Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

• US 6,660,480 B2
• Filed: 03/05/2002
• Issued: 12/09/2003
• Est. Priority Date: 04/28/1997
• Status: Expired due to Fees

• Current Assignee: UT-Battelle LLC
• Original Assignee: UT-Battelle LLC
• Inventors: Foote, Robert S., Ramsey, J. Michael
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/090,955
• Publication Number:
  US 20020160405A1
• Time in Patent Office: 644 Days
• Field of Search: 435/6, 435/285.2, 435/287.2, 436/94
• US Class Current: 435/6.19
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00831 Glass
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

Claims

1. A method for analyzing nucleic acids comprising the steps of:

a. providing a substrate having a microchannel structure, at least one nucleic acid probe being affixed to at least one portion of said microchannel structure;

b. electrokinetically transporting an analyte nucleic acid to said at least one probe; and

,

c. analyzing for the presence of said analyte nucleic acid hybridized to said at least one probe.

2. A method according to claim 1, wherein the step of electrokinetically transporting an analyte nucleic acid includes electroosmotically transporting a solution containing said analyte nucleic acid through said microchannel structure.

3. A method according to claim 1, wherein the step of electrokinetically transporting an analyte nucleic acid includes electrophoretically migrating said analyte nucleic acid through a solution contained in said microchannel structure.

4. A method according to claim 1, wherein said microchannel structure is in fluid communication with at least two fluid reservoirs and the step of electrokinetically transporting an analyte nucleic acid includes applying electrical voltages to said at least two fluid reservoirs, one of said reservoirs containing a solution of said analyte nucleic acid.

5. A method according to claim 1, wherein the step of analyzing for the presence of said analyte nucleic acid hybridized to said at least one probe includes electrokinetically transporting a flourescent label to the probes and detecting fluorescence emission from said label.

6. A method according to claim 4, wherein said fluorescent label is an intercalating dye.

7. A method according to claim 1, wherein said analyte nucleic acid is fluorescently labeled and the step of analyzing for the presence of said analyte nucleic acid hybridized to said at least one probe comprises detecting fluorescence emission from said label.

8. A method according to claim 1, additionally comprising the step of washing said at least one probe following the transport of analyte nucleic acids to said at least one probe.

9. A method according to claim 4, additionally comprising the step of washing said at least one probe following the transport of said fluorescent label to said at least one probe.

10. A method according to claim 8, wherein said washing step comprises electrokinetically transporting buffer through the microchannel containing said at least one probe.

11. A method according to claim 9, wherein said washing step comprises electrokinetically transporting buffer through the microchannel containing said at least one probe.

12. An apparatus for analyzing nucleic acid comprising:

a. a substrate with a microchannel structure formed therein, said microchannel structure having a probe channel and at least two end portions;

b. at least one nucleic acid probe affixed within said probe channel intermediate said two end portions of said microchannel structure;

c. a cover plate affixed to the substrate, said cover plate and said substrate cooperating to enclose said at least one nucleic acid probe;

d. at least two fluid reservoirs in fluid communication with said end portions of said microchannel structure, one of said reservoirs adapted to contain a fluid comprising at least one substance from the group consisting of analyte nucleic acid and fluorescent label and, optionally, a buffer;

e. a source of electrical voltage applied to said reservoirs for effecting electrokinetic transport of said at least one fluid from said one reservoir through said microchannel structure to a probe site adjacent to said at least one probe in said probe channel; and

f. a detector for detecting the presence of said at least one fluid at said probe site.

13. An apparatus according to claim 12, wherein said at least one fluid comprises an intercalating fluorescent dye, and said detector is operable to detect fluorescence emission from said dye.

14. An apparatus according to claim 12, wherein said at least two fluid reservoirs include a first reservoir containing a solution of said analyte nucleic acid and a second reservoir containing a solution of a fluorescent label.

15. An apparatus according to claim 14, wherein one end portion of said microchannel structure is connected to both said first reservoir and said second reservoir, said voltage source effecting transport of said fluids to said at least one nucleic acid probe in said probe channel.

16. An apparatus according to claim 14, wherein said at least two fluid reservoirs further include a third reservoir containing a wash buffer, one end portion of said microchannel structure being connected to said first, said second and said third reservoirs, said voltage source effecting transport of said fluids to said at least one nucleic acid probe in said probe channel, and transport of said wash buffer through said probe channel and past said at least one nucleic acid probe.

17. An apparatus according to claim 14, wherein said at least two fluid reservoirs further include a third reservoir containing a wash buffer, one end portion of said microchannel structure being connected to said first and said second reservoirs, the other end portion of said microchannel structure being connected to said third reservoir, said at least two fluid reservoirs further including at least two waste reservoirs, said waste reservoirs being connected to said probe channel at said opposite end portions, said voltage source, effecting transport of said analyte nucleic acid and said fluorescent label to said at least one nucleic acid probe in one direction in said probe channel, and transport of said wash buffer in the opposite direction through said probe channel and past said at least one nucleic acid probe.

18. An apparatus according to claim 12, wherein said at least two fluid reservoirs include a first reservoir containing a solution of said analyte nucleic acid, a second reservoir containing a solution of a fluorescent label, and at least one additional waste reservoir.

19. An apparatus according to claim 18, wherein one end portion of said microchannel structure is connected to both said first reservoir and said second reservoir, and another end portion of said microchannel structure is connected to said at least one additional waste reservoir, said voltage source effecting transport of said analyte nucleic acid and said fluorescent label through said probe channel and into said at least one additional waste reservoir.

20. An apparatus according to claim 12, wherein said at least two fluid reservoirs include a first reservoir containing a solution of said analyte nucleic acid, a second reservoir containing a solution of an intercalating dye providing a fluorescent label, and at least one additional waste reservoir, one end portion of said microchannel structure being connected to both said first reservoir and said second reservoir, and another end portion of said microchannel structure being connected to said at least one additional waste reservoir, said voltage source applying an electrical potential between said second reservoir and said at least one additional reservoir, effecting transport of said analyte nucleic acid and said dye through said probe channel and into said at least one additional waste reservoir.

21. An apparatus according to claim 20, wherein said at least one additional waste reservoir comprises an analyte waste reservoir and a separate dye waste reservoir.

22. An apparatus according to claim 20, wherein said at least one additional waste reservoir comprises a combined analyte and dye waste reservoir.

23. An apparatus according to claim 12, wherein said at least two fluid reservoirs include a first reservoir containing a solution of said analyte nucleic acid, a second reservoir containing a wash buffer, and at least one additional waste reservoir, one end portion of said microchannel structure being connected to both said first reservoir and said second reservoir, and another end portion of said microchannel structure being connected to said at least one additional waste reservoir, said voltage source applying an electrical potential between said second reservoir and said at least one additional reservoir, effecting transport of said analyte nucleic acid and said wash buffer through said probe channel and into said at least one additional waste reservoir.

24. An apparatus according to claim 23, wherein said at least one additional waste reservoir comprises an analyte nucleic acid waste reservoir and a separate wash buffer waste reservoir.

25. An apparatus according to claim 23, wherein said at least one additional waste reservoir comprises a combined analyte nucleic acid and wash buffer waste reservoir.

26. An apparatus for analyzing nucleic acids comprising:

a. a substrate having a microchannel structure formed therein said microchannel structure including at least two end portions and a number of different nucleic acid probes being immobilized at multiple probe sites within said microchannel structure, each of said different nucleic acid probes being immobilized at a discrete probe site;

b. a cover plate affixed to the substrate, said cover plate and said substrate cooperating to enclose said probe sites;

c. at least two fluid reservoirs in fluid communication with said end portions of said microchannel structure, one of said reservoirs adapted to contain a fluid comprising at least one substance from the group consisting of analyte nucleic acid and fluorescent label and, optionally, a buffer;

d. a source of electrical voltage applied to said reservoirs for effecting electrokinetic transport of said at least one fluid from said one reservoir through said microchannel structure to a nucleic acid probe site; and

e. a detector for detecting the presence of said at least one fluid at one of said nucleic acid probe sites.