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

• US 6,071,394 A
• Filed: 01/30/1998
• Issued: 06/06/2000
• Est. Priority Date: 09/06/1996
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: O'Connell, James P., Wu, Lei, Cheng, Jing, Sheldon, Edward L. III
• Primary Examiner(s): Warden, Sr., Robert J.
• Assistant Examiner(s): ALDAG, ANDREW J
• Application Number:
  US09/016,596
• Time in Patent Office: 858 Days
• Field of Search: 204/547, 204/545, 204/600, 204/643, 204/403, 204/409, 435/173.7, 435/173.9, 435/817, 435/306.1, 435/285.2, 435/287.2, 435/288.5, 422/63, 422/81, 422/50, 422/129
• 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 which prevents direct contact between any electrode and a sample introduced into the flow chamber. The permeation layer also helps to reduce cell adhesion at field minima, and enables immobilization of specific antibodies for specific cell capture. Specific cells from various cell mixtures were separated, lysed, and enzymatically digested on the chip.

Claims

1. An apparatus for at least one of separating, lysing, and enzymatically reacting biological samples, said apparatus comprising:

a channel-less flow chamber having a partition for establishing more than one subchamber within the flow chamber, said flow chamber for accepting a biological sample, and

a bioelectronic chip comprising a plurality of electrodes, wherein the electrodes are disposed in the flow chamber and are each capable of being biased, and wherein the chip and electrode surfaces in the flow chamber are coated with a permeation layer to prevent direct contact between said biological sample and said surfaces.

2. The apparatus of claim 1 wherein the plurality of electrodes comprises at least one array.

3. The apparatus of claim 2 wherein the plurality of electrodes comprises two arrays.

4. The apparatus of claim 1 wherein the flow chamber comprises a covered flow cell attached to the bioelectronic chip.

5. The apparatus of claim 1 wherein the permeation layer enables immobilization of a capture probe thereon.

6. The apparatus of claim 1 wherein the flow chamber has an approximate volume of about 10 microliters.

7. The apparatus of claim 1 wherein the electrodes are addressable in a checkerboard biasing format.

8. The apparatus of claim 1 wherein each electrode is addressable to enable biasing each electrode opposite from nearest neighboring electrodes.

9. The apparatus of claim 1 wherein the plurality of electrodes comprises groups of electrodes, and wherein each group is addressable to enable biasing each group of electrodes opposite from nearest neighboring groups of electrodes.

10. The apparatus of claim 1 further comprising at least one pair of counterelectrodes to enable electronic lysis through a series of electronic pulses between the electrodes and said counterelectrodes.

11. The apparatus of claim 10 comprising two pair of counterelectrodes.

12. The apparatus of claim 1 further comprising an immobilized capture probe on the permeation layer at the electrode surfaces in the flow chamber.

13. The apparatus of claim 12 wherein the capture probe comprises at least one of an antibody and ligand.

14. An apparatus for at least one of separating, lysing, and enzymatically reacting a biological sample, said apparatus comprising:

a channel-less flow chamber having a partition for establishing at least two subchambers within the flow chamber, said flow chamber for accepting a biological sample,

a bioelectronic chip comprising a plurality of electrodes, wherein the electrodes are disposed in the flow chamber and are each capable of being biased, and

wherein the flow chamber comprises a first subchamber and a second subchamber, and the plurality of electrodes comprise at least two electrode arrays, and wherein one electrode array is disposed in the first subchamber and one electrode array is disposed in the second subchamber.

15. The apparatus of claim 14 wherein the electrodes are addressable in a checkerboard biasing format.

16. The apparatus of claim 14 further comprising two pairs of counterelectrodes to enable electronic lysis through a series of electronic pulses between the electrodes and said counterelectrodes.

17. The apparatus of claim 14 wherein the flow chamber comprises a covered flow cell attached to the bioelectronic chip.

18. The apparatus of claim 17 wherein the chip and electrode surfaces in the flow chamber are coated with a permeation layer which enables immobilization of a capture probe thereon.

19. The apparatus of claim 18 wherein the capture probe comprises at least one of an antibody and ligand.

20. A method of manipulating biological samples comprising a mixture of desired cellular materials and undesired cellular materials, the manipulation being achieved in a dielectrophoretic system including a flow chamber having an array of electrodes, comprising the steps of:

introducing a cell mixture suspension into the flow chamber of the system;

subjecting the cell mixture suspension to an electric field by biasing the electrodes in a checkerboard format such that desired cells collect at the electrodes and undesired cells collect between the electrodes; and

washing the undesired cells out of the flow chamber.

21. The method of claim 20 comprising the additional step of lysing the desired cells retained in the flow chamber of the system by subjecting the cells to a series of electronic pulses having alternating polarity.

22. The method of claim 21 comprising the additional step of subjecting the lysed material in the flow chamber of the system to an enzymatic reaction.

23. A method of manipulating biological samples comprising a mixture of desired cellular materials and undesired cellular materials, the manipulation being achieved in a dielectrophoretic system including a flow chamber having a first array of electrodes for separating and lysing, and a second array of electrodes which include oligonucleotide capture probes for hybridizing, comprising the steps of:

introducing a cell mixture suspension into the flow chamber of the system;

subjecting the cell mixture suspension to an electric field by biasing the first array of electrodes in a checkerboard format such that desired cells collect at the electrodes and undesired cells collect between the electrodes;

washing the undesired cells out of the flow chamber;

lysing the desired cells retained in the flow chamber of the system by subjecting the cells to a series of electronic pulses having alternating polarity;

subjecting the lysed material in the flow chamber of the system to an enzymatic reaction; and

hybridizing the enzymatic reaction product by exposure to the second array of electrodes.

24. A method of manipulating biological samples comprising a mixture of desired cellular materials and undesired cellular materials, the manipulation being achieved in a dielectrophoretic system including a flow chamber having a first array of electrodes for separating and lysing, and a second array of electrodes which include oligonucleotide capture probes for hybridizing, comprising the steps of:

introducing a cell mixture suspension into the flow chamber of the system;

subjecting the cell mixture suspension to an electric field by biasing the first array of electrodes in a checkerboard format such that desired cells collect at the electrodes and undesired cells collect between the electrodes;

washing the undesired cells out of the flow chamber;

lysing the desired cells retained in the flow chamber of the system by subjecting the cells to a series of electronic pulses having alternating polarity;

subjecting the lysed material in the flow chamber of the system to an enzymatic reaction; and

exposing the enzymatic reaction product to the second array of electrodes for ligand binding.

25. The method of claim 24 wherein the capture probes are immobilized antibodies.