Systems and methods for monitoring the amplification and dissociation behavior of DNA molecules

• US 20080003594A1
• Filed: 11/30/2006
• Published: 01/03/2008
• Est. Priority Date: 06/30/2006
• Status: Active Grant

• Current Assignee: Canon USA, Inc. (Canon Inc.)
• Original Assignee: Canon USA, Inc. (Canon Inc.)
• Inventors: Hasson, Kenton C., Dale, Gregory A.
• Granted Patent: US 7,906,319 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2300/1811 using electromagnetic induction heating
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1877 using chemical reactions
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• 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 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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 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 21/71 thermally excited
• 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: G06T 2207/10004 : Still image; Photographic image
• CPC Class Codes: G06T 2207/30072 : Microarray; Biochip, DNA array; Well plate
• CPC Class Codes: G06T 7/0012 : Biomedical image inspection
• CPC Class Codes: G06T 7/11 : Region-based segmentation
• CPC Class Codes: Y10T 436/115831 : Condition or time responsive
• CPC Class Codes: Y10T 436/117497 : with a continuously flowing sample or carrier stream

Abstract

The present invention relates to systems and methods for monitoring the amplification of DNA molecules and the dissociation behavior of the DNA molecules. The present invention in one embodiment provides a system that includes a microfluidic channel comprising a PCR processing zone and an HRTm analysis zone; and an image sensor having a first image sensor region having a first field of view and a second image sensor region having a second field of view, wherein the second field of view is different than the first field of view, wherein at least a portion of the PCR processing zone is within the first field of view; and at least a portion of the HRTm analysis zone is within the second field of view.

Claims

1. A system, comprising:

a microfluidic channel comprising a PCR processing zone and an HRTm analysis zone; and

an image sensor having a first image sensor region having a first field of view and a second image sensor region having a second field of view, wherein the second field of view is different than the first field of view, wherein

at least a portion of the PCR processing zone is within the first field of view, and

at least a portion of the HRTm analysis zone is within the second field of view.

2. The system of claim 1, further comprising:

a first thermal generating apparatus configured to provide heat to and/or absorb heat from the PCR processing zone; and

a second thermal generating apparatus configured to provide heat to and/or absorb heat from the HRTm analysis zone.

3. The system of claim 2, wherein the first thermal generating apparatus is configured to cycle the temperature in the PCR processing zone when a bolus of solution containing real-time PCR reagents is within the PCR processing zone in order to achieve PCR.

4. The system of claim 3, wherein the first thermal generating apparatus is configured such that, after the bolus enters the PCR processing zone for PCR processing, the first thermal generating apparatus heats the bolus to a first temperature, cools the bolus to a second temperature, and heats the bolus to a third temperature.

5. The system of claim 4, wherein, the second thermal generating apparatus is configured to provide to the bolus a substantially steadily increasing amount of heat at a thermal ramp rate of between about 0.1 to 1 degree Celsius (C.) per second.

6. The system of claim 1, further comprising a lens disposed between the channel and the image sensor, wherein the lens is configured to focus onto the first image sensor region light coming from the PCR processing zone and focus onto the second image sensor region light coming from the HRTm analysis zone.

7. The system of claim 1, further comprising an excitation source for producing electromagnetic radiation directed at the channel.

8. The system of claim 1, wherein the HRTm analysis zone is operable to receive a plurality of spaced apart boluses of solution containing real-time PCR reagents.

9. The system of claim 8, further comprising an image sensor controller configured such that, for each of said bolus that undergoes HTRm processing in the HRTm analysis zone, the image sensor controller captures at least about 10 images per second from the second image sensor region for at least about 1 minute while the bolus undergoes the HRTm analysis.

10. The system of claim 9, wherein the image sensor controller is further configured so that, for each said bolus that undergoes PCR processing in the PCR processing zone, the controller captures at least about 1 image every 90 seconds from the first image sensor region for at least about 10 minutes while the bolus is undergoing the PCR processing.

11. The system of claim 9, wherein the image sensor controller is further configured so that, for each said bolus that undergoes PCR processing in the PCR processing zone, the controller captures not more than 1 image every 1 second from the first image sensor region for at least about 10 minutes while the bolus undergoes the PCR processing.

12. A system, comprising:

a channel for receiving a bolus of solution containing real-time PCR reagents, the channel comprising a DNA amplification zone and a DNA melting zone adjacent to the DNA amplification zone; and

an image sensor disposed in relation to the channel such that both the DNA melting zone and the DNA amplification zone are within the field of view of the sensor at the same time.

13. The system of claim 12, further comprising:

a first thermal generating apparatus configured to provide heat to and/or absorb heat from the DNA amplification zone; and

a second thermal generating apparatus configured to provide heat to and/or absorb heat from the DNA melting zone.

14. The system of claim 12, wherein the first thermal generating apparatus is configured such that when the bolus enters the DNA amplification zone for PCR processing, the first thermal generating apparatus cycles the temperature in the DNA amplification zone in order to achieve PCR.

15. The system of claim 14, wherein the first thermal generating apparatus is configured such that when the bolus enters the DNA amplification zone for PCR processing, the first thermal generating apparatus heats the processing zone to a first temperature, cools the DNA amplification zone to a second temperature, and heats the DNA amplification zone to a third temperature.

16. The system of claim 15, wherein the second thermal generating apparatus is configured such that, when the bolus enters the DNA melting zone, the second thermal generating apparatus provides to the DNA melting zone a substantially steadily increasing amount of heat at a thermal ramp rate of between about 0.1 to 1 degree Celsius (C.) per second.

17. The system of claim 12, further comprising a lens disposed between the channel and the image sensor, wherein the lens is configured to focus onto a first image sensor region of the image sensor light coming from the DNA amplification zone and focus onto a second image sensor region of the image sensor light coming from the DNA melting zone.

18. The system of claim 12, further comprising an excitation source for producing electromagnetic radiation directed at the channel.

19. The system of claim 12, wherein the DNA melting zone is operable to receive a plurality of spaced apart boluses of solution containing real-time PCR reagents.

20. The system of claim 19, further comprising an image sensor controller configured such that, for each said bolus that enters the DNA melting zone, the image sensor controller captures at least about 10 images per second from the second image sensor region for at least about 1 minute.

21. The system of claim 20, wherein the image sensor controller is further configured so that, for each said bolus that undergoes PCR processing in the DNA amplification zone, the controller captures at least about 1 image every 90 seconds from the first image sensor region for at least about 10 minutes while the bolus is undergoing the PCR processing.

22. The system of claim 20, wherein the image sensor controller is further configured so that, for each said bolus that undergoes PCR processing in the DNA amplification zone, the controller captures not more than 1 image every 1 second from the first image sensor region for at least about 10 minutes while the bolus undergoes the PCR processing.

23. In a system comprising (a) an image sensor and (b) a microfluidic channel comprising a PCR processing zone and a separate HRTm analysis zone, a method, comprising:

(a) using the PCR processing zone of the channel to achieve PCR;

(b) using the HRTm analysis zone of the channel to perform an HRTm process;

(c) while performing step (b), using the image sensor to obtain images of the HRTm analysis zone; and

(d) after performing step (c) and without moving the image sensor from the position it was in relative to the channel when step (c) was performed, using the image sensor to obtain images of and the PCR processing zone while performing step (a).

24. The method of claim 23, wherein the step of using the PCR processing zone to achieve PCR comprises:

moving a bolus of test solution containing real-time PCR reagents through the PCR processing zone; and

while the bolus is in the PCR processing zone, cycling the temperature of the bolus.

25. The method of claim 24, wherein the step of cycling the temperature of the bolus comprises heating the bolus to a first temperature, cooling the bolus to a second temperature, and heating the bolus to a third temperature.

26. The method of claim 24, wherein the step of using the HRTm analysis zone to perform an HRTm process comprises:

moving a bolus of test solution containing real-time PCR reagents through the HRTm analysis zone; and

while the bolus is in the HRTm analysis zone, steadily increasing the temperature of the bolus.

27. The method of claim 26, wherein:

the step of cycling the temperature of the bolus in order to achieve PCR comprises using a first thermal generating apparatus to cycle the temperature, and

the step of steadily increasing the temperature of the bolus comprises using a second thermal generating apparatus to steadily increase the temperature.

28. The method of claim 23, further comprising using a lens to focus onto a first portion of the image sensor light coming from the PCR processing zone and to focus onto a second portion of the image sensor light coming from the HRTm analysis zone.

29. The method of claim 23, further comprising using an excitation source to supply electromagnetic radiation to the PCR processing zone of the channel.

30. The method of claim 23, further comprising causing a bolus of solution containing real-time PCR reagents to move through the PCR processing zone and then through the HRTm analysis zone.

31. The method of claim 30, further comprising:

using the image sensor to capture at least about 10 images of the bolus per second for at least about 1 minute while the bolus is in the HRTm analysis zone.

32. The method of claim 31, further comprising:

using the image sensor to capture at least about 1 image of the bolus every 90 seconds for at least about 10 minutes while the bolus is in the PCR processing zone.

42. The method of claim 31, further comprising:

using the image sensor to capture at least about 1 image of the bolus every 90 seconds for at least about 10 minutes or at least about 20 PCR cycles while the bolus is in the first zone.

33. A method, comprising:

(a) performing a PCR process in a first zone of a microfluidic channel;

(b) performing an HRTm process in a second zone of said microfluidic channel;

(c) while performing step (a), focusing radiation from the first zone onto a first region of an image sensor, but not onto a second region of the image sensor; and

(d) while performing step (c), focusing radiation from the second zone onto the second region of the image sensor, but not onto the first region of the image sensor.

34. The method of claim 33, wherein the step of performing the PCR process in the first zone comprises:

moving a bolus of test solution containing real-time PCR reagents through the first zone; and

while the bolus is in the first zone, cycling the temperature of the bolus in order to achieve PCR.

35. The method of claim 34, wherein the step of cycling the temperature of the bolus comprises heating the bolus to a first temperature, cooling the bolus to a second temperature, and heating the bolus to a third temperature.

36. The method of claim 34, wherein the step of performing an HRTm process in the second zone comprises:

moving a bolus of test solution containing real-time PCR reagents through the second zone after moving the bolus through the first zone; and

while the bolus is in the second zone, steadily increasing the temperature of the bolus.

37. The method of claim 36, wherein:

the step of cycling the temperature of the bolus in order to achieve PCR comprises using a first thermal generating apparatus to cycle the temperature, and

the step of steadily increasing the temperature of the bolus comprises using a second thermal generating apparatus to steadily increase the temperature.

38. The method of claim 33, further comprising using a single lens to perform the focusing steps.

39. The method of claim 33, further comprising using an excitation source to supply electromagnetic radiation to the PCR processing zone of the channel.

40. The method of claim 33, further comprising causing a bolus of solution containing real-time PCR reagents to move through the first zone and then through the second zone.

41. The method of claim 40, further comprising:

using the image sensor to capture at least about 10 images of the bolus per second for at least about 1 minute while the bolus is in the second zone.