Nucleic acid sample preparation

• US 10,006,083 B2
• Filed: 06/19/2015
• Issued: 06/26/2018
• Est. Priority Date: 04/16/2012
• Status: Active Grant

• Current Assignee: Biological Dynamics, Inc.
• Original Assignee: Biological Dynamics, Inc.
• Inventors: Krishnan, Rajaram, Charlot, David J., Tu, Eugene, McCanna, James, Kumosa, Lucas, Swanson, Paul D., Turner, Robert, Yang, Kai, Dobrovolskaya, Irina, Liu, David, Hinestrosa Salazar, Juan Pablo, Menezes, Juscilene
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/320,730
• Publication Number:
  US 20170137870A1
• Time in Patent Office: 1,103 Days
• Field of Search: None
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: B03C 5/005 Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength B03C5/02 takes precedence; electrophoresis B01D57/02
• 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: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• 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 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 27/447 using electrophoresis

Abstract

The present invention includes methods, devices and systems for isolating a nucleic acid from a fluid comprising cells. In various aspects, the methods, devices and systems may allow for a rapid procedure that requires a minimal amount of material and/or results in high purity nucleic acid isolated from complex fluids such as blood or environmental samples.

Claims

1. A method for detecting a cell free biomarker, the method comprising:

a. applying a sample with a conductivity of greater than 100 mS/m to a device, the device comprising an array of electrodes capable of establishing an AC electrokinetic field region, the device further comprising at least two chambers, the sample applied to a first chamber;

b. applying known quantities of a cell-free biomarker standard to a second chamber;

c. establishing a first AC electrokinetic high field region in the first chamber, the first AC electrokinetic high field capable of isolating larger nanoparticulate molecular targets;

d. establishing a second AC electrokinetic low field in the first chamber, the second AC electrokinetic low field capable of concentrating cells or micron-sized entities that may be present in the sample;

e. establishing a third AC electrokinetic high field to the second chamber, the second AC electrokinetic high field isolating the molecular nucleic acid standard applied to the second chamber;

f. flushing cells and micron-sized entities that may be present in the sample from the first chamber;

g. detecting bound cell-free biomarker signal on the array in the first chamber and second chamber; and

h. quantifying the bound cell-free biomarker by comparing the detected signal from the first chamber to detected signal from the second chamber.

2. The method of claim 1, wherein the cell-free biomarker is chosen from the group consisting of high molecular weight DNA, nucleosomes, exosomes, aggregated proteins, cell membrane fragments, mitochondria, cellular vesicles, and cellular debris.

3. The method of claim 1, wherein the cell-free biomarker is chosen from the group consisting of mutations, deletions, rearrangements, loss of heterozygosity, methylated nucleic acids, micro RNA, RNA from microvesicles, and combinations thereof.

4. The method of claim 1, wherein detection of the cell-free biomarker provides information useful for cancer diagnosis, cancer prognosis, or treatment response in a patient.

5. The method of claim 4, wherein the cell-free biomarker is associated with CNS tumors, neuroblastoma, gliomas, breast cancer, endometrial tumors, cervical tumors, ovarian tumors, hepatocellular carcinoma, pancreatic carcinoma, esophageal tumors, Stoch tumors, colorectal tumors, head and neck tumors, nasopharyngeal carcinoma, thyroid tumors, lymphoma, leukemia, lung cancer, non-small cell lung carcinoma, small cell lung carcinoma, testicular tumors, kidney tumors, prostate carcinoma, skin cancer, malignant melanoma, squamous cell carcinoma, or combinations thereof.

6. The method of claim 4, wherein the cell-free biomarker is GFAP, VEGF, EGFR, b-FGF, KRAS, YKL-40, MMP-9, or combinations thereof.

7. The method of claim 1, wherein the cell-free biomarker is chosen from the group consisting of proteins, lipids, antibodies, high molecular weight DNA, exosomes, nucleosomes, and nanosomes.

8. The method of claim 1, further comprising eluting the bound cell-free biomarker from the first chamber for further characterization.

9. The method of claim 8, wherein nucleic acid from the eluted cell-free biomarker is amplified or sequenced.

10. The method of claim 1, wherein the sample is whole blood, serum, plasma, cerebrospinal fluid, body tissue, urine, or saliva.

11. The method of claim 1, wherein the sample is blood.

12. The method of claim 1, wherein the AC electrokinetic field is produced using an alternating current having a voltage of 1 volt to 40 volts peak-peak, and/or a frequency of 5 Hz to 5,000,000 Hz and duty cycles from 5% to 50%.

13. The method of claim 1, wherein the conductivity of the fluid is greater than 500 mS/m.

14. The method of claim 1, wherein the array of electrodes is spin-coated with a hydrogel having a thickness from about 0.1 microns to about 1 micron.

15. The method of claim 14, wherein the hydrogel comprises two or more layers of a synthetic polymer.

16. The method of claim 14, wherein the hydrogel has a viscosity from about 0.5 cP to about 5 cP prior to spin-coating.

17. The method of claim 14, wherein the hydrogel has a conductivity from about 0.1 S/m to about 1.0 S/m.

18. The method of claim 1, wherein the bound cell-free biomarker comprises less than about 10% non-nucleic acid cellular material or cellular protein by mass.

19. The method of claim 1, wherein the array of electrodes comprises a wavy line configuration, wherein the configuration comprises a repeating unit comprising the shape of a pair of dots connected by linker, wherein the linker tapers inward toward the midpoint between the pair of dots, wherein the diameters of the dots are the widest points along the length of the repeating unit, wherein the edge to edge distance between a parallel set of repeating units is equidistant, or roughly equidistant.

20. The method of claim 1, wherein the array of electrodes comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0.