Enzyme Assays for a Droplet Actuator

• US 20100041086A1
• Filed: 07/05/2009
• Published: 02/18/2010
• Est. Priority Date: 03/22/2007
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Rouse, Jeremy, Eckhardt, Allen E., Srinivasan, Vijay
• Granted Patent: US 8,202,686 B2
• Time in Patent Office: Days
• US Class Current: 435/18
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/1827 using resistive heater
• 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 7/54 using spatial temperature gradients
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 2021/035 Supports for sample drops
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)
• CPC Class Codes: G01N 2800/042 Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Abstract

The invention relates to a microfluidic platform and methods of using the platform for conducting enzyme assays using a droplet actuator. The enzyme assays of the invention are useful for, among other things, identifying and/or characterizing disorders resulting from conditions in which enzymes are defective or are produced in inappropriate amounts. Enzyme assays of the invention may, for example, be used to detect altered activity of a particular enzyme in a sample, which may serve as an indicator of a particular disease. Altered activity may, for example, be caused by conditions which result in the increased or reduced production of a certain enzyme or its substrate and/or conditions which result in defective enzymes and/or substrates exhibiting increased or decreased effectiveness relative to corresponding normal enzymes and/or substrates.

Claims

1. A method of conducting a droplet-based enzyme assay, the method comprising:

(a) providing an immiscible fluid comprising;

(i) a sample droplet comprising an enzyme of interest; and

(ii) one or more reagent droplets comprising;

(1) a substrate which is potentially modified in the presence of the enzyme yielding one or more signal-producing products; and

(2) optionally, other reagents sufficient to produce activity of the target enzyme;

(b) combining the sample droplet and the one or more reagent droplets in the immiscible fluid to yield a reaction droplet effecting an enzyme reaction in the immiscible fluid; and

(c) measuring any signal produced by the one or more signal producing products.

2. The method of claim 1 wherein steps 1(b) and 1(c) are accomplished without removing the sample droplet, one or more reagent droplets, or reaction droplet from the immiscible fluid.

3. The method of claim 1 wherein step 1(a) comprises activating one or more electrodes to mediate dispensing of the sample droplet.

4. The method of claim 1 wherein step 1(a)(ii) comprises activating one or more electrodes to mediate dispensing of the one or more reagent droplets.

5. The method of claim 1 wherein step 1(b) comprises activating one or more electrodes for combining the sample droplets and the one or more reagent droplets.

6. The method of claim 1 further comprising dividing the reaction droplet into two or more sub-droplets.

7. The method of claim 6 wherein dividing the reaction droplet comprises activating one or more electrodes to mediate the dividing of the reaction droplet into two or more sub-droplets.

8. The method of claim 7 further comprising:

(a) immediately combining one of the sub-droplets with a quenching droplet to yield a time 0 droplet; and

(b) detecting signal from the time 0 droplet.

9. The method of claim 8 further comprising:

(a) incubating one of the sub-droplets for a predetermined period of time;

(b) combining the incubating droplet with a quenching droplet to yield an endpoint droplet; and

(c) detecting signal from the endpoint droplet.

10. The method of claim 9 further comprising assessing modification of the substrate by the enzyme of interest based on differences in signal in the time 0 droplet and the endpoint droplet.

11. The method of claim 1 comprising:

(a) providing multiple sample droplets comprising an enzyme of interest;

(b) incubating two or more of the sample droplets for different periods of time;

(c) measuring endpoint signals for each of the two or more of the sample droplets;

(d) constructing a curve using the endpoint signals; and

(e) calculating enzyme activity based on the curve.

12. The method of claim 1 wherein the sample droplet has a volume which is less than about 1000 nL.

13. The method of claim 1 wherein the sample droplet has a volume which is less than about 500 nL.

14. The method of claim 1 wherein the sample droplet has a volume which is less than about 250 nL.

15. The method of claim 1 wherein the sample droplet has a volume which is less than about 100 nL.

16. The method of claim 1 wherein the one or more reagent droplets each has a volume which is less than about 1000 nL.

17. The method of claim 1 wherein the one or more reagent droplets each has a volume which is less than about 500 nL.

18. The method of claim 1 wherein the one or more reagent droplets each has a volume which is less than about 250 nL.

19. The method of claim 1 wherein the one or more reagent droplets each has a volume which is less than about 100 nL.

20. The method of claim 1 wherein the reaction droplet has a volume which is less than about 2500 nL.

21. The method of claim 1 wherein the reaction droplet has a volume which is less than about 1000 nL.

22. The method of claim 1 wherein the reaction droplet has a volume which is less than about 750 nL.

23. The method of claim 1 wherein the reaction droplet has a volume which is less than about 500 nL.

24. The method of claim 1 wherein the enzyme of interest exhibits altered enzyme activity as compared to a corresponding normal enzyme.

25. A method of providing diagnostic information, the method comprising:

(a) conducting an assay according to the method of claim 1, wherein the sample droplet comprises a clinical sample from a subject, the sample comprising the enzyme of interest; and

(b) providing diagnostic information based on the activity of the enzyme of interest from the human clinical sample.

26. The method of claim 25 wherein the diagnostic information comprises information diagnostically relevant to a glycogen storage disease.

27. The method of claim 26 wherein the glycogen storage disease comprises a lysosomal storage diseases involving a defect in a glycosidase.

28. The method of claim 26 wherein the glycogen storage disease comprises a deficiency in α

-glucosidase activity.

29. The method of claim 26 wherein the glycogen storage disease comprises a deficiency in α

-galactosidase activity.

30. The method of claim 25 wherein the assay is conducted and the diagnostic information is provided at a point of sample collection.

31. The method of claim 30 wherein the point of sample collection is in the presence of the subject.

32. The method of claim 25 wherein the clinical sample comprises a sample substance selected from the group consisting of:

blood, plasma, serum, tears, saliva, and urine.

33. The method of claim 25 wherein the clinical sample comprises a dried blood sample.

34. The method of claim 25 wherein the clinical sample comprises a fresh blood sample.

35. The method of claim 34 wherein the fresh blood sample is collected from the subject and immediately loaded onto a droplet actuator for conducting the assay.

36. The method of claim 25 wherein time from collection of the blood sample to providing diagnostic information is less than about 12 hours.

37. The method of claim 25 wherein time from collection of the blood sample to providing diagnostic information is less than about 6 hours.

38. The method of claim 25 wherein the clinical sample comprises a human clinical sample.

39. The method of claim 25 wherein the clinical sample comprises a non-human animal clinical sample.

40. The method of claim 1 wherein the substrate comprises a glycoside substrate.

41. The method of claim 1 wherein the substrate releases a fluorophore upon contact with the enzyme of interest.

42. The method of claim 41 wherein two or more assays are conducted simultaneously using different fluorophores for each enzyme tested.

43. The method of claim 41 wherein the fluorophore comprises 4-methylumbelliferyl.

44. The method of claim 1 wherein the substrate comprises a glycoside substrate which releases a fluorophore upon contact with the enzyme of interest.

45. The method of claim 1 wherein the substrate comprises a glycoside substrate comprising glucose, galactose, fucose, mannose, sialic acid, hexose, hexosamine and/or N-acetylated hexosamine.

46. The method of claim 45 wherein the substrate comprises a 4-methylumbelliferyl glycoside.

47. The method of claim 1 further comprising reducing or eliminating reaction contaminants associated with the substrate prior to yielding the assay droplet.

48. The method of claim 47 wherein the reducing or eliminating reaction contaminants comprises photobleaching the substrate prior to yielding the assay droplet.

49. The method of claim 39 wherein the photobleaching is effected prior to providing the droplet comprising the substrate on the droplet actuator.

50. The method of claim 39 wherein the photobleaching is effected after to providing the droplet comprising the substrate on the droplet actuator.

51. The method of claim 1 wherein the substrate comprises a 4-methylumbelliferyl glycoside substrate.

52. The method of claim 51 further comprising photobleaching the substrate prior to yielding the assay droplet.

53. The method of claim 1 wherein the immiscible fluid is a liquid and the liquid comprises a filler fluid.

54. The method of claim 1 wherein the immiscible fluid comprises a silicone oil.

55. The method of claim 53 wherein the filler fluid comprises a surfactant.

56. The method of claim 55 wherein the surfactant comprises nonionic low hydrophile-lipophile balanced (HLB) surfactant.

57. The method of claim 56 wherein the HLB is less than about 10.

58. The method of claim 56 wherein the HLB is less than about 5.

59. The method of claim 56 wherein the surfactant is selected from the group consisting of Triton X-15;

Span 85;

Span 65;

Span 83;

Span 80;

Span 60; and

fluorinated surfactants.

60. The method of claim 1 wherein:

(a) the sample droplet comprises a reconstituted blood sample;

(b) the blood sample is reconstituted using a single universal reconstitution solution;

(c) the blood sample is divided to yield two or more reaction droplets; and

(d) two or more of the reaction droplets are each combined with one or more sets of one or more reagent droplets, each such set comprising reagents selected for establishing reaction conditions for a different enzyme assay.

61. The method of claim 60 wherein the universal reconstitution solution comprises a saline solution.

62. The method of claim 60 wherein the universal reconstitution solution comprises water.

63. The method of claim 1 comprising combining each of two or more sample droplets with reagent droplets selected for establishing reaction conditions for a different enzyme assay.

64. The method of claim 63 wherein the sample droplets comprise fresh blood and/or reconstituted blood spot.

65. The method of claim 63 wherein the sample droplets comprise a dried blood spot reconstituted in water.

66. The method of claim 63 wherein the sample droplets comprise a dried blood spot reconstituted in a saline solution.

67. The method of claim 63 wherein the sample droplets comprise a dried blood spot reconstituted in a buffer.

68. The method of claim 63 wherein the sample droplets comprise a dried blood spot reconstituted in a solution comprising a surfactant.

69. The method of claim 68 wherein the surfactant has an HLB in the range of about 10 to about 20.

70. The method of claim 68 wherein the surfactant has an HLB in the range of about 15 to about 20.

71. The method of claim 63 wherein the sample droplets are from the same subject.

72. The method of claim 63 wherein the sample droplets are from different subjects.

73. The method of claim 63 wherein the wherein two or more of the two or more sample droplets are each combined with one or more reagent droplets selected for establishing reaction conditions for an enzyme assay for an enzyme deficiency selected from the group consisting of Pompe, Niemann-Pick, Fabry, Krabbe, and Gaucher.

74. The method of claim 1 wherein the enzyme assay is selected to provide diagnostic information about an enzyme deficiency.

75. The method of claim 74 wherein the enzyme deficiency is selected from lysosomal storage diseases.

76. The method of claim 74 wherein the enzyme deficiency is selected from the group consisting of Pompe, Niemann-Pick, Fabry, Krabbe, and Gaucher.

77. The method of claim 74 further comprising providing therapeutic treatment to a subject based on the diagnostic information.

78. The method of claim 1 wherein the sample droplet comprising an enzyme of interest comprises cultured cells and/or supernatant from a cell culture.

79. The method of claim 1 wherein the substrate comprises 4-methylumbelliferyl-α

-L-iduronide, 4-methylumbelliferyl-β

-D-galactoside, 4-methylumbelliferyl-β

-D-glucuronic acid, 4-methylumbelliferyl-α

-L-fucoside, 4-methylumbelliferyl-α

-mannoside, 4 methylumbelliferyl-β

-D-mannoside, 4-nitrocathecol sulfate, 4-methylumbelliferyl-β

-D-N-acetylglucosaminide, 4-methylumbelliferyl-β

-D-N-acetylglucosaminide sulfate, 4-methylumbelliferyl-β

-D-glucosaminide, 4-methylumbelliferyl-α

-D-galactoside, 4-methylumbelliferyl-α

-D-neuraminic acid, 4-methylumbelliferyl-α

-D-N-acetylgalactosaminide, phenolphthalein β

-D-glucuronic acid, and mixtures and derivatives thereof.

80. The method of claim 1 wherein the substrate comprises a fluorophoric moiety.

81. The method of claim 80 wherein the fluorophoric moiety comprises 4-methylumbelliferyl.

82. The method of claim 1 wherein the substrate comprises a chromophoric moiety.

83. The method of claim 82 wherein the chromophoric moiety comprises 4-nitrocathecol or phenolphthalein.

84. The method of claim 1 wherein the substrate comprises a radioactive moiety.

85. The method of claim 84 wherein the radioactive moiety comprises ¹⁴C sphingomyeline or ³H galactosylceramide.

86. The method of claim 1 comprising incubating the reaction droplet for a period of less than about 12 hours.

87. The method of claim 1 further comprising controlling the temperature of the reaction droplet.

88. The method of claim 1 wherein:

(a) the sample droplet is prepared by reconstituting a dried blood spot disc having a diameter of less than about 10 mm in less than about 1000 μ

L of solution; and

(b) the sample is dispensed into at least 2 sample droplets.

89. The method of claim 88 comprising reconstituting the dried blood spot disc in less than about 750 μ

L of solution.

90. The method of claim 88 comprising reconstituting the dried blood spot disc in less than about 500 μ

L of solution.

91. The method of claim 88 comprising reconstituting the dried blood spot disc in less than about 250 μ

L of solution.

92. The method of claim 88 comprising reconstituting the dried blood spot disc in solution having a volume ranging from about 25 μ

L to about 750 μ

L.

93. The method of claim 88 comprising reconstituting the dried blood spot disc in solution having a volume ranging from about 25 μ

L to about 500 μ

L.

94. The method of claim 88 comprising reconstituting the dried blood spot disc in solution having a volume ranging from about 25 μ

L to about 250 μ

L.

95. The method of claim 88 comprising reconstituting the dried blood spot disc in solution having a volume ranging from about 25 μ

L to about 150 μ

L.

96. The method of claim 88 wherein the sample is dispensed into at least 5 sample droplets.

97. The method of claim 88 wherein the sample is dispensed into at least 10 sample droplets.

98. The method of claim 88 wherein the sample is dispensed into at least 25 sample droplets.

99. The method of claim 88 wherein the dried blood spot disc has a diameter ranging from about 1 mm to about 10 mm.

100. The method of claim 88 wherein the dried blood spot disc has a diameter ranging from about 1 mm to about 8 mm.

101. The method of claim 88 wherein the dried blood spot disc has a diameter ranging from about 1 mm to about 6 mm.

102. The method of claim 88 wherein the dried blood spot disc has a diameter ranging from about 1 mm to about 4 mm.

103. The method of claim 88 wherein each sample droplet is used to conduct a different enzyme assay.

104. The method of claim 1 wherein the droplet comprising an enzyme of interest is prepared by reconstituting a dried blood spot disc having a diameter of less than about 4 mm in less than about 1000 μ

L of solution.

105. The method of claim 88 wherein the sample is dispensed into at least 10 sample droplets.

106. The method of claim 96 wherein each sample droplet is used to conduct a different enzyme assay.

107. The method of claim 88 wherein:

(a) the disc has a diameter of less than about 4 mm;

(b) the disk is reconstituted in less than about 1000 μ

L of solution;

(c) the sample is dispensed into at least 5 sub-droplets; and

(d) at least 5 sub-droplets are each used to conduct a different enzyme assay.

108. The method of claim 88 wherein:

(a) the disc has a diameter of less than about 8 mm;

(b) the disc is reconstituted in less than about 500 μ

L of solution;

(c) the sample is dispensed into at least 5 sub-droplets; and

(d) at least 5 sub-droplets are each used to conduct a different enzyme assay.

109. The method of claim 88 wherein:

(a) the disc has a diameter of less than about 8 mm;

(b) the disc is reconstituted in less than about 200 μ

L of solution;

(c) the sample is dispensed into at least 5 sub-droplets; and

(d) at least 5 sub-droplets are each used to conduct a different enzyme assay.

110. The method of claim 88 wherein:

(a) the disc has a diameter of less than about 4 mm;

(b) the disk is reconstituted in less than about 1000 μ

L of solution;

(c) the sample is dispensed into at least 10 sub-droplets; and

(d) at least 10 sub-droplets are each used to conduct a different enzyme assay.

111. The method of claim 88 wherein:

(a) the disc has a diameter of less than about 8 mm;

(b) the disc is reconstituted in less than about 500 μ

L of solution;

(c) the sample is dispensed into at least 10 sub-droplets; and

(d) at least 10 sub-droplets are each used to conduct a different enzyme assay.

112. The method of claim 88 wherein:

(a) the disc has a diameter of less than about 8 mm;

(b) the disc is reconstituted in less than about 200 μ

L of solution;

(c) the sample is dispensed into at least 10 sub-droplets; and

(d) at least 10 sub-droplets are each used to conduct a different enzyme assay.

113. The method of claim 88 wherein:

(a) the disc has a diameter ranging from about 1 mm to about 8 mm;

(b) the disc is reconstituted in solution ranging from about 25 μ

L to about 200 μ

L;

(c) the sample is dispensed into at least 5 sub-droplets; and

(d) at least 5 sub-droplets are each used to conduct a different enzyme assay.

114. The method of claim 113 wherein the disc is prepared from a dried blood spot made using less than about 10 mL of blood.

115. The method of claim 113 wherein the disc is prepared from a dried blood spot made using less than about 1 mL of blood.

116. The method of claim 114 wherein the blood is from a newborn human infant.

117. The method of claim 114 wherein the blood is from a premature human infant.

118. The method of claim 114 wherein the blood is from a laboratory animal.

119. The method of claim 1 wherein the volume of the sample droplet ranges from about 1 nL to about 1000 μ

L.

120. The method of claim 1 wherein the volume of the sample droplet ranges from about 1 nL to about 1000 nL.

121. The method of claim 1 wherein the volume of the sample droplet ranges from about 1 nL to about 500 nL.

122. The method of claim 1 wherein the volume of the sample droplet ranges from about 1 nL to about 250 nL.

123. The method of claim 1 wherein the volume of each of the one or more reagent droplets ranges from about 1 nL to about 1000 μ

L.

124. The method of claim 1 wherein the volume of each of the one or more reagent droplets ranges from about 1 nL to about 1000 nL.

125. The method of claim 1 wherein the volume of each of the one or more reagent droplets ranges from about 1 nL to about 500 nL.

126. The method of claim 1 wherein the volume of each of the one or more reagent droplets ranges from about 1 nL to about 250 nL.

127. The method of claim 1 wherein the volume of the reaction droplet ranges from about 1 nL to about 1000 μ

L.

128. The method of claim 1 wherein the volume of the reaction droplet ranges from about 1 nL to about 2000 nL.

129. The method of claim 1 wherein the volume of the reaction droplet ranges from about 1 nL to about 1000 nL.

130. The method of claim 1 wherein the volume of the reaction droplet ranges from about 1 nL to about 500 nL.

131. The method of claim 1 combining the reaction droplet with a quench droplet comprising one or more quenching reagents selected to substantially stop the enzyme reaction.

132. The method of claim 131 wherein multiple reaction droplets are used to effect multiple enzyme reaction types, and a common quench droplet formulation is used to substantially stop each of the enzyme reaction types.