Methods for localizing target molecules in a flowing fluid sample

• US 20050009066A1
• Filed: 05/17/2004
• Published: 01/13/2005
• Est. Priority Date: 05/15/2003
• Status: Abandoned Application

• Current Assignee: Integrated Nano-Technologies, LLC
• Original Assignee: Integrated Nano-Technologies, LLC
• Inventors: Connolly, Dennis M.
• Application Number:
  US10/847,196
• Publication Number:
  US 20050009066A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502776 specially adapted for focusing or laminating flows
• CPC Class Codes: B03C 5/02 Separators
• CPC Class Codes: G01N 1/40 Concentrating samples
• CPC Class Codes: G01N 2001/4038 electric methods, e.g. electromigration, electrophoresis, ionisation
• CPC Class Codes: G01N 27/44769 Continuous electrophoresis, i.e. the sample being continuously introduced, e.g. free flow electrophoresis [FFE]
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures

Abstract

The present invention relates to a device and method for concentrating and detecting target molecules in a flowing fluid sample. The device includes a housing defining a passage through which a fluid sample flows and a concentrating device positioned in the housing and including at least one electrode on a first side of the passage. The at least one electrode on the first side of the passage has a polarity which electrostatically attracts target molecules in the flowing fluid sample. The device also includes a detection device downstream of the concentrating device and including test structures having capture probes that are capable of specifically binding to the target molecules, if any, in the flowing fluid sample. The test structures are positioned in the housing on the first side of the passage.

Claims

1. A device for concentrating and detecting target molecules in a flowing fluid sample, said device comprising:

a housing defining a passage through which a fluid sample flows;

a concentrating device positioned in the housing and comprising at least one electrode on a first side of the passage, wherein the at least one electrode on the first side of the passage has a polarity which electrostatically attracts target molecules in the flowing fluid sample; and

a detection device downstream of the concentrating device and comprising test structures having capture probes that are capable of specifically binding to the target molecules, if any, in the flowing fluid sample, wherein said test structures are positioned in the housing on the first side of the passage.

2. The device according to claim 1, further comprising:

at least one electrode in the concentrating device on a second side of the passage opposite the first side of the passage.

3. The device according to claim 2, wherein the at least one electrode on the first side of the passage and the at least one electrode on the second side of the passage are oppositely charged.

4. The device according to claim 2, wherein the at least one electrode on the first side of the passage and the at least one electrode on the second side of the passage are different in size.

5. The device according to claim 1, wherein the test structures comprise pairs of electrodes spaced apart by a gap with that gap being bridged by a conductor when the flowing fluid sample contains target molecules to which the capture probes are specific.

6. The device according to claim 5, wherein the capture probes are attached to the spaced apart electrical conductors such that a gap exists between the capture probes on the electrical conductors with that gap being bridged by a conductor when the flowing fluid sample contains target molecules to which the capture probes are specific.

7. The device according to claim 1, wherein the detection device comprises a plurality of different groups of test structures.

8. The device according to claim 1, wherein the capture probes are oligonucleotides.

9. The device according to claim 1, wherein the capture probes are peptide nucleic acid analogs.

10. The device according to claim 1, wherein the capture probes are antibodies.

11. The device according to claim 1, wherein the capture probes specifically bind to target molecules that are nucleic acid molecules.

12. The device according to claim 1, wherein the capture probes specifically bind to target molecules that are proteins.

13. The device according to claim 1 further comprising:

a flow reversing device, coupled to said housing, for reversing direction of fluid flowing through the passage, whereby fluid can be made to flow from the detection device to the concentrating device.

14. The device according to claim 1 further comprising:

a reconcentrating device positioned in the housing downstream of the detection device and comprising at least one electrode on the first side of the passage, wherein the at least one electrode on the first side of the passage of the reconcentrating device has a polarity which electrostatically attracts target molecules in the flowing fluid sample; and

a flow reversing device, coupled to said housing, for reversing direction of fluid flowing through the passage, whereby fluid can be made to flow from the reconcentrating device to the detection device.

15. The device according to claim 14 further comprising:

at least one electrode in the reconcentrating device on a second side of the passage opposite the first side of the passage with a polarity opposite that of the first electrode within the reconcentrating device to enhance movement of target molecules toward the electrodes on the first side.

16. The device according to claim 15, wherein the at least one electrode on the first side of the passage of the reconcentrating device and the at least one electrode on the second side of the reconcentrating device are oppositely charged.

17. The device according to claim 15, wherein the electrodes of the reconcentrating device are different in size.

18. The device according to claim 1, wherein the at least one electrode of the concentrating device is coated with a material that prevents the target molecules from contacting the electrode.

19. A method for concentrating and detecting target molecules in a flowing fluid sample, said method comprising:

providing a device comprising;

a housing defining a passage through which a fluid sample flows;

a concentrating device positioned in the housing and comprising at least one electrode on a first side of the passage, wherein the at least one electrode on the first side of the passage has a polarity which electrostatically attracts target molecules in the flowing fluid sample; and

a detection device downstream of the concentrating device and comprising test structures having capture probes that are capable of specifically binding to the target molecules, if any, in the flowing fluid sample, wherein said test structures are positioned in the housing on the first side of the passage;

introducing a sample containing target molecules into said device;

applying an electric field to the at least one electrode on the first side of the passage of the concentrating device under conditions effective to electrostatically attract and concentrate the target molecules, if any, in the flowing fluid sample near the at least one electrode on the first side of the passage of the concentrating device; and

permitting the concentrated target molecules in the flowing fluid sample to specifically bind to the capture probes of the detection device, wherein the presence of the target molecules in the flowing fluid sample is detected.

20. The method according to claim 19, wherein the concentrating device further comprises:

at least one electrode on a second side of the passage opposite the first side of the passage.

21. The method according to claim 20, wherein the at least one electrode on the first side of the passage and the at least one electrode on the second side of the passage are oppositely charged.

22. The method according to claim 20, wherein the at least one electrode on the first side of the passage and the at least one electrode on the second side of the passage are different in size.

23. The method according to claim 19, wherein the test structures comprise pairs of electrodes spaced apart by a gap with that gap being bridged by a conductor when the flowing fluid sample contains target molecules to which the capture probes are specific.

24. The method according to claim 23, wherein the capture probes are attached to electrical conductors such that a gap exists between the capture probes on the electrical conductors with that gap being bridged by a conductor when the flowing fluid sample contains target molecules to which the capture probes are specific.

25. The method according to claim 19, wherein the detection device comprises a plurality of different groups of test structures.

26. The method according to claim 19, wherein the capture probes are oligonucleotides.

27. The method according to claim 26, wherein the oligonucleotides are complementary to the genetic material of a pathogenic bacteria.

28. The method according to claim 27, wherein the pathogenic bacteria is a biowarfare agent.

29. The method according to claim 27, wherein the pathogenic bacteria is a food borne pathogen.

30. The method according to claim 26, wherein the oligonucleotides are complementary to the genetic material of a virus.

31. A method according to claim 26, wherein the oligonucleotides are complementary to the genetic material of a human.

32. A method according to claim 26, wherein the oligonucleotides have a sequence which is complementary to a sequence containing a polymorphism.

33. The method according to claim 19, wherein the capture robes are peptide nucleic acid analogs.

34. The method according to claim 19, wherein the capture probes are antibodies.

35. The method according to claim 19, wherein the target molecules are nucleic acid molecules.

36. The method according to claim 19, wherein the target molecules are proteins.

37. The method according to claim 19, wherein the device further comprises a flow reversing device, coupled to said housing, for reversing direction of fluid flowing through the passage, said method further comprising:

recycling the flowing fluid from the detection device to the concentrating device in order to reconcentrate target molecules in the flowing fluid sample that did not bind to the capture probes of the detection device.

38. The method according to claim 19, wherein the device further comprises:

a reconcentrating device positioned in the housing downstream of the detection device and comprising at least one electrode on the first side of the passage, wherein the at least one electrode on the first side of the passage of the reconcentrating device has a polarity which electrostatically attracts target molecules in the flowing fluid sample and a flow reversing device for reversing direction of fluid flowing through the passage, said method further comprising;

applying an electric field to the at least one electrode on the first side of the passage of the reconcentrating device under conditions effective to electrostatically attract and concentrate the target molecules in the flowing fluid sample; and

recycling the flowing fluid sample from the reconcentrating device to the detection device.

39. The method according to claim 38, wherein the device further comprises:

at least one electrode in the reconcentrating device on a second side opposite the first side of the passage with a polarity opposite the at least one electrode on the second side of the reconcentrating device, whereby target molecules in the flowing fluid sample within the reconcentrating device are electrostatically repelled by the electrodes on the second side toward the electrodes on the first side.

40. The method according to claim 39, wherein the at least two electrodes of the reconcentrating device are oppositely charged.

41. The method according to claim 39, wherein the at least two electrodes of the reconcentrating device are different in size.

42. The method according to claim 19, wherein the at least one electrode of the concentrating device is coated with a material that prevents the target molecules from contacting the electrode.

43. The method according to claim 19, wherein flow of the fluid sample is continuous.

44. The method according to claim 19, wherein flow of the fluid sample is pulsed.

45. The method according to claim 19, wherein the sample contains an additive that increases or decreases electrical mobility of the flowing fluid sample.

46. The method according to claim 45, wherein the additive is a salt, acid, or base.

47. The method according to claim 19, wherein the flowing fluid sample contains an additive that minimizes variations in viscosity.

48. The method according to claim 47, wherein the additive is a solution containing uncharged polymers.

49. The method according to claim 19, further comprising:

coating the capture probes as well as the target molecule bound to the capture probes with a conductive material after said permitting.

50. A method according to claim 49, wherein the conductive material is silver.

51. A method according to claim 49, wherein the conductive material is gold.