Nucleic acid sample preparation
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.
1. A device for isolating a nucleic acid from a fluid comprising cells, the device comprising:
a. a first array of electrodes capable of establishing a first AC electrokinetic field region, wherein the first AC electrokinetic field region is a first dielectrophoretic low field region capable of concentrating and lysing a plurality of cells in a conductivity of fluid of 100 mS/m or more;
b. a second array of electrodes capable of establishing a second AC electrokinetic field, wherein the second AC electrokinetic field is a dielectrophoretic high field region capable of concentrating and isolating nucleic acid; and
c. a reservoir comprising a fluid and a device for flushing residual and lysed cellular material from the first array of electrodes.
2. The device of claim 1 , wherein the AC electrokinetic field produced by the first and second arrays of electrodes is 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%.
3. The device of claim 1 , wherein the first and second array of electrodes is a robust electrode structure of platinum, palladium or gold.
4. The device of claim 1 , wherein the electrodes are selectively energized to provide the first dielectrophoretic high field region and subsequently or continuously selectively energized to provide the second dielectrophoretic high field region.
5. The device of claim 1 , wherein the cells are lysed using a direct current, a chemical lysing agent, an enzymatic lysing agent, heat, pressure, sonic energy, or a combination thereof.
6. The device of claim 5 , wherein the first array of electrodes use a direct current with a voltage of 1-500 volts, a pulse frequency of 0.2 to 200 Hz with duty cycles from 10-50%, and a pulse duration of 0.01 to 10 seconds applied at least once to lyse the plurality of cells concentrated on the first array of electrodes.
7. The device of claim 1 , wherein the first array of electrodes is further capable of degrading residual proteins after cell lysis.
8. The device of claim 1 , wherein the first array of electrodes and second array of electrodes are spin-coated with a hydrogel having a thickness between about 0.1 microns and 1 micron.
9. The device of claim 8 , wherein the hydrogel comprises two or more layers of a synthetic polymer.
10. The device of claim 8 , wherein the hydrogel has a viscosity between about 0.5 cP to about 5 cP prior to spin-coating.
11. The device of claim 8 , wherein the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m.
12. The device of claim 1 , wherein the nucleic acid isolated on the second array of electrodes comprises less than 10% non-nucleic acid cellular material or cellular protein by mass.
13. The device of claim 1 , wherein the isolation of nucleic acids on the second array of electrodes is completed in less than 10 minutes.
14. The device of claim 1 , wherein the first 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 diameter 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.
15. The device of claim 1 , wherein the first array of electrodes comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0.
16. The device of claim 1 , further comprising a second reservoir comprising an eluant for eluting the concentrated and isolated nucleic acid on the second array of electrodes.
17. A device for isolating a nucleic acid from a fluid comprising cells, the device comprising:
a. a first array of electrodes capable of establishing a first AC electrokinetic field region, wherein the first AC electrokinetic field region is a first dielectrophoretic high field region capable of concentrating and lysing a plurality of cells in a conductivity of fluid 100 mS/m or more;
b. a second array of electrodes capable of establishing a second AC electrokinetic field, wherein the second AC electrokinetic field is a second diselectrophoretic high field region capable of concentrating and isolating nucleic acid;
c. means for flushing away the lysed plurality of cells from the first array of electrodes; and
d. a reservoir comprising an eluant for eluting the concentrated and isolated nucleic acid on the second array of electrodes.
18. The device of claim 17 , wherein the AC electrokinetic field produced by the first and second arrays of electrodes is 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%.
19. The device of claim 17 , wherein the first array of electrodes is a robust electrode structure of platinum, palladium or gold.
20. The device of claim 17 , wherein the first array of electrodes are selectively energized to provide the first dielectrophoretic high field region and subsequently or continuously selectively energized to provide the second dielectrophoretic high field region.
21. The device of 17 , further comprising degrading residual proteins on the array.
22. The device of any of claim 21 , wherein the residual proteins are degraded by one or more of a chemical degradant or an enzymatic degradant.
23. The device of claim 22 , wherein the residual proteins are degraded by Proteinase K.
24. The device of claim 17 , wherein the array of electrodes is spin-coated with a hydrogel having a thickness between about 0.1 microns and 1 micron.
25. The device of claim 24 , wherein the hydrogel comprises two or more layers of a synthetic polymer.
26. The device of claim 24 , wherein the hydrogel has a viscosity between about 0.5 cP to about 5 cP prior to spin-coating.
27. The device of claim 24 , wherein the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m.
28. The device of claim 17 , wherein the isolated nucleic acid comprises less than 10% non-nucleic acid cellular material or cellular protein by mass.
29. The device of claim 17 , wherein the method is completed in less than 10 minutes.
30. The device of claim 17 , wherein the first 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 diameter 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.
31. The device of claim 17 , wherein the array of electrodes comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0.