Thermal cycling or temperature control device and method using alumina plate

• US 6,228,634 B1
• Filed: 09/03/1999
• Issued: 05/08/2001
• Est. Priority Date: 03/03/1997
• Status: Expired due to Fees

• Current Assignee: Regents of The University of Minnesota (University of Minnesota)
• Original Assignee: Regents of The University of Minnesota (University of Minnesota)
• Inventors: Chaplin, Jonathan, Blumenfeld, Martin
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TAYLOR, JANELL E
• Application Number:
  US09/389,896
• Time in Patent Office: 613 Days
• Field of Search: 435/286.1, 435/287.1, 435/286.2, 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.33, 536/25.3, 935/76, 935/77, 935/86
• US Class Current: 435/286.1
• 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: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: G01N 1/28 Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Qmounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 ; laboratory gas handling apparatus B01L5/00

Abstract

A thermal cycling device comprising a ceramic sample plate, and method of use thereof, is provided. The device is adapted for in situ thermal processing of biological samples on planar substrates, in particular amplification of nucleic acids. The device comprises electrical heating means attached to the ceramic sample plate, and forced air cooling means.

Claims

1. A device for subjecting a plurality of biological samples disposed on at least one substantially flat substrate to thermal cycling, comprising:

a thermal sensing means placed on the surface of one substantially flat substrate and at least one substantially flat substrate lacking said thermal sensing means and comprising at least one biological sample;

a means for holding the plurality of substantially flat substrates, wherein the means for holding comprises a ceramic sample plate, and wherein the substantially flat substrates are disposed on the surface of said holding means;

a means for heating the lower surface of the means for holding, wherein the means for heating is positioned in close proximity to the means for holding, and wherein the means for heating comprises a plate which is parallel to the ceramic plate;

a means for cooling the surface of the means for heating, wherein the means for cooling comprises a rotating means for dispersing air; and

a means for controlling, wherein the controlling means is operatively connected to the means for thermal sensing, the means for heating and the means for cooling such that the temperature of the substrates can be rapidly and controllably increased and decreased by the control means in response to the temperature sensed by the means for sensing such that the biological sample can be subjected to rapid thermal cycling over a temperature range of at least 40°

C.

5. The device of claim 1, 2, 3 or 4 wherein the means for sensing comprises a thermocouple.

6. The device of claim 1, 2, 3 or 4 wherein the means for sensing comprises a means other than a thermocouple.

7. The device of claim 1, 2, 3 or 4 wherein the flat substrate is a glass microscope slide.

8. The device of claim 1, 2, 3 or 4 wherein the flat substrate comprises glass, plastic, nitrocellulose or nylon.

9. The device of claim 1 or 3 wherein the ceramic is alumina.

10. The device of claim 1 or 3 wherein the ceramic is other than alumina.

11. The device of claim 1, 2, 3 or 4 wherein the eating means is an etched foil heater.

12. The device of claim 1, 2, 3 or 4 further comprising a housing containing the means for holding, the means for cooling, the means for heating, and the means for sensing.

13. The device of claim 1 or 3 wherein the space which separates the ceramic plate from the parallel plate is less than about ⅛

of an inch.

14. The device of claim 1, 2, 3 or 4 wherein the parallel plate is formed from a conductive material.

15. The device of claim 14 wherein the material is ceramic, aluminum or copper.

16. A method for amplifying target nucleic acid comprising:

(a) contacting a biological sample, which comprises nucleic acid, that is disposed on a substantially flat substrate with an amount of PCR reagents so as to yield a mixture;

(b) subjecting the mixture to thermal cycling in the device of claim 1 or 2 so as to yield amplified nucleic acid.

17. A method for maintaining the temperature of a biological sample disposed on a substantially flat substrate, comprising contacting the substrate with the device of claim 1, 2, 3 or 4 so as to maintain the temperature of the biological sample on said substrate.

18. A method for in situ PCR amplification of target nucleic acid wherein said amplified nucleic acid is spatially confined to individual cells originally containing said target nucleic acid, comprising:

(a) contacting fixed cells suspected of containing the target nucleic acid with an amount of PCR reagents sufficient to amplify said target nucleic acid so as to form a mixture; and

(b) subjecting the mixture to thermal cycling in the device of claim 1 or 3 so as to yield amplified nucleic acid.

21. The method of claim 16 or 17 wherein said biological sample comprises cells which have been exposed to a fixative.

22. The method of claim 16 or 17 wherein said biological sample is a tissue section.

23. The method of claim 22 wherein the tissue section was previously incubated with antibodies comprising a detectable label.

26. The method of claim 16, 17 or 18 further comprising:

c) detecting said amplified nucleic acid.

2. A thermal cycling device useful for the amplification of nucleic acids, comprising:

a thermal sensing means placed on the surface of one substantially flat substrate and at least one substantially flat substrate lacking said thermal sensing means and comprising at least one biological sample;

a means for holding the plurality of substantially flat substrates, wherein the means for holding comprises an alumina sample plate, and wherein the substantially flat substrates are disposed on the surface of said holding means;

a means for heating the surface of the means for holding, wherein the means for heating is attached to the means for holding, and wherein the means for heating comprises a plate which is parallel to the alumina plate;

a means for cooling the surface of the means for heating, wherein the means for cooling comprises a rotating means for dispersing air; and

a means for controlling, wherein the controlling means is operatively connected to the means for thermal sensing, the means for heating and the means for cooling such that the temperature of the substrates can be rapidly and controllably increased and decreased by the control means in response to the temperature sensed by the means for sensing such that the biological sample can be subjected to rapid thermal cycling over a temperature range of at least 30°

C.

33. The device of claim 2 or 4 wherein the space which separates the alumina plate from the parallel plate is less than about ⅛

of an inch.

3. A device for maintaining the temperature of a plurality of biological samples which are disposed on at least one substantially flat substrate, comprising:

a thermal sensing means placed on the surface of one substantially flat substrate and at least one substantially flat substrate lacking said thermal sensing means and comprising at least one biological sample;

a means for holding the plurality of substantially flat substrates, wherein the means for holding comprises a ceramic sample plate, and wherein the substantially flat substrates are disposed on the surface of said holding means;

a means for heating the surface of the means for holding, wherein the means for heating is positioned in close proximity to the means for holding, wherein the means for heating comprises a plate which is parallel to the alumina plate;

a means for cooling the surface of the means for heating, wherein the means for cooling comprises a rotating means for dispersing air; and

a means for controlling, wherein the controlling means is operatively connected to the means for thermal sensing, the means for heating and the means for cooling such that the temperature of the substrates can be maintained at a particular temperature by the control means in response to the temperature sensed by the means for sensing such that the biological sample can be maintained at a particular temperature over a temperature range of at least 40°

C.

19. A method for in situ hybridization of a target nucleic acid wherein said target nucleic acid is spatially confined to a substantially flat surface, comprising:

(a) contacting the target nucleic acid with an amount of a labeled probe comprising a preselected DNA comprising the target nucleic acid sequence to as to form a mixture;

(b) maintaining the temperature of the mixture for a sufficient time to form binary complexes between at least a portion of said probe and said target nucleic acid, wherein the temperature is maintained on the device of claim 3 or 4; and

(c) detecting the absence or presence of said binary complexes.

20. A method for in situ hybridization of target nucleic acid wherein said target nucleic acid is spatially confined to individual cells originally containing said target nucleic acid, comprising:

(a) contacting fixed cells suspected of containing the target nucleic acid with with an amount of a labeled probe comprising a preselected DNA comprising the target nucleic acid sequence so as to form a mixture; and

(b) maintaining the temperate of the mixture for a sufficient time to form binary complexes between at least a portion of said probe and said target nucleic acid, wherein the temperature is mainlined on the device of claim 3 or 4; and

(c) detecting the absence or presence of said binary complexes.

24. The method of claim 20 wherein a tissue section provides the cells.

25. The method of claim 24 wherein the tissue section was previously incubated with antibodies comprising a detectable label.

4. A device useful for the in situ hybridization of nucleic acids, comprising:

a thermal sensing means placed on the surface of one substantially flat substrate and at least one substantially flat substrate lacking said thermal sensing means and comprising at least one biological sample;

a means for holding the plurality of substantially flat substrates, wherein the means for holding comprises an alumina sample plate, and wherein the substantially flat substrates are disposed on the surface of said holding means;

a means for heating the lower surface of the means for holding, wherein the means for heating is attached to the means for holding, wherein the means for heating is positioned in close proximity to the means for holding, and wherein the means for heating comprises a plate which is parallel to the alumina plate;

a means for cooling the lower surface of the means for heating, wherein the means for cooling comprises a rotating means for dispersing air; and

a means for controlling, wherein the controlling means is operatively connected to the means for thermal sensing, the means for heating and the means for cooling such that the temperature of the substrates can be maintained at a particular temperature by the control means in response to the temperature sensed by the means for sensing such that the biological sample can be maintained at a particular temperature over a temperature range of at least 30°

C.

27. A device for subjecting a biological sample to thermal cycling comprising:

a housing;

a flat substrate having a thermal sensor coupled to said flat substrate, said flat substrate having a biological sample disposed thereon;

a holder for said flat substrate, said holder attached to said housing, wherein said holder comprises a ceramic sample plate which is parallel to a second plate, wherein the sample plate and the second plate are separated by no more than about ⅛

of an inch, and wherein said flat substrate is disposed on the surface of said ceramic sample plate;

a cooler for said flat substrate, said cooler attached to said housing; and

a heater thermally coupled to the second plate.

29. The device of claim 27 or 28 wherein said holder holds a plurality of flat substrates.

30. The device of claim 27 or 28 wherein said cooler is a fan.

31. The device of claim 27 or 28 further comprising:

a controller operatively connected to the thermal sensor, the heater and the cooler such that the temperature of the flat substrate can be controllably increased and decreased by the controller in response to the temperature sensed by the means for thermal sensor.

32. The device of claim 27 or 28 wherein the biological sample can be subjected to rapid thermal cycling over a temperature range of at least 40°

C.

28. A device for maintaining the temperature of a biological sample comprising:

a housing;

a flat substrate having a thermal sensor coupled to said flat substrate, said flat substrate having a biological sample disposed thereon;

a holder for said flat substrate, said holder attached to said housing, wherein said holder comprises a ceramic sample plate which is parallel to a second plate, wherein the sample plate and the second plate are separated by no more than about ⅛

of an inch, and wherein said flat substrate is disposed on the surface of said ceramic sample plate;

a cooler for said flat substrate, said cooler attached to said housing; and

a heater thermally coupled to the second plate.