SYSTEM AND METHOD FOR SERIAL PROCESSING OF MULTIPLE NUCLEIC ACID ASSAYS

• US 20120052560A1
• Filed: 08/31/2011
• Published: 03/01/2012
• Est. Priority Date: 08/31/2010
• Status: Active Grant

• Current Assignee: Canon USA, Inc. (Canon Inc.)
• Original Assignee: Canon US Life Sciences Inc (Canon Inc.)
• Inventors: Knight, Ivor T., Hasson, Kenton C., Coursey, Johnathan S., Liang, Hongye, Owen, Gregory H., Corey, Scott, Lane, Ben, Flamm, Alex, Murphy, Brian, Schneider, Eric, Hanagata, Takayoshi, Zeng, Shulin, Bean, Brian, Kanderian, Sami, Cao, Weidong, Wang, Ying-Xin, Laskowski, Conrad, Inoue, Hiroshi, Regan, Franklin
• Granted Patent: US 9,114,399 B2
• Time in Patent Office: Days
• US Class Current: 435/286.1
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/028 Modular arrangements
• 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 2300/0663 Whole sensors
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• 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 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/77 by observing the effect on a chemical indicator
• CPC Class Codes: G01N 35/08 using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: G01N 35/1095 : for supplying the samples to flow-through analysers for a specific analyser see relevant groups, e.g. under G01N15/00, G01N21/00, G01N27/00, G01N30/00, H01J49/00

Abstract

The present invention relates generally to systems and methods for the rapid serial processing of multiple nucleic acid assays. More particularly, the present invention provides for the real time processing of nucleic acid during polymerase chain reaction (PCR) and thermal melt applications. According to an aspect of the invention, a system for the rapid serial processing of multiple nucleic acid assays is provided. In one embodiment, the system includes, but is not limited to: a microfluidic cartridge having microfluidic (flow-through) channels, a fluorescence imaging system, a temperature measurement and control system; a pressure measurement and control system for applying variable pneumatic pressures to the microfluidic cartridge; a storage device for holding multiple reagents (e.g., a well-plate); a liquid handling system comprising at least one robotic pipettor for aspirating, mixing, and dispensing reagent mixtures to the microfluidic cartridge; systems for data storage, processing, and output; and a system controller to coordinate the various devices and functions.

Claims

1. A system for serial processing of nucleic acid assays, comprising:

a microfluidic cartridge comprising an interface chip having at least one inlet port and microfluidic channel and a reaction chip having at least one microfluidic channel in fluid communication with an associated microfluidic channel of the interface chip;

a flow control module configured to control assay fluid flow through the at least one microfluidic channel and to selectively apply pneumatic pressures to the at least one microfluidic channel of the interface chip and the reaction chip;

said flow control module being configured to;

(a) draw a first fluid into the microfluidic channel of the interface chip via the inlet port of the interface chip;

(b) create a first fluid segment in the microfluidic channel of the reaction chip by drawing the first fluid from the associated microfluidic channel of the interface chip into the microfluidic channel of the reaction chip;

(c) draw a second fluid into the microfluidic channel of the interface chip via the inlet port of the interface chip while maintaining the position of the first fluid segment in the reaction chip; and

(d) create a second fluid segment in the microfluidic channel of the reaction chip by drawing the second fluid from the associated microfluidic channel of the interface chip into the microfluidic channel of the reaction chip;

a temperature measurement and control system configured to control and measure a temperature of one or more portions of the at least microfluidic channel of the reaction chip; and

a fluorescence imaging system comprising one or more excitation elements and an imaging sensor and configured to create images of fluorescent emissions from materials within the at least one microfluidic channel of the reaction chip.

2. The system of claim 1, wherein the fluorescence imaging system comprises:

a sensor element configured to generate a storable image of at least a portion of the microfluidic channel of the reaction chip; and

a plurality of illumination elements disposed with respect to the sensor element and configured to illuminate a portion of the reaction chip to be imaged by the sensor element.

3. The system of claim 2, wherein at least one of the illumination elements comprises an illumination assembly comprising:

an LED;

a mask disposed in front of the LED and having an opening formed therein so as to control an area illuminated by the illumination assembly;

a filter along an optic path of the illumination assembly for controlling the spectral content of light emitted by the illumination assembly, and

a lens for imaging an area with light emitted by the illumination assembly,

wherein the LED, the mask, the filter, and the lens are aligned along an optic axis of the illumination assembly.

4. The system of claim 2, wherein at least two of the illumination elements are configured to illuminate different portions of the reaction chip.

5. The system of claim 2, wherein the sensor element comprises a digital single lens reflex camera.

6. The system of claim 2, wherein each of the illumination elements comprises an LED.

7. The system of claim 2, wherein the fluorescence imaging system comprises four illumination elements disposed at 90-degree angular increments about the sensor element.

8. The system of claim 2, wherein the sensor element comprises a CMOS sensor.

9. The system of claim 2, wherein said sensor element includes a pixel array, and wherein said system further comprises logic elements configured to detect an image of only a portion of the pixels of the pixel array.

10. The system of claim 2, wherein said sensor element is configured to generate multiple images.

11. The system of claim 2, wherein said sensor element is configured to generate an image having a JPEG format.

12. The system of claim 2, wherein the fluorescence imaging system comprises at least one extension tube between the sensor and the microfluidic channel of the reaction chip.

13. The system of claim 2, wherein the fluorescence imaging system further comprises an emission filter positioned between the sensor and the microfluidic channel of the reaction chip and configured to allow light signals of only a selected wavelength to reach the sensor.

14. The system of claim 7, wherein the microfluidic channel of the reaction chip comprises a first zone and a second zone, and wherein a first one of the LEDs is positioned and oriented to illuminate the second zone;

a second and a third of the LEDs are spaced 180-degrees from each other and are disposed on opposed sides of the sensor and are positioned and oriented to illuminate the first zone; and

a fourth one of the LEDs is positioned and oriented to illuminate both the first zone and the second zone.

15. The system of claim 1, further comprising a liquid handling system comprising at least one pipettor configured to be accessible to the inlet port of the interface chip and configured to aspirate reagent fluids, mix the reagent fluids, and dispense a mixture of reagent fluids to the inlet port of the interface chip.

16. The system of claim 15, wherein the pipettor is configured to

(a) draw a first volume of a first fluid into the pipettor;

(b) draw a second volume of a second fluid into the pipettor;

(c) expel a droplet including the first and second fluids from the pipettor without releasing the droplet from the pipettor, wherein a volume of the droplet is greater than half the sum of the first and second volumes;

(d) draw the droplet back into the pipettor; and

(e) repeat steps (c) and (d) at least one time before dispensing a mixture of the first and second fluids to the inlet port of the interface chip.

17. The system of claim 15, wherein the pipettor includes at least one pipette tip having a docking feature configured to facilitate automatic alignment of the pipette tip with the inlet port of the interface chip.

18. The system of claim 17, wherein the inlet port of the interface chip includes a docking receptacle configured for cooperative engagement with the docking feature of the pipette tip to align the pipette tip with the inlet port

19. The system of claim 18, wherein the pipettor is configured to

engage the docking feature of the pipette tip containing the fluid mixture with the docking receptacle of the inlet port of the interface chip;

produce a bead of the fluid mixture that makes contact with the microfluidic channel of the interface chip while the flow control module pulls at least a portion of the fluid in the bead into the microfluidic channel of the microfluidic chip, wherein the docking feature and the docking receptacle are configured to position the pipette tip with respect to the inlet port such that the proximity of the pipette tip and the microfluidic channel allows a portion of the bead to contact the microfluidic channel while remaining attached to the pipette tip; and

disengage the docking feature of the pipette tip from the docking receptacle of the inlet port of the interface chip while removing the bead from contact with the microfluidic channel of the interface chip, leaving fluid only inside the microfluidic channel and not in the inlet port of the interface chip.

20. The system of claim 15, wherein the interface chip includes a plurality of inlet ports and a microfluidic channel associated with each inlet port and the reaction chip includes a plurality of microfluidic channels, each in fluid communication with an associated microfluidic channel of the interface chip, and wherein the pipettor is configured to simultaneously dispense a mixture of reagent fluids to two or more of the inlet ports of the interface chip.

21. The system of claim 1, wherein the flow control module comprises a first pumping system and a second pumping system, wherein the first pumping system is configured to control movement of fluid in the microfluidic channel of the interface chip, and the second pumping system is configured to control movement of fluid in the microfluidic channel of the reaction chip.

22. The system of claim 1, further comprising a system controller in communication with the flow control module, the temperature measurement and control system;

and the fluorescence imaging system and configured transmit control signals to control operation of the flow control module, the temperature measurement and control system; and

the fluorescence imaging system.

23. An instrument for serial processing of multiple nucleic acid assays, comprising:

a frame chassis;

a processing drawer configured to be moveable relative to the frame chassis between an open position and a closed position and including a microfluidic device support structure configured to hold a microfluidic device;

a cooling manifold assembly carried on a portion of said frame chassis adjacent said processing drawer and configured to be in an operative position with respect to the microfluidic device support structure when the processing drawer is in the closed position;

a liquid handling system supported by the frame chassis, wherein the liquid handling system is configured such that a microfluidic device mounted on the microfluidic device support structure is accessible to the liquid handling system when the processing drawer is in the closed position, and

an optical imaging system configured to create images of fluorescent emissions from materials within a microfluidic channel of a microfluidic device, the optical imaging system being carried on a portion of said frame chassis adjacent said processing drawer and configured to be in an operative position with respect to the microfluidic device support structure when the processing drawer is in the closed position.

24. The instrument of claim 23, wherein the cooling manifold assembly is positioned above the processing drawer.

25. The instrument of claim 23, wherein the optical imaging system is positioned below the processing drawer.

26. The instrument of claim 23, wherein the liquid handling system comprises at least one robotic pipettor supported by the frame chassis and configured for automated x, y, and z movement.

27. The instrument of claim 26, further comprising a pipette tip loading and cleaning mechanism supported on a pipette tip loading and cleaning mechanism support structure of the processing drawer, the pipette tip loading and cleaning mechanism including racks configured to removably hold a plurality of pipette tips in positions that are accessible to the robotic pipettor when the processing drawer is in the closed position.

28. The instrument of claim 23, further comprising a container support surface on said processing drawing and configured to support a fluid container in a position accessible to the liquid handling system when the processing drawer is in the closed position.

29. The instrument of claim 28, further comprising a removable tray configured to be removably supported on said processing drawer, wherein said container support surface and microfluidic device support structure are located on the removable tray.

30. The instrument of claim 23, further comprising a microfluidic device supported on the microfluidic device support structure of the processing drawer, the microfluidic device comprising an interface chip having at least one inlet port and microfluidic channel and a reaction chip having at least one microfluidic channel in fluid communication with an associated microfluidic channel of the interface chip.

31. The instrument of claim 30, wherein said reaction chip comprises:

a plurality of microchannels;

a plurality of resistive temperature detectors (RTDs) each adjacent to a portion of an associated one of the plurality of microchannels;

a plurality of individual electrodes, each connected to an associated one of the plurality of RTDs;

a first common electrode connected to each of the plurality of RTDs;

a second common electrode connected the first common electrode and to each of the plurality of RTDs; and

a temperature measurement circuit configured to sense a temperature of each of the plurality of RTDs.

32. The instrument of claim 31, further comprising a temperature control circuit configured to drive the plurality of RTDs with heater control signals transmitted to each RTD via the associated individual electrode.

33. The instrument of claim 28, further comprising a multi-well tray supported on the container support surface on the processing drawer.

34. The instrument of claim 23, wherein the optical imaging system comprises:

a sensor element configured to generate a storable image of at least a portion of a microfluidic device supported on the microfluidic device support structure; and

a plurality of illumination elements disposed with respect to the sensor element and configured to illuminate a portion of the microfluidic device to be imaged by the sensor element.

35. The instrument of claim 34, wherein at least one of the illumination elements comprises an illumination assembly comprising:

an LED;

a mask disposed in front of the LED and having an opening formed therein so as to control an area illuminated by the illumination assembly;

a filter along an optic path of the illumination assembly for controlling the spectral content of light emitted by the illumination assembly, and

a lens for imaging an area with light emitted by the illumination assembly,

wherein the LED, the mask, the filter, and the lens are aligned along an optic axis of the illumination assembly.

36. The instrument of claim 34, wherein at least two of the illumination elements are configured to illuminate different portions of a microfluidic device supported on the microfluidic device support structure.

37. The instrument of claim 34, wherein the sensor element comprises a digital single lens reflex camera.

38. The instrument of claim 34, wherein each of the illumination elements comprises an LED.

39. The instrument of claim 34, wherein the fluorescence imaging system comprises four illumination elements disposed at 90-degree angular increments about the sensor element.

40. The instrument of claim 23, wherein the liquid handling system comprising at least one pipettor configured to aspirate reagent fluids, mix the reagent fluids, and dispense a mixture of reagent fluids to an inlet port of a microfluidic device mounted on the microfluidic device support structure.

41. The instrument of claim 40, wherein the pipettor is configured to:

(a) draw a first volume of a first fluid into the pipettor;

(b) draw a second volume of a second fluid into the pipettor;

(c) expelling a droplet including the first and second fluids from the pipettor without releasing the droplet from the pipettor, wherein a volume of the droplet is greater than half the sum of the first and second volumes;

(d) drawing the droplet back into the pipettor; and

(e) repeat steps (c) and (d) at least one time before dispensing a mixture of the first and second fluids to the inlet port of the microfluidic device.

42. The instrument of claim 40, wherein the pipettor includes at least one pipette tip having a docking feature configured to facilitate automatic alignment of the pipette tip with the inlet port of the microfluidic device.

43. The instrument of claim 42, wherein the pipettor is configured to

engage the docking feature of the pipette tip containing a fluid mixture with a docking receptacle of an inlet port of the microfluidic device;

and

produce a bead of the fluid mixture that makes contact with a microfluidic channel of the microfluidic device, wherein the docking feature and the docking receptacle are configured to position the pipette tip with respect to the inlet port such that the proximity of the pipette tip and the microfluidic channel allows a portion of the bead to contact the microfluidic channel while remaining attached to the pipette tip; and

disengage the docking feature of the pipette tip from the docking receptacle of the inlet port of the microfluidic device while removing the bead from contact with the microfluidic channel of the microfluidic device, leaving fluid only inside the microfluidic channel and not in the inlet port of the microfluidic device.

44. The instrument of claim 40, wherein the pipettor includes two or more pipettes and is configured to simultaneously dispense a mixture of reagent fluids to two or more inlet ports of a microfluidic device.

45. The instrument of claim 23, wherein the cooling manifold assembly is configured to direct airflow to a microfluidic device supported on the microfluidic device support structure while isolating the airflow from one or more inlet ports of the microfluidic device, and wherein the cooling manifold assembly comprises:

an air inlet configured to receive the airflow;

an air outlet;

an inlet duct configured to direct the airflow from the air inlet to a portion of the microfluidic device; and

an outlet duct configured to direct the airflow from the microfluidic device to the air outlet.

46. The instrument of claim 23, wherein the cooling manifold assembly comprises a bi-level cooling manifold for cooling a microfluidic device having one or more inlet ports, the cooling manifold comprising:

a first duct comprising;

an upper confinement channel; and

a vertical channel connected to the upper confinement channel;

a second duct comprising;

a lower confinement channel, wherein at least a portion of the lower confinement channel is beneath the upper confinement channel; and

an opening;

wherein the cooling manifold is configured to isolate airflow in the first and second ducts from one or more inlet ports of the microfluidic device.

47. The instrument of claim 23, further comprising one or more gaskets disposed between the cooling manifold and the microfluidic device, each gasket being configured to provide a seal between the cooling manifold and the microfluidic device to substantially keep cooling air flowing in the cooling manifold from impinging on selected portions of the microfluidic device.