Channel-less separation of bioparticles on a bioelectronic chip by dielectrophoresis

• US 6,989,086 B2
• Filed: 07/13/2001
• Issued: 01/24/2006
• Est. Priority Date: 09/06/1996
• Status: Expired due to Fees

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Wu, Lei, Cheng, Jing, O'Connell, James P., Sheldon, Edward L. III
• Primary Examiner(s): Nguyen, Nam
• Assistant Examiner(s): BARTON, JEFFREY THOMAS
• Application Number:
  US09/905,755
• Publication Number:
  US 20010045359A1
• Time in Patent Office: 1,656 Days
• Field of Search: 204/547, 204/643, 435/173.7, 435/173.9, 435/285.2, 435/287.2, 435/306.1
• US Class Current: 204/547
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• 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 2219/00853 Employing electrode arrangements
• CPC Class Codes: B01J 2219/00914 by dielectrophoresis
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• 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 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0496 Travelling waves, e.g. in combination with electrical or acoustic forces
• CPC Class Codes: B01L 2400/0644 rotary valves
• 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: 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 3/502761 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: B03C 5/026 : using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• CPC Class Codes: B03C 5/028 : using travelling electric fields, i.e. travelling wave dielectrophoresis [TWD]
• CPC Class Codes: C12N 13/00 : Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: H01L 29/6656 : using multiple spacer layers, e.g. multiple sidewall spacers

Abstract

The present invention comprises devices and methods for performing channel-less separation of cell particles by dielectrophoresis, DC high voltage-pulsed electronic lysis of separated cells, separation of desired components from crude mixtures such as cell lysates, and/or enzymatic reaction of such lysates, all of which can be conducted on a single bioelectronic chip. A preferred embodiment of the present invention comprises a cartridge (10) including a microfabricated silicon chip (12) on a printed circuit board (14) and a flow cell (16) mounted to the chip (12) to form a flow chamber. The cartridge (10) also includes output pins (22) for electronically connecting the cartridge (10) to an electronic controller. The chip (12) % includes a plurality of circular microelectrodes (24) which are preferably coated with a protective permeation layer. Specific cells from various cell mixtures were separated, lysed, and enzymatically digested on the chip.

Claims

1. An assay system, comprising:

a reaction apparatus comprising

a microfabricated chip having a plurality of electrodes, each electrode having a surface coated with a permeation layer, the chip being mounted on a printed circuit board,

a flow chamber comprising a first reaction area and a second reaction area within the flow chamber, wherein the first reaction area and the second reaction area are partially separated by a partition that is impermeable to a fluid, and wherein the flow chamber is mounted over the chip,

a plurality of tubes coupled to the flow chamber, and

an output connector for electrically coupling the reaction apparatus to a controller,

a controller electrically coupled to the reaction apparatus, and

a pump coupled to the plurality of tubes.

2. The system of claim 1 wherein the flow chamber further comprises a detection window.

3. The system of claim 1 further comprising:

a waveform generator electrically coupled to the electrodes to generate signals from the electrodes, and

an oscillator to monitor the signals from the electrodes.

4. The system of claim 1 wherein the plurality of electrodes comprises a first array of electrodes and a second array of electrodes, the first reaction area corresponds to the first array of electrodes, and the second reaction area corresponds to the second array of electrodes.

5. The system of claim 1 wherein the electrodes are disposed in a checkerboard biasing format.

6. The system of claim 1 wherein the electrodes comprise a first array of electrodes and a second array of counterelectrodes, the counterelectrodes being less in number than the electrodes.

7. The system of claim 1 further comprising capture probes immobilized on the permeation layer of the electrodes.

8. The system of claim 1 wherein the flow chamber has a volume of substantially 10 μ

l.

9. The system of claim 1 further comprising a fluid reservoir coupled to the pump.

10. The system of claim 1 wherein the flow chamber is substantially U-shaped, and the plurality of electrodes comprises a first array of electrodes situated within the first reaction area of the flow chamber and a second array of electrodes situated within the second reaction area of the flow chamber.

11. The system of claim 10 wherein the plurality of tubes comprises a first tube, a second tube, and a third tube, wherein the first and second tubes are coupled to the first reaction area and the third tube is coupled to the second reaction area.

12. The system of claim 10 wherein the first array of electrodes is greater in number than the second array of electrodes.

13. An assay system, comprising:

a reaction apparatus comprising;

a microfabricated chip having a plurality of electrodes, each electrode having a surface coated with a permeation layer, the chip being mounted on a printed circuit board,

a flow chamber comprising a first reaction area and a second reaction area within the flow chamber, wherein the first reaction area and the second reaction area are partially separated by a partition that is impermeable to a fluid, and wherein the flow chamber is mounted over the chip,

a plurality of tubes coupled to the flow chamber, and

an output connector for electrically coupling the reaction apparatus to a controller,

a controller electrically coupled to the reaction apparatus,

a pump coupled to the plurality of tubes, and

a laser subsystem configured to deliver excitation energy to the electrodes.

14. The system of claim 13 wherein the laser subsystem comprises a He—

Ne laser component and an optic fiber coupled to the laser component, wherein the optic fiber is capable of delivering excitation energy to the electrodes.

15. The system of claim 13 further comprising a cooled color charge-coupled device capable of detecting signals from the electrodes, the signals being generated after the delivery of excitation energy to the electrodes.

16. The system of claim 13 wherein the flow chamber has a volume of substantially 10 μ

l.

17. A method of manipulating a biological sample comprising a mixture of desired and undesired cellular materials within a single apparatus including a chip, a first and second array of electrodes coated with a permeation layer, a channel-less flow chamber mounted over the chip and having a first reaction area corresponding to the first array of electrodes and a second reaction area corresponding to the second array of electrodes, and a laser subsystem configured to deliver excitation energy, wherein the first reaction area and the second reaction area are partially separated by a partition that is impermeable to a fluid, the method comprising:

pumping separation buffer into the flow chamber,

introducing the sample into the flow chamber,

applying an electric current to the first array of electrodes to bias the electrodes in a checkerboard format, and to provide field maxima at each electrode and field minima between the electrodes, wherein desired materials are collected at the field maxima and undesired materials are collected at the field minima,

washing away the undesired materials,

staining the desired materials, and

generating an image of the desired materials by using the laser subsystem to deliver excitation energy to the desired materials.

18. The method of claim 17 wherein the washing away of the undesired materials step is performed while the application of the electric current to the first array of electrodes is maintained.

19. The method of claim 17 wherein the staining step is accomplished with a fluorescent dye.

20. The method of claim 17 further comprising:

lysing the desired materials by subjecting the materials to a series of electronic pulses having alternate polarity, wherein the polarity of the pulses is alternated between the first and second array of electrodes.