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 method for isolating a nucleic acid from a fluid comprising cells, the method comprising:
a. applying the fluid to a device, the device comprising an array of electrodes capable of establishing an AC electrokinetic field region;
b. concentrating a plurality of cells in a first AC electrokinetic field region, wherein the first AC eletrokinetic field region is a first dielectrophoretic high field region and the conductivity of the fluid is less than 500 mS/m;
c. lysing the cells on the array; and
d. isolating nucleic acid in a second AC electrokinetic field region, wherein the second AC electrokinetic field is a second dielectrophoretic high field region.
2. 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%.
3. The method of claim 1 , wherein the conductivity of the fluid is less than 300 mS/m.
4. The method 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 method of any 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 method of claim 5 , wherein the cells are lysed using 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.
7. The method of claim 1 , wherein the method further comprises degradation of residual proteins after cell lysis.
8. The method of claim 1 , wherein the array of electrodes is spin-coated with a hydrogel having a thickness between about 0.1 microns and 1 micron.
9. The method of claim 8 , wherein the hydrogel comprises two or more layers of a synthetic polymer.
10. The method of claim 8 , wherein the hydrogel has a viscosity between about 0.5 cP to about 5 cP prior to spin-coating.
11. The method of claim 8 , wherein the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m.
12. The method of claim 1 , wherein the isolated nucleic acid comprises less than 10% non-nucleic acid cellular material or cellular protein by mass.
13. The method of claim 1 , wherein the method is completed in less than 10 minutes.
14. 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.
15. 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.
16. A method for isolating a nucleic acid from a fluid comprising cells, the method comprising:
a. applying the fluid to a device, the device comprising an array of electrodes capable of establishing an AC electrokinetic field region;
b. concentrating a plurality of cells in a first AC electrokinetic field region, wherein the first AC electrokinetic field region is a first dielectrophoretic low field region and the conductivity of the fluid is greater than 300 mS/m;
c. isolating nucleic acid in a second AC electrokinetic field region, wherein the second AC electrokinetic field is a second eletrophoretic high field region; and
d. flushing cells away from the array.
17. The method of claim 16 , 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%.
18. The method of claim 16 , wherein the conductivity of the fluid is greater than 500 mS/m.
19. The method of claim 16 , 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.
20. The method of 16 , further comprising degrading residual proteins on the array.
21. The method of any of claim 20 , wherein the residual proteins are degraded by one or more of a chemical degradant or an enzymatic degradant.
22. The method of claim 21 , wherein the residual proteins are degraded by Proteinase K.
23. The method of claim 16 , wherein the array of electrodes is spin-coated with a hydrogel having a thickness between about 0.1 microns and 1 micron.
24. The method of claim 23 , wherein the hydrogel comprises two or more layers of a synthetic polymer.
25. The method of claim 23 , wherein the hydrogel has a viscosity between about 0.5 cP to about 5 cP prior to spin-coating.
26. The method of claim 23 , wherein the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m.
27. The method of claim 16 , wherein the isolated nucleic acid comprises less than about 10% non-nucleic acid cellular material or cellular protein by mass.
28. The method of claim 16 , wherein the method is completed in less than 10 minutes.
29. The method of claim 16 , 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.
30. The method of claim 16 , wherein the array of electrodes comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0.