

injecting a diluent into the microdroplet; and

flowing the microdroplet through a microfluidic channel on which an electric field is being applied, under conditions in which the microdroplet is split.

106. The method according to any of claims 88-105, wherein the subject is mammalian.

107. The method according to any of claims 88-106, wherein the subject is human.

108. The method according to any of claims 88-107, wherein the subject has been diagnosed with cancer.

109. A microfluidic device comprising:

a cell encapsulation device for encapsulating a cell obtained from a subject'"'"'s blood sample in a microdroplet;

a first chamber in fluidic communication with the cell encapsulation device, the first chamber comprising a first reagent injector element for adding a first reagent to the microdroplet, and a heating element;

a second chamber in fluidic communication with the first chamber, the second chamber comprising a second reagent injector element for adding a second reagent to the microdroplet, and a heating element, wherein the heating element is configured to heat the microdroplet at two or more temperatures; and

a detection region, in fluidic communication with the second chamber, which detects the presence or absence of reaction products from the second chamber.

110. The microfluidic device as set forth in claim 109, wherein the heating element of the second chamber comprises a Peltier plate, heat sink, and control computer.

111. The microfluidic device as set forth in claim 109, wherein the microfluidic device comprises one or more liquid electrodes.

112. A single cell RT-PCR microfluidic device comprising:

an input microchannel coupled to a drop maker for introducing microdroplets into the microfluidic device;

a pairing microchannel in fluidic communication with the input microchannel;

a dilution buffer drop maker in fluidic communication with the pairing microchannel, for producing drops of dilution buffer that are larger in volume than the microdroplets and for pairing a single drop of dilution buffer with a single microdroplet;

a merging microchannel in fluidic communication with the pairing microchannel, for accepting a paired drop of dilution buffer and microdroplet from the pairing microchannel;

a first electric field generator positioned along the merging microchannel for producing an electric field that is capable of merging a paired drop of dilution buffer and microdroplet in the merging microchannel to form a diluted microdroplet;

a mixing microchannel in fluidic communication with the merging microchannel, for receiving the diluted microdroplet from the merging channel and mixing the contents of the diluted microdroplet;

a drop sampler in fluidic communication with the mixing microchannel, for extracting a sample of the diluted microdroplet,

a picoinjection microchannel in fluidic communication with the drop sampler, wherein the picoinjection microchannel comprises a picoinjector and is for receiving the sample of the diluted microdroplet and picoinjecting RT-PCR reagents into the sample;

a second electric field generator, wherein the second electric field generator is positioned along the picoinjection microchannel to create an electric field sufficient to allow for the picoinjection of the RT-PCR reagents into the sample;

a thermocycler heating element in fluidic communication with the picoinjection microchannel for carrying out an RT-PCR reaction on the sample picoinjected with the RT-PCR reagents.

113. The microfluidic device of claim 112, further comprising an encapsulating chamber in fluidic communication with the input microchannel, for encapsulating a cell and lysis reagent into a microdroplet.

114. The microfluidic device of claim 112, wherein the first and/or second electric field generators are liquid electrodes connected to a power supply or high voltage amplifier.

115. The microfluidic device of claim 112, comprising ridges in one or more walls of a microfluidic flow channel downstream of the input microchannel, wherein the ridges are configured to trap a layer of oil and prevent wetting of the one or more walls of the flow channel.

116. The microfluidic device of claim 112, comprising ridges in one or more walls of a microfluidic flow channel downstream of the pairing microchannel, wherein the ridges are configured to trap a layer of oil and prevent wetting of the one or more walls of the flow channel.

117. The microfluidic device of claim 112, comprising ridges in one or more walls of a microfluidic flow channel downstream of the picoinjection microchannel, wherein the ridges are configured to trap a layer of oil and prevent wetting of the one or more walls of the flow channel.

118. The microfluidic device of claim 112, wherein the pioinjection microchannel is configured to receive a sample that has undergone an RT-PCR reaction in the sampler and picoinject the sample with PCR reagents.

119. The microfluidic device of claim 118, wherein the thermocycler is configured for performing a PCR reaction on a sample picoinjected with the PCR reagents.

120. The microfluidic device of claim 118, wherein the PCR reagents and the RT-PCR reagents comprise the same primers.

121. The microfluidic device of claim 118, wherein the PCR reagents and the RT-PCR reagents comprise different primers.

122. The microfluidic device of claim 112, wherein the RT-PCR reagents comprises a bead conjugated with a fluorescent dye and a nucleic acid probe.

123. The microfluidic device of claim 112, wherein the RT-PCR reagents comprises a fluorescent DNA probe.

124. A single cell RT-PCR microfluidic device comprising:

an input microchannel coupled to a flow focus drop maker for introducing microdroplets into the microfluidic device, wherein the flow focus drop maker spaces the microdroplets in the input microchannel by a volume of oil and wherein each microdroplet comprising a cell lysate sample;

a pairing microchannel in fluidic communication with the input microchannel;

a dilution buffer drop maker in fluidic communication with the pairing microchannel, for producing a drop of dilution buffer that is larger in volume than a microdroplet and for pairing a single drop of dilution buffer with a single microdroplet;

a merging microchannel in fluidic communication with the pairing microchannel, for accepting a paired drop of dilution buffer and microdroplet from the pairing microchannel;

a first electric field generator positioned along the merging microchannel for producing an electric field across the merging channel that is capable of merging a paired drop of dilution buffer and microdroplet in the merging microchannel to form a diluted microdroplet;

a mixing microchannel in fluidic communication with the merging microchannel, for receiving the diluted microdroplet from the merging channel and mixing the contents of the diluted microdroplet;

a drop sampler in fluidic communication with the mixing microchannel, for extracting a sample of the diluted microdroplet,

a picoinjection microchannel in fluidic communication with the drop sampler, wherein the picoinjection microchannel comprises a picoinjector and is for receiving the sample of the diluted microdroplet and picoinjecting RT-PCR reagents into the sample;

a second electric field generator, wherein the second electric field generator is positioned along the picoinjection microchannel to create an electric field across the picoinjection microchannel sufficient to allow for the picoinjection of the RT-PCR reagents into the sample;

a thermocycler heating element in fluidic communication with the picoinjection microchannel for carrying out an RT-PCR reaction on the sample picoinjected with the RT-PCR reagents.

125. The microfluidic device of claim 124, further comprising an encapsulating chamber in fluidic communication with the input microchannel, for encapsulating a cell and lysis reagent into a microdroplet.

126. The microfluidic device of claim 124, wherein the first and/or second electric field generators are liquid electrodes connected to a power supply or high voltage amplifier.

127. The microfluidic device of claim 124, comprising ridges in one or more walls of a microfluidic flow channel downstream of the input microchannel, wherein the ridges are configured to trap a layer of oil and prevent wetting of the one or more walls of the flow channel.

128. The microfluidic device of claim 124, comprising ridges in one or more walls of a microfluidic flow channel downstream of the pairing microchannel, wherein the ridges are configured to trap a layer of oil and prevent wetting of the one or more walls of the flow channel.

129. The microfluidic device of claim 124, comprising ridges in one or more walls of a microfluidic flow channel downstream of the picoinjection microchannel, wherein the ridges are configured to trap a layer of oil and prevent wetting of the one or more walls of the flow channel.

130. The microfluidic device of claim 124, wherein the pioinjection microchannel is configured to receive a sample that has undergone an RT-PCR reaction in the sampler and picoinject the sample with PCR reagents.

131. The microfluidic device of claim 130, wherein the thermocycler is configured for performing a PCR reaction on a sample picoinjected with the PCR reagents.

132. The microfluidic device of claim 130, wherein the PCR reagents and the RT-PCR reagents comprise the same primers.

133. The microfluidic device of claim 130, wherein the PCR reagents and the RT-PCR reagents comprise different primers.

134. The microfluidic device of claim 124, wherein the RT-PCR reagents comprises a bead conjugated with a fluorescent dye and a nucleic acid probe.

135. The microfluidic device of claim 124, wherein the RT-PCR reagents comprises a fluorescent DNA probe.

138. A microfluidic device comprising:

a flow channel, a microfluidic junction fluidically connected to the flow channel, and ridges in one or more walls of the microfluidic flow channel immediately downstream of the microfluidic junction.

139. The microfluidic device of claim 138, wherein the ridges trap a layer of oil and prevent wetting of the one or more walls of the flow channel.

140. The microfluidic device of claim 138, wherein the base of each of the one or more ridges is from about 10 microns to about 20 microns in length.

141. The microfluidic device of claim 138, wherein, the peak of each of the one or more ridges has a width of about 1 to about 10 microns.

142. The microfluidic device of claim 138, wherein the height of each of the one or more ridges is from about 5 microns to about 15 microns.

143. The microfluidic device of claim 138, wherein the ratio of the base of each of the one or more ridges to the height of each of the one or more ridges is from about 1.0:

0.75 to about 0.75;

1.0.

144. The microfluidic device of claim 138, wherein the base of each of the one or more ridges to the height of each of the one or more ridges to the width of the peak of the one or more ridges is about 1:

0.75;

0.5.

145. The microfluidic device of claim 138, wherein the ridges extend for a distance along the channel wall of from about 50 microns to about 500 microns.

146. The microfluidic device of claim 138, wherein the ridges extend for a distance along the channel wall, wherein the ratio between the distance along the channel wall and the width of the channel is from about 10:

1 to about 1;

2.