Molecular analyzer and method of use

• US 5,840,573 A
• Filed: 10/03/1996
• Issued: 11/24/1998
• Est. Priority Date: 02/01/1994
• Status: Expired due to Fees

• Current Assignee: Igene, Inc.
• Original Assignee: Robert E. Fields
• Inventors: Fields, Robert E.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/687,589
• Time in Patent Office: 782 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/287.2, 435/286.6, 435/286.7, 261/42, 261/158, 261/159, 422/3, 422/105, 422/109, 422/99, 422/106, 422/101, 422/102, 422/103, 422/104
• US Class Current: 435/287.2
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• 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 9/06 Test-tube stands; Test-tube holders
• CPC Class Codes: B01L 9/065 specially adapted for capillary tubes
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• 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 21/763 Bioluminescence

Abstract

A molecular analyzer and method of use for sensitive, rapid and accurate detection of specific, or target, molecules or specific parts of molecules in a liquid sample to be detected without the use of radioisotopes. Apparatus for sealingly holding any number of sample tubes (23) containing a test sample and having pre-incorporated, internally contained detection reagents for removing unbound molecules from the sample tubes, as well as means for rapidly changing the temperature of the test sample and means for quantitating the target molecules without allowing molecules in the sample tube to contact the environment is provided. Alternatively, the apparatus may be adapted to receive detection reagents that are not preincorporated without exposing them or the test sample to the atmosphere.

Claims

1. A molecular analyzer comprising a sample tube holder having a rigid frame with oppositely disposed sides, at least one internal chamber contained inside one of said sides of said frame, at least one sample tube, at least one internal conduit for providing communication between said at least one internal chamber and said at least one sample tube, characterized by;

said sample tube holder being further comprised of elastomeric members fitted to and held in close connection to said rigid frame;

said at least one sample tube being sealingly held between said elastomeric members;

said at least one internal chamber being filled with a detection reagent;

at least one port provided in one of said elastomeric members, said at least one port providing sealable access for a test sample to be loaded into said at least one sample tube;

an evacuated chamber contained within another of said sides of said frame and being in communication with said at least one sample tube;

means for blocking and unblocking said at least one internal conduit such that when said at least one internal conduit is blocked no communication between said at least one internal chamber and said at least one sample tube occurs, and when said at least one internal conduit is unblocked communication between said at least one internal chamber and said at least one sample tube occurs, whereby said detection reagent can flow through said at least one internal conduit, said at least one sample tube and into said evacuated chamber; and

means for controlling a temperature of said test sample.

2. The molecular analyzer of claim 1 wherein said means for controlling a temperature of said test sample comprises:

a sample compartment, said sample compartment being provided with means for holding at least one said sample tube holder and a first fan for recirculating air at a first temperature within said sample compartment;

a hot air compartment, said hot air compartment being provided with a heating element and a second fan for recirculating hot air at a second temperature within said hot air compartment;

a cold air compartment, said cold air compartment being provided with a cooling element and a third fan for recirculating cold air at a third temperature within said cold air compartment;

a first door located between said sample compartment and said hot air compartment, said first door being moveable between an closed position of said first door and at least one open position of said first door;

a second door located between said sample compartment and said cold air compartment, said second door being moveable between a closed position of said second door and at least one open position of said second door;

wherein;

when said first door is in said closed position of said first door and said second door is in said closed position of said second door, no air circulates between said sample compartment and said hot air compartment and no air circulates between said sample compartment and said cold air compartment;

when said first door is in said at least one open position of said first door and said second door is in said closed position of said second door, air from said sample compartment and said hot air compartment circulates between said sample compartment and said hot air compartment, and no air circulates between said sample compartment and said cold air compartment; and

when said first door is in said closed position of said first door and said second door is in said at least one open position of said second door, air from said sample compartment and said cold air compartment circulates between said sample compartment and said cold air compartment, and no air circulates between said sample compartment and said hot air compartment.

3. The molecular analyzer of claim 2 wherein said sample compartment is further provided with a first section maintained at said first temperature, a second section maintained at said third temperature by said cold air recirculating between said cold air compartment and said second section of said sample compartment, and a base for holding said at least one sample tube holder comprised of a thermal conductive material dividing said sample compartment into said first and second sections;

wherein;

said detection reagents are maintained at said third temperature.

4. The molecular analyzer of claim 2 wherein said first temperature is a temperature optimal for DNA polymerase activity.

5. The molecular analyzer of claim 4 wherein said first temperature is lowered to a temperature optimal for DNA annealing when said second door is moved from said closed position of said second door to said at least one open position of said second door.

6. A molecular analyzer comprising a sample tube holder having a frame having two pairs of opposing sides and being provided with at least one pair of internal cavities formed within each of one of said pairs of opposing sides of said frame, and at least one sample tube having an internal volume communicating with each of said at least one pair of internal cavities, characterized by:

said at least one sample tube being sealingly held between one of said pairs of opposing sides of said frame;

at least one pair of orifices, each of said pair of orifices being provided in each of one of said pairs of opposing sides of said frame, said at least one pair of orifices providing sealable access to said at least one pair of internal cavities;

means for filling said at least one sample tube with a test sample wherein said test sample is loaded into one of said at least one pair of orifices and flows into said at least one sample tube;

means for passing at least one detection reagent through said at least one sample tube, wherein said at least one detection reagent is loaded into one of said at least one pair of orifices, flows through said at least one sample tube, and is removed from said other of said at least one pair of orifices; and

means for controlling the temperature of said test sample.

7. The molecular analyzer of claim 6 wherein said means for controlling the temperature of said test sample comprises:

a sample compartment, said sample compartment being provided with means for holding at least one said sample tube holder and a first fan for recirculating air at a first temperature within said sample compartment;

a hot air compartment, said hot air compartment being provided with a heating element and a second fan for recirculating hot air at a second temperature within said hot air compartment;

a cold air compartment, said cold air compartment being provided with a cooling element and a third fan for recirculating cold air at a third temperature within said cold air compartment;

a first door located between said sample compartment and said hot air compartment, said first door being moveable between a closed position of said first door and at least one open position of said first door;

a second door located between said sample compartment and said cold air compartment, said second door being moveable between a closed position of said second door and at least one open position of said second door;

wherein;

when said first door is in said closed position of said first door and said second door is in said closed position of said second door, no air circulates between said sample compartment and said hot air compartment and no air circulates between said sample compartment and said cold air compartment;

when said first door is in said at least one open position of said first door and said second door is in said closed position of said second door, air from said sample compartment and said hot air compartment circulates between said sample compartment and said hot air compartment, and no air circulates between said sample compartment and said cold air compartment; and

when said first door is in said closed position of said first door and said second door is in said at least one open position of said second door, air from said sample compartment and said cold air compartment circulates between said sample compartment and said cold air compartment, and no air circulates between said sample compartment and said hot air compartment.

8. The molecular analyzer of claim 7 wherein said sample compartment is further provided with a first section maintained at said first temperature, a second section maintained at said third temperature by said cold air recirculating between said cold air compartment and said second section of said sample compartment, and a base for holding said at least one sample tube holder comprised of a thermal conductive material dividing said sample compartment into said first and second sections;

wherein;

said detection reagents are maintained at said third temperature.

9. A molecular analyzer comprising a sample tube holder comprised of a rigid frame provided with oppositely disposed recesses, and at least one sample tube, characterized by:

said recesses receiving a pair of elastomeric strips;

said at least one sample tube being sealingly held between said elastomeric strips, said elastomeric strips and said at least one sample tube being together removable from said frame;

at least one internal cavity formed within one of said elastomeric strips and located behind each of said at least one sample tube holder, said at least one internal cavity communicating with an internal volume of said at least one sample tube;

at least one pair of orifices, each of said pair of orifices being provided in each of one of said pair of elastomeric strips, said at least one pair of orifices providing sealable access to said at least one internal cavity;

means for filling said at least one sample tube with a test sample wherein said test sample is loaded into one of said at least one pair of orifices and flows into said at least one sample tube;

means for passing at least one detection reagent through said at least one sample tube, wherein said at least one detection reagent is loaded into one of said at least one pair of orifices, flows through said at least one sample tube, and is removed from said other of said at least one pair of orifices; and

means for controlling the temperature of said test sample.

10. The molecular analyzer of claim 9 wherein said means for controlling the temperature of said test sample comprises:

a sample compartment, said sample compartment being provided with means for holding at least one said sample tube holder and a first fan for recirculating air at a first temperature within said sample compartment;

a hot air compartment, said hot air compartment being provided with a heating element and a second fan for recirculating hot air at a second temperature within said hot air compartment;

a cold air compartment, said cold air compartment being provided with a cooling element and a third fan for recirculating cold air at a third temperature within said cold air compartment;

a first door located between said sample compartment and said hot air compartment, said first door being moveable between a closed position of said first door and at least one open position of said first door;

a second door located between said sample compartment and said cold air compartment, said second door being moveable between a closed position of said second door and at least one open position of said second door;

wherein;

when said first door is in said closed position of said first door and said second door is in said closed position of said second door, no air circulates between said sample compartment and said hot air compartment and no air circulates between said sample compartment and said cold air compartment;

when said first door is in said at least one open position of said first door and said second door is in said closed position of said second door, air from said sample compartment and said hot air compartment circulates between said sample compartment and said hot air compartment, and no air circulates between said sample compartment and said cold air compartment; and

when said first door is in said closed position of said first door and said second door is in said at least one open position of said second door, air from said sample compartment and said cold air compartment circulates between said sample compartment and said cold air compartment, and no air circulates between said sample compartment and said hot air compartment.

11. The molecular analyzer of claim 10 wherein said sample compartment is further provided with a first section maintained at said first temperature, a second section maintained at said third temperature by said cold air recirculating between said cold air compartment and said second section of said sample compartment, and a base for holding said at least one sample tube holder comprised of a thermal conductive material dividing said sample compartment into said first and second sections;

wherein;

said detection reagents are maintained at said third temperature.

12. A molecular analyzer comprising a sample tube holder comprised of a rigid frame, and at least one sample tube, characterized by:

said sample tube holder being further provided with a pair of oppositely disposed elastomeric strips;

said at least one sample tube being sealingly held between said elastomeric strips;

at least one internal chamber filled with a detection reagent, said at least one internal chamber being contained inside one of said elastomeric strips;

at least one port provided in one of said elastomeric strips, said at least one port providing sealable access for a test sample to be loaded into said at least one sample tube;

an evacuated chamber located within another of said elastomeric strips;

means for providing a pathway between said at least one internal chamber and said evacuated chamber whereby said detection reagent flows from said internal chamber through said at least one sample tube and into said evacuated chamber; and

means for controlling the temperature of said test sample.

13. The molecular analyzer of claim 12 wherein said means for controlling the temperature of said test sample comprises:

a sample compartment, said sample compartment being provided with means for holding at least one said sample tube holder and a first fan for recirculating air within said sample compartment;

a hot air compartment, said hot air compartment being provided with a heating element and a second fan for recirculating hot air within said hot air compartment;

a cold air compartment, said cold air compartment being provided with a cooling element and a third fan for recirculating cold air within said cold air compartment;

a first door located between said sample compartment and said hot air compartment, said first door being moveable between a closed position of said first door and at least one open position of said first door;

a second door located between said sample compartment and said cold air compartment, said second door being moveable between a closed position of said second door and at least one open position of said second door;

wherein;

when said first door is in said closed position of said first door and said second door is in said closed position of said second door, no air circulates between said sample compartment and said hot air compartment and no air circulates between said sample compartment and said cold air compartment;

when said first door is in said at least one open position of said first door and said second door is in said closed position of said second door, air from said sample compartment and said hot air compartment circulates between said sample compartment and said hot air compartment, and no air circulates between said sample compartment and said cold air compartment; and

when said first door is in said closed position of said first door and said second door is in said at least one open position of said second door, air from said sample compartment and said cold air compartment circulates between said sample compartment and said cold air compartment, and no air circulates between said sample compartment and said hot air compartment.

14. The molecular analyzer of claim 13 wherein said sample compartment is further provided with a first section, a second section maintained at the same temperature as said cold air compartment by said cold air recirculating between said cold air compartment and said second section of said sample compartment, and said means for holding said at least one sample tube holder is comprised of a thermal conductive base dividing said sample compartment into said first and second sections;

whereby;

said detection reagents are maintained at the said third temperature.

15. A molecular analyzer having a plurality of sample tubes, characterized by:

a pair of oppositely disposed elastomeric members;

said plurality of sample tubes being sealingly held between said elastomeric members;

at least one internal chamber, said at least one internal chamber being filled with a detection reagent and contained inside one of said elastomeric members;

at least one internal conduit for providing communication between said at least one internal chamber and said at least one sample tube;

at least one internal cavity formed behind each of said at least one sample tube, said at least one internal cavity communicating with an internal volume of said at least one sample tube;

at least one port provided in one of said elastomeric members, said at least one port providing sealable access for a test sample to be loaded into said at least one internal cavity;

an evacuated chamber located within another of said elastomeric members and being in communication with said at least one sample tube;

means for blocking and unblocking said at least one internal conduit such that when said at least one internal conduit is blocked no communication between said at least one internal chamber and said at least one sample tube occurs, and when said at least one internal conduit is unblocked communication between said at least one internal chamber and said at least one sample tube occurs; and

means for controlling the temperature of said test sample.

16. The molecular analyzer of claim 15 wherein said means for controlling the temperature of said test sample comprises:

a sample compartment, said sample compartment being provided with means for holding at least one said sample tube holder and a first fan for recirculating air within said sample compartment;

a hot air compartment, said hot air compartment being provided with a heating element and a second fan for recirculating hot air within said hot air compartment;

a cold air compartment, said cold air compartment being provided with a cooling element and a third fan for recirculating cold air within said cold air compartment;

a first door located between said sample compartment and said hot air compartment, said first door being moveable between a closed position of said first door and at least one open position of said first door;

a second door located between said sample compartment and said cold air compartment, said second door being moveable between a closed position of said second door and at least one open position of said second door;

wherein;

when said first door is in said closed position of said first door and said second door is in said closed position of said second door, no air circulates between said sample compartment and said hot air compartment and no air circulates between said sample compartment and said cold air compartment;

when said first door is in said at least one open position of said first door and said second door is in said closed position of said second door, air from said sample compartment and said hot air compartment circulates between said sample compartment and said hot air compartment, and no air circulates between said sample compartment and said cold air compartment; and

when said first door is in said closed position of said first door and said second door is in said at least one open position of said second door, air from said sample compartment and said cold air compartment circulates between said sample compartment and said cold air compartment, and no air circulates between said sample compartment and said hot air compartment.

17. The molecular analyzer of claim 16 wherein said sample compartment is further provided with a first section, a second section maintained at the same temperature as said cold air compartment by said cold air recirculating between said cold air compartment and said second section of said sample compartment, and said means for holding said at least one sample tube holder is comprised of a thermal conductive base dividing said sample compartment into said first and second sections;

whereby;

said detection reagents are maintained at said third temperature.

18. A thermal controller for a molecular analyzer comprising:

a sample compartment, said sample compartment being provided with means for holding at least one sample tube holder and a first fan for recirculating air at a first temperature within said sample compartment;

a hot air compartment, said hot air compartment being provided with a heating element and a second fan for recirculating hot air at a second temperature within said hot air compartment;

a cold air compartment, said cold air compartment being provided with a cooling element and a third fan for recirculating cold air at a third temperature within said cold air compartment;

a first door located between said sample compartment and said hot air compartment, said first door being moveable between a closed position of said first door and at least one open position of said first door;

a second door located between said sample compartment and said cold air compartment, said second door being moveable between a closed position of said second door and at least one open position of said second door;

wherein;

when said first door is in said closed position of said first door and said second door is in said closed position of said second door, no air circulates between said sample compartment and said hot air compartment and no air circulates between said sample compartment and said cold air compartment;

when said first door is in said at least one open position of said first door and said second door is in said closed position of said second door, air from said sample compartment and said hot air compartment circulates between said sample compartment and said hot air compartment, and no air circulates between said sample compartment and said cold air compartment; and

when said first door is in said closed position of said first door and said second door is in said at least one open position of said second door, air from said sample compartment and said cold air compartment circulates between said sample compartment and said cold air compartment, and no air circulates between said sample compartment and said hot air compartment.

19. The thermal controller of claim 18 wherein said first temperature is a temperature optimal for DNA polymerase activity.

20. The thermal controller of claim 19 wherein said second temperature is maintained at 200°

Celsius, and said third temperature is maintained at 0°

Celsius.

21. The thermal controller of claim 20 wherein said first temperature is raised to approximately 96°

Celsius when said first door is moved from said closed position of said first door to said at least one open position of said first door.

22. The thermal controller of claim 19 wherein said first temperature is lowered to a temperature optimal for DNA annealing when said second door is moved from said closed position of said second door to said at least one open position of said second door.

23. The thermal controller of claim 18 wherein said sample compartment is further provided with a first section, a second section maintained at said third temperature by said cold air recirculating between said cold air compartment and said second section of said sample compartment, and said means for holding said at least one sample tube holder is comprised of a thermal conductive base dividing said sample compartment into said first and second sections;

whereby;

at least one detection reagent contained within said sample tube holder is maintained at said third temperature.

24. A molecular analyzer comprising a sample tube holder comprised of a rigid frame having two pairs of opposing sides, at least one sample tube held between one pair of opposing sides, one side being provided with at least one well formed in said side and at least one internal cavity formed within the other side of said pair of opposing sides of said frame and located behind and communicating with an internal volume of said at least one sample tube, characterized by:

said at least one sample tube being sealingly held between one of said pairs of opposing sides of said frame;

at least one sample port provided within said side of said other of said one of said pairs of opposing sides of said frame, said at least one sample port providing sealable access for a test sample to be loaded into said at least one sample tube;

at least one reagent port provided within said side of said other of said one of said pairs of opposing sides of said frame, said at least one reagent port providing sealable access for at least one detection reagent to be loaded into said at least one sample tube;

an evacuated block provided with an interior hollow chamber and at least one stud for engaging said at least one well such that when said at least one stud is received by said at least one well, a fluid path is provided between said at least one internal cavity and said interior hollow chamber; and

means for controlling the temperature of said test sample.

25. The molecular analyzer of claim 24 wherein said means for controlling the temperature of said test sample comprises:

a sample compartment, said sample compartment being provided with means for holding at least one said sample tube holder and a first fan for recirculating air within said sample compartment;

a hot air compartment, said hot air compartment being provided with a heating element and a second fan for recirculating hot air within said hot air compartment;

a cold air compartment, said cold air compartment being provided with a cooling element and a third fan for recirculating cold air within said cold air compartment;

a first door located between said sample compartment and said hot air compartment, said first door being moveable between a closed position of said first door and at least one open position of said first door;

a second door located between said sample compartment and said cold air compartment, said second door being moveable between a closed position of said second door and at least one open position of said second door;

wherein;

when said first door is in said closed position of said first door and said second door is in said closed position of said second door, no air circulates between said sample compartment and said hot air compartment and no air circulates between said sample compartment and said cold air compartment;

when said first door is in said at least one open position of said first door and said second door is in said closed position of said second door, air from said sample compartment and said hot air compartment circulates between said sample compartment and said hot air compartment, and no air circulates between said sample compartment and said cold air compartment; and

when said first door is in said closed position of said first door and said second door is in said at least one open position of said second door, air from said sample compartment and said cold air compartment circulates between said sample compartment and said cold air compartment, and no air circulates between said sample compartment and said hot air compartment.

26. The molecular analyzer of claim 25 wherein said sample compartment is further provided with a first section, a second section maintained at the same temperature as said cold air compartment by said cold air recirculating between said cold air compartment and said second section of said sample compartment, and said means for holding said at least one sample tube holder is comprised of a thermal conductive base dividing said sample compartment into said first and second sections;

whereby;

said detection reagents are maintained at said third temperature.

27. A molecular analyzer comprising a sample tube holder comprised of a rigid frame having two pairs of opposing sides, and at least one sample tube sealingly held between one pair of said opposing sides, characterized by:

a reagent chamber having an internal volume and slidably engageable with a side of one of said pairs of opposing sides, said reagent chamber being provided with at least one reagent port providing sealable access for at least one reagent to be loaded into said reagent chamber;

an evacuated chamber having an internal volume and slidably engageable with another side of one of said pairs of opposing sides, whereby a fluid passageway is provided between said internal volume of said reagent chamber, said at least one sample tube and said internal volume of said evacuated chamber when said reagent chamber and said evacuated chamber are engaged with said frame; and

means for controlling the temperature of a test sample disposed within said at least one sample tube.

28. The molecular analyzer of claim 27 wherein said means for controlling the temperature of said test sample comprises:

a sample compartment, said sample compartment being provided with means for holding at least one said sample tube holder and a first fan for recirculating air within said sample compartment;

a hot air compartment, said hot air compartment being provided with a heating element and a second fan for recirculating hot air within said hot air compartment;

a cold air compartment, said cold air compartment being provided with a cooling element and a third fan for recirculating cold air within said cold air compartment;

a first door located between said sample compartment and said hot air compartment, said first door being moveable between a closed position of said first door and at least one open position of said first door;

a second door located between said sample compartment and said cold air compartment, said second door being moveable between a closed position of said second door and at least one open position of said second door;

wherein;

when said first door is in said closed position of said first door and said second door is in said closed position of said second door, no air circulates between said sample compartment and said hot air compartment and no air circulates between said sample compartment and said cold air compartment;

when said first door is in said at least one open position of said first door and said second door is in said closed position of said second door, air from said sample compartment and said hot air compartment circulates between said sample compartment and said hot air compartment, and no air circulates between said sample compartment and said cold air compartment; and

when said first door is in said closed position of said first door and said second door is in said at least one open position of said second door, air from said sample compartment and said cold air compartment circulates between said sample compartment and said cold air compartment, and no air circulates between said sample compartment and said hot air compartment.

29. The molecular analyzer of claim 28 wherein said sample compartment is further provided with a first section, a second section maintained at the same temperature as said cold air compartment by said cold air recirculating between said cold air compartment and said second section of said sample compartment, and said means for holding said at least one sample tube holder is comprised of a thermal conductive base dividing said sample compartment into said first and second sections;

whereby;

said detection reagents are maintained at said third temperature.