High density electrophoresis device and method

• US 20040178074A1
• Filed: 12/08/2003
• Published: 09/16/2004
• Est. Priority Date: 07/16/1999
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Kowallis, Reid B., Woudenberg, Timothy M.
• Granted Patent: US 7,491,305 B2
• Time in Patent Office: Days
• US Class Current: 204/542
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• CPC Class Codes: B01L 2300/044 pierceable, e.g. films, membranes
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• 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 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: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44782 of a plurality of samples
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 35/00069 : whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk

Abstract

The present invention provides, in one aspect, an apparatus or electrophoretic separation of analytes. In one aspect, the apparatus comprises a disc-shaped substrate defining (1) a central reservoir region, (2) a plurality of electrophoretic channels in fluid communication with, and emanating substantially radially from, the central reservoir region, the channels being coplanar with each other, and each channel having (i) a proximal end which is linked to the reservoir region, and (ii) a distal end, and preferably (3) for each channel, at least one chamber, and preferably three chambers, linked by a passageway in fluid communication with the distal end of that channel. Preferably, each passageway leads from each chamber in a direction that is initially away from the central reservoir region, whereby centrifugation of the substrate about a central axis that is perpendicular to the channels is effective to disperse liquid from the central reservoir region into the channels and chambers, such that any air bubbles in the chambers, channels, and passageways are forced towards the axis of rotation, when such liquid is present in the central reservoir region.

Claims

1. Apparatus for electrophoretic separation of analytes, the device comprising:

a substrate including

(1) a central reservoir region,

(2) a plurality of electrophoretic channels in fluid communication with, and emanating substantially radially from, the central reservoir region, the channels being coplanar with each other, and each channel having (i) a proximal end which is linked to the reservoir region, and (ii) a distal end, and

(3) for each channel one or more chambers that are each linked by a passageway in fluid communication with the distal end of that channel,

wherein each passageway leads from each chamber in a direction that is initially away from the central reservoir region, whereby centrifugation of the substrate about a central axis that is perpendicular to the plane of the channels is effective to disperse liquid from the central reservoir region into said channels and chambers such that any air bubbles in the chambers, channels, and passageways are forced towards the axis of rotation, when such liquid is present in the central reservoir region.

2. The apparatus of claim 1, wherein the distal end of each channel is linked by passageways to two chambers.

3. The apparatus of claim 1, wherein the distal end of each channel is linked by passageways to a sample chamber, a sample-receiving chamber, and a running buffer chamber.

4. The apparatus of claim 1, which further includes electrodes for applying a voltage potential between said chambers and the central reservoir.

5. The apparatus of claim 1, which further includes a detector for detecting selected components which may be present in one or more of the channels.

6. The apparatus of claim 5, wherein the detector is a fluorescence or chemiluminescence detector.

7. The apparatus of claim 5, wherein the detector is rotatable about a central axis within the central reservoir region, for detecting signal emission from each of the channels at a selected distance from the axis.

8. The apparatus of claim 5, which further includes a mechanism for rotating the substrate about a central axis such that the channels pass sequentially by the detector, for detecting one or more components that may be present in the channels.

9. The apparatus of claim 1, wherein said chambers are defined in part by an annular septum that covers the chambers and permits needle-access to the chambers for delivery of liquid to the chambers.

10. The apparatus of claim 1, wherein at least one of said channels contains an electrophoresis medium.

11. The apparatus of claim 10, wherein the electrophoresis medium is a flowable medium.

12. The apparatus of claim 10, wherein the electrophoresis medium is a covalently crosslinked medium.

13. The apparatus of claim 1, wherein the substrate defines at least 20 of said channels.

14. The apparatus of claim 1, wherein said channels have cross-sectional diameters between 1 and 100 μ

m.

15. The apparatus of claim 12, wherein said channels have cross-sectional diameters between 2 and 50 μ

m.

16. A method for preparing a plurality of electrophoretic paths which are substantially bubble-free, comprising

providing an apparatus as defined in claim 1 such that the reservoir region either contains a liquid or is in fluid communication with a liquid source,

centrifuging the substrate about a central axis that is perpendicular to the channels so that the liquid is dispersed from the central reservoir region into said channels and chambers, such that any air bubbles in the chambers, channels, and/or passageways are forced towards the axis of rotation, yielding a plurality of bubble-free electrophoretic paths between said reservoir and said chambers.

17. A method for preparing a plurality of electrophoretic paths which are substantially bubble-free, comprising

providing an apparatus as defined in claim 1 such that the reservoir region, and optionally the channels, passageways, and/or chambers contain a liquid,

centrifuging the substrate about a central axis that is perpendicular to the channels so that the liquid is dispersed from the central reservoir region into said channels and chambers, such that any air bubbles in the chambers, channels, and/or passageways are forced towards the axis of rotation, yielding a plurality of bubble-free electrophoretic paths between said reservoir and said chambers.

18. A method for analyzing a plurality of samples comprising:

providing an apparatus in accordance with claim 1, such that the central reservoir region, channels, and chambers contain a liquid medium suitable for electrophoresis of such samples,

applying an electric field under conditions effective to cause migration of sample through at least one of said channels towards the central reservoir region, and

interrogating at least one of the channel(s) to detect one or more sample components in the channel(s).

19. The method of claim 18, wherein the components to be detected are nucleic acids.

20. The method of claim 19, wherein the nucleic acids to be detected are fluorescently labeled.

21. The method of claim 19, wherein the samples are prepared by polymerase chain reaction amplification of one or more selected target sequences.

22. The method of claim 19, wherein the samples are prepared by ligating at least two oligonucleotides when the oligonucleotides are bound to adjacent regions of a target polynucleotide that are complementary to the at least two oligonucleotides.

23. The method of claim 18, wherein the components to be detected are polypeptides.