Inline-injection microdevice and microfabricated integrated DNA analysis system using same

• US 20090035770A1
• Filed: 10/25/2007
• Published: 02/05/2009
• Est. Priority Date: 10/25/2006
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Blazej, Robert, Kumaresan, Palani, Mathies, Richard A.
• Granted Patent: US 8,841,116 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• 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 2300/0803 Disc shape
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• 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: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 27/44743 Introducing samples

Abstract

Methods and microfluidic circuitry for inline injection of nucleic acids for capillary electrophoresis analysis are provided. According to various embodiments, microfabricated structures including affinity-based capture matrixes inline with separation channels are provided. The affinity-based capture matrixes provide inline sample plug formation and injection into a capillary electrophoresis channel. Also provided are methods and apparatuses for a microbead-based inline injection system for DNA sequencing.

Claims

1. A microfabricated structure for inline injection of a sample plug into a separation channel, comprising:

a sample channel region for containing an unpurified sample of an analyte;

a capture channel region containing a capture matrix for forming a concentrated sample plug, wherein said capture matrix has a selective affinity for the analyte; and

a separation channel region to receive the sample plug and separate the analyte, wherein the capture channel and separation channel regions are contiguous and/or arranged in a line.

2. The microfabricated structure of claim 1 wherein the capture matrix supports a capture compound having a selective affinity for the analyte.

3. The microfabricated structure of claim 1 wherein the capture matrix forms a purified and concentrated sample plug.

4. The microfabricated structure of claim 1 further comprising a waste port located downstream of the capture channel region and upstream of the separation channel region.

5. The microfabricated structure of claim 1 further comprising a cathode upstream of the capture channel region and an anode downstream of the separation channel region.

6. The microfabricated structure of claim 1 wherein the capture matrix comprises at least one of a gel, immobilized beads, a porous monolith, dense posts, pillars and weirs.

7. The microfabricated structure of claim 1 wherein the capture matrix comprises a UV-polymerized gel.

8. The microfabricated structure of claim 1 wherein the sample channel region includes a thermal cycler chamber to produce extension fragments from a sequencing template.

9. The microfabricated structure of claim 1 wherein the analyte comprises extension fragments from a DNA or RNA template and the capture matrix supports an oligonucleotide complementary to a portion of the extension fragments.

10. The microfabricated structure of claim 1 wherein the sample channel region has a volume between about 10-1000 nanoliters.

12. The microfabricated structure of claim 1 wherein the capture channel region has a volume between about 1-1000 nanoliters.

13. The microfabricated structure of claim 1 wherein the capture channel region has a volume between about 1-100 nanoliters.

14. The microfabricated structure of claim 1 further comprising:

a radial array of inline injection and separation elements, each element comprising;

a sample channel region for containing an unpurified sample of an analyte;

a capture channel region containing a capture matrix for forming a concentrated sample plug, wherein said capture matrix has a selective affinity for the analyte;

a separation channel region to receive the sample plug and separate the analyte, wherein the capture and separation channel regions are contiguous and/or are arranged in a line;

a reagent distribution channel linking the sample channel regions;

a capture matrix distribution channel linking the capture channel regions; and

an anode common to all elements.

15. The microfabricated structure of claim 14 wherein the reagent distribution channel is configured to distribute microreactor elements each of which carries multiple copies of a unique clonal sequencing template into the sample channel regions such that only one microreactor element will pass into one sample channel region.

16. A microfabricated structure for inline injection of a sample plug into a separation channel comprising:

sample region means for containing an unpurified sample of an analyte;

capture matrix means for forming a concentrated sample plug;

means for inline injecting the sample plug into a separation channel; and

the separation channel means for receiving the sample plug and separating the analyte.

17. A microfabricated structure for paired-end sequencing comprising a plurality of sequencing elements, each element comprising:

a thermal cycling reactor for producing forward and reverse extension fragments from a sequencing template;

a forward capture channel region containing a forward capture matrix for concentrating forward extension fragments, wherein said forward capture matrix supports an oligonucleotide that selectively hybridizes to the forward extension fragments;

a reverse capture channel region containing a reverse capture matrix for concentrating reverse extension fragments, wherein said reverse capture matrix supports an oligonucleotide that selectively hybridizes to the reverse extension fragments;

a forward separation channel to separate the forward extension fragments, wherein the forward capture channel region and the forward separation channel region are contiguous and/or arranged in a line; and

a reverse separation channel to separate the reverse extension fragments, wherein the reverse capture channel region and the reverse separation channel region are contiguous and/or arranged in a line.

18. The microfabricated structure of claim 17, further comprising:

a reagent distribution channel to distribute microreactor elements carrying multiple copies of a clonal sequencing template into the plurality of thermal cycling chambers, wherein each thermal cycling chamber receives exactly one unique clonal sequencing template; and

a forward capture matrix distribution channel to distribute forward capture matrix material into the forward capture channel region.

19. The microfabricated structure of claim 18, further comprising:

a reverse capture matrix distribution channel to distribute reverse capture matrix material into the reverse capture channel region.

20. The microfabricated structure of claim 18, further comprising a transfer channel connecting the forward and reverse capture channel regions.

21. The microfabricated structure of claim 18 further comprising passive valving to distribute the microreactor elements into the plurality of thermal cycling chambers.

22. The microfabricated structure of claim 18 further comprising active valving to distribute the microreactor elements into the plurality of thermal cycling chambers.

23. A microfabricated structure comprising:

a distribution channel configured to distribute microreactor elements carrying multiple copies of a clonal sequencing template into each of a plurality of channels such that only one microreactor element will pass into one channel, wherein each channel comprises a thermal cycling chamber connected to a purification chamber connected to a component separation channel, wherein;

thermal cycling extension fragments are produced from a microreactor element in the thermal cycling chambers;

the purification chambers are configured to capture and concentrate the extension fragments; and

the component separation are configured to analyze the extension fragments, and further wherein each component separation channel is contiguous with and/or is arranged in a line with a purification chamber and configured to receive the concentrated extension fragments via inline injection from that purification chamber.

24. A system for performing sequencing comprising:

means for shearing DNA into DNA fragments;

means for ligating the DNA fragments to form a mixture of desired circular and contaminating linear products;

means for exonuclease degradation for selectively removing the contaminating linear products;

emulsion PCR reaction means for generating microspheres carrying multiple clonal copies of a single DNA sequencing template;

fluorescent activated cell sorting (FACS) means for selecting which microspheres have a DNA sequencing template;

microfluidic distribution channel means for distributing a selected microsphere with a DNA sequencing template into a thermal cycling chamber;

valving means for ensuring that statistically only one microsphere will flow into one thermal cycling chamber;

Sanger extension means, including the thermal cycling chambers, for producing thermal cycling extension fragments from the microspheres carrying multiple copies of the DNA sequencing template;

capture means for capturing, purifying and concentrating the extension fragments into a sample plug;

means for releasing the sample plug from the capture means;

means for inline injecting the sample plug into a capillary array electrophoresis channel; and

separation means, including a capillary array electrophoresis channel, for analyzing the extension fragments.

25. A device comprising:

a) a channel defining a flow path between a first end and a second end comprising;

i. a capture channel region comprising an affinity capture matrix;

ii. a separation channel region, wherein the capture channel region and the separation channel regions are contiguous and/or arranged in a line;

b) a first electrode in electrical communication with a first end of the channel;

c) a waste port in fluid communication with the capture channel region;

d) a second electrode in electrical communication with the waste port, wherein a voltage applied between the first and second electrodes moves charged molecules through the capture channel region to the waste port; and

e) a third electrode in electrical communication with a second end of the sample channel, wherein a voltage applied between the first and third electrodes moves charged molecules from the capture channel region through the separation channel region.

26. A process of introducing an analyte in a sample to a separation channel comprising:

introducing a sample containing an analyte to a sample channel region;

driving the analyte in the sample to a capture channel region containing a capture matrix, said matrix having a selective affinity for the analyte;

forming a concentrated sample plug in the capture channel region; and

inline injecting the concentrated sample plug from the capture channel region into the separation channel.

27. The process of claim 26 further comprising forming a purified sample plug in the capture channel region.

28. The process of claim 26 wherein the sample channel region comprises a thermal cycling reactor and introducing a sample containing an analyte to the sample channel region comprises forming extension fragments from a sequencing template in the thermal cycling reactor.

29. The process of claim 26 wherein the analyte comprises extension fragments produced from a sequencing template and forming a concentrated sample plug in the capture channel region comprises selective hybridization of at least some of the extension fragments to oligonucleotides in the capture matrix.

30. The process of claim 26 further comprising thermally releasing the sample plug from the capture matrix prior to injection.

31. A process for performing sequencing comprising:

distributing microreactor elements with DNA sequencing templates into thermal cycling chambers, wherein each microreactor element has multiple clonal copies of a single unique sequencing template;

producing thermal cycling extension fragments from the microreactor elements carrying multiple copies of a sequencing template;

forming a concentrated sample plug of the extension fragments in a capture channel region comprising a capture matrix;

inline injecting the sample plug from the capture matrix into a separation channel; and

separating the extension fragments in the separation channel.

32. The process of claim 31 further comprising introducing capture matrix material into the capture channel region and photo-polymerizing at least a portion of the capture matrix material to produce the capture matrix.

33. The process of claim 31 wherein the microreactor element includes a microcarrier element which carries the multiple copies of the clonal sequencing template.

34. The process of claim 31 wherein the microreactor element is a bolus or a microemulsion droplet.

35. The process of claim 31 wherein the microreactor element includes a microbead carrying the multiple copies of the clonal sequencing template.

36. The process of claim 31 wherein the distributing step is done such that only one microreactor element will pass into one thermal cycling chamber.

37. The process of claim 36 further including using a valve at an exit port of the thermal cycling chambers to ensure that only one microreactor element will flow into one thermal cycling chamber.

38. The process of claim 31 further comprising opening a single microvalve in a distribution channel to distribute reagent containing microelements to the thermal cycling chambers via the distribution channel.

39. The process of claim 31 further comprising sensing the presence of a microelement having multiple clonal copies of DNA sequencing template in a distribution channel and directing that microelement to a thermal cycling reactor.

40. A process for performing sequencing comprising:

distributing microreactor elements with DNA sequencing templates into thermal cycling chambers, wherein each microreactor element has multiple clonal copies of a single unique sequencing template;

producing thermal cycling extension fragments from the microreactor elements carrying multiple copies of a sequencing template;

forming a concentrated sample plug of the extension fragments in a capture channel region comprising a capture matrix;

injecting the sample plug from the capture matrix into a separation channel, wherein at least about 50% of the extension fragments produced are injected into the separation channel.

41. The process for performing sequencing of claim 40, wherein at least about 70% of the extension fragments produced are injected into the separation channel.

42. The process for performing sequencing of claim 40, wherein at least about 80% of the extension fragments produced are injected into the separation channel.

43. The process for performing sequencing of claim 40, wherein at least about 90% of the extension fragments produced are injected into the separation channel.

44. A method comprising:

a) providing in a sample port a sample comprising analyte molecules and non-analyte molecules;

b) moving the sample to a sample channel region;

c) applying an electrical potential across a fluid path comprising the sample channel region, a capture channel region and a waste port, wherein the capture channel region comprises an affinity capture matrix configured to capture analyte molecules and wherein non-analyte molecules are moved by the electrical potential to the waste port;

d) releasing captured analyte molecules from the affinity capture matrix;

e) applying an electrical potential across a second fluid path comprising the capture channel region and a separation channel region of a separation channel, wherein the separation channel region is contiguous with and/or arranged in a line with the capture channel and wherein analyte molecules are moved by the electrical potential through the separation channel, whereby analyte molecules are resolved; and

f) detecting the resolved analyte molecules in the separation channel.

45. The method of claim 44 wherein releasing comprises increasing the temperature of the affinity capture matrix.