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

• US 8,841,116 B2
• Filed: 10/25/2007
• Issued: 09/23/2014
• 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: Mathies, Richard A., Blazej, Robert, Kumaresan, Palani
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US11/978,224
• Publication Number:
  US 20090035770A1
• Time in Patent Office: 2,525 Days
• Field of Search: 435/6.1, 435/283.1, 435/287.2, 435/288.5, 435/288.6, 422/68.1, 204/451, 204/453, 204/604
• US Class Current: 435/283.1
• 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 comprising:

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

i. a capture channel region comprising a capture matrix;

ii. a separation channel region, wherein the structure of the capture channel region and the separation channel region is characterized by at least one of the following;

     1) the capture channel region contacts that separation channel region without intervening channels or

     2) the capture channel region and at least a portion of the separation channel region are arranged in a continuous line along the sample channel, said line being a straight line or a line that deviates from 180°

by no more than about +/−

45°

;

b) a first electrode in electrical communication with the first location;

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 the second location, wherein a voltage applied between the first and third electrodes moves charged molecules from the capture channel region through the separation channel region, wherein the sample channel, first electrode, second electrode and third electrode are all integrated into the same microfabricated structure.

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 wherein the waste port is connected to a sidearm located downstream of the capture channel region.

5. 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.

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

7. The microfabricated structure of claim 1 further comprising a sample channel region configured to contain an unpurified sample of an analyte, wherein the sample channel region includes a thermal cycler chamber to produce extension fragments from a sequencing template.

8. 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.

9. The microfabricated structure of claim 1 further comprising a sample channel region configured to contain an unpurified sample of an analyte, wherein the sample channel region has a volume between about 10-1000 nanoliters.

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

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

12. 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 structure of the capture channel region and the separation channel region is characterized by at least one of the following;

     1) the capture channel region contacts that separation channel region without intervening channels or

     2) the capture channel region and at least a portion of the separation channel region are arranged in a continuous line along the sample channel, said line being a straight line or a line that deviates from 180°

by no more than about +/−

45°

;

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.

13. The microfabricated structure of claim 12 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.

14. The microfabricated structure of claim 1, further 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; and

a reverse separation channel to separate the reverse extension fragments, wherein the forward and reverse capture channel regions and forward and reverse separation channel regions are all integrated into the same microfabricated structure.

15. The microfabricated structure of claim 14, 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.

16. The microfabricated structure of claim 15, further comprising:

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

17. The microfabricated structure of claim 15, further comprising a transfer channel connecting the forward and reverse capture channel regions.

18. The microfabricated structure of claim 15 further comprising passive valving to distribute the microreactor elements into the plurality of thermal cycling chambers.

19. The microfabricated structure of claim 15 further comprising active valving to distribute the microreactor elements into the plurality of thermal cycling chambers.

20. The microfabricated structure of claim 1 further 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 channels are configured to analyze the extension fragments.

21. The microfabricated structure of claim 1 wherein the capture channel region and the separation channel region are part of the same etched main channel.

22. The microfabricated structure of claim 1 wherein the capture channel region and the separation channel region share a common axis.

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

providing a microfabricated structure comprising;

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

i. a capture channel region comprising a capture matrix;

ii. a separation channel region, wherein the structure of the capture channel region and the separation channel region is characterized by at least one of the following;

     1) the capture channel region contacts that separation channel region without intervening channels or

     2) the capture channel region and at least a portion of the separation channel region are arranged in a continuous line along the sample channel, said line being a straight line or a line that deviates from 180°

by no more than about +/−

45°

;

b) a first electrode in electrical communication with the first location;

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 the second location, wherein a voltage applied between the first and third electrodes moves charged molecules from the capture channel region through the separation channel region;

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

driving the analyte in the sample to the capture channel region containing the 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 region.

24. The process of claim 23 further comprising forming a purified sample plug in the capture channel region.

25. The process of claim 23 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.

26. The process of claim 23 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.

27. The process of claim 23 further comprising thermally releasing the sample plug from the capture matrix prior to injection.

28. A process for performing sequencing comprising:

providing a microfabricated structure comprising;

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

i. a capture channel region comprising a capture matrix;

ii. a separation channel region, wherein the structure of the capture channel region and the separation channel region is characterized by at least one of the following;

     1) the capture channel region contacts that separation channel region without intervening channels or

     2) the capture channel region and at least a portion of the separation channel region are arranged in a continuous line along the sample channel, said line being a straight line or a line that deviates from 180°

by no more than about +/−

45°

;

b) a first electrode in electrical communication with the first location

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 location, wherein a voltage applied between the first and third electrodes moves charged molecules from the capture channel region through the separation channel region;

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 the capture channel region comprising the capture matrix;

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

separating the extension fragments in the separation channel.

29. The process of claim 28 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.

30. The process of claim 28 wherein the microreactor element includes a microcarrier element which carries the multiple copies of the clonal sequencing template.

31. The process of claim 28 wherein the microreactor element is a bolus or a microemulsion droplet.

32. The process of claim 28 wherein the microreactor element includes a microbead carrying the multiple copies of the clonal sequencing template.

33. The process of claim 28 wherein the distributing step is done such that only one microreactor element will pass into one thermal cycling chamber.

34. The process of claim 33 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.

35. The process of claim 28 further comprising opening a single microvalve in a distribution channel to distribute reagent containing microelements to the thermal cycling chambers via the distribution channel.

36. The process of claim 28 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.

37. A process for performing sequencing comprising:

providing a microfabricated structure comprising;

a) a plurality of sample channels each comprising;

i. a thermal cycling chamber;

ii. a capture channel region comprising a capture matrix;

iii. a separation channel region, wherein the structure of the capture channel region and the separation channel region is characterized by at least one of the following;

     1) the capture channel region contacts that separation channel region without intervening channels or

     2) the capture channel region and at least a portion of the separation channel region are arranged in a continuous line along the sample channel, said line being a straight line or a line that deviates from 180°

by no more than about +/−

45°

;

b) a first electrode in electrical communication with a first end of each of the plurality of sample channels;

c) one or more waste ports in fluid communication with the capture channel regions;

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

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

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; and

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.

38. The process for performing sequencing of claim 37, wherein at least about 70% of the extension fragments produced are injected into the separation channel.

39. The process for performing sequencing of claim 37, wherein at least about 80% of the extension fragments produced are injected into the separation channel.

40. The process for performing sequencing of claim 37, wherein at least about 90% of the extension fragments produced are injected into the separation channel.

41. A method comprising:

providing a microfabricated structure comprising;

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

i. a capture channel region comprising a capture matrix;

ii. a separation channel region, wherein the structure of the capture channel region and the separation channel region is characterized by at least one of the following;

     1) the capture channel region contacts that separation channel region without intervening channels or

     2) the capture channel region and at least a portion of the separation channel region are arranged in a continuous line along the sample channel, said line being a straight line or a line that deviates from 180°

by no more than about +/−

45°

;

b) a first electrode in electrical communication with the first location;

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 the second location, wherein a voltage applied between the first and third electrodes moves charged molecules from the capture channel region through the separation channel region;

providing in the sample channel a sample comprising analyte molecules and non-analyte molecules;

applying an electrical potential across a fluid path comprising a capture channel region and the waste port, wherein non-analyte molecules are moved by the electrical potential to the waste port;

releasing captured analyte molecules from the capture matrix;

applying an electrical potential across a second fluid path comprising the capture channel region and the separation channel region to move analyte molecules through the separation channel, whereby analyte molecules are resolved; and

detecting the resolved analyte molecules.

42. The method of claim 41 wherein releasing comprises increasing the temperature of the affinity capture matrix.