SCANNING REAL-TIME MICROFLUIDIC THERMOCYCLER AND METHODS FOR SYNCHRONIZED THERMOCYCLING AND SCANNING OPTICAL DETECTION

• US 20140045186A1
• Filed: 10/15/2013
• Published: 02/13/2014
• Est. Priority Date: 04/15/2011
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Handique, Kalyan, Ganesan, Karthik, Drummond, Daniel M., Gubatayao, Thomas Catalino
• Granted Patent: US 9,765,389 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1838 using fluid heat transfer medium
• 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 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2035/00366 Several different temperatures used
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/278 Constitution of standards
• 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
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 35/00693 Calibration
• CPC Class Codes: G01N 35/026 having blocks or racks of reaction cells or cuvettes

Abstract

Systems and methods for performing simultaneous nucleic acid amplification and detection. The systems and methods comprise methods for managing a plurality of protocols in conjunction with directing a sensor array across each of a plurality of reaction chambers. In certain embodiments, the protocols comprise thermocycling profiles and the methods may introduce offsets and duration extensions into the thermocycling profiles to achieve more efficient detection behavior.

Claims

1. A apparatus for performing real-time nucleic acid amplification and detection, comprising:

a detector head comprising a plurality of photodetector and light source pairs, said detector head being mounted on a rail, wherein the detector and light source pairs are aligned into a first row and a second row;

a receptacle for a microfluidic cartridge comprising a plurality of independent reaction chambers aligned in adjacent columns of a first row and a second row; and

an aperture plate, the aperture plate configured to be positioned over the microfluidic cartridge when the cartridge is present in the receptacle, the detector head located over the aperture plate,

wherein the aperture plate comprises a plurality of apertures that are each aligned over each of the plurality of reaction chambers when the receptacle is holding the microfluidic cartridge,

wherein the detector head is moveable along the rail, such that each of the plurality of photodetector and light source pairs in the first row can be positioned over each aperture in the first row of the aperture plate, and each of the plurality of photodetector and light source pairs in the second row can be positioned over each aperture in the second row of the aperture plate.

2. The apparatus of claim 1, further comprising

a second detector head comprising a plurality of photodetector and light source pairs, said second detector head being mounted on the rail, wherein the second detector and light source pairs are aligned into a first row and a second row;

a second receptacle for a microfluidic cartridge comprising a plurality of independent reaction chambers aligned in adjacent columns of a first row and a second row; and

a second aperture plate, the second aperture plate configured to be positioned over the second microfluidic cartridge when the second cartridge is present in the second receptacle, the second detector head located over the aperture plate,

wherein the second aperture plate comprises a plurality of apertures that are each aligned over each of the plurality of reaction chambers of the second microfluidic cartridge when the second receptacle is holding the second microfluidic cartridge,

wherein the second detector head is moveable along the rail, such that each of the plurality of photodetector and light source pairs in the first row of the second detector head can be positioned over each aperture in the first row of the second aperture plate, and each of the plurality of photodetector and light source pairs in the second row of the second detector head can be positioned over each aperture in the second row of the second aperture plate.

3. The apparatus of claim 1, wherein the photodetector and light source pairs comprise at least six different photodetector and light source pairs operating in six different wavelengths.

4. The apparatus of claim 3, wherein the six different wavelengths comprise a light source emitting a green colored light, a light source emitting a yellow colored light, a light source emitting an orange colored light, a light source emitting a red colored light, and a a light source emitting a crimson colored light.

5. The apparatus of claim 1, the detector head comprises at least N rows of photodetector and light source pairs, and the detector is configured to move to at least M+N−

1 positions over an aperture plate comprising M rows of apertures.

6. The apparatus of claim 1, wherein the aperture plate comprises steel.

7. The apparatus of claim 6, wherein the aperture plate comprises a thickness of approximately 0.25 inches

8. The apparatus of claim 1, wherein at least part of the aperture plate is electrochemically oxidized to be darker than when the aperture plate is not electrochemically oxidized.

9. The apparatus of claim 1, wherein the aperture plate provides substantially uniform pressure across the area of the microfluidic cartridge, when the cartridge is present within the receptacle.

10. The apparatus of claim 1, further comprising a heater plate, wherein the heater plate is positioned underneath the microfluidic cartridge, if present in the receptacle

11. The apparatus of claim 10, wherein the aperture plate provides substantially uniform pressure substantially across the area of the microfluidic cartridge when the cartridge is present within the receptacle, and wherein the substantially uniform pressure facilitates substantially uniform thermal contact between the reaction chambers and the heater plate.

12. The apparatus of claim 1, wherein the aperture plate comprises at least one of aluminum, zinc or nickel, the aperture plate further comprising a colorant.

13. The apparatus of claim 1, wherein the reaction chamber is configured to receive light at a glancing angle from the light source relative to the photodetector.

14. The apparatus of claim 12, wherein the heater plate comprises a plurality of heating elements, wherein each of the plurality of heating elements is positioned such that when the microfluidic cartridge is present in the receptacle, the plurality of heating elements are in thermal communication with each of the plurality of reaction chambers, respectively.

15. A method implemented on one or more computer processors for optimizing protocols for simultaneously performing a plurality of thermal cycling reactions, wherein each thermal cycling reaction comprises one or more detection steps, and wherein said thermal cycling reactions are performed in a plurality of reactors, the method comprising:

determining or providing or accessing a detection cycle time for each of said plurality of reactors;

receiving or accessing a protocol step, the step associated with a step duration, the step comprising a time for detection; and

determining a first adjustment to the step such that the step duration is a multiple of the detection cycle time.

16. The method of claim 15, further comprising determining a second adjustment to the step, wherein the time for detection is a multiple of the detection cycle time when the step is adjusted by the first adjustment and by the second adjustment.

17. The method of claim 15, further comprising determining a starting offset adjustment based on a position of a reaction chamber associated with the protocol.

18. The method of claim 15, wherein the detection cycle time comprises the amount of time required for a detector head to perform a predetermined plurality of detections for a reactor.

19. The method of claim 18, wherein the detection cycle time further comprises a time required for movement of the detector head to each of a plurality of reactors and movement of the detector head to the start position.

20. The method of claim 15, wherein the protocol comprises a polymerase chain reaction (PCR) protocol.

21. The method of claim 15, further comprising initiating the protocol.

22. A non-transitory computer-readable medium comprising instructions, the instructions configured to cause one or more processors to perform the following method:

determining or providing or accessing a detection cycle time;

receiving or accessing a protocol step, the step associated with a step duration, the step comprising a time for detection;

determining a first adjustment to the step such that the step duration is a multiple of the detection cycle time.

23. The non-transitory computer-readable medium of claim 22, wherein the protocol step is associated with a protocol from a plurality of protocols, each of the plurality of protocols associated with at least one of a plurality of thermal cycling reactions, wherein each thermal cycling reaction comprises one or more detection steps, and wherein the determining a first adjustment is based at least in part on a timing of one or more detection steps associated with the thermal cycling reactions of at least two or more of the plurality of protocols when the two or more of the plurality of protocols are simultaneously run.

24. The non-transitory computer-readable medium of claim 22, the method further comprising determining a second adjustment to the step, wherein the time for detection is a multiple of the detection cycle time when the step is adjusted by the first adjustment and by the second adjustment.

25. The non-transitory computer-readable medium of claim 22, the method further comprising determining a starting offset adjustment based on a position of a reaction chamber associated with the protocol.

26. The non-transitory computer-readable medium of claim 22, wherein the detection cycle time comprises the amount of time required for a detector head to perform a predetermined plurality of detections for a reaction chamber.

27. The non-transitory computer-readable medium of claim 26, wherein the detection cycle time further comprises a time required for movement of the detector head to each of a plurality of reaction chamber detection positions and movement of the detector head to a start position.

28. The non-transitory computer-readable medium of claim 22, wherein the protocol comprises a polymerase chain reaction (PCR) protocol.

29. The non-transitory computer-readable medium of claim 22, the method further comprising initiating the protocol.

30. A system for optimizing protocols for a plurality of reaction chambers, the system comprising:

a processor configured to perform the following;

determining or providing or accessing a detection cycle time;

receiving or accessing a protocol step, the step associated with a step duration, the step comprising a time for detection; and

determining a first adjustment to the step such that the step duration is a multiple of the detection cycle time.

31. The system of claim 30, wherein the protocol step is associated with a protocol from a plurality of protocols, each of the plurality of protocols associated with at least one of a plurality of thermal cycling reactions, wherein each thermal cycling reaction comprises one or more detection steps, and wherein the determining a first adjustment is based at least in part on a timing of one or more detection steps associated with the thermal cycling reactions of at least two or more of the plurality of protocols when the two or more of the plurality of protocols are simultaneously run.

32. The system of claim 30, wherein the processor is further configured to determine a second adjustment to the step, wherein the time for detection is a multiple of the detection cycle time when the step is adjusted by the first adjustment and by the second adjustment.

33. The system of claim 30, wherein the processor is further configured to determine a starting offset adjustment based on a position of a reaction chamber associated with the protocol.

34. The system of claim 30, wherein the detection cycle time comprises the amount of time required for a detector head to perform a predetermined plurality of detections for a reaction chamber.

35. The system of claim 34, wherein the detection cycle time further comprises a time required for movement of the detector head to each of a plurality of reaction chamber detection positions and movement of the detector head to the start position.

36. The system of claim 30, wherein the protocol comprises a polymerase chain reaction (PCR) protocol.

37. The system of claim 30, the processor is further configured to initiate the protocol.

38. A method for simultaneously performing real-time PCR in a plurality of PCR reaction chambers, comprising:

(a) providing a scan time sufficient for a detector assembly to perform a scan cycle during which it can scan each of the plurality of PCR reaction chambers for at least one detectable signal and become ready to repeat the scan;

(b) providing a reaction protocol for each of the PCR reaction chambers that includes multiple cycles, each cycle comprising a cycle time that includes at least one heating step, at least one cooling step, and at least one temperature plateau that includes a reading cycle period during which the detector assembly is to scan the reaction chamber for at least one detectable signal;

(c) determining, using a processor, whether the cycle time for that reaction chamber is the same as or an integer multiple of the scan time, and if not, adjusting the scan time or the cycle time so that the cycle time is the same as or an integer multiple of the scan time;

(d) performing at least steps (b) and (c) for the reaction protocol for each of the plurality of PCR reaction chambers so that the cycle time for each reaction protocol is the same as or an integer multiple of the scan time; and

(e) under direction of a processor, performing real time PCR on each of the reaction chambers using the reaction protocol for each of the reaction chambers, including performing multiple scan cycles with the detector assembly, wherein each PCR reaction chamber is scanned by the detector assembly during each reading cycle period for that reaction chamber.

39. The method of claim 38, further comprising phase adjusting the cycle time of the reaction protocol for at least one of the reaction chambers.

40. The method of claim 38, wherein at least one said reaction protocol is different from another said reaction protocol.

41. The method of claim 40, wherein at least one cycle time in one reaction protocol is different from the cycle time in another reaction protocol.