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

• US 6,280,590 B1
• Filed: 04/13/2000
• Issued: 08/28/2001
• Est. Priority Date: 09/06/1996
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Cheng, Jing, OʼConnell, James P., Wu, Lei, Sheldon, Edward L. III
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): Starsiak, John S.
• Application Number:
  US09/548,522
• Time in Patent Office: 502 Days
• Field of Search: 204/643, 204/547, 204/600, 204/450, 204/403, 204/409, 435/306.1, 435/287.2, 435/285.2, 435/173.7, 435/173.9
• US Class Current: 204/463
• 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 to divide the chamber into more than one reaction area,

said flow chamber for accepting a biological sample,

each of said more than one reaction area comprising an electrode array having a plurality of electrodes, wherein the electrodes are disposed in the flow chamber and are capable of being biased, and wherein electrode surfaces in the flow chamber are coated with a permeation layer to prevent direct contact between said biological sample and said surfaces, and

a plurality of fluid tubes attached to the flow chamber, wherein at least one fluid tube is in operable connection with each reaction area of the flow chamber.

2. The apparatus of claim 1 wherein the electrodes are disposed in a checkerboard biasing format.

3. The apparatus of claim 1 wherein the electrode arrav further comprises at least one pair of counterelectrodes to enable electronic lysis through a series of electronic pulses between the electrodes and said counterelectrodes, the pulses being generated by a generator.

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

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

a channel-less flow chamber for accepting a biological sample,

said flow chamber comprising a first reaction area, a second reaction area, and a partition therebetween,

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

wherein the plurality of electrodes comprise at least two electrode arrays, and wherein one electrode array is disposed in the first reaction area and one electrode array is disposed in the second reaction area,

a first fluid tube operably connected to the first reaction area of the flow chamber, and

a second fluid tube operably connected to the second reaction area of the flow chamber,

wherein the first fluid tube and the second fluid tube are independently operable relative to each other.

6. The apparatus of claim 5 wherein the electrodes are disposed in a checkerboard biasing arrangement.

7. The apparatus of claim 5 wherein the chip and electrode surfaces in the flow chamber are coated with a permeation layer which prevents direct contact between the biological sample and said surfaces.

8. A method of manipulating a biological sample comprising a mixture of desired and undesired cellular materials within a single apparatus including a flow chamber having a first subchamber, a second subchamber, a first array of electrodes disposed in the first subchamber, a second array of electrodes disposed in the second subchamber, a first set of fluid tubes operably connected to the first subchamber, and a second set of fluid tubes operably connected to the second subchamber, the first and second set of fluid tubes being independently operable, comprising the steps of:

opening the first set of fluid tubes such that a fluid is capable of being introduced therein,

closing the second set of fluid tubes such that a fluid is prevented from being introduced therein,

introducing a biological sample into the first subchamber via the first set of fluid tubes,

subjecting the sample to an electric field by biasing the first array of electrodes within the first subchamber in a checkerboard biasing format, causing desired cellular materials to collect at the electrodes and undesired cellular materials to collect between the electrodes,

lysing the desired cellular materials by subjecting the desired cellular materials to a series of electronic pulses having alternating polarity;

closing the first set of fluid tubes such that a fluid is prevented from being introduced therein,

opening the second set of fluid tubes such a fluid is capable of being introduced therein, and

applying an electrical charge to the array of second electrodes to move the desired cellular materials out of the first subchamber and into the second subchamber.

9. The method of claim 8 wherein the electric field comprises field maxima and field minima, and the subjecting the sample to an electric field step comprises the steps of:

providing electric field maxima at each electrode of the first array of electrodes, and

providing electric field minima between each electrode of the first array of electrodes.

10. The method of claim 8 flirther comprising the step of:

subjecting the desired cellular materials in the second subchamber to an enzymatic reaction by introducing an enzymatic reactant into the second subchamber via the second set of fluid tubes.

11. The method of claim 8 wherein the second array of electrodes include oligonucleotide capture probes disposed on the array for hybridization, and the method further comprises the steps of:

introducing an enzymatic reactant into the second subchamber via the second set of fluid tubes,

producing an enzymatic reaction product by subjecting the desired cellular materials in the second subchamber to the enzymatic reactant, and

hybridizing the enzymatic reaction product by exposure to the oligonucleotide capture probes disposed on the second array of electrodes.

12. The method of claim 11 wherein the oligonucleotide capture probes are immobilized antibodies.