System for high throughput sperm sorting

• US 9,757,726 B2
• Filed: 03/14/2013
• Issued: 09/12/2017
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Inguran LLC
• Original Assignee: Inguran LLC
• Inventors: Sharpe, Johnathan Charles, Sedoglavich, Nemanya, Buchanan, Kris, Morad, Blair, Perrault, Jr., Donald Francis, Koksal, Erin
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): FISHER, BRITTANY I
• Application Number:
  US13/830,316
• Publication Number:
  US 20140273192A1
• Time in Patent Office: 1,643 Days
• Field of Search: 422 73
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• 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 2400/0439 ultrasonic vibrations, vibrating piezo elements
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502776 specially adapted for focusing or laminating flows
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

This disclosure relates to a system for sorting sperm cells in a microfluidic chip. In particular, various features are incorporated into the system for aligning and orienting sperm in flow channels, as well as, for determining sperm orientation and measuring relative DNA content for analysis and/or sorting.

Claims

1. A sperm sorting system comprising:

a substrate;

at least one flow channel formed in the substrate, the flow channel having a sample inlet that receives sample, the flow channel further comprising;

an inspection region, a first outlet, a second outlet, and a core stream forming geometry, the core stream forming geometry further comprising;

a lateral fluid focusing region on the same plane as the sample inlet, a first vertical fluid focusing channel, and a second vertical fluid focusing channel, wherein one of the first fluid focusing channel or the second vertical fluid focusing channel is in fluid communication with the flow channel from a second plane above the inlet and the other is in fluid communication with the flow channel from a third plane below the inlet, and wherein the second vertical fluid focusing channel is downstream of the first vertical focusing channel in the flow channel;

at least one diverting mechanism in communication with the at least one flow channel to selectively divert sperm in the at least one flow channel away from the first outlet;

an electromagnetic radiation source for illuminating sperm at the inspection region of the at least one flow channel;

an analyzer in communication with the detector to determine sperm characteristics;

a controller in communication with the analyzer for selectively activating the diverting mechanism based on measured sperm characteristics; and

a collection vessel in communication with the second outlet.

2. The system of claim 1, wherein the at least one flow channel comprises multiple flow channels formed on a microfluidic chip.

3. The system of claim 2, wherein the multiple flow channels comprises between 4 and 512 flow channels.

4. The system of claim 2, wherein either sperm characterized as viable X-chromosome bearing sperm or sperm characterized as viable Y-chromosome bearing sperm are deflected to the second outlet of each flow channel.

5. The system of claim 4, wherein the collection vessel comprises a common collection vessel in fluid communication with the second outlet of the multiple flow channels.

6. The system of claim 1, wherein each flow channel further comprises a third outlet.

7. The system of claim 6, wherein the sperm cells characterized as viable X-chromosome bearing sperm are diverted to one of the second outlet or the third outlet and sperm characterized as viable Y-chromosome bearing sperm are diverted to the other of the second outlet and the third outlet.

8. The system of claim 6, wherein each second outlet of the flow channels are connected to a first common collection vessel.

9. The system of claim 6, wherein each third outlet of the flow channels are connected to a second common collection vessel.

10. The system of claim 1, further comprising a passive collection vessel in communication with the first outlet.

11. The system of claim 1, further comprising a sheath inlet that receives sheath fluid.

12. The system of claim 11, further comprising a sheath fluid recycling system comprising:

a transport mechanism in fluid communication with the passive collection vessel;

a fluid path providing fluid communication between the passive collection vessel and the sheath inlet; and

a particle concentrating device or a fluid removing system in the fluid path providing fluid communication between the passive collection vessel and the sheath inlet.

13. The system of claim 1, wherein the at least one flow channel comprises multiple flow channels formed on a microfluidic chip and wherein at least a portion of the diverting mechanism is embedded within the microfluidic chip.

14. The system of claim 1, wherein the at least one flow channel comprises multiple flow channels formed on a microfluidic chip and wherein at least a portion of the diverting mechanism is positioned on the exterior of the microfluidic chip.

15. The system of claim 1, wherein the diverting mechanism comprises a side passage in fluid communication with the flow channel and in fluid communication with a volume of fluid through a flexible interface.

16. The system of claim 15, wherein the fluid comprises one selected from the group consisting of:

a gel, a liquid, and a gas.

17. The system of claim 15, further comprising an actuator contacting a portion of the flexible interface, wherein the actuator is in communication with the controller.

18. The system of claim 17, wherein the actuator is movable between a resting position and two or more activation positions while maintaining contact with the flexible interface.

19. The system of claim 18, further comprising a third outlet and wherein particles passively flow to the second outlet and wherein actuator movement between the resting position and the first active position diverts particles to the first outlet and wherein actuator movement between the resting position and the second active position diverts particles to a third outlet.

20. The system of claim 18, wherein the actuator is attached to the flexible interface.

21. The system of claim 18, wherein the actuator is preloaded onto the flexible interface.

22. The system of claim 15, further comprising a bimorph piezoelectric element.

23. The system of claim 22, wherein the bimorph piezoelectric element comprises the flexible interface.

24. The system of claim 22, wherein the bimorph piezoelectric element contacts the flexible interface.

25. The system of claim 22, wherein the bimorph piezoelectric element is configured for deflection in two directions to divert sperm in the flow channel two directions.

26. The system of claim 1, wherein the diverting mechanism comprises a transducer coupled to the flow channel.

27. The system of claim 26, wherein the transducer comprises an ultrasonic transducer for diverting particles in the flow channel.

28. The system of claim 27, wherein the ultrasonic transducer comprise an array of ultrasonic transducers and further comprising a driving element which times the activation of each transducer in the array to achieve the desired deflection.

29. The system of claim 28, comprising a second array of ultrasonic transducers, wherein each array of ultrasonic transducers is located on opposite sides of the flow channel.

30. The system of claim 28, wherein the array of ultrasonic transducers is configured to produce multiple standing waves.

31. The system of claim 28, wherein the array of ultrasonic transducers are configured to maintain the trajectory of a sperm cell in the flow path towards the first outlet, deflect the trajectory of a sperm cell in a flow path towards the second outlet, or deflect the trajectory of a sperm cell in a flow path towards a third outlet.

32. The system of claim 26, wherein the transducer is at least partially embedded in the substrate adjacent to the flow channel.

33. The system of claim 26, wherein the transducer is placed in contact with an exterior surface of the substrate.

34. The system of claim 1, further comprising one or more sources of electromagnetic radiation for deflecting sperm in the flow channel.

35. The system of claim 1, further comprising beam shaping optics for manipulating electromagnetic radiation produced from the electromagnetic radiation source to inspect sperm at each inspection region of the at least one flow channel.

36. The system of claim 35, wherein the at least one flow channel comprises a plurality of flow channels, and wherein the beam shaping optics comprise a beam splitting device for directing substantially equivalent beams to the inspection region of each of the plurality of flow channels.

37. The system of claim 36, wherein the beam splitting device comprises a reflective surface or refractive material for reflecting portions of the beam profile as beam segments or for dividing a beam intensity among beams having the same profile.

38. The system of claim 35, wherein the beam shaping optics further comprise a beam shaping optics for establishing a top hat beam profile.

39. The system of claim 1, wherein each flow channel has an associated reflective surface or an associated refractive element that redirects a side fluorescence produced by sperm in the flow channel.

40. The system of claim 39, wherein the associated reflective surface or associated refractive element redirects a side fluorescence in a direction substantially parallel to a first fluorescence.

41. The system of claim 39, wherein the first fluorescence comprises a forward fluorescence.

42. The system of claim 39, wherein the first fluorescence comprises a back fluorescence.

43. The system of claim 39, wherein the reflective surface is formed by a surface on the substrate.

44. The system of claim 39, wherein the reflective surface is formed by a surface of the flow channel.

45. The system of claim 39, wherein each flow channel is separated by a light blocking element.

46. The system of claim 39, wherein the reflective surface further comprises a reflective element embedded in the substrate.

47. The system of claim 39, wherein the reflective surface comprises an exterior surface of the substrate formed by cutaway portion adjacent to the inspection region, wherein the refractive index difference in the cutaway portion provides a reflective property.

48. The system of claim 47, wherein the cutaway portion provides a reflective surface at about 45 degrees relative to the surface of the substrate and/or the desired plane or orientation of the sperm.

49. The system of claim 47, further comprising a second reflective surface comprising a second exterior surface of the substrate formed by a second cutaway portion adjacent to the inspection region for producing a second side fluorescence.

50. The system of claim 1, wherein the detector comprises a forward fluorescence detector.

51. The system of claim 50, further comprising a first side fluorescence detector.

52. The system of claim 51, further comprising a second side fluorescence detector.

53. The system of claim 52, wherein the first and the second side fluorescence detectors are located about 90 degrees apart.

54. The system of claim 51, further comprising an array of first side fluorescence detectors of measuring a first side fluorescence value in each of a plurality of flow channels and an array of second side fluorescence detectors.

55. The system of claim 1, further comprising collection optics for collecting fluorescence from one or more flow channels.

56. The system of claim 55, wherein the collection optics comprise a single collection lens for collecting fluorescence from multiple channels.

57. The system of claim 55, further comprising an array of lens for collecting fluorescence from each flow channel.

58. The system of claim 55, further comprising a fiber array for collecting fluorescence from each flow channel.

59. The system of claim 55, further comprising epi-illumination forward collection optics.

60. The system of claim 59, further comprising a dichroic mirror positioned to reflect electromagnetic radiation from the electromagnetic radiation source onto the inspection region and through which fluorescence emissions in the back direction travel to a detector.

61. The system of claim 1, wherein the first vertical fluid focusing channel and the second vertical fluid focusing channel are in communication with the flow channel in opposite vertical positions.

62. The system of claim 1, wherein the first vertical fluid focusing channel provides a first vertical influence and wherein the second vertical fluid focusing channel provides a second vertical influence in the opposite direction as the first vertical influence.

63. The system of claim 1 further comprising a series of transducers configured to produce a standing pressure wave along the flow channel.

64. The system of claim 1, wherein the at least one flow channel comprises orienting features.

65. The system of claim 64, wherein the orienting features comprise an interior channel geometry dimensioned to orient sperm cells.

66. The system of claim 65, wherein the channel geometry further comprises a planar channel geometry.

67. The system of claim 65, wherein the channel geometry further comprises a nozzle geometry.

68. The system of claim 65, wherein the channel geometry further comprises one or more of the following channel features:

a chevron, a gentle ramp, an abrupt ramp, a decompression-compression zone, a step, or one or more undulations.

69. The system of claim 64, wherein the orienting features further comprise:

a magnet for producing a magnetic field in the orienting region of each flow channel.

70. The system of claim 1, wherein the flow channel further comprises a sheath inlet that receives sheath fluid, wherein the sample inlet is positioned within a sheath flow created by the sheath inlet to facilitate a co-axial flow of sheath and sample.

71. The system of claim 70, wherein the sample inlet comprises an inlet which is beveled, flattened, or has a rectangular cross-section.

72. The system of claim 70, wherein the flow channel comprises a first width and a first height at the sample inlet.

73. The system of claim 72, wherein the flow channel comprises a second width and a second height at a first transition point.

74. The system of claim 73, wherein the width of the flow channel is reduced between the sample inlet and the first transition point.

75. The system of claim 73, wherein the flow channel comprises a third width and a third height at a second transition point.

76. The system of claim 74, wherein the width remains constant between the first transition point and the second transition point and the height is reduced between the first transition point and the second transition point.

77. The system of claim 75, wherein the third height and the third width are maintained through the inspection region.

78. The system of claim 75, wherein the flow channel transitions from square cross section to a rectangular cross section.

79. The system of claim 75, wherein the flow channel transitions from a circular cross section to an elliptical cross section.

80. The system of claim 1, further comprising at least one mask.

81. The system of claim 80, wherein the at least one mask comprises an illumination mask positioned in the path of electromagnetic radiation directed to the inspection region.

82. The system of claim 81, wherein the illumination mask comprises a first region and a second region along the flow path.

83. The system of claim 82, wherein the first region provides an opening configured to produce a sufficient waveform pulse to differentiate viable X-chromosome bearing sperm from viable Y-chromosome bearing sperm, when orientated.

84. The system of claim 82, wherein the second region comprises a series of openings configured to produce a series of waveform pulses which differentiate oriented sperm cells from unoriented sperm cells.

85. The system of claim 82, wherein the second region comprises a series of openings with differing transverse profiles along the flow path.

86. The system of claim 82, wherein the second region comprises a first spaced pair of openings followed by a second spaced pair of openings, wherein the spacing is differed between the first pair of openings and the second pair of openings.

87. The system of claim 85, wherein the second region comprises a sequential series of openings along the flow path, each opening having a different transverse position along the flow path.

88. The system of claim 81, wherein the at least one mask comprises at least one detection mask.

89. The system of claim 88, wherein a first detection mask is placed in the path of emitted forward fluorescence and a second detection mask is placed in the path of emitted side fluorescence.

90. The system of claim 89, wherein the first detection mask and the second detection masks have differing profiles of slits, and wherein each mask is in communication with the same detector.

91. The system of claim 90, wherein the analyzer is in communication with the detector and is configured to deconvolve a first waveform pulses representing the forward fluorescence and a second waveform pulse representing the side fluorescence based on the profile of slits in each of the first detection mask and the second detection mask.

92. The system of claim 80, wherein the mask is positioned in free space.

93. The system of claim 80, wherein the mask is located on the substrate.

94. The system of claim 1, wherein the detector comprises a first detector and the system further comprises a second detector.

95. The system of claim 94, wherein the first detector comprises an absorption detector and the second detector comprises a fluorescence detector.

96. The system of claim 95, further comprises a neutral density filter in the light path of the absorption detector.