Microfluidic DNA sample preparation method and device

• US 6,352,838 B1
• Filed: 04/07/2000
• Issued: 03/05/2002
• Est. Priority Date: 04/07/1999
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Gascoyne, Peter R. C., Krulevitch, Peter A., Mariella, Raymond P., Balch, Joseph W., Miles, Robin R., Wang, Xiao-Bo
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/545,150
• Time in Patent Office: 697 Days
• Field of Search: 435/306.1, 435/261, 435/308.1, 435/252.31, 435/34, 204/547, 204/643, 536/23.1
• US Class Current: 435/34
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Manipulation of DNA molecules in solution has become an essential aspect of genetic analyses used for biomedical assays, the identification of hazardous bacterial agents, and in decoding the human genome. Currently, most of the steps involved in preparing a DNA sample for analysis are performed manually and are time, labor, and equipment intensive. These steps include extraction of the DNA from spores or cells, separation of the DNA from other particles and molecules in the solution (e.g. dust, smoke, cell/spore debris, and proteins), and separation of the DNA itself into strands of specific lengths. Dielectrophoresis (DEP), a phenomenon whereby polarizable particles move in response to a gradient in electric field, can be used to manipulate and separate DNA in an automated fashion, considerably reducing the time and expense involved in DNA analyses, as well as allowing for the miniaturization of DNA analysis instruments. These applications include direct transport of DNA, trapping of DNA to allow for its separation from other particles or molecules in the solution, and the separation of DNA into strands of varying lengths.

Claims

1. A method of separating a target material comprising the steps of:

a. flowing a bulk sample comprising target material and contaminant material through a microdevice;

b. exerting a dielectrophoretic force upon said bulk sample within the microdevice to selectively separate said target material from said contaminant material, said target material adhering to a dielectric outer layer of an electrode contained in said microdevice, and said dielectric outer layer having a thickness of less than about 2000 angstroms.

2. The method of claim 1 where said target material is selected from the group consisting of DNA, spores, bacteria, and polystyrene beads.

3. The method of claim 2 wherein said spores comprise Bacillus globigii spores, and said bacteria comprose Erwinia herbicola bacteria.

10. The method of claim 1 wherein said target material adhering on said dielectric outer layer for a predetermined interval.

11. The method of claim 10 wherein said target material comprises DNA.

17. The method of claim 1 wherein said dielectric outer layer has a thickness of about 300 to about 500 angstroms.

4. A microfluidic DNA sample preparation method including the steps of:

(a) separating DNA by dielectrophoretic forces from a bulk sample containing DNA and other materials, said DNA adhering to a dielectric outer layer of thickness less than 1000 angstroms of an electrode exerting said dielectrophoretic forces, and (b) concentrating said DNA from said other materials in said bulk sample to form an extracted DNA.

5. The microfluidic DNA sample preparation method of claim 4, wherein said separating step (a) is performed using dielectrophoresis (DEP) to selectively capture said DNA up to 25 microns above said outer layer of said electrode at frequencies below 1000 Hz.

12. The method of claim 4 further comprising (c) adding a polymerase chain replication (PCR) master mix to said extracted DNA.

13. The method of claim 12 wherein said other materials are selected from the group consisting of proteins, RNA, and interference sources of PCR.

16. The method of claim 4 wherein said DNA adhering on said dielectric outer layer for a predetermined interval.

18. The method of claim 4 wherein said dielectric outer layer has a thickness of about 300 to about 500 angstroms.

6. A microfluidic DNA sample preparation device comprising:

a. a glass wafer substrate;

b. an interdigitated electrode set patterned on said glass wafer substrate, said electrode set comprising electrodes having a dielectric outer layer of thickness less than 1000 angstroms;

c. means for applying an alternating voltage to said interdigitated electrode set;

d. Separation means having a thickness of about 10 to about 15 microns placed over said electrode set; and

e. an additional wafer substrate placed over said separation means, and

wherein a first orifice for fluid access, and a second orifice for fluid access is contained on said first substrate and/or on said additional wafer substrate.

7. The device of claim 6 wherein said first orifice is positioned perpendicularly to the interdigitations of said electrode set, and electrode set is capable of collecting a target portion of DNA on said dielectric outer layer of said electrode.

19. The device of claim 6 wherein said dielectric outer layer has a thickness of about 300 to about 500 angstroms.

8. An electrode set comprising:

a. a left electrode set;

b. a right electrode set; and

c. an electrical connection to each of the two electrode sets;

wherein the two electrode sets form an interdigitated pattern of about 10 to 150 micron widths and spaces and comprise electrodes constructed to apply alternating current (AC), said electrodes comprising a dielectric outer layer of thickness less than about 2000 angstroms.

9. The electrode set of claim 8 wherein said electrodes further comprise:

a. a glass wafer substrate;

b. a first conductive layer placed on a top surface of the glass wafer substrate;

c. a second conductive layer placed on the first conductive layer; and

d. a thin layer of said dielectric outer layer placed on the second conductive layer.

14. The set of claim 9 wherein said dielectric is selected from the group consisting of silicon nitride, silicon carbide, paralene, Teflon, and silicon oxide.

15. The set of claim 8 wherein said dielectric outer layer has a dielectric constant of at least 50.

20. The set of claim 8 wherein said dielectric outer layer has a thickness of about 300 to about 500 angstroms.