Apparatus for high throughput chemical reactions

• US 8,252,581 B2
• Filed: 01/22/2008
• Issued: 08/28/2012
• Est. Priority Date: 01/22/2007
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: WaferGen, Inc.
• Inventors: Joseph, Victor, Huda, Amjad, Shivji, Alnoor
• Primary Examiner(s): Boyer, Randy
• Application Number:
  US12/009,953
• Publication Number:
  US 20080176290A1
• Time in Patent Office: 1,680 Days
• Field of Search: 422/50, 422/68.1, 422 8205- 82, 435/287.2
• US Class Current: 435/287.2
• 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 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: B01L 7/5255 by moving sample containers
• CPC Class Codes: B01L 7/54 using spatial temperature gradients
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 21/0332 with temperature control control of temperature G05D23/00; cryostats F17C3/08
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00

Abstract

Apparatus, systems, chips, and methods of performing a large number of simultaneous chemical reactions are provided herein. The chips of the invention comprise addressable units that can be addressed according to the temperature of the reaction to be run. The subject apparatus, systems, and chips are particularly suited for performing polymerase chain reactions on thousands of nucleic acid sequences, up to and including sequences of an entire genome of an organism of interest.

Claims

1. An apparatus comprising at least one heating element, configured to be in thermal contact with a chip said chip comprising a substrate and an array of nanowells, wherein the number of nanowells is greater than 30,000, and wherein the array of nanowells comprises a plurality of individually controllable temperature zones corresponding to a plurality of heating elements, each temperature zone comprising a plurality of nanowells in the substrate, wherein the substrate has a thermal conductivity value higher than 1 W/mK, and each temperature zone being thermally isolated from one another.

2. The apparatus of claim 1, wherein the at least one heating element is in thermal contact with the chip from above and below the chip, and wherein the heating element in thermal contact from below the chip is set at a temperature lower than the temperature of the heating element in thermal contact from above the chip.

3. The apparatus of claim 1, wherein the chip comprises an upper surface and a bottom surface and wherein a first series of nanowells is arranged along one orientation on the upper surface and a second series of nanowells is oriented perpendicular to the first series of nanowells.

4. The apparatus claim 1, wherein the heating element is positioned above or below a stationary chip comprising an array of nanowells.

5. The apparatus of claim 1, wherein the heating element is capable of heating and cooling.

6. The apparatus of claim 1, wherein each temperature zone corresponds to a predetermined annealing temperature for one or more samples within the nanowells within the temperature zone.

7. The apparatus of claim 6, wherein the plurality of temperature zones provides a range from about 52°

C. to about 95°

C.

8. The apparatus of claim 7, wherein the plurality of temperature zones provide a temperature gradient.

9. The apparatus of claim 6, wherein at least one of the temperature zones is set at a temperature ranging from about 52°

C. to about 65°

C. and at least one other temperature zone is set at a temperature ranging from about 90°

C. to about 95°

C.

10. The apparatus of claim 9, further comprising at least one other temperature zone set at temperature ranging from about 68°

C. to about 72°

C.

11. The apparatus of claim 1, wherein the at least one heating element is configured to provide an output comprising a spike waveform of temperature over time.

12. The apparatus of claim 1,wherein an individual nanowell in said array has a dimension of about 250 μ

m in length, about 250 μ

m in width, and a depth of about 525 μ

m, or less.

13. The apparatus of claim 1, wherein the chip is operatively coupled to an optical system that detects optical signals.

14. The apparatus of claim 13, wherein the optical system comprises a plurality of optical detectors.

15. The apparatus of claim 1, wherein the at least one heating element is configured to move relative to the chip.

16. The apparatus of claim 1, wherein the nanowells are configured to contain about 100 nl.

33. The apparatus of claim 1 wherein the nanowell is sealed.

34. The apparatus of claim 1 wherein a cover is placed on the nanowell to encompass the peripheral dimensions that define the open surface of the well.

35. The apparatus of claim 1 wherein the nanowells are addressed to indicate the predetermined temperature for running chemical reactions.

36. The apparatus of claim 1 wherein a given assay is assigned to a nanowell belonging to a temperature zone that most closely corresponds with the given assay.

48. A method of conducting a chemical reaction comprising:

providing a reaction sample to the apparatus of claim 1;

providing the plurality of heating elements; and

conducting the chemical reaction in the reaction sample by varying the temperature of the chip, wherein said varying the temperature is effected by varying the temperature of the plurality of heating elements.

49. The method of claim 48 wherein the chemical reaction is a nucleic acid amplification reaction.

50. The method of claim 48 wherein the chemical reaction is a PCR reaction.

17. A chip for running a reaction comprising an array of thermally communicating addressable units in a substrate, wherein the substrate has a thermal conductivity value higher than 1 W/mK, each unit being configured for running a chemical reaction, wherein the array of thermally communicating addressable units is configured to correspond to a predetermined temperature zone, and wherein an individual unit in said array is dimensioned to hold a chemical reaction mixture of less than about 1 μ

l; and

one or more sensors to determine the position of a heating element relative to the chip.

18. The chip of claim 17 comprising a plurality of arrays.

19. The chip of claim 17 comprising a plurality of arrays, each of which corresponds to a different temperature zone.

20. The chip of claim 17 comprising a plurality of arrays wherein at least one of the arrays is set at an annealing temperature for supporting a nucleic acid amplification reaction and at least one other array is set at a denaturing temperature for supporting a nucleic acid amplification reaction.

21. The chip of claim 17, wherein the zone is addressed to indicate the predetermined temperature zones.

22. The chip of claim 17, wherein the array of addressable units are configured to correspond to six or more predetermined temperature zones.

23. The chip of claim 17, wherein the chip is in thermal contact with a heating element.

25. The apparatus of claim 17 or claim 24, wherein the array of addressable units is greater than about 30,000.

30. The apparatus of claim 17 or claim 24, wherein an individual unit within the array comprises a nanowell for receiving and confining a sample, said well being sealed when filled with the sample.

31. The apparatus of claim 17 or claim 24, wherein the chemical reaction is a nucleic acid amplification reaction.

32. The apparatus of claim 31, wherein the predetermined temperature of a unit is configured to yield at least 90% of homogeneous product from the chemical reaction.

37. The apparatus of claim 17 wherein the chip is made up of a set of smaller chips with a thermal buffer between each of the smaller chips.

38. The apparatus of claim 37 wherein each of the smaller chips correspond to a different temperature zone.

39. The apparatus of claim 17 wherein the chip is configured to analyze a whole genome of an organism.

40. The apparatus of claim 17 wherein the addressable units are coated with a hydrophobic or hydrophilic coating.

41. The apparatus of claim 40 wherein the coating covalently adheres to the surface, or attaches via non-covalent interactions.

42. The apparatus of claim 17 wherein at least one addressable unit contains a solution comprising one of the following:

PCR primer, reverse PCR primer, or PCR probe.

51. A method of conducting a chemical reaction comprising:

providing a reaction sample to the chip of claim 17 or claim 24;

providing the heating element; and

conducting the chemical reaction in the reaction sample by varying the temperature of the chip, wherein said varying the temperature is effected by varying the temperature of the heating element.

52. The method of claim 51 wherein the chemical reaction is a nucleic acid amplification reaction.

53. The method of claim 51 wherein the chemical reaction is a PCR reaction.

24. An apparatus for conducting a chemical reaction requiring cycling at least two temperature levels, comprising:

(a) chip for running a reaction comprising an array of addressable units in a substrate, wherein the substrate has a thermal conductivity value higher than 1 W/mK, each unit being configured for a chemical reaction, wherein the array of the addressable units is configured to correspond to a predetermined temperature zone having a thermal buffer between an adjacent temperature zone, and wherein an individual unit in said array is dimensioned to hold a chemical reaction mixture of less than about 1 μ

l; and

(b) a heating element in thermal contact with the chip.

26. The apparatus of claim 24, further comprising (c) an optical system operatively coupled to the chip, wherein the optical system detects an optical signal coming from an addressed thermo-controllable unit.

27. The apparatus of claim 26, wherein the optical system comprises a plurality of optical detectors.

28. The apparatus of claim 24, further comprising a plurality of heating elements.

29. The apparatus of claim 28, wherein the plurality of heating elements comprises six or more heating elements.

43. The apparatus of claim 24 wherein the addressable unit is 100 μ

m to 1 mm in length, 100 μ

m to 1 mm in width, and 100 μ

m to 1 mm in depth.

44. The apparatus of claim 24 wherein the heating element is controlled to effect cycling at least two temperature levels.

45. The apparatus of claim 44 wherein the heating element is controlled by a predetermined algorithm stored on a computer readable medium linked to the heating element.

46. The apparatus of claim 24 further comprising a top cover slide with a slide heater.

47. The apparatus of claim 46 wherein the slide heater is an indium tin oxide heater that balances the temperature of the surface of the chip so that condensation does not occur.