Apparatus and methods for correcting for variable velocity in microfluidic systems

• US 20030165960A1
• Filed: 02/05/2003
• Published: 09/04/2003
• Est. Priority Date: 06/09/1997
• Status: Abandoned Application

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Cohen, Claudia B., Chow, Andrea W., Parce, John Wallace, Kopf-Sill, Anne R., Sundberg, Steven A.
• Application Number:
  US10/359,395
• Publication Number:
  US 20030165960A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/0095 Control aspects
• CPC Class Codes: B01J 2219/0097 Optical sensors
• CPC Class Codes: B01J 2219/00995 Mathematical modeling
• CPC Class Codes: B01L 2200/14 Process control and prevention of errors
• CPC Class Codes: B01L 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 3/563 Joints or fittings in general F16L; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
• CPC Class Codes: B01L 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/557 using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays

Abstract

Electrokinetic devices having a computer for correcting for electrokinetic effects are provided. Methods of correcting for electrokinetic effects by establishing the velocity of reactants and products in a reaction in electrokinetic microfluidic devices are also provided. These microfluidic devices can have substrates with channels, depressions, and/or wells for moving, mixing and monitoring precise amounts of analyte fluids.

Claims

1. A method for determining the rate or extent of a reaction or assay in a microfluidic system, comprising:

converting a first reaction or assay component having a first velocity (U₁) into a product having a second velocity (U_p) in a microfluidic channel;

determining at least one velocity selected from the group consisting of U₁ and U_p;

determining the concentration of the reaction or assay product in a portion of the microfluidic channel, whereby determining the at least one velocity and the concentration of the reaction or assay product provides for determination of the rate or extent of the reaction or assay.

2. The method of claim 1, wherein the first reaction or assay component is converted into the product by exposing the product to heat, light, acid, or base.

3. The method of claim 1, wherein the first reaction or assay component is converted into the product by contacting the first reaction or assay component with a second reaction or assay component.

7. The method of claim 1, 4 or 5, the first reactant or product further comprising a detectable label.

11. The method of claim 1, 4 or 5, further comprising determining the reaction rate constant (k) for the formation of the product.

17. The method of claim 1, 4, 5 or 16, further comprising the step of injecting one or more fluorescent dyes or other flow markers into the microfluidic channel to generate a flow profile versus time mask file.

18. The method of claim 1, 4, 5 or 16, further comprising the step of injecting one or more labeled size markers into the microfluidic channel to generate a fluorescence intensity versus time mask file.

19. The method of claim 1, 4, 5 or 16, further deconvolution of a complex signal with a time mask file.

20. The method of claim 1, 4, 5 or 16, further comprising baseline subtraction by injecting a series of blanks into the microfluidic channel in a control experiment to measure a time dependent baseline.

21. The method of claim 1, 4, 5 or 16, further comprising injecting at least one flow marker into the microfluidic channel, sampling signal from the flow marker and generating a flow profile versus time mask file.

4. A method for determining the rate or extent of a reaction or assay in a microfluidic system, comprising:

contacting a first reaction or assay component having a first velocity (U₁) to a second reaction or assay component having a second velocity (U₂) in a microfluidic channel, thereby permitting formation of a reaction or assay product with a third velocity (U_p);

determining at least one velocity selected from the group consisting of U₁, U₂, and U_p;

determining the concentration of the reaction or assay product in a portion of the microfluidic channel, whereby determining the at least one velocity and the concentration of the reaction or assay product provides for determination of the rate or extent of the reaction or assay.

8. The method of claim 4 or 5, wherein the velocity of U₁ or U₂ is zero.

9. The method of claim 4 or 5, further comprising measuring the velocity of the first reaction component or the second reaction component and determining U_p.

10. The method of claim 4 or 5, further comprising measuring U_p.

12. The method of claim 4 or 5, wherein the second velocity U₂ and the third velocity U_p are different.

56. The method of claim 4, 5 or 55, further comprising measuring the concentration of the product in a microfluidic channel, and, optionally, measuring the concentration of the first reaction or assay component in a portion of the microfluidic channel and, optionally, measuring the concentration of the second reaction or assay component in a portion of the microfluidic channel.

57. The method of claim 4, 5 or 55, further comprising measuring a length of time for travel of the first reaction component or the second reaction component along a selected length of the microfluidic channel.

58. The method of claim 4, 5 or 55, further comprising measuring a length of time for travel of the product along a selected length of the microfluidic channel.

59. The method of claim 4, 5 or 55, wherein the first component, the second component, and the product are soluble in an aqueous solvent, wherein the microchannel comprises said aqueous solvent.

60. The method of claim 4, 5 or 55, further comprising providing an electrokinetic microfluidic device having the microfluidic channel;

and, applying an electric field along the length of the microchannel.

61. The method of claim 4, 5 or 55, wherein the first and second component have a K_a of between about 10⁵ and 10¹⁵.

5. A method for determining the rate or extent of a reaction or assay in an electrokinetic microfluidic system, comprising:

providing an electrokinetic microfluidic device having a microfluidic channel;

applying an electric field along the length of the microfluidic channel;

contacting a first reaction or assay component having a first charge mass ratio (CM₁) and a first velocity (U₁) to a second reaction or assay component having a second charge mass ratio (CM₂) and a second velocity (U₂) in the microchannel, thereby permitting formation of a reaction or assay product with a third charge mass ratio (CM_p) and a third velocity (U_p);

determining at least one velocity selected from the group consisting of U₁, U₂, and U_p;

determining the concentration of the reaction or assay product in a portion of the microfluidic channel, whereby determining the at least one velocity and the concentration of the reaction or assay product provides for a determination of the rate or extent of the reaction.

6. The method of claim 5, wherein U₁ is proportional to CM₁, U₂ is proportional to CM₂, and U_p is proportional to CM_P.

13. A method of determining concentration of a reaction or assay product (C_p) in a microfluidic device, the method comprising the steps of:

(i) converting a labeled first reactant or assay component having a velocity (U_r) and a label (L_r), the labeled first reactant or assay component producing a signal (S_as) in a signal detection system, to a reaction or assay product comprising a label L_p, having a velocity (U_p), wherein (U_r) does not equal (U_p) and wherein L_p comprises component elements of L_r; and

,

(ii) detecting a resulting change in S_as, wherein the change in S_as is an indicator of C_p.

14. The method of claim 13, wherein the resulting change in S_as is an indicator of U_r.

15. The method of claim 13, wherein L_r comprises a fluorophore.

16. The method of claim 13, wherein the first reactant or assay component is contacted by the second reactant or assay component in microfluidic channel in a first microfluidic channel region and wherein S_as is detected by monitoring an output from a label detection device which is mounted to view a second microfluidic channel region in fluid communication with the first microfluidic channel region.

22. The method of claim 13, further comprising baseline subtraction of reactant signal (S_r) produced by the labeled first reactant from S_as to provide a normalized signal (S_n) produced by the product.

23. The method of claim 13, wherein the step of converting the labeled first reactant or assay component to a reaction or assay product is performed by contacting the labeled first reactant or assay component with a second reactant or assay component to form a reaction or assay product comprising a label L_p having a velocity (U_p), wherein (U_r) does not equal (U_p) and wherein L_p comprises component elements of L_r.

24. The method of claim 13 wherein L_p is formed from the components of L_r by treating the first reactant with a label modifier selected from light, heat, electrical charge, a polymerization agent, and a catalyst.

25. The method of claim 13, wherein L_p and L_r comprise the same label moiety.

26. The method of claim 13, wherein L_p and L_r comprise different label moieties.

27. The method of claim 4, 5 or 13, wherein the assay or reaction is in a continuous flow format.

28. The method of claim 4, 5 or 13, wherein flux is conserved in the assay or reaction.

29. The method of claim 4, 5 or 13, wherein the reaction or assay is a non-fluorogenic reaction or assay.

30. The method of claim 4, 5 or 13, wherein the first reactant or assay component is contacted to the second reactant or assay component in a microfluidic channel.

31. The method of claim 4, 5 or 13, wherein the first reactant flows down a first channel and the second reactant is periodically injected into the first channel to contact the first reactant.

32. The method of claim 4, 5 or 13, wherein the first reactant or assay component flows down a first microfluidic channel and the second reactant or assay component is periodically injected into the first microfluidic channel, whereby the first reactant or assay component contacts the second reactant or assay component in the first microfluidic channel.

33. The method of claim 4, 5 or 13, wherein the second reactant or assay component is injected into a microfluidic channel comprising the first reactant for a duration of from 0.001 to 10 min.

34. The method of claim 4, 5 or 13, wherein the second reactant or assay component is reacted with the first reactant or assay component in a non-fluorogenic continuous flow mode.

35. The method of claim 4, 5 or 13, wherein the first reactant or assay component comprises a moiety derived from an antibody, an antigen, a ligand, a receptor, an enzyme, an enzyme substrate, an amino acid, a peptide, a protein, a nucleoside, a nucleotide, a nucleic acid, a fluorophore, a chromophore, biotin, avidin, an organic molecule, a monomer, a polymer, a drug, a polysaccharide, a lipid, a liposome, a micelle, a toxin, a biopolymer, a therapeutically active compound, a I molecule from a biological source, a blood constituent, or a cell.

36. The method of claim 4, 5 or 13, wherein the first assay component is a component of a biological assay.

37. The method of claim 4, 5 or 13, wherein the first assay component is a component of a non-biological assay.

38. The method of claim 4, 5 or 13, wherein the first assay component is a component of a chemical synthetic reaction.

39. The method of claim 4, 5 or 13, further comprising contacting the first or second reactant or assay component with at least one additional reactant.

40. The method of claim 4, 5 or 13, further comprising the formation of at least one additional reactant or product.

41. The method of claim 4, 5 or 13, further comprising determining the velocity of an additional reactant, assay component, or product.

42. The method of claim 4, 5 or 13, further comprising the step of injecting a series of blanks into a channel comprising the first reactant to determine a time-dependent baseline.

43. The method of claim 4, 5 or 13, wherein the first or second reactant or assay component is dissolved in an aqueous buffer.

44. The method of claim 4, 5 or 13, wherein the first or second reactant or assay component is dissolved in an aqueous buffer having a pH between 3 and 11.

45. The method of claim 4, 5 or 13, further comprising determining the flux for the first reaction component, the second reaction component and the product.

46. The method of claim 4, 5 or 13, wherein the first reaction component and the second reaction component are mixed at a first pH which facilitates reaction of the first and second reaction component, wherein unreacted first component, unreacted second component or product are subsequently electrokinetically transported at a second pH which inhibits reaction of the first and second components.

47. The method of claim 4, 5 or 13, wherein the first reaction component is an enzyme.

48. The method of claim 4, 5 or 13, wherein the first reactant or assay component is an enzyme, the second reactant or assay component is a substrate and the product is formed by conversion of the substrate by the enzyme into the product.

49. The method of claim 4, 5 or 13, wherein the first reactant or assay component and the second reactant or assay component hybridize to form the product, which product has a velocity faster than either the first component or the second component.

50. The method of claim 4, 5 or 13, wherein the first reactant or assay component and the second reactant or assay component hybridize to form the product, which product has a velocity slower than either the first component or the second component.

51. The method of claim 4, 5 or 13, further comprising measuring the concentration of the product spectrophotometrically, or optically.

52. The method of claim 4, 5 or 13, wherein the first reactant or assay component and the second reactant or assay component comprise a ligand and a ligand binder, wherein the first component hybridizes to the second component.

53. The method of claim 4, 5 or 13, wherein the first reactant or assay component and the second reactant or assay component comprise a ligand and a ligand binder wherein the first reactant or assay component hybridizes to the second reactant or assay component, and the ligand and ligand binder are selected from the group consisting of:

a first nucleic acid and a second nucleic acid;

an antibody and an antibody ligand;

a receptor and a receptor ligand;

biotin and avidin;

a protein and a complementary protein; and

, a carbohydrate and a carbohydrate binding moiety.

54. The method of claim 4, 5 or 13, the first reactant or assay component further comprising a biotin moiety, the second reactant or assay component further comprising a streptavidin moiety and the product further comprising the biotin moiety hybridized to the streptavidin moiety.

55. The method of claim 13, wherein the step of converting the labeled first reactant or assay component to a reaction or assay product is performed by contacting the labeled first reactant or assay component with a second reactant or assay component to form a reaction or assay product comprising a label L_p having a velocity (U_p), wherein (U_r) does not equal (U_p) and wherein L_p comprises component elements of L_r, wherein the first reactant or assay component and the second reactant or assay component are contacted in a microfluidic channel.

62. A method of detecting a product formed by contacting a first and second component of a reaction comprising:

contacting the first and second reactant in a microfluidic channel, wherein the first reactant comprises a detectable label, thereby producing a product comprising 5 the detectable label, which product has a different electrokinetic mobility than the first or second reactant;

flowing the product and any first or second reactant remaining in the channel subsequent to said contacting step past a detector, wherein the label on the first reactant and the label on the product comprise the same detectable moiety; and

,

determining at least one of;

concentration of the product, rate of product formation, or amount of product produced.

63. The method of claim 62, wherein the detectable label is a fluorophore.

64. The method of claim 62, wherein the detectable label is a fluorophore and the reaction is non-fluorogenic.

65. The method of claim 62, the method further comprising measuring or calculating the velocity of the first reactant, the second reactant, or the product.

66. The method of claim 62, wherein flux of the detectable label is conserved.

67. The method of claim 62, wherein the first or second reactant is periodically injected into the channel.

68. The method of claim 62, wherein the first reactant, the second reactant and the product are flowed continuously in the channel.

69. The method of claim 62 further comprising detecting phase shift of reactant and product waves.

70. A method of dispensing representative mixtures in a microfluidic system, comprising:

(i) introducing a first mixture into a first microfluidic channel, the mixture comprising at least first and second materials;

(iii) transporting the first and second materials through the first channel, wherein the first and second mixtures travel at different velocities in the channel;

(iv) gating an aliquot of first and second materials into the second channel for a selected period of time, the relative amount of first and second materials in the aliquot being proportional to the flux of first and second materials in the first mixture in the first channel, thereby dispensing a representative mixture of the first and second components.

71. The method of claim 70, wherein flux is conserved in the system.

72. The method of claim 70, wherein the first and second compounds have different fluxes during electrokinetic movement.

73. The method of claim 70, wherein the first or second material is labeled, the method comprising measuring signal from the aliquot of first or second labeled material, wherein the amount of labeled material is determined by measuring the signal.

74. The method of claim 70, comprising providing a microfluidic device comprising a body structure having at the first channel and at least a second channel disposed therein, the first and second channels communicating at a first intersection.

75. The method of claim 74, wherein the first and second channels communicate at a crossing intersection.

76. The method of claim 70, wherein the first and second materials are moved electrokinetically in the first channel.

77. The method of claim 70, further comprising measuring the amount of first or second material in the aliquot.

78. The method of claim 70, wherein the first material is a reactant and the second material is a product of a reaction of the reactant.

79. The method of claim 70, wherein a separation of the first and second materials occurs in the second channel within the aliquot.

80. The method of claim 70, wherein the aliquot is injected into the second channel by a voltage change.

81. The method of claim 70, wherein the aliquot is injected into the second channel by a current change.

82. The method of claim 70, the method further comprising detecting the first or second material using a total amount detector which measures label across the entire aliquot.

83. The method of claim 70, the method further comprising detecting the first or second material with a label detector comprising a wide photomultiplier tube slit and a photomultiplier tube.

84. The method of claim 70, the method further comprising detecting the first or second material by total photobleaching, a long window fluorescent detector or an electrochemical detector which samples the entire aliquot.

85. A method of correcting data in a microfluidic system for effects of stacking of charged molecules in a microfluidic channel comprising:

injecting at least one labeled blank into the microfluidic channel;

monitoring control signal from the labeled blank in the channel to determine the signal of the blank over time; and

,

subtracting the control signal of the blank over time from experimental data from an analyte in the microfluidic channel.

86. The method of claims 85, further comprising injecting at least one flow marker into the microfluidic channel, sampling signal from the flow marker and generating a flow profile versus time mask file.

88. The method of claims 85 or 87 wherein the label is fluorescent.

87. A method of correcting data in a microfluidic system for effects of stacking of charged molecules in a microfluidic channel comprising:

injecting a series of labeled control molecules in discreet high-salt buffer control plugs into the microfluidic channel to characterize timing of the control plugs as they pass the detection point;

creating a control label intensity versus time data mask file; and

,

correlating the label intensity versus time mask file to experimental data from an analyte in the microfluidic channel to determine which times from the experimental data correlate with a sample plug.

89. A method of regulating a flowing reaction in a microfluidic channel comprising:

mixing a plurality of reaction components in a first buffer, thereby providing a mixture of reaction components;

electrokinetically transporting the mixture of reaction components in a microfluidic channel, thereby permitting the mixture of reaction components to react;

applying a reaction inhibitor to at least a portion of the reaction mixture, thereby inhibiting further reaction of the reaction components in the portion.

90. The method of claim 89, wherein the inhibitor is selected from:

an aliquot of high pH buffer;

an aliquot of low pH buffer;

an aliquot of buffer comprising an ion chelator;

an aliquot of high temperature buffer, an aliquot of low temperature buffer, heat, and light.

91. The method of claim 89, wherein the inhibitor is applied to selected regions of the flowing mixture of reaction components, wherein the selected regions bracket regions which are not selected in which the inhibitor is not applied.

92. The method of claim 89, wherein the inhibitor is added in a time-gated aliquot.

93. The method of claim 89, wherein the mixture of reaction components are electrokinetically flowed for a selected period of time.

94. The method of claim 89, wherein the mixture of reaction components are electrokinetically flowed for a selected distance in the microfluidic channel.

95. A microfluidic apparatus for determining a rate of formation of a moving analyte on an electrokinetic microfluidic substrate comprising:

a microfluidic substrate holder for receiving a microfluidic substrate during operation of the apparatus, which substrate holder has a microfluidic substrate viewing region;

an analyte detector mounted proximal to the substrate viewing region to detect the moving analyte in a portion of the substrate viewing region; and

,

a computer operably linked to the analyte detector, which computer determines the rate of formation of the analyte, correcting for the effects of the motion of the analyte.

98. The microfluidic apparatus of claim 95, 96, wherein the apparatus is in use and further comprises a microfluidic substrate mounted in the microfluidic substrate holder.

103. The microfluidic apparatus of claim 95, 96 or 99, wherein the apparatus comprises an optical or fluorescent detection system for viewing the analyte.

104. The apparatus of claim 95, 96 or 99 comprising an electrokinetic fluid direction system.

105. The apparatus of claim 104, comprising an electrode disposed within a well formed in the top portion of the body.

106. The apparatus of claim 95, 96 or 99 further comprising a microscope.

96. A microfluidic apparatus for determining a rate of formation of a moving analyte on an electrokinetic microfluidic substrate comprising:

a microfluidic substrate holder for receiving a microfluidic substrate during operation of the apparatus, which substrate holder has a microfluidic substrate viewing region;

analyte movement means for imparting velocity to the analyte in a channel of the microfluidic substrate during operation of the apparatus;

detection means for detecting the moving analyte in the substrate viewing region; and

,

correction means for correcting the observed rate of formation of the moving analyte for the effects of the velocity of the analyte, which means are operably linked to the means for detecting the moving analyte.

97. The microfluidic apparatus of claim 96, wherein the correction means comprise a computer operably linked to the detection means, which computer determines the rate of formation of the analyte, and which computer corrects for the effects of the motion of the analyte.

99. A microfluidic apparatus, comprising:

a microfluidic substrate comprising a body having a top portion, a bottom portion and an interior portion;

the interior portion comprising at least two intersecting channels, wherein at least one of the two intersecting channels has at least one cross sectional dimension between about 0.1 μ

m and 500 μ

m;

a detection zone for detecting the analyte in at least one of the two intersecting channels, when the analyte is in motion; and

,

a data analyzer which determines a rate of formation of the analyte in motion, wherein the analyzer comprises a processor which calculates the flux or velocity of the analyte in the detection zone.

100. The microfluidic apparatus of claim 99, wherein the apparatus is formed by etching at least two intersecting groves in a top surface of the bottom portion, the top portion being fused to the top surface of the bottom portion, thereby forming the interior portion.

101. The microfluidic apparatus of claim 99, the data analyzer comprising a computer with software for determining the rate of formation of moving analytes on a microfluidic device in which flux is conserved.

102. The microfluidic apparatus of claim 99, the top portion of the device further comprising a plurality of wells in fluid communication, and an electrokinetic fluid direction system comprising a plurality-of electrodes adapted to fit into the plurality of wells.

107. An apparatus for determining the concentration of a product in a non-fluorogenic format, comprising:

conversion means for converting a labeled first reactant or assay component to a second labeled component;

signal detection means for detecting signal amplitude from the labeled first reactant or assay component and second labeled component;

concentration calculation means for calculating the concentration of the product by measuring a change in signal amplitude which results from converting the first reactant or assay component into the second labeled component.

108. The apparatus of claim 107, wherein the signal detection means comprises an optical detector for detecting a light signal.

109. The apparatus of claim 107, wherein the signal detection means comprises an optical detector for detecting a fluorescent signal.

110. The apparatus of claim 107, wherein the concentration calculation means comprises a digital computer.

111. The apparatus of claim 107, wherein the conversion means comprises a microfluidic substrate having at least two intersecting channels fabricated therein.

115. The apparatus of claim 107 or 112, further comprising an electrokinetic fluid control means.

116. The apparatus of claim 107 or 112, comprising a microfluidic substrate with a reaction channel fabricated therein, wherein, during use of the apparatus, first and second reactants are contacted in the reaction channel, wherein the reaction channel is in fluid communication with a first reagent introduction channel, wherein the second reactant is introduced into the reaction channel from the first reagent introduction channel by time gated injection.

112. An apparatus for determining the concentration of a product in a non-fluorogenic format, comprising:

a microfluidic substrate holder for receiving a microfluidic substrate, the holder comprising a substrate viewing region;

a signal detector mounted proximal to the substrate viewing region;

a signal output processor which converts variations in signal amplitude from the signal detector into concentration measurements for at least one of a plurality of moving analytes comprising one or more labels which have the same signal output, which plurality of analytes are detected by the signal detector.

113. The apparatus of claim 112, wherein the signal detector detects one or more label selected from the group of a fluorescent label, a calorimetric label, and, a radioactive label.

114. The apparatus of claim 112, comprising:

a microfluidic substrate having a plurality of microchannels fabricated therein, the substrate mounted in the substrate holder, wherein the apparatus in use comprises a first analyte comprising a first label, and a second analyte comprising the same first label, wherein the mobility of the first and second analyte are different.