UNIVERSAL SAMPLE PREPARATION SYSTEM AND USE IN AN INTEGRATED ANALYSIS SYSTEM

• US 20110005932A1
• Filed: 06/07/2010
• Published: 01/13/2011
• Est. Priority Date: 06/05/2009
• Status: Active Grant

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Vangbo, Mattias, El-Sissi, Omar, Nielsen, William D., Van Gelder, Ezra, Cohen, David S., Recknor, Michael, Majlof, Lars, JOVANOVICH, Stevan B.
• Granted Patent: US 8,394,642 B2
• Time in Patent Office: Days
• US Class Current: 204/453
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• 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 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/527 for a plurality of reagents
• 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: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01N 1/31 Apparatus therefor
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 2035/00148 : Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: G01N 2035/00247 : Microvalves
• CPC Class Codes: G01N 21/64 : Fluorescence; Phosphorescence
• CPC Class Codes: G01N 2201/06113 : Coherent sources; lasers
• CPC Class Codes: G01N 2201/062 : LED's
• CPC Class Codes: G01N 27/44704 : Details; Accessories
• CPC Class Codes: G01N 27/44721 : by optical means
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 27/745 : for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01N 35/00029 : provided with flat sample substrates, e.g. slides G01N35/028 takes precedence
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The invention provides a system that can process a raw biological sample, perform a biochemical reaction and provide an analysis readout. For example, the system can extract DNA from a swab, amplify STR loci from the DNA, and analyze the amplified loci and STR markers in the sample. The system integrates these functions by using microfluidic components to connect what can be macrofluidic functions. In one embodiment the system includes a sample purification module, a reaction module, a post-reaction clean-up module, a capillary electrophoresis module and a computer. In certain embodiments, the system includes a disposable cartridge for performing analyte capture. The cartridge can comprise a fluidic manifold having macrofluidic chambers mated with microfluidic chips that route the liquids between chambers. The system fits within an enclosure of no more than 10 ft³. and can be a closed, portable, and/or a battery operated system. The system can be used to go from raw sample to analysis in less than 4 hours.

Claims

1. A system that fits within an enclosure of no more than 10 ft.³ comprising:

(a) a sample preparation module adapted to capture an analyte from a non-microfluidic volume on a capture particle and route the captured analyte through a microfluidic channel;

(b) a reaction module comprising a reaction chamber in fluidic communication with the microfluidic channel adapted to immobilized the captured analyte and perform a biochemical reaction on the analyte in a non-microfluidic volume to produce a reaction product; and

(c) an analysis module in fluidic communication with the reaction chamber adapted to perform an analysis on the reaction product.

2. The system of claim 1 configured to capture the analyte, perform a biochemical reaction on the analyte, and perform an analysis on the product in less than 4 hours.

3. The system of claim 1 further comprising a data analysis module configured to receive data about the analysis from the analysis module and comprising executable code that transforms the data and outputs a result of the analysis.

4. The system of claim 1 further comprising a processing module in fluidic communication with the reaction chamber and the analysis module and adapted (1) to route the reaction product through a second microfluidic channel into a non-microfluidic processing chamber;

(2) process the reaction product and (3) route the processed reaction product into the analysis module.

5. The system of claim 1 wherein the system fits within an enclosure of no more than 8 ft³.

6. The system of claim 1 wherein the system fits within an enclosure of no more than 5 ft³.

7. The system of claim 1 wherein the system fits within an enclosure of no more than 2½

ft³.

8. The system of claim 1 wherein the sample preparation module is adapted to release the analyte from a cell.

9. The system of claim 1 wherein the capture particle is a magnetically responsive capture particle and the reaction module comprises a source of magnetic force configured to immobilized the captured analyte.

10. The system of claim 1 wherein the reaction module is adapted to perform thermal cycling.

11. The system of claim 1, wherein the system is a closed system.

12. The system of claim 1, wherein the system is battery operated.

49. The method of claim 37, wherein the method is carried out by a system as described in any of claim 1-15 or 26-35.

13. A system comprising a cartridge cover, a cartridge and a pneumatic manifold wherein the cartridge can be releaseably engaged with the cartridge cover and the pneumatic manifold,

wherein the cartridge comprises one or more pneumatically actuated valves and one or more microfluidic channels,

wherein the pneumatic manifold and the cartridge cover are each fluidically connected to at least one pressure source, and

wherein the pneumatic manifold and the cartridge cover are each adapted to control fluid flow within the cartridge.

14. The system of claim 13, wherein the pneumatic manifold is adapted to actuate the pneumatically actuated valves and the cartridge cover is adapted to apply pressure to one or more chambers in the cartridge.

15. A system comprising:

a) a disposable cartridge comprising at least one set of fluidic chambers including a sample chamber, a mixing chamber and a thermal cycling chamber in fluid communication with each other, and a reagent card comprising reagents for performing a chemical reaction involving thermal cycling, wherein the reagent card is configured to be carried on the cartridge in a closed configuration and to be moved into fluid communication with the at least one set of fluidic chambers;

b) an actuator assembly configured to move fluids between chambers when the cartridge is engaged with the actuator assembly;

c) a thermal cycler configured to cycle temperature in the thermal cycling chamber when the cartridge is engaged with the actuator assembly;

d) a capillary electrophoresis assembly configured to accept a sample from cartridge when the cartridge is engaged with the actuator assembly and to perform capillary electrophoresis on the sample; and

(e) a computerized control system configured to control the actuator assembly, the thermal cycler and the capillary electrophoresis assembly.

16. A cartridge comprising:

a) a fluidic manifold comprising a fluidic side and a reagent card side wherein the fluidic manifold comprises;

(i) at least one set of fluidic chambers, each chamber comprising a port on the fluidic side;

(ii) at least one of thermal cycling chamber comprising at least one port;

(iii) at least one of exit port;

(iv) a slot on the reagent card side adapted to engage a reagent card, wherein the slot comprises a plurality of slot channels comprising cannulae on the reagent card side and communicating between the two sides;

b) at least one microfluidic chip comprising;

(i) at least one fluidic circuit;

(ii) a plurality of ports in fluid communication with the fluidic circuit;

(iii) at least one pneumatically activated diaphragm valve configured to regulate fluid flow within the fluidic circuit;

wherein the at least one chip is engaged with the fluidic manifold so that the ports in the at least one chip are in fluid communication with the ports of the chambers and the slot channels wherein each fluidic chamber is in fluid communication with at least one other fluidic chamber and each cannula is in communication with a fluidic chamber; and

c) a reagent card engaged with the slot, wherein the card comprises a plurality of reagent chambers comprising reagents, each aligned with at least one of the cannulae and adapted to take a first engagement position wherein the reagent chambers are not punctured by the cannulae and a second engagement position wherein the reagent chambers are punctured by the cannulae, thereby putting the reagent chambers in fluid communication with the fluidic circuit.

17. The cartridge of claim 16 wherein the reagents comprise reagents for performing PCR.

18. The cartridge of claim 17 wherein the reagents comprise primers for amplifying a plurality of short tandem repeats.

19. The cartridge of claim 16 wherein the at least one set of fluidic chambers is a plurality of sets of fluidic chambers.

20. The cartridge of claim 16 wherein the fluidics manifold further comprises at least one auxiliary fluidic channel on the fluidic side of the manifold, the at least one chip is a plurality of chips and fluidic circuits in each of the plurality of chips are in fluidic communication with fluidic circuits of at least one other chip through the auxiliary fluidic channel.

21. The cartridge of claim 20 wherein further comprising a gasket between the chips and the manifold, wherein the gasket seals the channels on the manifold.

22. The cartridge of claim 16 wherein the fluidic chambers comprise a distribution chamber, a capture chamber, a sample chamber, and a clean-up chamber.

23. The cartridge of claim 16 wherein at least one fluidic chamber comprises magnetically responsive particles.

24. The cartridge of claim 16 wherein the at least one set of fluidic chambers is at least 4 sets or at least 8 sets.

25. The cartridge of claim 16 wherein the chips comprise at least one diaphragm valve.

26. A system comprising:

a) a pneumatic assembly comprising;

(i) a pneumatic manifold adapted to removably engage the cartridge of claim 16 on the fluidic side, wherein the pneumatic manifold comprises a plurality of pneumatic ports configured to engage pneumatic channels in the at least one microfluidic chip and activate the diaphragm valves; and

(ii) a pressure source configured to supply positive or negative pressure to the pneumatic channels;

b) a cartridge activation assembly adapted to engage the cartridge on the reagent card side;

wherein the cartridge activation assembly comprises;

(i) a reagent pneumatic manifold comprising a pneumatic side and reagent card side, wherein the reagent pneumatic manifold comprises reagent pneumatic manifold channels communicating between the two sides and comprising a cannula on the reagent card side;

(ii) a pressure source configured to supply positive or negative pressure to the reagent pneumatic manifold channels; and

(iii) a clamp configured to move the reagent card from the first engagement position to the second engagement position, wherein clamping results in the cannulae of the reagent pneumatic manifold puncturing the reagent chambers and putting the reagent chambers in communication with the pressure source;

c) a thermal cycler configured to cycle temperature in the at least one thermal cycling chamber when the cartridge is clamped;

d) a capillary electrophoresis assembly comprising;

(i) at least one separation channel fluidically engaged with the exit port when the cartridge is clamped; and

(ii) an optical sub-assembly configured to detect signal from the at least one separation channel; and

(e) a computerized control system configured to control pneumatic assembly, the cartridge activation assembly, the thermal cycler and the capillary electrophoresis assembly.

27. The system of claim 26 wherein clamping seals the chamber ports and the slot channels with the at least one microfluidic chip.

28. The system of claim 26 wherein the cartridge activation assembly further comprises at least one heater configured to heat at least one of the fluidic chambers when the reagent pneumatic manifold is engaged with the cartridge.

29. The system of claim 26 wherein the cartridge activation assembly further comprises movable magnets configured to move into and out of a position wherein the magnets exert a magnetic force on at least one fluidic chamber.

30. The system of claim 26 wherein the cartridge activation assembly further comprises sensors configured to detect the presence of a sample in a sample chamber of the fluidic manifold.

31. The system of claim 26 wherein the thermal cycler comprises a Peltier device.

32. The system of claim 26 comprised in a portable case.

33. The system of claim 32 wherein the case has an internal volume of no more than 10 ft³.

34. The system of claim 32 wherein the case has an internal volume of no more than 8 ft³.

35. The system of claim 32 wherein the case has an internal volume of no more than 2½

ft³.

36. An article in computer readable form comprising code for operating the system of claim 26.

58. The method of claim 51 wherein the system is a system of claim 26.

37. A method comprising:

producing, from a sample comprising at lest one cell comprising DNA, a computer file identifying a plurality of STR markers in the DNA, wherein the method is performed in less than 4 hours.

38. The method of claim 37 wherein the method is performed in less than 3 hours.

39. The method of claim 37 wherein the method is performed in less than 2 hours.

40. The method of claim 37 wherein producing comprises extracting the DNA from the at least one cell, amplifying the STR markers from the DNA, performing capillary electrophoresis on the amplified markers, detecting the amplified markers, and performing computer analysis on the detected amplified markers to identify the markers.

41. The method of claim 37 wherein the plurality of STR markers is at least 5 STR markers.

42. The method of claim 37 wherein the plurality of markers are CODIS STR markers.

43. The method of claim 42 wherein the plurality of STR markers is at least 5, 10, or 13 CODIS STR markers.

44. The method of claim 37 wherein the at least one cell is a plurality of cells.

45. The method of claim 37 wherein the sample is a forensic sample.

46. The method of claim 37 performed at the site of a sample collection.

47. The method of claim 37 wherein the sample comprises blood.

48. The method of claim 37 wherein the sample comprises a cheek swab.

50. A system configured to perform a method, wherein the method comprises:

producing, from a sample comprising at least one cell comprising DNA, a computer file identifying a plurality of STR markers in the DNA, wherein the method is performed in less than 4 hours.

51. A method comprising:

a) providing a system comprising;

i) a disposable cartridge comprising at least one set of fluidic chambers including a sample chamber, a mixing chamber and a thermal cycling chamber in fluid communication with each other, and a reagent card comprising reagents for performing a chemical reaction involving thermal cycling, wherein the reagent card is configured to be carried on the cartridge in a closed configuration and to be moved into fluid communication with the at least one set of fluidic chambers;

ii) an actuator assembly configured to move fluids between chambers when the cartridge is engaged with the actuator assembly;

iii) a thermal cycler configured to cycle temperature in the thermal cycling chamber when the cartridge is engaged with the actuator assembly;

iv) a capillary electrophoresis assembly configured to accept a sample from cartridge when the cartridge is engaged with the actuator assembly and to perform capillary electrophoresis on the sample; and

v) a computerized control system configured to control the actuator assembly, the thermal cycler and the capillary electrophoresis assembly;

b) moving of the reagent card into fluid communication with at least one set of fluidic chambers;

c) providing a sample comprising a nucleic acid molecule to a sample chamber; and

d) operating the system to amplify and detect at least one nucleic acid sequence in the sample.

52. The method of claim 51 comprising providing each of a plurality of samples to a different sample chamber.

53. The method of claim 51 wherein the time it takes to go from step b) to step d) is less than 4 hours.

54. The method of claim 51 comprising amplifying and detecting a plurality of nucleic acid sequences in the sample.

55. The method of claim 54 wherein the plurality of nucleic acid sequences comprise short tandem repeats.

59. The method of claim 51 wherein the sample is a forensic sample.

60. The method of claim 51 wherein the sample is selected from a buccal swab, blood, hair or semen.

61. The method of claim 51 wherein the sample is a raw sample.

62. The method of claim 51 further comprising transporting the system to a forensic site.

56. The method of claim 56 wherein the short tandem repeats comprise a plurality of Combined DNA Index System (CODIS) markers.

57. The method of claim 56 wherein the CODIS markers comprise a plurality of markers selected from AMEL, D3S1358, THO1, D21s11, D18s51, D5s818, D13s317, D7s820, D16s539, CSF1PO, vWA, D8S1179, TPOX and FGA.

63. An optical system comprising:

a) a plurality of optically transparent channels;

b) a light source configured to direct to the plurality of optically transparent channels;

c) a dispersive element that disperses light passing through the optically transparent channels in a wavelength dependent manner; and

d) a detector configured to receive the dispersed light.

64. The system of claim 63, wherein the plurality of optically transparent channels comprises at least eight capillaries.

65. The system of claim 63, wherein the optically transparent channels are aligned in a first plane and the dispersive element disperses light along a second plane, wherein the first plane and the second plane are different.

66. The system of claim 65, wherein the first plane is orthogonal to the second plane.

68. The optical system of claim 63 wherein the excitation light comprises light of a wavelength between 0.3 microns and 1 micron.

67. An optical system comprising:

a) an excitation source configured to direct excitation light to an object;

b) a carrier for an object, wherein the object emits light other than excitation light when excited by the excitation energy;

c) a rejection filter configured to filter out excitation energy and to allow transmission of the emitted light;

d) an imaging lens configured to focus the emitted light;

e) a dichroic mirror substantially transparent to the excitation energy and configured to reflect emitted light to a detector;

f) a focusing system comprising at least one lens configured to focus light reflected from the dichroic mirror; and

g) a photodetector (CCD camera) configured to receive the reflected light.

69. The optical system of claim 67 wherein the carrier comprises an array of capillary tubes and the object comprises a fluorescent species.

70. The optical system of claim 67wherein the mirror reflects emitted light and an angle between about 5 degrees and about 10 degrees off an incident angle.

71. The optical system of claim 67wherein the dichroic mirror further comprises a portion that transmits substantially all light.

72. The optical system of claim 67wherein the focusing system comprises at least one folding mirror.

73. The optical system of claim 67wherein the photodetector comprises a CCD camera.

74. The optical system of claim 67 further comprising a prism located between the object and the imaging lens.

75. An optical system comprising:

a) an array of capillary tubes aligned substantially parallel and substantially in a plane;

b) an excitation assembly comprising an excitation source and configured to deliver excitation light from the excitation source to the array, wherein the light delivery assembly is configured (i) to deliver a thin band of light that covers the array and (ii) to deliver the light to the array at an angle other than 90 degrees to the plane;

c) a collection lens configured to collect light emitted from the array by objects in the array excited by the excitation light;

wherein the excitation assembly and the collection lens are configured with respect to the array so that excitation light passing through the array substantially avoids collection by the collection lens.

76. The optical system of claim 75 wherein the angle is between about 10 degrees and about 85 degrees.

77. An instrument comprising:

a) a microfluidic component comprising a plurality of intersecting microfluidic channels and at least one controllable valve configured to regulate flow of fluid between the intersecting channels; and

b) a non-microfluidic component comprising a plurality of non-microfluidic chambers, wherein each non-microfluidic chamber is fluidically connected to at least one of the microfluidic channels;

wherein the instrument is configured to flow fluid from at least one non-microfluidic chamber into another non-microfluidic chamber through a microfluidic channel and flow is regulated by at least one valve.

78. The instrument of claim 77 wherein the plurality of non-microfluidic chambers comprises at least three chambers and the at least one valve selectively directs fluid from one chamber to either of the at least two other chambers.

79. The instrument of claim 77 further comprising a pump to pump fluid from a non-microfluidic chamber into a microfluidic channel.

80. The instrument of claim 77 wherein at least one valve is a diaphragm valve.

81. The instrument of claim 80 wherein the pump is a diaphragm pump comprising a series of three diaphragm valves.

82. The instrument of claim 77 wherein the microfluidic component comprises a monolithic device.

83. The instrument of claim 77 wherein the combination of the microfluidic component and the non-microfluidic component define a fluidic circuit and the instrument comprises a plurality of fluidic circuits.

84. The instrument of claim 77 wherein the non-microfluidic component further comprises a particulate capture agent.

85. The instrument of claim 84 wherein the particles are responsive to a magnetic field and the instrument further comprises a magnet configured to immobilize the particles.

86. A device comprising a plurality of non-microfluidic chambers fluidically connected to a common microfluidic channel.

87. A method comprising:

a) providing a device comprising;

i) a microfluidic component comprising a plurality of intersecting microfluidic channels and at least one controllable valve configured to regulate flow of fluid between the intersecting channels; and

ii) a non-microfluidic component comprising a plurality of non-microfluidic chambers, wherein each non-microfluidic chamber is fluidically connected to at least one of the microfluidic channels;

wherein the instrument is configured to flow fluid from at least one non-microfluidic chamber into another non-microfluidic chamber through a microfluidic channel and flow is regulated by at least one valve;

and wherein a first non-microfluidic chamber comprises a first volume of sample comprising an analyte;

b) providing an amount of particulate capture agent in the first non-microfluidic chamber to bind a selected amount of analyte from the sample;

c) moving the particulate capture agent bound to the analyte through a microfluidic channel in the microfluidic device to a second non-microfluidic chamber;

d) contacting the particulate capture agent bound to the analyte with a reagent in a second non-microfluidic chamber; and

e) performing a chemical reaction on the analyte using the reagent.

88. The method of claim 87 wherein contacting comprises flowing the reagent from a third non-microfluidic chamber through a microfluidic channel in the microfluidic device into the second non-microfluidic chamber.

89. The method of claim 87 wherein the particles are response to magnetic force and the method further comprises immobilizing the particulate capture agent bound to the analyte in the instrument with a magnetic force.

90. The method of claim 87 comprising suspending the particulate capture agent bound to the analyte in the instrument in a volume at least an order of magnitude smaller than the sample volume.

91. A method comprising:

a) performing a first chemical reaction on an analyte in a first chamber which is a non-microfluidic chamber to produce a first reaction product; and

b)moving the first reaction product through a microfluidic channel into a second chamber which is a non-microfluidic chamber and performing a second chemical reaction on the first product to create a second reaction product.

94. The method of any of claims 91-93 comprising cleaning the first reaction product before moving it to the second chamber.

95. The method of any of claims 91-93 further comprising, at least once, moving a reaction product through a microfluidic channel into a subsequent non-microfluidic chamber and performing a subsequent chemical reaction on the reaction product to create a subsequent reaction product.

96. The method of any of claims 91-93 further comprising, at least once and before moving a reaction product into a non-microfluidic chamber, moving a reaction product through a microfluidic channel into a microfluidic chamber and performing a subsequent chemical reaction on the reaction product to create a subsequent reaction product.

92. A method comprising:

a) performing a first chemical reaction on an analyte in a first chamber which is a non-microfluidic chamber to produce a first reaction product; and

b) moving the first reaction product through a microfluidic channel into a second chamber which is a microfluidic chamber and performing a second chemical reaction on the first product to create a second reaction product.

93. A method comprising:

a) performing a first chemical reaction on an analyte in a first chamber which is a microfluidic chamber to produce a first reaction product; and

b) moving the first reaction product through a microfluidic channel into a second chamber which is a non-microfluidic chamber and performing a second chemical reaction on the first product to create a second reaction product.

97. A device comprising:

a) a sample channel having a channel inlet and a channel outlet;

b) an electrophoresis capillary having a capillary inlet and a capillary outlet, wherein the capillary comprises an electrically conductive medium and is in communication with the sample channel at a point of connection;

c) an anode and a cathode configured to apply a voltage across the capillary inlet and capillary outlet, wherein one of the anode or cathode comprises a forked electrode wherein the forks are in electrical communication with the sample channel on different sides of the point of connection; and

d) a second electrode in electrical communication with the sample channel substantially opposite the point of connection.

98. The device of claim 97 wherein the second electrode is comprised as a third fork in the forked electrode.