NUCLEIC ACID SAMPLE PREPARATION

专利摘要:
nucleic acid sample preparation. The present invention relates to methods, devices and systems for isolating a nucleic acid from a fluid comprising cells. in many respects, the methods, devices and systems can allow 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.
公开号:BR112014025695B1
申请号:R112014025695-0
申请日:2013-04-16
公开日:2021-08-03
发明作者:Paul Swanson；Robert Turner；Kai Yang；Irina DOBROVOLSKAYA；David Liu；Rajaram Krishnan；David CHARLOT；Eugene Tu；James MCCANNA；Lucas Kumosa
申请人:Biological Dynamics, Inc；
IPC主号:

专利说明:

REFERENCE REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 61/624,897, filed April 16, 2012, which application is incorporated herein by reference. BACKGROUND OF THE INVENTION
[0002] Exponentially rapid progress has been made in the field of DNA sequencing in recent years. Methods such as pyrosequencing, ionic semiconductor sequencing, and polony sequencing aim to reduce costs to the point where sequencing a complete genome becomes routine. This is expected to transform fields as diverse as medicine, renewable energy, biosafety and agriculture to name a few. However, techniques for isolating DNA suitable for sequencing have not kept pace and there is a risk that this will become a limitation. SUMMARY OF THE INVENTION
[0003] In some cases, the present invention satisfies a need for improved methods of isolating nucleic acid from biological samples. Particular attributes of some aspects provided here include a total sample preparation time of less than about one hour, with effective working time of less than about one minute. In some embodiments, the present invention can be used to isolate DNA from dilute and/or complex fluids such as blood or environmental samples. In other aspects, the present invention can use small amounts of starting material, obtain highly purified nucleic acids, and is amenable to multiplexed and high-throughput operation.
Described herein, in some embodiments, is 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 generating an AC electrokinetic field; B. concentrating a plurality of cells in a first AC electrokinetic field region; ç. lyse cells in the first AC electrokinetic field region; and d. isolating the nucleic acid in a second AC electrokinetic field region, where the fluid is in a conductivity capable of concentrating a plurality of cells in the first AC electrokinetic field region. In some embodiments, the first AC electrokinetic region is a dielectrophoretic field region, where the second AC electrokinetic field region is a dielectrophoretic field region, or a combination thereof. In some embodiments, the first AC electrokinetic field region is a first region of low dielectrophoretic field and the second AC electrokinetic field region is a second region of high dielectrophoretic field, where the fluid conductivity is greater than 300 mS/m. In some embodiments, the first AC electrokinetic field region is a high dielectrophoretic first field region and the second AC electrokinetic field region is a second high dielectrophoretic field region where the fluid conductivity is less than 300 mS/m. In some embodiments, the nucleic acid is concentrated in the second AC electrokinetic field region. In some embodiments, the method further comprises discarding residual material from the array and isolated nucleic acid. In some embodiments, the method further comprises degrading a residual protein. In some embodiments, the method further comprises discarding degraded array proteins and isolated nucleic acid. In some embodiments, the method further comprises collecting the nucleic acid. In some embodiments, the first AC electrokinetic field region is produced by an alternating current. In some embodiments, the first AC electrokinetic field region is produced using an alternating current that has a peak-to-peak voltage of 1 volt to 40 volts; and/or a frequency from 5 Hz to 5,000,000 Hz, and duty cycles from 5% to 50%. In some embodiments, the second AC electrokinetic field region is a different region from the electrode array of the first AC electrokinetic field region. In some embodiments, the second AC electrokinetic field region is the same region of the electrode array as the first AC electrokinetic field region. In some embodiments, the second AC electrokinetic field region is produced by an alternating current. In some embodiments, the second AC electrokinetic field region is produced using an alternating current that has a voltage of 1 volt to 50 volts peak-to-peak; and/or a frequency from 5 Hz to 5,000,000 Hz, and duty cycles from 5% to 50%. In some embodiments, electrodes are selectively energized to provide the first region of AC electrokinetic field and selectively energized subsequently or continuously to provide the second region of AC electrokinetic field. In some embodiments, cells are lysed by applying a direct current to the cells. In some embodiments, the direct current used to lyse the cells has a voltage of 1 to 500 volts; and a duration of .01 to 10 seconds applied once or as multiple pulses. In some embodiments, the direct current used to lyse a cell is a direct current pulse or a plurality of direct current pulses applied at a frequency suitable for lysing the cells. In some modes, the pulse has a frequency of 0.2 to 200 Hz with duty cycles of 10 to 50%. In some embodiments, cells are lysed in the device using a direct current, a chemical lysing agent, an enzymatic lysing agent, heat, osmotic pressure, sonic energy, or a combination of these. In some embodiments, the waste material comprises lysed cell material. In some embodiments, the lysed cell material comprises residual protein released from the plurality of cells upon lysis. In some embodiments, the electrode array is coated with a hydrogel. In some embodiments, the hydrogel comprises two or more layers of a synthetic polymer. In some embodiments, the hydrogel is coated by rotation on the electrodes. In some embodiments, the hydrogel has a viscosity of between about 0.5 cP to about 5 cP prior to rotational coating. In some embodiments, the hydrogel is between about 0.1 micron and 1 micron thick. In some embodiments, the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m. In some modalities, the electrode array is in a point configuration. In some embodiments, the orientation angle between the points is from about 25° to about 60°. In some embodiments, the electrode array is in a wavy or non-linear line configuration, where the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a ligand, where the dots and the ligand define the electrode boundaries, where the ligand tapers inward towards or at the midpoint between the pair of points, where the diameters of the points are the largest points along the length of the repeat unit, where the edge distance the border between a parallel set of repeating units is equidistant, or nearly equidistant. In some embodiments, the electrode array comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0. In some embodiments, the method further comprises amplifying the isolated nucleic acid by polymerase chain reaction. In some embodiments, the nucleic acid comprises DNA, RNA, or any combination thereof. In some embodiments, the isolated nucleic acid comprises less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30% , less than about 20%, less than about 10%, less than about 5%, or less than about 2% cellular material without nucleic acid and/or protein by mass. In some embodiments, the isolated nucleic acid comprises more than about 99%, more than about 98%, more than about 95%, more than about 90%, more than about 80%, more than about 70% , more than about 60%, more than about 50%, more than about 40%, more than about 30%, more than about 20%, or more than about 10% nucleic acid by mass. In some embodiments, the method is completed in less than about an hour. In some modalities, no centrifugation is used. In some embodiments, residual proteins are degraded by one or more of chemical degradation and enzymatic degradation. In some embodiments, residual proteins are degraded by Proteinase K. In some embodiments, residual proteins are degraded by an enzyme, the method further comprising inactivating the enzyme after protein degradation. In some embodiments, the enzyme is inactivated by heat (eg, 50 to 95°C for 5 to 15 minutes). In some embodiments, waste material and degraded proteins are discarded in separate or simultaneous steps. In some embodiments, isolated nucleic acid is collected by (i) turning off the second AC electrokinetic field region; and (ii) eluting the nucleic acid from the array into an eluent. In some embodiments, the nucleic acid is isolated in a form suitable for sequencing. In some embodiments, nucleic acid is isolated in a fragmented form suitable for shotgun sequencing. In some embodiments, the fluid comprising cells has a low conductivity or a high conductivity. In some embodiments, the fluid comprises a bodily fluid, blood, serum, plasma, urine, saliva, a food, a beverage, a growth medium, an environmental sample, a liquid, water, clonal cells, or a combination thereof. In some embodiments, cells comprise clonal cells, pathogen cells, bacterial cells, viruses, plant cells, animal cells, insect cells, and/or combinations thereof. In some embodiments, the method further comprises sequencing the isolated nucleic acid. In some embodiments, nucleic acid is sequenced by Sanger sequencing, pyrosequencing, ionic semiconductor sequencing, polony sequencing, bond sequencing, DNA nanosphere sequencing, bond sequencing, or single molecule sequencing. In some embodiments, the method further comprises performing a reaction on the DNA (e.g., fragmentation, restriction digestion, ligation). In some embodiments, the reaction takes place in or near the array or device. In some embodiments, the cell-comprising fluid comprises no more than 10,000 cells.
Described herein, in some embodiments, is 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 generating an AC electrokinetic field; B. concentrating a plurality of cells in a first electrokinetic AC (eg, dielectrophoretic); ç. isolating the nucleic acid in a second AC electrokinetic field region (eg, dielectrophoretic); and d. discard cells, in which the fluid is in a conductivity capable of concentrating a plurality of cells in the first region of AC electrokinetic field. In some embodiments, the first AC electrokinetic field region is a dielectrophoretic field region. In some embodiments, the first AC electrokinetic field region is a region of low dielectrophoretic field, and in which the fluid conductivity is greater than 300 mS/m. In some embodiments, the second AC electrokinetic field region is a dielectrophoretic field region. In some embodiments, the method further comprises degrading residual proteins after step (e). In some embodiments, the method further comprises discharging degraded nucleic acid proteins. In some embodiments, the method further comprises collecting the nucleic acid. In some embodiments, the first AC electrokinetic field region is produced by an alternating current. In some embodiments, the first AC electrokinetic field region is produced using an alternating current that has a voltage of 1 volt to 40 volts peak-to-peak; and/or a frequency from 5 Hz to 5,000,000 Hz, and duty cycles from 5% to 50%. In some embodiments, the second AC electrokinetic field region is a different region from the electrode array of the first AC electrokinetic field region. In some embodiments, the second AC electrokinetic field region is the same region of the electrode array as the first AC electrokinetic field region. In some embodiments, the second AC electrokinetic field region is produced by an alternating current. In some embodiments, the second AC electrokinetic field region is a high dielectrophoretic field region. In some embodiments, the second AC electrokinetic field region is produced using an alternating current that has a voltage of 1 volt to 50 volts peak-to-peak; and/or a frequency from 5 Hz to 5,000,000 Hz, and duty cycles from 5% to 50%. In some embodiments, electrodes are selectively energized to provide the first region of AC electrokinetic field and selectively energized subsequently or continuously to provide the second region of AC electrokinetic field. In some embodiments, the electrode array is coated with a hydrogel. In some embodiments, the hydrogel comprises two or more layers of a synthetic polymer. In some embodiments, the hydrogel is coated by rotation on the electrodes. In some embodiments, the hydrogel has a viscosity of between about 0.5 cP to about 5 cP prior to rotational coating. In some embodiments, the hydrogel is between about 0.1 micron and 1 micron thick. In some embodiments, the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m. In some modalities, the electrode array is in a point configuration. In some embodiments, the orientation angle between the points is from about 25° to about 60°. In some embodiments, the electrode array is in a wavy or non-linear line configuration, where the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a ligand, where the dots and the ligand define the electrode boundaries, where the ligand tapers inward towards or at the midpoint between the pair of points, where the diameters of the points are the largest points along the length of the repeat unit, where the edge distance the border between a parallel set of repeating units is equidistant, or nearly equidistant. In some embodiments, the electrode array comprises a passivation layer with one having a relative electrical permittivity from about 2.0 to about 4.0. In some embodiments, the method further comprises amplifying the isolated nucleic acid by polymerase chain reaction. In some embodiments, the nucleic acid comprises DNA, RNA, or any combination thereof. In some embodiments, the isolated nucleic acid comprises less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30% , less than about 20%, less than about 10%, less than about 5%, or less than about 2% non-nucleic acid and/or protein cellular material by mass. In some embodiments, the isolated nucleic acid comprises more than about 99%, more than about 98%, more than about 95%, more than about 90%, more than about 80%, more than about 70% , more than about 60%, more than about 50%, more than about 40%, more than about 30%, more than about 20%, or more than about 10% nucleic acid by mass. In some embodiments, the method is completed in less than about an hour. In some modalities, no centrifugation is used. In some embodiments, residual proteins are degraded by one or more of chemical degradation and enzymatic degradation. In some embodiments, residual proteins are degraded by Proteinase K. In some embodiments, residual proteins are degraded by an enzyme, the method further comprising inactivating the enzyme after protein degradation. In some embodiments, the enzyme is inactivated by heat (eg, 50 to 95°C for 5 to 15 minutes). In some embodiments, waste material and degraded proteins are discarded in separate or simultaneous steps. In some embodiments, isolated nucleic acid is collected by (i) turning off the second AC electrokinetic field region; and (ii) eluting the nucleic acid from the array into an eluent. In some embodiments, the nucleic acid is isolated in a form suitable for sequencing. In some embodiments, nucleic acid is isolated in a fragmented form suitable for shotgun sequencing. In some embodiments, the fluid comprising cells has a low conductivity or a high conductivity. In some embodiments, the fluid comprises a bodily fluid, blood, serum, plasma, urine, saliva, a food, a beverage, a growth medium, an environmental sample, a liquid, water, clonal cells, or a combination thereof. In some embodiments, cells comprise clonal cells, pathogen cells, bacterial cells, viruses, plant cells, animal cells, insect cells, and/or combinations thereof. In some embodiments, the method further comprises sequencing the isolated nucleic acid. In some embodiments, nucleic acid is sequenced by Sanger sequencing, pyrosequencing, ionic semiconductor sequencing, polony sequencing, bond sequencing, DNA nanosphere sequencing, bond sequencing, or single molecule sequencing. In some embodiments, the method further comprises performing a reaction on the DNA (e.g., fragmentation, restriction digestion, ligation). In some embodiments, the reaction takes place in or near the array or device. In some embodiments, the cell-comprising fluid comprises no more than 10,000 cells.
In some embodiments, a device for isolating a nucleic acid from a fluid comprising cells is described herein, the device comprising: a. an accommodation; B. a heater and/or a reservoir comprising a protein degrading agent; and c. a plurality of alternating current (AC) electrodes within the housing, the AC electrodes configured to be selectively energized in order to establish regions of high electrokinetic field and low electrokinetic field, with this the AC electrokinetic effects provide cell concentration in regions from the device's low field. In some embodiments, the plurality of electrodes are configured to selectively energize so as to establish regions of high dielectrophoretic field and low dielectrophoretic field. In some embodiments, the electrode array is coated with a hydrogel. In some embodiments, the hydrogel comprises two or more layers of a synthetic polymer. In some embodiments, the hydrogel is coated by rotation on the electrodes. In some embodiments, the hydrogel has a viscosity of between about 0.5 cP to about 5 cP prior to rotational coating. In some embodiments, the hydrogel is between about 0.1 micron and 1 micron thick. In some embodiments, the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m. In some modalities, the electrode array is in a point configuration. In some embodiments, the orientation angle between the points is from about 25° to about 60°. In some embodiments, the electrode array is in a wavy or non-linear line configuration, where the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a ligand, where the dots and the ligand define the electrode boundaries, where the ligand tapers inward towards or at the midpoint between the pair of points, where the diameters of the points are the largest points along the length of the repeat unit, where the edge distance the border between a parallel set of repeating units is equidistant, or nearly equidistant. In some embodiments, the electrode array comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0. In some embodiments, the protein degrading agent is Proteinase K. In some embodiments, the device further comprises a second reservoir that comprises an eluent.
In some embodiments, a system for isolating a nucleic acid from a fluid comprising cells is described herein, the system comprising: a. a device comprising a plurality of alternating current (AC) electrodes, the AC electrodes configured to be selectively energized so as to establish regions of high AC electrokinetic field and low AC electrokinetic field, thereby AC electrokinetic effects provide cell concentration in high-field regions of the device, where the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a ligand, where the dots and the ligand define the electrode boundaries, where the ligand tapers inward towards or midpoint between the pair of stitches, where the stitch diameters are the largest points along the length of the repeating unit, where the edge-to-edge distance between a parallel set of repeating units is equidistant, or nearly equidistant; and b. a module capable of sequencing DNA by Sanger sequencing or next-generation sequencing methods; ç. a software program capable of controlling the device comprising a plurality of AC electrodes, the module capable of sequencing DNA or a combination thereof; and d. a fluid comprising cells. In some embodiments, the plurality of electrodes are configured to be selectively energized so as to establish regions of high dielectrophoretic field and low dielectrophoretic field.
[0008] A device is described here, in some modalities, comprising: a. a plurality of alternating current (AC) electrodes, the AC electrodes configured to be selectively energized so as to establish regions of high AC electrokinetic field and low AC electrokinetic field, where the electrode array is in a wavy line configuration or not linear, in which the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a ligand, in which the dots and the ligand define the boundaries of the electrode, in which the ligand tapers inwards towards or in the midpoint between the pair of points, where the diameters of the points are the largest points along the length of the repeating unit, where the edge-to-edge distance between a parallel set of repeating units is equidistant, or nearly equidistant; and b. a module capable of thermocycling and amplifying nucleic acids. In some embodiments, the plurality of electrodes are configured to be selectively energized so as to establish regions of high dielectrophoretic field and low dielectrophoretic field. In some embodiments, the device is capable of isolating nucleic acids from a fluid comprising cells and carrying out amplification of the isolated nucleic acids. In some embodiments, the isolated nucleic acid is DNA or mRNA. In some embodiments, nucleic acid is isolated and amplification is performed in a single chamber. In some embodiments, nucleic acid is isolated and amplification is performed in multiple regions of a single chamber. In some embodiments, the device further comprises using at least one of an elution tube, a chamber and a reservoir to carry out the amplification. In some embodiments, nucleic acid amplification is polymerase chain reaction (PCR) based. In some embodiments, nucleic acid amplification is performed in a microchannel coil that comprises a plurality of temperature zones. In some embodiments, amplification is performed on aqueous droplets trapped in immiscible fluids (ie, digital PCR). In some embodiments, thermocycling comprises convection. In some embodiments, the device comprises a contact surface or proximal to the electrodes, where the surface is functionalized with biological ligands that are capable of selectively capturing biomolecules. In some embodiments, the electrode array is coated with a hydrogel. In some embodiments, the hydrogel comprises two or more layers of a synthetic polymer. In some embodiments, the hydrogel is coated by rotation on the electrodes. In some embodiments, the hydrogel has a viscosity of between about 0.5 cP to about 5 cP prior to rotational coating. In some embodiments, the hydrogel is between about 0.1 micron and 1 micron thick. In some embodiments, the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m. In some embodiments, the electrode array comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0. In some embodiments, the surface selectively captures biomolecules by: a. nucleic acid hybridization; B. antibody-antigen interactions; ç. biotin-avidin interactions; d. ionic or electrostatic interactions; or e. any combination of these. In some embodiments, the surface is functionalized to reduce and/or inhibit non-specific binding interactions by: a. polymers (for example polyethylene glycol PEG); B. ionic or electrostatic interactions; ç. surfactants; or d. any combination of these. In some embodiments, the device comprises a plurality of microelectrode devices oriented (a) flat side by side, (b) faced vertically, or (c) faced horizontally. In some modalities, the device comprises a module capable of performing Sanger sequencing. In some embodiments, the module capable of performing Sanger sequencing comprises a module capable of capillary electrophoresis, a module capable of multicolor fluorescence detection, or a combination thereof.
[0009] A device is described here, in some modalities, which comprises: a. a plurality of alternating current (AC) electrodes, the AC electrodes configured to be selectively energized so as to establish regions of high AC electrokinetic field and low AC electrokinetic field, where the electrode array is in a wavy line configuration or not linear, in which the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a ligand, in which the dots and the ligand define the boundaries of the electrode, in which the ligand tapers inwards towards or in the midpoint between the pair of points, where the diameters of the points are the largest points along the length of the repeating unit, where the edge-to-edge distance between a parallel set of repeating units is equidistant, or nearly equidistant; and b. a module capable of sequencing. In some embodiments, the plurality of electrodes are configured to be selectively energized to establish regions of high electrokinetic field and low electrokinetic field. In some embodiments, the device comprises a contact surface or proximal to the electrodes, where the surface is functionalized with biological ligands that are capable of selectively capturing biomolecules. In some embodiments, the electrode array is coated with a hydrogel. In some embodiments, the hydrogel comprises two or more layers of synthetic polymer. In some embodiments, the hydrogel is coated by rotation on the electrodes. In some embodiments, the hydrogel has a viscosity of between about 0.5 cP to about 5 cP prior to rotational coating. In some embodiments, the hydrogel is between about 0.1 micron and 1 micron thick. In some embodiments, the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m. In some embodiments, the electrode array comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0. In some embodiments, the surface selectively captures biomolecules by: a. nucleic acid hybridization; B. antibody-antigen interactions; ç. biotin-avidin interactions; d. ionic or electrostatic interactions; or e. any combination of these. In some embodiments, the surface is functionalized to reduce and/or inhibit non-specific binding interactions by: a. polymers (for example polyethylene glycol PEG); B. ionic or electrostatic interactions; ç. surfactants; or d. any combination of these. In some embodiments, the device comprises a plurality of microelectrode devices oriented (a) flat side by side, (b) faced vertically, or (c) faced horizontally. In some embodiments, the device comprises a module capable of performing next-generation sequencing. In some embodiments, the module capable of performing next-generation sequencing is capable of performing pyrosequencing, ionic semiconductor sequencing, polony sequencing, bond sequencing, DNA nanosphere sequencing, or single-molecule sequencing.
Described herein, in some embodiments, is a method of isolating a nucleic acid from a fluid comprising cells, comprising a) performing a method described herein; b) performing PCR amplification on nucleic acid, or a cDNA version of the nucleic acid, to produce a PCR product; c) isolating the PCR product in a third AC electrokinetic region; d) performing Sanger chain termination reactions on the PCR product to produce a nucleic acid sequencing product; and e) performing the electrophoretic separation of the nucleic acid sequencing product. In some embodiments, the third AC electrokinetic region is a dielectrophoretic field region. In some modalities, the third AC electrokinetic region is a region with a high dielectrophoretic field. In some embodiments, the electrode array is in a wavy or non-linear line configuration, where the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a ligand, where the dots and the ligand define the electrode boundaries, where the ligand tapers inward towards or at the midpoint between the pair of points, where the diameters of the points are the largest points along the length of the repeat unit, where the edge distance the border between a parallel set of repeating units is equidistant, or nearly equidistant. In some embodiments, the electrophoretic separation of the nucleic acid sequencing product is capillary electrophoresis. In some embodiments, the method further comprises using multicolor fluorescence detection to analyze the nucleic acid sequencing product. In some modalities, all steps are performed on a single chip. In some embodiments, the cell-comprising fluid comprises no more than 10,000 cells. MERGER AS REFERENCE
[0011] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in the same way as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by way of reference. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The new features of the invention are presented with particularity in the appended claims. A better understanding of the characteristics and advantages of the present invention will be obtained by reference to the following detailed description which presents illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings, in which:
[0013] Figure 1 shows a top view (A), a bottom view (B) and a cross-sectional view (C) of an exemplary device.
[0014] Figure 2 shows the electrodes associated with various amounts of genomic DNA.
[0015] Figure 3 shows the isolation of fluorescent green E. coli in an array. Panel (A) shows a brightfield view. Panel (B) shows a fluorescent green view of the electrodes prior to DEP activation. Panel (C) shows E. coli on electrodes after one minute at 10 kHz, 20 Vp-p in 1xTBE buffer. Panel (D) shows E. coli on electrodes after one minute at 1 MHz, 20 Vp-p in 1xTBE buffer.
Figure 4 shows a comparison between the methods of the present invention (upper right panel) and the Epicentre™ WaterMaster™ DNA purification procedure (upper left panel). Pie charts consist of the distribution of 10,000 Illumina™ BLAST sequencing reads examined in the MEGAN™ database. As shown, a similar percentage of sequencing reads originate from the E. coli sequence for both methods. The table in the bottom panel shows the sequencing coverage and quality of E. Coli operated via the chip and compared to a control run off-chip according to the manufacturer's protocol.
Figure 5 shows an exemplary method for isolating nucleic acids from cells.
Figure 6 shows an exemplary method for isolating extracellular nucleic acids from a fluid comprising cells.
[0019] Figure 7 exemplifies ACEK (AC Electrokinetic) forces that result from the methods and devices described here. With the use of the relationship between forces on particles due to Dielectrophoresis (DEP), Electrothermal AC flux (ACET) and Electro-osmosis AC, (ACEO), in some embodiments, size cuts are used for the isolation and purification of nucleic acid . Isolation relies on flux vortices bringing nucleic acids closer to an electrode edge due to ACET and ACEO depending on fluid conductivity. Effective Stokes.
[0020] Figure 8 exemplifies a waved electrode configuration, as described here. The edge-to-edge distance between the electrodes is usually equidistant. A wavy electrode configuration increases the electrode surface area while maintaining the alternating non-uniform electric field to induce the ACEK gradient to allow for DEP, ACEO, ACET, and other ACEK forces.
[0021] Figure 9 exemplifies how the E-field gradient in a dielectric layer corner based on the thickness of silicon nitride. A lower and lower thickness K resulted in a larger E-field gradient (bending) in a dielectric layer corner.
[0022] Figure 10 exemplifies the capture of DNA in an electrode with a layer of hydrogel deposited by steam. Vapor-phase coatings of activated monomers form uniform thin-film coatings on a variety of substrates. Hydrogels such as pHEMA were deposited at various thicknesses (100, 200, 300, 400 nm) and crosslink densities (5, 25, 40%) on electrode chips by GVD Corporation (Cambridge, MA). The hydrogel films were tested using a standard ACE protocol (no pretreatment, 7Vp-p, 10KHz, 2 minutes, 0.5XPBS, 500ng/ml gDNA labeled with Sybr Green 1). Fluorescence on the electrodes was captured by imaging. The device with a thickness of 100nm, 5% crosslinking gel was considered to have strong DNA capture. Optionally, the process could be optimized by altering the rate of deposition or growth of attachment to the surface of the microelectrode array (ie, to the passivation layer and exposed electrodes), using an adhesion promoter such as a silane derivative. DETAILED DESCRIPTION OF THE INVENTION
Methods, devices and systems suitable for isolating or separating particles or molecules from a fluid composition are described herein. In specific embodiments, provided herein are methods, devices and systems for isolating or separating a nucleic acid from a fluid comprising cells or other particulate material. In some aspects, methods, devices and systems can allow for rapid separation of particles and molecules in a fluid composition. In other aspects, the methods, devices and systems can allow rapid isolation of particle molecules in a fluid composition. In many respects, the methods, devices and systems can allow for a rapid procedure that requires a minimal amount of material and/or results in high purity DNA isolated from complex fluids such as blood or environmental samples.
[0024] Provided herein in some embodiments are methods, devices and systems for isolating or separating particles or molecules from a fluid composition, methods, devices and systems comprising applying the fluid to a device comprising an array of electrodes and is capable of generating AC electrokinetic forces (eg when electrode array is energized). In some embodiments, the dielectrophoretic field is a component of AC electrokinetic force effects. In other embodiments, the AC electrokinetic force effect component is AC electroosmosis or AC electrothermal. In some modalities, the AC electrokinetic force, including dielectrophoretic fields, comprises high field regions (positive DEP, ie, area where there is a strong concentration of electric field lines due to a non-uniform electric field) and/or low regions field (negative DEP, ie area where there is a weak concentration of electric field lines due to a non-uniform electric field).
[0025] In specific cases, particles or molecules (eg, nucleic acid) are isolated (eg, isolated or separated from cells) in a field region (eg, a high-field region) of the dielectrophoretic field. In some embodiments, the method, device, or system additionally includes one or more of the following steps: concentrating the cells of interest in a first dielectrophoretic field region (eg, a high DEP field region), lysing the cells in the first region of dielectrophoretic field, and/or concentrating nucleic acid in a first or second dielectrophoretic field region. In other embodiments, the method, device, or system includes one or more of the following steps: concentrating cells in a first dielectrophoretic field region (eg, a low DEP field region), concentrating nucleic acid in a second field region dielectrophoresis (eg, a region of high DEP field), and wash the cells and waste material. The method also optionally includes devices and/or systems capable of performing one or more of the following steps: washing or otherwise removing residual (e.g., cellular) material from the nucleic acid (e.g., washing the array with water or buffer while the nucleic acid is concentrated and maintained within a high DEP field region of the array), degrade residual proteins (eg, residual proteins from lysed cells and/or other sources, such degradation occurs according to any suitable mechanism, as with heat, a protease, or a chemical), discard the degraded nucleic acid proteins, and collect the nucleic acid. In some embodiments, the result of the methods, operation of the devices, and operation of the systems described herein is an isolated nucleic acid, optionally of adequate quantity and purity for DNA sequencing.
[0026] In some cases, it is advantageous that the methods described here are performed in a short period of time, devices are operated in a short period of time, and systems are operated in a short period of time. In some embodiments, the time period is short with reference to the "procedural time" measured from the time between adding fluid to the device and obtaining isolated nucleic acid. In some embodiments, the procedure time is less than 3 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, or less than 5 minutes.
[0027] In another aspect, the period of time is short with reference to "effective working time" measured as the cumulative amount of time a person must participate in the procedure from the time between adding fluid to the device and obtaining acid isolated nucleic. In some modalities, the effective working time is less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds.
[0028] In some cases, it is advantageous that the devices described herein comprise a single container, the systems described herein comprise a device comprising a single container, and the methods described herein can be performed in a single container, for example, in a device dielectrophoretic as described here. In some aspects, this single container modality reduces the number of fluid handling steps and/or is accomplished in a short period of time. In some cases, the present method, devices and systems are contrasted with methods, devices and systems that use one or more steps of centrifugation and/or medium exchanges. In some cases, centrifugation increases the amount of effective working time required to isolate nucleic acids. In another aspect, the single container procedure or device isolates nucleic acids using a minimal amount of consumable reagents. DEVICES AND SYSTEM
In some embodiments, devices for collecting a nucleic acid from a fluid are described herein. In one aspect, described herein are devices for collecting a nucleic acid from a fluid comprising cells or other particulate material. In other aspects, the devices described herein are capable of collecting and/or isolating nucleic acid from a fluid comprising cellular material or protein. In other cases, the devices described herein are capable of collecting and/or isolating nucleic acid from cellular material.
In some embodiments, a device for isolating a nucleic acid from a fluid comprising cells or other particulate material is described herein, the device comprising: a. an accommodation; B. a heater and/or a reservoir comprising a protein degrading agent; and c. a plurality of alternating current (AC) electrodes within the housing, the AC electrodes configured to be selectively energized in order to establish regions of high electrokinetic field and low electrokinetic field, with this the AC electrokinetic effects provide the cell concentration in regions of low field of the device. In some embodiments, the plurality of electrodes are configured to be selectively energized so as to establish regions of high electrokinetic field and low electrokinetic field. In some embodiments, the protein degrading agent is a protease. In some embodiments, the protein degrading agent is Proteinase K. In some embodiments, the device further comprises a second reservoir that comprises an eluent.
[0031] In some embodiments, a device is described here comprising: a. a plurality of alternating current (AC) electrodes, the AC electrodes are configured to be selectively energized so as to establish regions of high AC electrokinetic field and low AC electrokinetic field; and b. a module capable of thermocycling and performing PCR or other enzymatic reactions. In some embodiments, the plurality of electrodes are configured to be selectively energized so as to establish regions of high electrokinetic field and low electrokinetic field. In some embodiments, the device is capable of isolating DNA from a fluid comprising cells and performing PCR amplification or other enzymatic reactions. In some embodiments, DNA is isolated and PCR or another enzymatic reaction is performed in a single chamber. In some embodiments, DNA is isolated and PCR or another enzymatic reaction is performed in multiple regions of a single chamber. In some embodiments, DNA is isolated and PCR or another enzymatic reaction is performed in multiple chambers.
[0032] In some embodiments, the device further comprises at least one of an elution tube, a chamber and a reservoir to perform PCR amplification or other enzymatic reaction. In some embodiments, PCR amplification or other enzymatic reaction is performed in a microchannel coil comprising a plurality of temperature zones. In some embodiments, PCR amplification or other enzymatic reaction is performed on aqueous droplets trapped in immiscible fluid (ie, digital PCR). In some embodiments, thermocycling comprises convection. In some embodiments, the device comprises a contact surface or proximal to the electrodes, where the surface is functionalized with biological ligands that are capable of selectively capturing biomolecules.
In some embodiments, a system for isolating a nucleic acid from a fluid comprising cells or other particulate material is described herein, the system comprising: a. a device comprising a plurality of alternating current (AC) electrodes, the AC electrodes are configured to be selectively energized in order to establish regions of high AC electrokinetic field and low AC electrokinetic field, whereby the AC electrokinetic effects provide the concentration of cells in high-field regions of the device; and b. a sequencer, thermocycler, or other device for performing enzymatic reactions on isolated or collected nucleic acid. In some embodiments, the plurality of electrodes are configured to be selectively energized so as to establish regions of high electrokinetic field and low electrokinetic field.
[0034] In various modalities, DEP fields are created or capable of being created by selectively energizing an electrode array as described here. The electrodes are optionally made of any suitable corrosion resistant material, including metals such as noble metals (eg, platinum, iridium-platinum alloy, palladium, gold, and the like). In various embodiments, electrodes are of any suitable size, of any suitable orientation, of any suitable spacing, energized or capable of being energized in any way, and the like so that DEP and/or other suitable electrokinetic fields are produced.
[0035] In some embodiments, methods, devices and systems are described here in which the electrodes are placed in separate chambers and positive DEP regions and negative DEP regions are created inside an inner chamber by passing the DEP AC field through pore structures or holes. Various geometries are used to form the positive (high field) and negative (low field) DEP regions suitable for performing cell, microparticle, nanoparticle, and nucleic acid separations. In some embodiments, the pore or bore structures contain (or are filled with) porous material (hydrogels) or are coated with porous membrane structures. In some embodiments, by segregating the electrodes into separate chambers, these pore/hole structure DEP devices reduce the electrochemical effects, heating, or chaotic fluid movement that occur in the internal separation chamber during the DEP process.
[0036] In one aspect, a device comprising electrodes is described here, in which the electrodes are placed in separate chambers and DEP fields are created within an inner chamber by passing through pore structures. The exemplary device includes a plurality of electrodes and electrode-containing chambers within a housing. A device controller independently controls the electrodes, as further described in PCT patent publication WO 2009/146143 A2, which is incorporated herein for that description.
[0037] In some embodiments, chamber devices are created with a variety of pore and/or hole structures (nanoscale, microscale and even macroscale) and contain membranes, gels or filtering materials that control, confine or prevent cells, nanoparticles or other entities diffuse or be transported into the inner chambers while AC/DC electric fields, solute molecules, buffer and other small molecules can pass through the chambers.
[0038] In various embodiments, a variety of device configurations is possible. For example, a device comprising a larger array of electrodes, for example, in a square or rectangular pattern configured to create a non-uniform electric field to allow for AC electrokinetics. For illustrative purposes only, a suitable electrode array may include, but is not limited to, a 10x10 electrode configuration, a 50x50 electrode configuration, a 10x100 electrode configuration, a 20x100 electrode configuration, or a 20x80 electrode configuration.
[0039] These devices include, but are not limited to, multiplex electrode and chamber devices, devices that allow reconfigurable electric field patterns to be created, devices that combine fluidic and DC electrophoretic processes; sample preparation devices, sample preparation, enzymatic manipulation of isolated nucleic acid molecules, and diagnostic devices that include subsequent detection and analysis, laboratory-on-a-chip-type devices, remote laboratory, and other clinical diagnostic systems or versions.
[0040] In some embodiments, a flat platinum electrode array device comprises a housing through which a sample fluid flows. In some embodiments, fluid flows from an inlet end to an outlet end, optionally comprising a side analyte outlet. The example device includes multiple AC electrodes. In some embodiments, the sample consists of a combination of micron-sized entities or cells, larger nanoparticles and smaller nanoparticles or biomolecules. In some cases, larger nanoparticles are cellular debris dispersed in the sample. In some embodiments, the smaller nanoparticles are proteins, fragments of DNA, RNA and smaller cells. In some embodiments, the planar electrode array device is a 60x20 electrode array that is optionally divided into three 20x20 arrays that can be separately controlled but simultaneously operated. Optional auxiliary DC electrodes can be switched to positive charge, while optional DC electrodes are switched to negative for electrophoretic purposes. In some cases, each AC and DC controlled system is used in a continuous and/or pulsed fashion (for example, each can be pulsed on and off at relatively short time intervals) in various modalities. Optional flat electrode arrays along the sides of the sample stream, when layered with nanoporous material (eg, a synthetic polymer hydrogel), are optionally used to generate DC electrophoretic forces as well as AC DEP. Additionally, microelectrophoretic separation processes are optionally carried out within the nanopore layers using flat electrodes in the array and/or auxiliary electrodes in dimensions x-y-z.
[0041] In various modalities, these methods, devices and systems are operated in the AC frequency range of 1000 Hz to 100 MHz, at voltages that can vary from approximately 1 volt to 2000 volts pk-pk; at DC voltages from 1 volt to 1000 volts, at flow rates from 10 microliters per minute to 10 milliliters per minute, and in temperature ranges from 1°C to 120°C. In some embodiments, methods, devices and systems are operated in AC frequency ranges from about 3 to about 15 kHz. In some embodiments, methods, devices and systems are operated at voltages from 5 to 25 volts pk-pk. In some embodiments, the methods, devices and systems are operated at voltages from about 1 to about 50 volts/cm. In some embodiments, the methods, devices, and systems are operated at DC voltages from about 1 to about 5 volts. In some embodiments, methods, devices and systems are operated at a flow rate of about 10 microliters to about 500 microliters per minute. In some embodiments, the methods, devices and systems are operated in temperature ranges from about 20°C to about 60°C. In some modalities, methods, devices and systems are operated in AC frequency ranges from 1000 Hz to 10 MHz. In some modalities, methods, devices and systems are operated in AC frequency ranges from 1000 Hz to 1 MHz. modalities, methods, devices and systems are operated in AC frequency bands from 1000 Hz to 100 kHz. In some modalities, methods, devices and systems are operated in AC frequency bands from 1000 Hz to 10 kHz. In some modalities, methods, devices and systems are operated in AC frequency ranges from 10 kHz to 100 kHz. In some embodiments, methods, devices, and systems are operated in AC frequency ranges from 100 kHz to 1 MHz. In some embodiments, methods, devices, and systems are operated in voltages from approximately 1 volt to 1500 volts pk-pk. In some embodiments, methods, devices and systems are operated at voltages from approximately 1 volt to 1500 volts pk-pk. In some embodiments, methods, devices and systems are operated at voltages from approximately 1 volt to 1000 volts pk-pk. In some embodiments, methods, devices and systems are operated at voltages from approximately 1 volt to 500 volts pk-pk. In some embodiments, methods, devices, and systems are operated at voltages from approximately 1 volt to 250 volts pk-pk. In some embodiments, methods, devices, and systems are operated at voltages from approximately 1 volt to 100 volts pk-pk. In some embodiments, methods, devices and systems are operated at voltages from approximately 1 volt to 50 volts pk-pk. In some embodiments, methods, devices and systems are operated on DC voltages from 1 volt to 1000 volts. In some embodiments, methods, devices and systems are operated on DC voltages from 1 volt to 500 volts. In some embodiments, methods, devices and systems are operated on DC voltages from 1 volt to 250 volts. In some embodiments, methods, devices and systems are operated on DC voltages from 1 volt to 100 volts. In some embodiments, methods, devices and systems are operated on DC voltages from 1 volt to 50 volts. In some embodiments, methods, devices and systems are operated at flow rates from 10 microliters per minute to 1 ml per minute. In some embodiments, methods, devices and systems are operated at flow rates from 10 microliters per minute to 500 microliters per minute. In some embodiments, methods, devices and systems are operated at flow rates from 10 microliters per minute to 250 microliters per minute. In some embodiments, methods, devices and systems are operated at flow rates from 10 microliters per minute to 100 microliters per minute. In some embodiments, methods, devices and systems are operated in temperature ranges from 1°C to 100°C. In some embodiments, methods, devices and systems are operated in temperature ranges from 20°C to 95°C. In some embodiments, methods, devices and systems are operated in temperature ranges from 25°C to 100°C. In some embodiments, methods, devices and systems are operated at room temperature.
[0042] In some modes, the controller independently controls each electrode. In some embodiments, the controller is externally connected to the device such as a socket and plug connection, or is integrated with the device housing.
[0043] Also described here are sectioned scaled arrays (x-y dimensional) of robust electrodes and strategically positioned arrays (x-y-z dimensional) of auxiliary electrodes that combine DEP, electrophoretics, and fluidic forces, and their use. In some modalities, clinically relevant volumes of blood, serum, plasma, or other samples are more directly analyzed under conditions of increased ionic strength and/or conductance. Described here is the superposition of robust electrode structures (eg, platinum, palladium, gold, etc.) with one or more porous layers of materials (natural or synthetic porous hydrogels, membranes, controlled nanopore materials, and dielectric materials in thin layers) to reduce the effects of any electrochemical reactions (electrolysis), heating, and chaotic fluid movement that may occur on or near the electrodes, and still allow for the effective separation of cells, bacteria, viruses, nanoparticles, DNA, and other biomolecules is carried out. In some embodiments, in addition to using AC frequency crossing points to perform higher resolution separations, in-device (array) DC microelectrophoresis is used for secondary separations. For example, the separation of nanoparticulate DNA fragments (20-50 kb), high molecular weight DNA (5-20 kb), intermediate molecular weight DNA (1-5 kb), and lower molecular weight DNA (0 ,1 -1kb) can be performed using DC microelectrophoresis in the array. In some embodiments, the device is sub-sectioned, optionally for purposes of simultaneous separations of different blood cells, bacteria and viruses, and DNA performed simultaneously on that device.
[0044] In some embodiments, the device comprises a housing and a heater or thermal source and/or a reservoir comprising a protein degrading agent. In some embodiments, the heater or thermal source is capable of increasing the fluid temperature to a desired temperature (for example, to a temperature suitable for degrading proteins, about 30°C, 40°C, 50°C, 60°C , 70°C, or the like). In some embodiments, the heater or thermal source is suitable for operation as a PCR thermal cycler. In other embodiments, the heater or thermal source is used to maintain a constant temperature (isothermal conditions). In some embodiments, the protein degrading agent is a protease. In other embodiments, the protein degrading agent is Proteinase Keo heater or thermal source is used to inactivate the protein degrading agent.
[0045] In some embodiments, the device also comprises a plurality of alternating current (AC) electrodes within the housing, the AC electrodes are capable of being selectively energized to establish regions of high dielectrophoretic field (DEP) and low dielectrophoretic field (DEP ), with this the AC electrokinetic effects provide the cell concentration in low field regions of the device. In some embodiments, electrodes are selectively energized to provide the first region of AC electrokinetic field and selectively energized subsequently or continuously to provide the second region of AC electrokinetic field. For example, a further description of electrodes and cell concentration in DEP fields is found in PCT patent publication WO 2009/146143 A2, which is incorporated herein for that description.
[0046] In some embodiments, the device comprises a second reservoir comprising an eluent. The eluent is any fluid suitable for eluting the isolated nucleic acid from the device. In some cases, the eluent is water or a buffer. In some cases, the eluent comprises reagents required for a DNA sequencing method.
[0047] Also provided herein are systems and devices comprising a plurality of alternating current (AC) electrodes, the AC electrodes are configured to be selectively energized in order to establish regions of high dielectrophoretic field (DEP) and low dielectrophoretic field ( DEP) . In some cases, AC electrokinetic effects provide cell concentration in low-field regions and/or concentration (or collection or isolation) of molecules (eg, macromolecules such as nucleic acid) in high-field regions of the DEP field.
[0048] Also provided herein are systems and devices comprising a plurality of direct current (DC) electrodes. In some embodiments, the plurality of DC electrodes comprises at least two rectangular electrodes, spread across the array. In some modalities, the electrodes are located at the edges of the array. In some modalities, DC electrodes are interspersed between AC electrodes.
In some embodiments, a system or device described herein comprises a means for manipulating nucleic acid. In some embodiments, a system or device described here includes a means of performing enzymatic reactions. In other embodiments, a system or device described herein includes a means of performing polymerase chain reaction, isothermal amplification, binding reactions, restriction analysis, nucleic acid cloning, transcription or translation assays, or other molecular biology assay of enzyme base.
In some embodiments, a system or device described herein comprises a nucleic acid sequencer. The sequencer is optionally any suitable DNA sequencing device that includes, but is not limited to, a Sanger sequencer, pyrosequencer, ionic semiconductor sequencer, polony sequencer, device bond sequencing, device DNA nanosphere sequencing, sequencing by device binding, or single-molecule device sequencing.
[0051] In some embodiments, a system or device described here is capable of maintaining a constant temperature. In some embodiments, a system or device described here is capable of cooling the array or chamber. In some embodiments, a system or device described here is capable of heating the array or chamber. In some embodiments, a system or device described herein comprises a thermocycler. In some embodiments, the devices described herein comprise a localized temperature control element. In some embodiments, the devices described here are capable of capturing and controlling temperature.
[0052] In some embodiments, the devices additionally comprise heating or thermal elements. In some embodiments, a heating or thermal element is located under an electrode. In some embodiments, the heating or thermal elements comprise a metal. In some embodiments, the heating or thermal elements comprise tantalum, aluminum, tungsten, or a combination thereof. Generally, the temperature reached by a heating or thermal element is proportional to the current flowing through it. In some embodiments, the devices described here comprise localized cooling elements. In some embodiments, heat resistant elements are placed directly under the exposed electrode array. In some embodiments, the devices described herein are capable of reaching and maintaining a temperature between about 20°C and about 120°C. In some embodiments, the devices described herein are capable of reaching and maintaining a temperature between about 30°C and about 100°C. In other embodiments, the devices described here are capable of reaching and maintaining a temperature between about 20oC and about 95oC. In some embodiments, the devices described here are capable of reaching and maintaining a temperature between about 25oC and about 90oC, between about 25oC and about 85oC, between about 25oC and about 75oC, between about 25oC and about 65oC or between about 25oC and about 55oC. In some embodiments, the devices described herein are capable of reaching and maintaining a temperature of about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, about 70 °C, about 80°C, about 90°C, about 100°C, about 110°C, or about 120°C. ELECTRODES
[0053] The plurality of alternating current electrodes are optionally configured in any manner suitable for the separation processes described here. For example, a further description of the system or device that includes electrodes and/or cell concentration in DEP fields is found in PCT patent publication WO 2009/146143, which is incorporated herein for that description.
[0054] In some embodiments, the electrodes described here can comprise any suitable metal. In some embodiments, electrodes may include, but are not limited to: aluminum, copper, carbon, iron, silver, gold, palladium, platinum, iridium, platinum-iridium alloy, ruthenium, rhodium, osmium, tantalum, titanium, tungsten , polysilicon, and indium tin oxide, or combinations thereof, as well as silicate materials such as platinum silicate, titanium silicate, gold silicate, or tungsten silicate. In some embodiments, the electrodes may comprise a conductive ink capable of being screen-printed.
[0055] In some embodiments, the edge-to-edge (E2E) to diameter ratio of an electrode is about 0.5 mm to about 5 mm. In some embodiments, the E2E to diameter ratio is about 1 mm to about 4 mm. In some embodiments, the E2E to diameter ratio is about 1 mm to about 3 mm. In some embodiments, the E2E to diameter ratio is about 1 mm to about 2 mm. In some embodiments, the E2E to diameter ratio is about 2 mm to about 5 mm. In some embodiments, the E2E to diameter ratio is about 1 mm. In some embodiments, the E2E to diameter ratio is about 2 mm. In some embodiments, the E2E to diameter ratio is about 3 mm. In some embodiments, the E2E to diameter ratio is about 4 mm. In some embodiments, the E2E to diameter ratio is about 5 mm.
[0056] In some embodiments, the electrodes described here are dry embossed. In some modalities, the electrodes are wet embossed. In some embodiments, the electrodes undergo a combination of dry embossing and wet embossing.
[0057] In some modalities, each electrode is individually site-controlled.
[0058] In some embodiments, an electrode array is controlled as a unit.
[0059] In some embodiments, a passivation layer is employed. In some embodiments, a passivation layer can be formed from any suitable material known in the art. In some embodiments, the passivation layer comprises silicon nitride. In some embodiments, the passivation layer comprises silicon dioxide. In some embodiments, the passivation layer has a relative electrical permittivity of about 2.0 to about 8.0. In some embodiments, the passivation layer has a relative electrical permittivity of about 3.0 to about 8.0, about 4.0 to about 8.0, or about 5.0 to about 8.0. In some embodiments, the passivation layer has a relative electrical permittivity of about 2.0 to about 4.0. In some embodiments, the passivation layer has a relative electrical permittivity of about 2.0 to about 3.0. In some embodiments, the passivation layer has a relative electrical permittivity of about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0. passivation is from about 0.1 micron to about 10 microns thick. In some embodiments, the passivation layer is between about 0.5 microns and 8 microns thick. In some embodiments, the passivation layer is between about 1.0 micron and 5 microns thick. In some embodiments, the passivation layer is between about 1.0 micron and 4 microns thick. In some embodiments, the passivation layer is between about 1.0 micron and 3 microns thick. In some embodiments, the passivation layer is between about 0.25 microns and 2 microns thick.
[0060] In some embodiments, the passivation layer is between about 0.25 microns and 1 micron thick.
[0061] In some embodiments, the passivation layer is comprised of any suitable low-k insulating dielectric material, including, but not limited to, silicon nitride or silicon dioxide. In some embodiments, the passivation layer is selected from the group consisting of polyamides, carbon, doped silicon nitride, carbon doped silicon dioxide, fluorine doped silicon nitride, fluorine doped silicon dioxide, silicon dioxide porous, or any combinations thereof. In some embodiments, the passivation layer may comprise a dielectric ink capable of being screen-printed. ELECTRODE GEOMETRY
[0062] In some embodiments, the electrodes described here can be arranged anyway to practice the methods described here.
[0063] In some embodiments, the electrodes are in a point configuration, for example, the electrodes comprise a generally circular or round configuration. In some embodiments, the orientation angle between the points is from about 25° to about 60°. In some embodiments, the orientation angle between the points is from about 30° to about 55°. In some embodiments, the orientation angle between the points is from about 30° to about 50°. In some embodiments, the orientation angle between the points is from about 35° to about 45°. In some modalities, the orientation angle between the points is about 25°. In some modalities, the orientation angle between the points is about 30°. In some embodiments, the orientation angle between the points is about 35°. In some embodiments, the orientation angle between the points is about 40°. In some embodiments, the orientation angle between the points is about 45°. In some embodiments, the orientation angle between the points is about 50°. In some embodiments, the orientation angle between the points is about 55°. In some embodiments, the orientation angle between the points is about 60°.
[0064] In some embodiments, the electrodes are in a substantially elongated configuration.
[0065] In some embodiments, the electrodes are in a configuration similar to wavy or non-linear lines. In some embodiments, the electrode array is in a wavy or non-linear line configuration, where the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a ligand, where the dots and the ligand define the electrode boundaries, where the ligand tapers inward towards or at the midpoint between the pair of points, where the diameters of the points are the largest points along the length of the repeat unit, where the edge distance the edge between a parallel set of repeating units is equidistant, or nearly equidistant. In some embodiments, the electrodes are strips that look like wavy lines, as shown in Figure 8. In some embodiments, the edge-to-edge distance between the electrodes is equidistant, or nearly equidistant, along the wavy line configuration. In some embodiments, the use of wavy line electrodes, as described here, results in an increased DEP field gradient.
[0066] In some embodiments, the electrodes described here are in a flat configuration. In some embodiments, the electrodes described here are in a non-flat configuration.
[0067] In some modalities, the devices described here selectively superficially capture the biomolecules on their surface. For example, the devices described herein can capture biomolecules, such as nucleic acids, by, for example, a. nucleic acid hybridization; B. antibody-antigen interactions; ç. biotin-avidin interactions; d. ionic or electrostatic interactions; or e. any combination of these. The devices described herein, therefore, may incorporate a functionalized surface that includes capture molecules such as complementary nucleic acid probes, antibodies or other protein captures capable of capturing biomolecules (such as nucleic acids), biotin or other fixation captures capable of capture complementary target molecules such as avidin, capture molecules capable of capturing biomolecules (such as nucleic acids) by ionic or electrostatic interactions, or any combination of these.
[0068] In some embodiments, the surface is functionalized to reduce and/or inhibit non-specific binding interactions by: a. polymers (for example polyethylene glycol PEG); B. ionic or electrostatic interactions; ç. surfactants; or d. any combination of these. In some embodiments, the methods described here include the use of additives that reduce non-specific binding interactions by interfering with these interactions, such as Tween 20 and the like, bovine serum albumin, non-specific immunoglobulins, etc.
[0069] In some embodiments, the device comprises a plurality of microelectrode devices oriented (a) flat side by side, (b) faced vertically, or (c) faced horizontally. In other embodiments, the electrodes are in a sandwiched configuration, for example, stacked on top of each other in a vertical format. HYDROGELS
[0070] Overlapping electrode structures with one or more layers of materials can reduce harmful electrochemical effects, including, but not limited to, electrolysis reactions, heating, and chaotic fluid movement that can occur at or near the electrodes , and still allow the effective separation of cells, bacteria, viruses, nanoparticles, DNA, and other biomolecules to be carried out. In some embodiments, the layered materials on the electrode structures can be one or more porous layers. In other embodiments, one or more porous layers consists of a polymer layer. In other embodiments, the one or more porous layers is a hydrogel.
[0071] In general, the hydrogel must have sufficient mechanical strength and be relatively chemically inert so that it is able to prolong the electrochemical effects on the electrode surface without disfigurement or decomposition. In general, the hydrogel is sufficiently permeable to small aqueous ions, but keeps the biomolecules away from the electrode surface.
[0072] In some embodiments, the hydrogel is a single layer, or coating.
[0073] In some embodiments, the hydrogel comprises a porosity gradient, wherein the lower part of the hydrogel layer has greater porosity than the upper part of the hydrogel layer.
[0074] In some embodiments, the hydrogel comprises multiple layers or coatings. In some embodiments, the hydrogel comprises two coatings. In some embodiments, the hydrogel comprises three coats. In some embodiments, the bottom (first) coating has greater porosity than subsequent coatings. In some embodiments, the top coat has less porosity than the first coat. In some embodiments, the top coating has an average pore diameter that works as a size reduction for particles larger than 100 picometers in diameter.
In some embodiments, the hydrogel has a conductivity of about 0.001 S/m to about 10 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.01 S/m to about 10 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.1 S/m to about 10 S/m. In some embodiments, the hydrogel has a conductivity of from about 1.0 S/m to about 10 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.01 S/m to about 5 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.01 S/m to about 4 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.01 S/m to about 3 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.01 S/m to about 2 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.1 S/m to about 5 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.1 S/m to about 4 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.1 S/m to about 3 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.1 S/m to about 2 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.1 S/m to about 1.5 S/m. In some embodiments, the hydrogel has a conductivity of from about 0.1 S/m to about 1.0 S/m.
[0076] In some embodiments, the hydrogel has a conductivity of about 0.1 S/m. In some embodiments, the hydrogel has a conductivity of about 0.2 S/m. In some embodiments, the hydrogel has a conductivity of about 0.3 S/m. In some embodiments, the hydrogel has a conductivity of about 0.4 S/m. In some embodiments, the hydrogel has a conductivity of about 0.5 S/m. In some embodiments, the hydrogel has a conductivity of about 0.6 S/m. In some embodiments, the hydrogel has a conductivity of about 0.7 S/m. In some embodiments, the hydrogel has a conductivity of about 0.8 S/m. In some embodiments, the hydrogel has a conductivity of about 0.9 S/m. In some embodiments, the hydrogel has a conductivity of about 1.0 S/m.
[0077] In some embodiments, the hydrogel has a thickness of about 0.1 micron to about 10 microns. In some embodiments, the hydrogel is from about 0.1 micron to about 5 microns thick. In some embodiments, the hydrogel is from about 0.1 micron to about 4 microns thick. In some embodiments, the hydrogel is from about 0.1 micron to about 3 microns thick. In some embodiments, the hydrogel is from about 0.1 micron to about 2 microns thick. In some embodiments, the hydrogel is from about 1 micron to about 5 microns thick. In some embodiments, the hydrogel is from about 1 micron to about 4 microns thick. In some embodiments, the hydrogel is from about 1 micron to about 3 microns thick. In some embodiments, the hydrogel is from about 1 micron to about 2 microns thick. In some embodiments, the hydrogel is from about 0.5 micron to about 1 micron thick.
[0078] In some embodiments, a hydrogel solution before rotational coating ranges from about 0.5 cP to about 5 cP. In some embodiments, a single coating of hydrogel solution has a viscosity between about 0.75 cP and 5 cP prior to rotational coating. In some embodiments, in a multicoated hydrogel, the first hydrogel solution has a viscosity of about 0.5 cP to about 1.5 cP prior to rotational coating. In some embodiments, the second hydrogel solution has a viscosity of about 1 cP to about 3 cP. The viscosity of the hydrogel solution is based on the polymer concentration (0.1% -10%) and polymer molecular weight (10,000 to 300,000) in the solvent and the starting solvent viscosity.
[0079] In some embodiments, the first hydrogel coating has a thickness between about 0.5 micron and 1 micron. In some embodiments, the first hydrogel coating is between about 0.5 micron and 0.75 micron thick. In some embodiments, the first hydrogel coating is between about 0.75 and 1 micron thick. In some embodiments, the second hydrogel coating is between about 0.2 microns and 0.5 microns thick. In some embodiments, the second hydrogel coating is between about 0.2 and 0.4 microns thick. In some embodiments, the second hydrogel coating is between about 0.2 and 0.3 microns thick. In some embodiments, the second hydrogel coating is between about 0.3 and 0.4 microns thick.
[0080] In some embodiments, the hydrogel comprises any suitable synthetic polymer that forms a hydrogel. In general, any sufficiently hydrophilic and polymerizable molecule can be used in producing a synthetic polymer hydrogel for use as described herein. Polymerizable moieties in monomers can include alkenyl moieties which include, but are not limited to, substituted or unsubstituted α,β,unsaturated carbonyls where the double bond is directly attached to a carbon that has a double bond to an oxygen and a single bond to another oxygen, nitrogen, sulfur, halogen, or carbon; vinyl, in which the double bond is uniquely attached to an oxygen, nitrogen, halogen, phosphorus, or sulfur; allyl, in which the double bond is uniquely bonded to a carbon that is bonded to an oxygen, nitrogen, halogen, phosphorus, or sulfur; homoallyl, in which the double bond is uniquely bonded to a carbon that is uniquely bonded to another carbon that is then uniquely bonded to an oxygen, nitrogen, halogen, phosphorus, or sulfur; alkynyl moieties in which there is a triple bond between two carbon atoms. In some embodiments, acryloyl or acrylamide monomers such as acrylates, methacrylates, acrylamides, methacrylamides, etc., are useful for forming hydrogels as described herein. The most preferred acrylamide monomers include acrylamides, N-substituted acrylamides, N-substituted methacrylamides, and methacrylamide. In some embodiments, a hydrogel comprises polymers such as epoxide-based polymers, vinyl-based polymers, allyl-based polymers, homoallyl-based polymers, cyclic anhydride-based polymers, ester-based polymers, based polymers of ether, alkylene glycol based polymers (e.g., polypropylene glycol), and the like.
[0081] In some embodiments, the hydrogel comprises polyhydroxyethylmethacrylate (pHEMA), cellulose acetate, cellulose acetate phthalate, cellulose acetate butyrate, or any suitable acrylamide or vinyl-based polymer, or a derivative thereof.
[0082] In some embodiments, the hydrogel is applied by vapor deposition.
[0083] In some embodiments, the hydrogel is polymerized through radical atomic transfer polymerization (ATRP).
[0084] In some embodiments, the hydrogel is polymerized through chain transfer polymerization and reversible addition fragmentation (RAFT).
[0085] In some embodiments, additives are added to a hydrogel to increase the conductivity of the gel. In some embodiments, hydrogel additives are conductive polymers (eg, PEDOT: PSS), salts (eg, copper chloride), metals (eg, gold), plasticizers (eg, PEG200, PEG 400, or PEG 600), or co-solvents.
[0086] In some embodiments, the hydrogel also comprises compounds or materials that help maintain the stability of DNA hybrids, including, but not limited to, histidine, histidine peptides, polyhistidine, lysine, lysine peptides, and other cationic compounds or substances. DIELECTROPHORETIC FIELDS
[0087] In some embodiments, devices and systems described herein provide a mechanism to collect, separate, or isolate cells, particles and/or molecules (such as nucleic acid) from a fluid material (which optionally contains other materials such as contaminants, cellular material residual, or similar).
[0088] In some embodiments, an AC electrokinetic field is generated to collect, separate or isolate biomolecules such as nucleic acids. In some embodiments, the AC electrokinetic field is a dielectrphoretic field. Consequently, in some embodiments dielectrophoresis (DEP) is used at various steps of the methods described here.
[0089] In some embodiments, the devices and systems described here are capable of generating DEP fields, and the like. In specific embodiments, DEP is used to concentrate cells and/or nucleic acids (eg, simultaneously or at different times). In some embodiments, the methods described herein further comprise energizing the electrode array to produce the first, second, and any additional optional DEP fields. In some embodiments, the devices and systems described here are capable of being powered to produce the first, second, and any additional optional DEP fields.
[0090] DEP is a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a non-uniform electric field. Depending on the step of the methods described herein, the aspects of the devices and systems described herein, and the like, the dielectric particle in various embodiments herein is a biological cell and/or a molecule, such as a nucleic acid molecule. Steps other than the methods described herein or aspects of the devices or systems described herein can be used to isolate and separate different components, such as intact cells or other particular material; additionally, field regions other than the DEP field may be used in steps other than the methods or aspects of the devices and systems described here. This dielectrophoretic force does not require the particle to be charged. In some cases, the strength of the force depends on the medium and electrical properties of the specific particles, the shape and size of the particles, as well as the frequency of the electric field. In some cases, fields of a particular frequency selectively manipulate particles. In some aspects described here, these processes allow for the separation of cells and/or smaller particles (such as molecules, including nucleic acid molecules) from other components (eg, in a fluid medium) or from each other.
[0091] In various embodiments provided here, a method described here comprises producing a first DEP field region and a second DEP field region with the array. In various embodiments provided herein, a device or system described herein is capable of producing a first DEP field region and a second DEP field region with the array. In some cases, the first and second field regions are part of a single field (for example, the first and second field regions are present at the same time but are found in different locations within the device and/or over the array) . In some modalities, the first and second field regions are different fields (for example, the first region is created by energizing the electrodes in a first moment, and the second region is created by energizing the electrodes in a second moment). In specific aspects, the first DEP field region is suitable for concentrating or isolating cells (for example, in a region of low DEP field). In some embodiments, the second DEP field region is suitable for concentrating smaller particles such as molecules (eg, nucleic acid), for example, in a region of high DEP field. In some cases, a method described here optionally excludes the use of the first or second DEP field region.
[0092] In some embodiments, the first DEP field region is in the same chamber of a device as described here as the second DEP field region. In some modalities, the first DEP field region and the second DEP field region occupy the same area of the electrode array.
[0093] In some embodiments, the first DEP field region is in a separate chamber of a device as described here, or a device entirely separate from the second DEP field region. FIRST REGION OF CAMPO DEP
[0094] In some aspects, for example, high conductance buffers (>100 mS/m), the method described here comprises applying a fluid comprising cells or other particulate material to a device comprising an array of electrodes, and with this, concentrate the cells in a first DEP field region. In some aspects, the devices and systems described herein are capable of applying a fluid comprising cells or other particulate material to the device comprising an electrode array, and thereby concentrating the cells in a first DEP field region. Subsequent or simultaneous second, third, and fourth DEP regions can collect or isolate other fluid components, including biomolecules such as nucleic acids.
[0095] The first DEP field region can be any field region suitable for concentrating cells in a fluid. For this application, cells are usually concentrated close to the electrode array. In some embodiments, the first DEP field region is a low dielectrophoretic field region. In some embodiments, the first DEP field region is a region of high dielectrophoretic field. In some aspects, for example, low conductance buffers (<100 mS/m), the method described here comprises applying a fluid comprising cells to a device comprising an array of electrodes, and thereby concentrating the cells or other particulate material in a first DEP field region.
[0096] In some aspects, the devices and systems described here are capable of applying a fluid comprising cells or other particulate material to the device comprising an array of electrodes, and concentrating the cells in a first DEP field region. In various embodiments, the first DEP field region can be any field region suitable for concentrating cells in a fluid. In some modalities, cells are concentrated on the electrode array. In some modalities, cells are trapped in a region of high dielectrophoretic field. In some modalities, cells are trapped in a region of low dielectrophoretic field. High versus low field capture is generally dependent on fluid conductivity, where generally the crossover point is between about 300-500 mS/m. In some embodiments, the first DEP field region is a region of low dielectrophoretic field realized at fluid conductivity greater than about 300 mS/m. In some embodiments, the first DEP field region is a region of low dielectrophoretic field realized at fluid conductivity less than about 300 mS/m. In some embodiments, the first DEP field region is a region of high dielectrophoretic field realized at fluid conductivity greater than about 300 mS/m. In some embodiments, the first DEP field region is a region of high dielectrophoretic field realized at fluid conductivity less than about 300 mS/m. In some embodiments, the first DEP field region is a region of low dielectrophoretic field realized at fluid conductivity greater than about 500 mS/m. In some embodiments, the first DEP field region is a region of low dielectrophoretic field realized at fluid conductivity less than about 500 mS/m. In some embodiments, the first DEP field region is a region of high dielectrophoretic field realized at fluid conductivity greater than about 500 mS/m. In some embodiments, the first DEP field region is a region of high dielectrophoretic field realized at fluidic conductivity less than about 500 mS/m.
[0097] In some embodiments, the first dielectrophoretic field region is produced by an alternating current. Alternating current has any suitable amperage, voltage, frequency, and the like to concentrate cells. In some embodiments, the first dielectrophoretic field region is produced using an alternating current that has an amperage of 0.1 microamps - 10 amps; a voltage of 1-50 Volts peak to peak; and/or a frequency of 1 - 10,000,000 Hz. In some embodiments, the first DEP field region is produced using an alternating current that has a voltage of 5 to 25 volts peak-to-peak. In some embodiments, the first DEP field region is produced using an alternating current that has a frequency of 3 to 15 kHz. In some embodiments, the first DEP field region is produced using an alternating current that has an amperage of 1 milliamps to 1 amp. In some embodiments, the first DEP field region is produced using an alternating current that has an amperage of 0.1 micro amps - 1 amp. In some embodiments, the first DEP field region is produced using an alternating current that has an amperage of 1 micro amps - 1 amp. In some embodiments, the first DEP field region is produced using an alternating current that has an amperage of 100 microamps to 1 amp. In some embodiments, the first DEP field region is produced using an alternating current that has an amperage of 500 micro amps to 500 milli amps. In some embodiments, the first DEP field region is produced using an alternating current that has a voltage of 1 to 25 Volts peak-to-peak. In some embodiments, the first DEP field region is produced using an alternating current that has a voltage of 1 to 10 volts peak-to-peak. In some embodiments, the first DEP field region is produced using an alternating current that has a voltage of 25 to 50 volts peak-to-peak. In some embodiments, the first DEP field region is produced using a frequency of 10 to 1,000,000 Hz. In some embodiments, the first DEP field region is produced using a frequency of 100 to 100,000 Hz. In some embodiments, the first DEP field region is produced using a frequency of 100 to 10,000 Hz. In some embodiments, the first DEP field region is produced using a frequency of 10,000 to 100,000 Hz. In some embodiments, the first DEP field region is produced using a frequency of 100,0001,000,000 Hz.
[0098] In some embodiments, the first dielectrophoretic field region is produced by a direct current. Direct current has any suitable amperage, voltage, frequency, and the like to concentrate cells. In some embodiments, the first dielectrophoretic field region is produced using a direct current that has an amperage of 0.1 microamps to 1 amps; a voltage of 10 milli Volts to 10 Volts; and/or a pulse width of 1 millisecond to 1000 seconds and a pulse frequency of 0.001 to 1000 Hz. In some embodiments, the first DEP field region is produced using a forward current that has an amperage of 1 micro amps to 1 Amperes. In some embodiments, the first DEP field region is produced using a forward current that has an amperage of 100 micro amps -500 milli amps. In some embodiments, the first DEP field region is produced using a forward current that has an amperage of 1 milli amps to 1 amps. In some embodiments, the first DEP field region is produced using a forward current that has an amperage of 1 micro amps to 1 milli amps. In some embodiments, the first DEP field region is produced using a forward current that has a pulse width of 500 milliseconds-500 seconds. In some embodiments, the first DEP field region is produced using a forward current that has a pulse width of 500 milliseconds to 100 seconds. In some embodiments, the first DEP field region is produced using a direct current that has a pulse width of 1 second to 1000 seconds. In some embodiments, the first DEP field region is produced using a forward current that has a pulse width of 500 milliseconds to 1 second. In some embodiments, the first DEP field region is produced using a pulse frequency from 0.01 to 1000 Hz. In some embodiments, the first DEP field region is produced using a pulse frequency from 0.1 to 100 Hz. In some embodiments, the first DEP field region is produced using a pulse frequency from 1 to 100 Hz. In some embodiments, the first DEP field region is produced using a pulse frequency from 100 to 1000 Hz.
[0099] In some embodiments, the fluid comprises a mixture of cell types. For example, blood comprises red blood cells and white blood cells. Environmental samples comprise many types of cells and other particulate material over a wide range of concentrations. In some embodiments, a cell type (or any number of cell types less than the total number of cell types comprising the sample) is preferably concentrated in the first DEP field. Without limitation, this modality is beneficial for focusing the nucleic acid isolation procedure on a particular environmental contaminant, such as fecal coliform bacteria, so that DNA sequencing can be used to identify the source of the contaminant. In another non-limiting example, the first DEP field is operated in such a way that it specifically concentrates viruses and not cells (eg, in a fluid with a conductivity greater than 300 mS/m, viruses concentrate in a region of high DEP field, whereas larger cells will concentrate in a region of low DEP field).
[0100] In some embodiments, a method, device, or system described here is suitable for isolating or separating specific cell types. In some embodiments, the DEP field of the method, device, or system is specifically set to allow for the separation or concentration of a specific cell type in a field region of the DEP field. In some embodiments, a method, device, or system described herein provides more than one field region where more than one cell type is isolated or concentrated. In some embodiments, a method, device, or system described herein is adjustable to allow for the isolation or concentration of different cell types within the DEP field regions thereof. In some embodiments, a method provided here further comprises adjusting the DEP field. In some embodiments, a device or system provided here is capable of having the DEP field set. In some cases, this adjustment can serve to provide a DEP particularly suited for the intended purpose. For example, modifications to the array, energy, or other parameter are optionally used to adjust the DEP field. Finer resolution adjustment parameters include electrode diameter, edge-to-edge distance between electrodes, voltage, frequency, fluid conductivity, and hydrogel composition.
[0101] In some modalities, the first DEP field region comprises the entirety of an electrode array. In some embodiments, the first DEP field region comprises a portion of an electrode array. In some modalities, the first DEP field region comprises about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 25% , about 20%, or about 10% of an electrode array. In some embodiments, the first DEP field region comprises about a third electrode array. SECOND REGION OF CAMPO DEP
[0102] In one aspect, after cell lysis (as provided below), the methods described here involve concentrating the nucleic acid in a second region of the DEP field. In another aspect, the devices and systems described herein are capable of concentrating nucleic acid in a second DEP field region. In some embodiments, the second DEP field region is any field region suitable for concentrating nucleic acids. In some embodiments, nucleic acids are concentrated onto the electrode array. In some embodiments, the second DEP field region is a high dielectrophoretic field region. The second DEP field region is optionally the same as the first DEP field region.
[0103] In some embodiments, the second dielectrophoretic field region is produced by an alternating current. In some embodiments, alternating current has any suitable amperage, voltage, frequency, and the like to concentrate the nucleic acids. In some embodiments, the second dielectrophoretic field region is produced using an alternating current that has an amperage of 0.1 microamps to 10 amps; a voltage of 1 to 50 Volts peak-to-peak; and/or a frequency of 1 to 10,000,000 Hz. In some embodiments, the second DEP field region is produced using an alternating current that has an amperage of 0.1 microamps to 1 amp. In some embodiments, the second DEP field region is produced using an alternating current that has an amperage of 1 microamps to 1 amps. In some embodiments, the second DEP field region is produced using an alternating current that has an amperage of 100 micro amps to 1 amp. In some embodiments, the second DEP field region is produced using an alternating current that has an amperage of 500 micro amps to 500 milli amps. In some embodiments, the second DEP field region is produced using an alternating current that has a voltage of 1 to 25 Volts peak-to-peak. In some embodiments, the second DEP field region is produced using an alternating current that has a voltage of 1 to 10 volts peak-to-peak. In some embodiments, the second DEP field region is produced using an alternating current that has a voltage of 25 to 50 volts peak-to-peak. In some embodiments, the second DEP field region is produced using a frequency of 10 to 1,000,000 Hz. In some embodiments, the second DEP field region is produced using a frequency of 100 to 100,000 Hz. In some embodiments, the second DEP field region is produced using a frequency of 100 to 10,000 Hz. In some embodiments, the second DEP field region is produced using a frequency of 10,000 to 100,000 Hz. In some embodiments, the second DEP field region is produced using a frequency from 100,000 to 1,000,000 Hz.
[0104] In some embodiments, the second dielectrophoretic field region is produced by a direct current. In some embodiments, direct current has any suitable amperage, voltage, frequency, and the like to concentrate the nucleic acids. In some embodiments, the second dielectrophoretic field region is produced using a direct current that has an amperage of 0.1 microamps to 1 amps; a voltage of 10 milli Volts to 10 Volts; and/or a pulse width of 1 milliseconds to 1000 seconds and a pulse frequency of 0.001 to 1000 Hz. In some embodiments, the second DEP field region is produced using an alternating current that has a voltage of 5 to 25 volts peak to peak. In some embodiments, the second DEP field region is produced using an alternating current that has a frequency of 3 to 15 kHz. In some embodiments, the second DEP field region is produced using an alternating current that has an amperage of 1 milliamps to 1 amp. In some embodiments, the second DEP field region is produced using a forward current that has an amperage of 1 microamps to 1 amps. In some embodiments, the second DEP field region is produced using a forward current that has an amperage of 100 micro amps to 500 milli amps. In some embodiments, the second DEP field region is produced using a forward current that has an amperage of 1 milli amps to 1 amps. In some embodiments, the second DEP field region is produced using a forward current that has an amperage of 1 micro amps to 1 milli amps. In some embodiments, the second DEP field region is produced using a forward current that has a pulse width of 500 milliseconds to 500 seconds. In some embodiments, the second DEP field region is produced using a forward current that has a pulse width of 500 milliseconds to 100 seconds. In some embodiments, the second DEP field region is produced using a direct current that has a pulse width of 1 second to 1000 seconds. In some embodiments, the second DEP field region is produced using a forward current that has a pulse width of 500 milliseconds to 1 second. In some embodiments, the second DEP field region is produced using a pulse frequency of 0.01 to 1000 Hz. In some embodiments, the second DEP field region is produced using a pulse frequency of 0.1 to 100 Hz. In some embodiments, the second DEP field region is produced using a pulse frequency from 1 to 100 Hz. In some embodiments, the second DEP field region is produced using a pulse frequency from 100 to 1000 Hz.
[0105] In some embodiments, the second DEP field region comprises the entirety of an electrode array. In some embodiments, the second DEP field region comprises a portion of an electrode array. In some embodiments, the second DEP field region comprises about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 25% , about 20%, or about 10% of an electrode array. In some embodiments, the second DEP field region comprises about one-third of an electrode array. NUCLEIC ACID INSULATION
[0106] In one aspect, described herein is a method for isolating a nucleic acid from a fluid comprising cells. In some embodiments, nucleic acids are initially inside cells. As seen in Figure 5, the method comprises concentrating cells close to a high-field region in some cases. In some embodiments, a method for isolating a nucleic acid from a fluid comprising cells is described herein, the method comprising: a. applying the fluid to a device, the device comprises an array of electrodes; B. concentrating a plurality of cells in a first electrokinetic (e.g., dielectrophoretic) AC field region; ç. isolating the nucleic acid in a second AC electrokinetic field region (eg, dielectrophoretic); and d. discard the cells. In some cases, cells are lysed in the high-field region. After lysis, nucleic acids remain in the high-field region and/or are concentrated in the high-field region. In some cases, residual cellular material is concentrated near the low-field region. In some embodiments, waste material is washed from the device and/or washed away from nucleic acids. In some embodiments, the nucleic acid is concentrated in the second AC electrokinetic field region.
[0107] In one aspect, described herein is a method for isolating a nucleic acid from a fluid comprising cells or other particulate material. In some embodiments, nucleic acids are not inside cells (eg, cell-free DNA in fluid). In some embodiments, a method for isolating a nucleic acid from a fluid comprising cells or other particulate material is described herein, the method comprising: a. applying the fluid to a device, the device comprising an array of electrodes; B. concentrating a plurality of cells in a first electrokinetic (e.g., dielectrophoretic) field region; ç. isolating the nucleic acid in a second AC electrokinetic field region (eg, dielectrophoretic); and d. discard the cells. In some embodiments, the method further comprises degrading residual proteins after discarding the cells. Figure 6 shows an exemplary method for isolating extracellular nucleic acids from a fluid comprising cells. In some embodiments, cells are concentrated in or near a low-field region and nucleic acids are concentrated in or near a high-field region. In some cases, cells are removed from the device and/or removed from nucleic acids.
[0108] In one aspect, the methods, systems and devices described herein isolate nucleic acid from a fluid comprising cells or other particulate material. In one aspect, dielectrophoresis is used to concentrate cells. In some embodiments, the fluid is a liquid, optionally water or an aqueous solution or dispersion. In some embodiments, the fluid is any suitable fluid that includes a bodily fluid. Exemplary bodily fluids include blood, serum, plasma, bile, milk, cerebrospinal fluid, gastric juice, ejaculation, mucus, peritoneal fluid, saliva, sweat, tears, urine, and the like. In some embodiments, nucleic acids are isolated from bodily fluids using the methods, systems or devices described herein as part of a therapeutic or diagnostic medical procedure, device or system. In some embodiments, the fluid consists of tissues and/or cells solubilized and/or dispersed in a fluid. For example, the tissue can be a cancerous tumor from which nucleic acid can be isolated using the methods, devices or systems described herein.
[0109] In some embodiments, the fluid is an environmental sample. In some embodiments, the environmental sample is analyzed or monitored for the presence of a particular nucleic acid sequence indicative of a particular incidence of contamination, infestation, or the like. The environmental sample can also be used to determine the source of a particular incidence of contamination, infestation or the like using the methods, devices or systems described herein. Exemplary environmental samples include municipal wastewater, industrial wastewater, water or fluid used or produced as a result of various manufacturing processes, lakes, rivers, oceans, aquifers, water table, stormwater, plants or parts of plants, animals or animal parts, insects, municipal water supply, and the like.
[0110] In some embodiments, the fluid is a food or drink. The food or beverage can be analyzed or monitored for the presence of a particular nucleic acid sequence indicative of a particular incidence of contamination, infestation or the like. The food or beverage can also be used to determine the source of a particular incidence of contamination, infestation or the like using the methods, devices or systems described herein. In various embodiments, the methods, devices, and systems described herein can be used with one or more bodily fluids, environmental samples, and foods and beverages to monitor public health or respond to adverse public health incidents.
[0111] In some embodiments, fluid is a growth medium. The growth medium can be any medium suitable for culturing cells, for example, lysogeny broth (LB) for culturing E. coli, Ham tissue culture medium for culturing mammalian cells, and the like. The medium can be rich medium, minimal medium, selective medium, and the like. In some embodiments, the medium comprises or consists essentially of a plurality of clonal cells. In some embodiments, the medium comprises a mixture of at least two species.
[0112] In some embodiments, the fluid is water.
[0113] Cells consist of any cell suitable for isolating nucleic acids as described herein. In various embodiments, cells are eukaryotic or prokaryotic. In various embodiments, the cells consist essentially of a plurality of clonal cells or can comprise at least two species and/or at least two strains.
[0114] In various embodiments, cells are pathogen cells, bacterial cells, plant cells, animal cells, insect cells, algal cells, cyanobacterial cells, organelles and/or combinations of these. as used herein, "cells" include intact viruses and other pathogenic microorganisms. Cells can be microorganisms or cells of multicellular organisms. In some cases, cells originate from a solubilized tissue sample.
[0115] In various modalities, cells are wild-type or genetically engineered. In some cases, cells comprise a library of mutant cells. In some embodiments, cells are randomly mutagenized by being subjected to chemical mutagenesis, radiation mutagenesis (eg, UV radiation), or a combination of these. In some embodiments, cells have been transformed with a library of mutant nucleic acid molecules.
[0116] In some embodiments, the fluid may also comprise other particulate material. Such particulate material can be, for example, inclusion bodies (for example, ceroids or Mallory bodies), cell cylinders (for example, granular cylinders, hyaline cylinders, cell cylinders, waxy cylinders and pseudo cylinders), Pick bodies, bodies of Lewy, fibrillar tangles, fibril formations, cell debris and other particulate matter. In some embodiments, the particulate material is an aggregated protein (eg, beta-amyloid).
[0117] The fluid can have any conductivity including high or low conductivity. In some embodiments, the conductivity is between about 1 µS/m to about 10 mS/m. In some embodiments, the conductivity is between about 10 µS/m to about 10 mS/m. In other embodiments, the conductivity is between about 50 µS/m to about 10 mS/m. In still other embodiments, the conductivity is between about 100 μS/m to about 10 mS/m, between about 100 μS/m to about 8 mS/m, between about 100 μS/m to about 6 mS/m, between about 100 µS/m at about 5 mS/m, between about 100 µS/m at about 4 mS/m, between about 100 µS/m at about 3 mS/m, between about 100 µS/m at about of 2 mS/m, or between about 100 μS/m to about 1 mS/m.
[0118] In some embodiments, the conductivity is about 1 μS/m. In some embodiments, the conductivity is about 10 µS/m. In some embodiments, the conductivity is about 100 µS/m. In some embodiments, the conductivity is about 1 mS/m. In other embodiments, the conductivity is about 2 mS/m. In some embodiments, the conductivity is about 3 mS/m. In still other modalities, the conductivity is about 4 mS/m. In some embodiments, the conductivity is about 5 mS/m. In some embodiments, the conductivity is about 10 mS/m. In still other modalities, the conductivity is about 100 mS/m. In some embodiments, the conductivity is about 1 S/m. In other embodiments, the conductivity is about 10 S/m.
[0119] In some embodiments, the conductivity is at least 1 μS/m. In still other embodiments, the conductivity is at least 10 µS/m. In some embodiments, the conductivity is at least 100 µS/m. In some embodiments, the conductivity is at least 1 mS/m. In additional embodiments, the conductivity is at least 10 mS/m. In still other embodiments, the conductivity is at least 100 mS/m. In some embodiments, the conductivity is at least 1 S/m. In some embodiments, the conductivity is at least 10 S/m. In some embodiments, the conductivity is a maximum of 1 µS/m. In some embodiments, the conductivity is a maximum of 10 μS/m. In other embodiments, the conductivity is a maximum of 100 µS/m. In some embodiments, the conductivity is at most 1 mS/m. In some embodiments, the conductivity is a maximum of 10 mS/m. In some embodiments, the conductivity is a maximum of 100 mS/m. In still other modalities, the conductivity is at most 1 S/m. In some embodiments, the conductivity is a maximum of 10 S/m.
[0120] In some embodiments, fluid is a small volume of fluid that includes less than 10 ml. In some embodiments, the fluid is less than 8 ml. In some embodiments, the fluid is less than 5 ml. In some embodiments, the fluid is less than 2 ml. In some embodiments, the fluid is less than 1 ml. In some embodiments, the fluid is less than 500 µl. In some embodiments, the fluid is less than 200 µl. In some embodiments, the fluid is less than 100 µl. In some embodiments, the fluid is less than 50 µl. In some embodiments, the fluid is less than 10 µl. In some embodiments, the : fluid is less than fluid is less than 1 µl. 5 µl. In some
[0121] In some embodiments, the amount of fluid applied to the device or used in the method comprises less than about 100,000,000 cells. In some embodiments, the fluid comprises less than about 10,000,000 cells. In some embodiments, the fluid comprises less than about 1,000,000 cells. In some embodiments, the fluid comprises less than about 100,000 cells. In some embodiments, the fluid comprises less than about 10,000 cells. In some embodiments, the fluid comprises less than about 1,000 cells.
[0122] In some embodiments, isolation of nucleic acid from a fluid comprising cells or other particulate material with the devices, systems and methods described herein takes less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, or less than about 1 minute. In other embodiments, isolation of nucleic acid from a fluid comprising cells or other particulate material with the devices, systems and methods described herein takes no more than 30 minutes, no more than about 20 minutes, no more than about 15 minutes , no more than about 10 minutes, no more than about 5 minutes, no more than about 2 minutes, or no more than about 1 minute. In additional embodiments, isolation of nucleic acid from a fluid comprising cells or other particulate material with the devices, systems and methods described herein takes less than about 15 minutes, preferably less than about 10 minutes or less than about 5 minutes.
[0123] In some cases, extracellular DNA or other nucleic acid (outside of cells) is isolated from a fluid that comprises cells of other particulate material. In some embodiments, the fluid comprises cells. In some embodiments, the fluid does not comprise cells. CELL LYSIS
[0124] In one aspect, after concentrating the cells in a first dielectrophoretic field region, the method involves releasing the nucleic acids from the cells. In another aspect, the devices and systems described herein are capable of releasing nucleic acids from cells. In some embodiments, nucleic acids are released from cells in the first DEP field region.
[0125] In some embodiments, the methods described herein release nucleic acids from a plurality of cells by cell lysis. In some embodiments, the devices and systems described herein are capable of releasing nucleic acids from a plurality of cells by cell lysis. One method of cell lysis involves applying a direct current to cells after isolating the cells in the array. Direct current has any amperage, adequate voltage, and the like suitable for cell lysis. In some embodiments, the current has a voltage from about 1 Volt to about 500 Volts. In some embodiments, the current has a voltage of about 10 Volts to about 500 Volts. In other embodiments, the current has a voltage of about 10 Volts to about 250 Volts. In still other embodiments, the current has a voltage of about 50 Volts to about 150 Volts. Voltage is usually the activator of cell lysis, as high electrical fields result in failures in membrane integrity.
[0126] In some embodiments, the direct current used for lysis comprises one or more pulses that have any duration, frequency, and the like suitable for cell lysis. In some embodiments, a voltage of about 100 volts is applied for about 1 millisecond for cell lysis. In some embodiments, a voltage of about 100 volts is applied 2 or 3 times to a second source.
[0127] In some embodiments, the frequency of direct current depends on volts/cm, pulse width, and fluid conductivity. In some modes, the pulse has a frequency from about 0.001 to about 1000 Hz. In some modes, the pulse has a frequency from about 10 to about 200 Hz. In other modes, the pulse has a frequency of about about 10 to about 200 Hz. .01 Hz to 1000 Hz. In still other modes, the pulse has a frequency of about 0.1 Hz to 1000 Hz, about 1 Hz to 1000 Hz, about 1 Hz to 500 Hz, about 1 Hz to 400 Hz, about 1 Hz to 300 Hz, or about 1 Hz to about 250 Hz. In some modes, the pulse has a frequency of about 0.1 Hz. In other modes, the pulse has a frequency of about 1 Hz. In still other modes, the pulse has a frequency of about 5 Hz, about 10 Hz, about 50 Hz, about 100 Hz, about 200 Hz, about 300 Hz, about 400 Hz, about 500 Hz, about 600 Hz, about 700 Hz, about 800 Hz, about 900 Hz, or about 1000 Hz.
[0128] In other modes, the pulse has a duration of about 1 millisecond (ms) to 1000 seconds (s) . In some modalities, the pulse has a duration of about 10 ms to 1000 s. In still other embodiments, the pulse has a duration of about 100 ms to 1000 s, about 1 s to 1000 s, about 1 s to 500 s, about 1 s to 250 s, or about 1 s to 150 s. In some modes, the pulse has a duration of about 1 ms, about 10 ms, about 100 ms, about 1 s, about 2 s, about 3 s, about 4 s, about 5 s, about 6 s, about 7 s, about 8 s, about 9 s, about 10 s, about 20 s, about 50 s, about 100 s, about 200 s, about 300 s, about 500s or about 1000s. In some modes, the pulse has a frequency of 0.2 to 200 Hz with duty cycles of 10 to 50%.
[0129] In some modes, direct current is applied once, or as multiple pulses. Any suitable number of pulses can be applied including about 1 to 20 pulses. There is any suitable period of time between pulses including about 1 millisecond to 1000 seconds. In some modes, the pulse duration is .01 to 10 seconds.
[0130] In some embodiments, cells are lysed using other methods in combination with a direct current applied to isolated cells. In still other modalities, cells are lysed without the use of direct current. In various aspects, devices and systems are capable of lysing cells with direct current in combination with other means, or they may be able to lyse cells without the use of direct current. Any cell lysis method known to those skilled in the art may be suitable including, but not limited to, application of a chemical lysing agent (eg an acid), an enzymatic lysing agent, heat, pressure, shear force, sonic energy, osmotic shock, or combinations of these. Lysozyme is an example of an enzymatic lysing agent. REMOVAL OF WASTE MATERIAL
[0131] In some embodiments, after concentrating the nucleic acids in the second DEP field region, the method optionally includes eliminating residual nucleic acid material. In some embodiments, the devices or systems described herein are optionally capable of and/or comprise a reservoir that comprises a fluid suitable for eliminating residual nucleic acid material. In some embodiments, the nucleic acid is held close to the electrode array, as in the second DEP field region, continuing to energize the electrodes. "Residual material" is anything originally present in the fluid, originally present in the cells, added during the procedure, created through any step in the process including, but not limited to, cell lysis (ie, residual cellular material), and the like. For example, waste material includes unlysed cells, cell wall fragments, proteins, lipids, carbohydrates, minerals, salts, buffers, plasma, and unwanted nucleic acids. In some embodiments, the lysed cell material comprises residual protein released from the plurality of cells upon lysis. It is possible that not all of the nucleic acid is concentrated in the second DEP field. In some embodiments, a certain amount of nucleic acid is purged with the waste material.
[0132] In some embodiments, waste material is purged in any suitable fluid, eg, in water, TBE buffer, or the like. In some embodiments, the waste material is purged with any suitable volume of fluid, purged for any suitable period of time, purged with more than one fluid, or any other variation. In some embodiments, the method of purging the waste material is related to the desired level of nucleic acid isolation, with the higher purity nucleic acid requiring more stringent purging and/or washing. In other embodiments, the method of purging waste material is related to the particular starting material and its composition. In some cases, a starting material that has a high lipid content requires a purging procedure that involves a suitable hydrophobic fluid to solubilize lipids.
[0133] In some embodiments, the method includes degrading waste material including waste protein. In some embodiments, devices or systems are capable of degrading waste material including waste protein. For example, proteins are degraded by one or more of chemical degradation (eg, acid hydrolysis) and enzymatic degradation. In some embodiments, the enzymatic degradation agent is a protease. In other embodiments, the protein degrading agent is Proteinase K. The optional waste material degradation step is performed at any suitable time, temperature, and the like. In some embodiments, degraded waste material (including degraded proteins) is purged from the nucleic acid.
[0134] In some embodiments, the agent used to degrade the waste material is inactivated or degraded. In some embodiments, the devices or systems are capable of degrading or inactivating the agent used to degrade the waste material. In some embodiments, an enzyme used to degrade the waste material is heat inactivated (eg, 50 to 95°C for 5 to 15 minutes). For example, enzymes that include proteases, (eg, Proteinase K) are degraded and/or inactivated using heat (typically 15 minutes, 70°C). In some embodiments where residual proteins are degraded by an enzyme, the method further comprises inactivating the degraded enzyme (e.g., Proteinase K) after protein degradation. In some embodiments, heat is provided by a heating module in the device (temperature range, for example, 30 to 95°C).
[0135] The order and/or combination of certain method steps may be varied. In some embodiments, devices or methods are capable of performing certain steps in any order or combination. For example, in some embodiments, waste material and degraded proteins are purged in separate or simultaneous steps. That is, the waste material is purged, followed by degradation of waste proteins, followed by purging of degraded proteins from the nucleic acid. In some embodiments, a material first degrades the waste proteins, and then purges the waste material and degraded proteins from the nucleic acid in a combined step.
[0136] In some embodiments, the nucleic acid is retained in the device and optionally used in additional procedures such as PCR or other procedures that manipulate or amplify the nucleic acid. In some embodiments, devices and systems are capable of performing PCR or other optional procedures. In other embodiments, nucleic acids are collected and/or eluted from the device. In some embodiments, the devices and systems are capable of allowing the collection and/or elution of nucleic acid from the device or system. In some embodiments, isolated nucleic acid is collected by (i) turning off the second dielectrophoretic field region; and (ii) eluting the nucleic acid from the array into an eluent. Exemplary eluents include water, TE, TBE and L-Histidine buffer. NUCLEIC ACIDS AND THEIR YIELD
[0137] In some embodiments, the method, device, or system described herein is optionally used to obtain, isolate, or separate any desired nucleic acid that can be obtained from such method, device, or system. Nucleic acids isolated by the methods, devices, and systems described herein include DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and combinations thereof. In some embodiments, the nucleic acid is isolated in a form suitable for sequencing or further manipulation of the nucleic acid, including amplification, ligation or cloning.
[0138] In various embodiments, an isolated or separated nucleic acid is a composition comprising nucleic acid that is free of at least 99% by mass of other materials, free of at least 99% by mass of residual cellular material (e.g., of lysed cells from which the nucleic acid is obtained), free of at least 98% by mass of other materials, free of at least 98% by mass of residual cellular material, free of at least 95% by mass of other materials , free from at least 95% by mass of residual cellular material, free from at least 90% by mass of other materials, free from at least 90% by mass of residual cellular material, free from at least 80% by mass of cellular material residual, free from at least 70% by mass of other materials, free from at least 70% by mass of residual cellular material, free from at least 60% by mass of other materials, free from at least 60% by mass of cellular material residual, free from at least 5 0% by mass of other materials, free from at least 50% by mass of residual cellular material, free from at least 30% by mass of other materials, free from at least 30% by mass of residual cellular material, free from at least 10% by mass of other materials, free from at least 10% by mass of residual cellular material, free from at least 5% by mass of other materials, or free from at least 5% by mass of residual cellular material.
[0139] In various embodiments, the nucleic acid has any suitable purity. For example, if a DNA sequencing procedure can work with nucleic acid samples that have about 20% residual cellular material, then isolation of the nucleic acid at 80% is adequate. In some embodiments, the isolated nucleic acid comprises less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30% , less than about 20%, less than about 10%, less than about 5%, or less than about 2% cellular material without nucleic acid and/or protein by mass. In some embodiments, the isolated nucleic acid comprises more than about 99%, more than about 98%, more than about 95%, more than about 90%, more than about 80%, more than about 70% , more than about 60%, more than about 50%, more than about 40%, more than about 30%, more than about 20%, or more than about 10% nucleic acid by mass.
[0140] Nucleic acids are isolated in any suitable form including unmodified, derivatized, fragmented, unfragmented, and the like. In some embodiments, nucleic acid is collected in a form suitable for sequencing. In some embodiments, nucleic acid is collected in a fragmented form suitable for shotgun sequencing, amplification, or other manipulation. Nucleic acid may be collected from the device in a solution comprising reagents used, for example, in a DNA sequencing procedure, such as nucleotides as used in sequencing by synthetic methods.
[0141] In some embodiments, the methods described herein result in an isolated nucleic acid sample that is approximately representative of the starting sample nucleic acid. In some embodiments, the devices and systems described herein are capable of isolating nucleic acid from a sample that is approximately representative of the nucleic acid from the starting sample. That is, the population of nucleic acids collected by the method, or capable of being collected by the device or system, is substantially proportional to the population of nucleic acids present in the cells in the fluid. In some embodiments, this aspect is advantageous in applications where the fluid is a complex mixture of many cell types and the practitioner desires a nucleic acid-based procedure to determine the relative populations of the various cell types.
[0142] In some embodiments, nucleic acid isolated using the methods described herein or capable of being isolated by the devices described herein is of high quality and/or suitable for use directly in downstream procedures such as DNA sequencing, nucleic acid amplification, such as PCR, or other nucleic acid manipulation, such as additional binding, cloning or translation or transformation tests. In some embodiments, the collected nucleic acid comprises at most 0.01% protein. In some embodiments, the collected nucleic acid comprises at most 0.5% protein. In some embodiments, the collected nucleic acid comprises at most 0.1% protein. In some embodiments, the collected nucleic acid comprises at most 1% protein. In some embodiments, the collected nucleic acid comprises at most 2% protein. In some embodiments, the collected nucleic acid comprises at most 3% protein. In some embodiments, the collected nucleic acid comprises a maximum of 4% protein. In some embodiments, the collected nucleic acid comprises at most 5% protein.
In some embodiments, nucleic acid isolated by the methods described herein or capable of being isolated by the devices described herein has a concentration of at least 0.5 ng/mL. In some embodiments, nucleic acid isolated by the methods described herein or capable of being isolated by the devices described herein has a concentration of at least 1 ng/ml. In some embodiments, nucleic acid isolated by the methods described herein or capable of being isolated by the devices described herein has a concentration of at least 5 ng/ml. In some embodiments, nucleic acid isolated by the methods described herein or capable of being isolated by the devices described herein has a concentration of at least 10 ng/ml.
[0144] In some embodiments, about 50 picograms of nucleic acid are isolated from about 5,000 cells using the methods, systems, or devices described herein. In some embodiments, the methods, systems, or devices described herein produce at least 10 picograms of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 20 picograms of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 50 picograms of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 75 picograms of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 100 picograms of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 200 picograms of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 300 picograms of nucleic acid from about 5,000 cells. In some embodiments, nucleic acid methods, systems, or picograms from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 500 picograms of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 1,000 picograms of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems, or devices described herein produce at least 10,000 picograms of nucleic acid from about 5,000 cells. TESTS AND APPLICATIONS
[0145] In some embodiments, the methods described herein further comprise optionally amplifying the isolated nucleic acid by polymerase chain reaction (PCR). In some embodiments, the PCR reaction is performed on or near the electrode array or device. In some embodiments, the device or system comprises a heater and/or temperature control mechanism suitable for thermocycling.
[0146] PCR is optionally performed using traditional thermocycling by placing the chemical reaction analytes between two efficient thermoconductive elements (eg aluminum or silver) and regulating the reaction temperatures using TECs. Additional designs optionally use infrared heating through optically transparent material such as glass or thermopolymers. In some cases, the designs use smart polymers or smart glass that comprise conductive wiring connected through the substrate. This conductive wiring allows for rapid thermal conductivity of materials and (by applying appropriate DC voltage) provides the temperature changes and gradients required to keep PCR reactions efficient. In some cases, heating is applied using chip resistive heaters and other resistive elements that will change temperature quickly and in proportion to the amount of current that passes through them.
[0147] In some modalities, used in conjunction with traditional fluorometry (ccd, pmt, other optical detector, and optical filters), the relative amplification is monitored in real time or at a timed interval. In some cases, quantification of final relative amplification is reported through optical detection converted to AFU (arbitrary fluorescence units correlated to analyze duplication) or translated to electrical signal through impedance measurement or other electrochemical detection.
[0148] Given the small size of the microelectrode array, these elements are optionally added on top of the microelectrode array and the PCR reaction will be carried out in the main sample processing chamber (over the DEP array) or the analytes that will be amplified are optionally transported through fluid mechanics to another chamber within the fluidic cartridge to allow for Lab-On-Chip in-cartridge processing
[0149] In some cases, light distribution schemes are used to provide optical excitation and/or emission and/or relative amplification detection. In some embodiments, this includes using flow cell materials (thermal polymers such as cyclic olefin polymer (COP), acrylic (PMMA), cyclic olefin copolymer, (COC), etc.) as optical waveguides to eliminate the need for use external components. Furthermore, in some cases, light sources - light emitting diodes - LEDs, vertical cavity surface emitting lasers - VCSELs, and other lighting schemes are either integrated directly into the flow cell or incorporated directly into the surface of the microelectrode array for have internally controlled and powered light sources. Miniature PMTs, CCDs, or CMOS detectors can also be incorporated into the flow cell. This minimization and miniaturization allows for compact devices capable of rapid signal distribution and detection while reducing the footprint of similar traditional devices (ie, a standard PCR/QPCR/Fluorimeter bench). AMPLIFICATION IN CHIP
[0150] In some cases, silicon microelectrode arrays can support the thermal cycling required for PCR. In some applications, on-chip PCR is advantageous as small amounts of target nucleic acids can be lost during the transfer steps. In some embodiments of devices, systems or processes described herein, any one or more of multiple PCR techniques is optionally used, such techniques optionally including any one or more of the following: thermal cycling directly in the flow cell; move material through microchannels with different temperature zones; and moving the volume into a PCR tube that can be amplified in the system or transferred to a PCR machine. In some cases, droplet PCR is performed if the output contains a T junction that contains an immiscible fluid and interfacial stabilizers (surfactants, etc). In some embodiments, droplets are thermally cycled by any suitable method.
[0151] In some embodiments, amplification is performed using an isothermal reaction, for example, transcription-mediated amplification, nucleic acid sequence-based amplification, RNA technology signal-mediated amplification, strand displacement amplification, circle amplification rolling, isothermal DNA circular amplification, isothermal multiple displacement amplification, helicase-dependent amplification, isothermal amplification using a single primer, or helicase-dependent circular amplification.
[0152] In various embodiments, amplification is performed in homogeneous solution or as a heterogeneous system with anchored primer(s). In some modalities of the latter, the resulting amplicons are directly attached to the surface for a greater degree of multiplexing. In some embodiments, the amplicon is denatured to provide single-chain products at or near the electrodes. Hybridization reactions are then optionally performed to interrogate genetic information such as single nucleotide polymorphisms (SNPs), Short Tandem Repeats (STRs), mutations, insertions/deletions, methylation, etc. Methylation is optionally determined by parallel analysis where one DNA sample is treated with bisulfite and another not. Unmodified C bisulphite depurines have become U. C methylated is unaffected in some cases. In some modalities, the allele-specific base extension is used to report the base of interest.
[0153] Instead of specific interactions, the surface is optionally modified with non-specific holds for capture. For example, surfaces could be modified with polycations, i.e., polylysine, to capture DNA molecules that can be released by reverse polarization (—V) . In some embodiments, surface modifications are uniform over the surface or specifically shaped to functionalize electrodes or electrodeless regions. In some modalities, this is accomplished with photolithography, electrochemical activation, marking, and the like.
[0154] In some applications, where multiple chip designs are employed, it is advantageous to have a chip sandwich where the two devices are facing, separated by a spacer, to form the flow cell. In various embodiments, devices are operated sequentially or in parallel. For next-generation sequencing and sequencing (NGS), fragmentation and size selection have ramifications for sequencing efficiency and quality. In some embodiments, multi-chip designs are used to reduce the size range of collected material by creating a band-pass filter. In some cases, current chip geometry (eg, 80 um diameter electrodes in a 200 um (80/200) center-to-center pitch acts as a 500 bp cut-off filter (eg using voltage conditions and frequency around 10 Vpp and 10 kHz). In these cases, a nucleic acid of more than 500 bp is captured, and a nucleic acid of less than 500 bp is not. Alternative electrode diameter and pitch geometries have different cut sizes so that a combination of chips provides a desired fragment size. In some cases, a 40 µm diameter electrode at a 100 µm center-to-center pitch (40/100) has a lower cut-off limit, while a 40 µm diameter electrode. 160 µm diameter in a 400 µm center-to-center pitch (160/400) has a higher cutting edge than the 80/200 geometry under similar conditions. are combined to select fragments or particles of special size. specific. For example a 600 bp cut-off chip could leave less than 600 bp nucleic acid in solution, so this material is optionally recaptured with a 500 bp cut-off chip (which is opposite to the 600 bp chip). This leaves a population of nucleic acid comprising 500 to 600 bp in solution. That population is then optionally amplified in the same chamber, a side chamber, or any other configuration. In some embodiments, size selection is performed using a single electrode geometry, where >500 bp nucleic acid is isolated on the electrodes, followed by washing, followed by ACEK high field resistance reduction (voltage change, frequency , conductivity) to release nucleic acids of <600 bp, resulting in a supernatant nucleic acid population between 500 to 600 bp.
[0155] In some embodiments, the chip device is oriented vertically with a heater on the lower edge that creates a column of temperature gradient. In some cases, the lower part is at the denaturation temperature, the middle part at the quench temperature, the upper part at the extension temperature. In some cases, convection continuously activates the process. In some embodiments, methods or systems are provided here that comprise an electrode design that specifically provides electrothermal fluxes and process acceleration. In some embodiments, this design is optionally on the same device or on a separate device properly positioned. In some cases, active or passive cooling at the top, through fins or fans, or the like, provides a high temperature gradient. In some cases, the device or system described here comprises, or a method described here uses, temperature sensors in the device or reaction chamber to monitor temperature and these sensors are optionally used to adjust the temperature on a power base. In some cases, these sensors are coupled to materials that have different heat transfer properties to create continuous and/or discontinuous gradient profiles.
[0156] In some embodiments, the amplification proceeds at a constant temperature (ie, isothermal amplification).
[0157] In some embodiments, the methods described herein further comprise sequencing the isolated nucleic acid as described herein. In some embodiments, nucleic acid is sequenced by Sanger sequencing or next-generation sequencing (NGS). In some embodiments, next-generation sequencing methods include, but are not limited to, pyrosequencing, ionic semiconductor sequencing, polony sequencing, bond sequencing, DNA nanosphere sequencing, bond sequencing, or single molecule sequencing.
[0158] In some embodiments, the isolated nucleic acids described herein are used in Sanger sequencing. In some embodiments, Sanger sequencing is performed within the same device as nucleic acid isolation (Lab-on-Chip). The Lab-on-Chip workflow of sample preparation and Sanger sequencing results could incorporate the following steps: a) sample extraction using ACE chips; b) carry out the amplification of target sequences on the chip; c) capture PCR products by ACE; d) performing cycle sequencing to enrich the target strand; e) capture the enriched target chains; f) carry out the Sanger chain termination reactions; perform the electrophoretic separation of target sequences by capillary electrophoresis with on-chip multicolor fluorescence detection. Nucleic acid washing, reagent addition, and voltage shutdown are performed as needed. Reactions can be carried out on a single chip with a plurality of capture zones or on separate chips and/or reaction chambers.
[0159] In some embodiments, the method described here further comprises performing a reaction on the nucleic acids (for example, fragmentation, restriction digestion, DNA or RNA ligation). In some embodiments, the reaction takes place in or near the array or in a device, as described here. OTHER TESTS
[0160] The isolated nucleic acids described herein can further be used in a variety of assay formats. For example, devices that are designed with nucleic acid probes or amplicons can be used in dot blot or reverse dot blot analysis, base stacking single nucleotide polymorphism (SNP) analysis, SNP analysis with electron stringency, or in analysis STR. In addition, those devices described herein can be used in formats for enzymatic nucleic acid modification, or protein-nucleic acid interaction, such as, for example, gene expression analysis with enzymatic reporting, anchored nucleic acid amplification, or other nucleic acid modifications. suitable for solid phase formats that include restriction endonuclease cleavage, endoor exonuclease cleavage, minor groove binding protein assays, terminal transferase reactions, polynucleotide kinase or phosphatase reactions, ligase reactions, topoisomerase reactions, and other nucleic acid binding or modification protein reactions.
[0161] Furthermore, the devices described here may be useful in immunoassays. For example, in some modalities, device locations can be associated with antigens (eg, peptides, proteins, carbohydrates, lipids, proteoglycans, glycoproteins, etc.) to analyze antibodies in a body fluid sample by sandwich assay, competitive essay, or other formats. Alternatively, device locations can be engineered with antibodies to detect antigens in a sample by sandwich assay, competitive assay, or other assay formats. Since the isoelectric point of antibodies and proteins can be easily determined by experimentation or pH/charge computations, the electronic addressing and electronic concentration advantages of the devices can be utilized simply by adjusting the pH of the buffer so that the targeted species or analyte are ordered.
[0162] In some embodiments, isolated nucleic acids are useful for use in immunoassay-type arrays or nucleic acid arrays. EXEMPLARY COMPARISON
[0163] Approximately 100 ng of input E. coli genome is required for conventional manual methods, (eg 50 ng of input DNA is required for Nextera, assuming 50% recovery (Epicentre WaterMaster kit claims recovery of about approx. 30 to 60%) purification by DNA extraction). This is equivalent to about 20 million bacteria. In some embodiments of the present invention, an input of less than 10,000 bacteria is sufficient (for example, since the chip is self-contained and involves fewer transfers, the efficiency is greater). In some modalities this equates to at least a 100-fold reduction in input, this can be important for applications where sample size is limited, such as tumor biopsies.
[0164] Table 1 below describes exemplary steps involved from E. coli to DNA suitable for sequencing. In some cases, conventional methods require centrifugation, multiple temperatures, washing steps, and numerous transfer steps that are inefficient. In contrast, as described herein, in some embodiments the same steps are allowed to be performed by a device that reduces pipette transfers and exposure to large virgin surfaces with varying degrees of non-specific binding properties. In some cases, the device is temperature controlled to provide proper reaction conditions. In some cases, PCR, sequencing preamp cycle-to-cycle PCR, or full PCR (evaluation criteria, real-time or digital) is performed off-device or on-device. The term outside the device includes not within the device, but within the same cartridge assembly, connections through fluid channels or conduits. Furthermore, in some cases, PCR amplification is performed in the device's flow cell chamber, in a PCR tube that is in the cartridge, or through fluid channels that have heat zones for temperature cycling. In some cases, the device chamber eluate is combined with the side channel(s) prepared with non-aqueous miscible fluid, eg, oil, and other droplet stabilizers to perform droplet amplification. In some embodiments, temperature cycling mechanics are as described above.
[0165] In Table 1, the amount of starting material for conventional processing is 2-5 x 107 E. Colí cells in approximately 1 ml of water and the total amount was concentrated on the filters. Using the chip, as described here, only 1 x 104 E.Coli cells in approximately 50 microliters were applied to the flow cell. That's 3 orders of magnitude smaller than the starting material. TABLE 1: COMPARISON OF METHODS FOR NUCLEIC ACID ISOLATION.


[0166] In various embodiments (ie, depending on AC electrokinetic parameters), cells or other micron-scale particles are concentrated in the low- or high-field regions. In some cases, the crossover frequency that determines whether a particle moves into or out of the high-field region can be tuned by varying the AC frequency, voltage, media conductivity, tampered particle polarizability (such as clamping or binding of materials with different DEP characteristics), or electrode geometry. In some cases, nanoscale particles are limited to concentration in the high-field region. In some cases, Brownian motion and other hydrodynamic forces limit the ability to concentrate in low-field regions. DEFINITIONS AND ABBREVIATIONS
[0167] The articles "a", "an" and "o" are non-limiting. For example, "the method" includes the broadest definition of the meaning of the phrase, which can be more than one method.
[0168] "Vp-p" is the peak-to-peak voltage.
[0169] "TBE" is a buffer solution containing a mixture of Tris base, boric acid and EDTA.
[0170] “TE” is a buffer solution that contains a mixture of Tris base and EDTA.
[0171] "L-Histidine Buffer" is a solution that contains L-histidine.
[0172] "DEP" is an abbreviation for dielectrophoresis. EXAMPLES EXAMPLE 1: HYDROGEL FORMATION BY ROTATIONAL COATING (TWO COATINGS)
[0173] For a hydrogel layer, approximately 70 microliters of hydrogel is used to coat a 10 x 12 mm chip.
[0174] A low concentration (^1% solids by volume) of cellulose acetate solution is dissolved in a solvent such as acetone, or a mixture of acetone and ethanol and applied to an electrode array chip as described here. The chip is rotated at a low rpm rate (1000-3000) . The low rpm rate ensures that the gel height is in the range of 500nm or more.
[0175] The first (bottom) coating of cellulose acetate is dried at room temperature, in a convection oven, or a vacuum oven. Optionally, the second spin-coated cellulose acetate layer is immediately added.
[0176] The second layer of cellulose acetate comprises a high concentration (12%) of cellulose acetate dissolved in a solvent such as acetone, or a mixture of acetone and ethanol. After a second layer of cellulose acetate is added, the chip is rotated at a high rpm rate (9000-12000). The high rpm rate will ensure that the gel height is in the range of 300nm or less.
[0177] The chip with two layers of cellulose acetate is then dried at room temperature, in a convection oven, or in a vacuum oven. EXAMPLE 2: FORMATION OF HYDROGEL WITH ADDITIVES BY ROTATIONAL COATING (TWO COATINGS)
[0178] For a hydrogel layer, approximately 70 microliters of hydrogel is used to coat a 10 x 12 mm chip.
[0179] A low concentration (^1% solids by volume) of cellulose acetate solution is dissolved in a solvent such as acetone, or a mixture of acetone and ethanol and applied to an electrode array chip as described here. The chip is rotated at a low rpm rate (1000-3000) . The low rpm rate ensures that the gel height is in the range of 500 nm or more.
[0180] The first (bottom) coating of cellulose acetate is dried at room temperature, in a convection oven, or a vacuum oven. Optionally, the second spin-coated cellulose acetate layer is immediately added.
[0181] The second layer of cellulose acetate comprises a high concentration (12%) of cellulose acetate dissolved in a solvent such as acetone, or a mixture of acetone and ethanol. A low concentration (1-15%) of conductive polymer (PEDOT:PSS or similar) is added to the second cellulose acetate solution. After a second layer of cellulose acetate is added, the chip is rotated at a high rpm rate (9000 to 12000). The high rpm rate will ensure that the gel height is in the range of 300 nm or less.
[0182] The chip with two layers of cellulose acetate is then dried at room temperature, in a convection oven, or in a vacuum oven. EXAMPLE 3: HYDROGEL FORMATION WITH ADDITIVES BY ROTATIONAL COATING (THREE COATINGS)
[0183] For a hydrogel layer, approximately 70 microliters of hydrogel are used to coat a 10 x 12 mm chip.
[0184] A low concentration (^1% solids by volume) of cellulose acetate solution is dissolved in a solvent such as acetone, or a mixture of acetone and ethanol and applied to an electrode array chip as described here. The chip is rotated at a high rpm rate (90012000). The low rpm rate ensures that the gel height is in the range of 300 nm or less.
[0185] The first (bottom) coating of cellulose acetate is dried at room temperature, in a convection oven, or a vacuum oven. Optionally, the second spin-coated cellulose acetate layer is immediately added.
[0186] The second layer of cellulose acetate comprises a high concentration (^2%) of cellulose acetate dissolved in a solvent such as acetone, or a mixture of acetone and ethanol. A low concentration (1-15%) of conductive polymer (PEDOT:PSS or similar) is added to the second cellulose acetate solution. After a second layer of cellulose acetate is added, the chip is rotated at a low rpm rate (1000 to 3000). The low rpm rate will ensure the gel height is in the range of 500 nm or more.
[0187] The second cellulose acetate coating is dried at room temperature, in a convection oven, or a vacuum oven. Optionally, the third spin-coated cellulose acetate layer is immediately added.
[0188] The third layer of cellulose acetate comprises a high concentration (>2%) of cellulose acetate dissolved in a solvent such as Acetone, or a mixture of Acetone and Ethanol. The chip is rotated at a high rpm rate (9000 to 12000) . The low rpm rate ensures that the gel height is in the range of 300 nm or less.
[0189] The chip with three layers of cellulose acetate is then dried at room temperature, in a convection oven, or a vacuum oven. EXAMPLE 4: CHIP CONSTRUCTION
[0190] In Figures 2 and 3: An array of 45x20 circular platinum microelectrodes 80μm in diameter in a 200 µm center-to-center pitch was fabricated based on the previous results (see references 1 to 3, below). All 900 microelectrodes are activated together and polarized by AC to form a gridded field geometry. The positive DEP regions occur directly on the microelectrodes, and the low-field negative regions occur between the microelectrodes. The array is coated with a 200nm to 500nm thick porous poly-Hema hydrogel layer (Procedure: 12% pHema in ethanol stock, purchased from PolySciences Inc., which is diluted to 5% using ethanol. 70uL of ethanol). 5% solution is spun on the above chip at a rotating speed of 6K RPM using a rotational coater. The chip+hydrogel layer is then placed in an oven at 60°C for 45 minutes) and confined in a microfluidic cartridge , forming a 50μL sample chamber covered with an acrylic window (Figure 1). Electrical connections to microelectrodes are accessed from Molex connectors on the PCB board in the flow cell. A function generator (HP 3245A) provides sinusoidal electrical signal at 10KHz and peak-to-peak 10 to 14V, depending on the conductivity of the solution. Images were captured with a fluorescent microscope (Leica) and an EGFP cube (band-pass filters of 485 nm emission and 525 nm excitation). The excitation source is a PhotoFluor II 200W Hg arc lamp.
[0191] [1] R. Krishnan, B.D. Sullivan, R.L. Mifflin, S.C. Esener, and M.J. Heller, "Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions". Electrophoresis, vol. 29, pages 1765-1774, 2008.
[0192] [2] R. Krishnan and M.J. Heller, “An AC electrokinetic method for enhanced detection of DNA nanoparticles”. J. Biophotonics, vol. 2, pages 253-261, 2009.
[0193] [3] R. Krishnan, D.A. Dehlinger, G.J. Gemmen, R.L. Mifflin, S.C. Esener, and M.J. Heller, "Interaction of nanoparticles at the DEP microelectrode interface under high conductance conditions" Electrochem. Comm., vol. 11, pages 1661-1666, 2009. EXAMPLE 5: ISOLATION OF HUMAN GENOMIC DNA
[0194] Human Genomic DNA (gDNA) was purchased from Promega (Promega, Madison, WI) and was sized for 20 to 40kbp. (Sizing gel not shown) . gDNA was diluted in DI water at the following concentrations: 50 nanograms, 5 nanograms, 1 nanogram, and 50 picograms. The gDNA was stained using the 1x SYBR Green I fluorescent green double-stranded DNA dye purchased from Invitrogen (Life Technologies, Carlsbad, CA). This mixture was then inserted into the microelectrode arrays and operated at 14 Volts peak-to-peak (Vp-p), in a 10kHz sinusoidal wave for 1 minute. At the end of 1 minute, a photo of the microelectrode pads was taken using a CCD camera with a 10x objective under a microscope using green fluorescence filters (FITC) so that the gDNA could be visualized (Figure 2) The chip was able to identify up to 50pg of gDNA in 50μL of water, ie, 1ng/mL concentration. Additionally, at 50 picograms, each microelectrode averaged ~60 fentograms of DNA since there are 900 microelectrodes in the array. The low-level concentrating capability of the ACE device is within the range of 1-10ng/mL required to identify Cfc-DNA biomarkers in plasma and serum (see references 4 to 6 below).
[0195] [4] T.L. Wu et al, "Cell-free DNA: measurement in various carcinomas and establishment of normal reference range." Clin Chim Acta., vol. 21, pages 77-87, 2002.
[0196] [5] R.E. Board et al, "DNA Methylation in Circulating Tumor DNA as a Biomarker for Cancer", Biomarker Insights, vol. 2, pages 307-319, 2007.
[0197] [6] O. Gautschi et al, "Circulating deoxyribonucleic acid as a prognostic marker in non-small cell lung cancer patients for chemotherapy." J Clin Oncol., vol. 22, pages 4157-4164, 2004. EXAMPLE 6: ISOLATION OF DNA FROM E.COLI
[0198] Using the Chip and the methods described in Examples 4 and 5, approximately 5000 green fluorescent E. coli cells in 50uL of fluid were inserted into a chip and operated using the protocol described in the title of Figure 3. The panel (A) shows a clear field view. Panel (B) shows a fluorescent green view of the electrodes prior to DEP activation. Panel (C) shows E. coli on electrodes after one minute at 10 kHz, 20 Vp-p in 1xTBE buffer. Panel (D) shows E. coli on electrodes after one minute at 1 MHz, 20 Vp-p in 1xTBE buffer.
[0199] The E. coli shown in Figure 3 was lysed using a 100-millisecond 100V DC pulse that uses the HP 3245A function generator. Lysed particulates were then collected on the electrode surface using 10kHz, 10Vp-p and the Illumina Nextera Protocol was used to prepare libraries for sequencing while the DNA is on the chip (by inserting the appropriate buffers at the appropriate times on the chip) to mark the DNA for Sequencing. The DNA was then eluted in 50uL of 1x TBE buffer and then amplified by PCR for 9 to 12 cycles (using the Nextera Protocol) in a Bio-Rad PCR machine. The amplified DNA was then subjected to an Illumina GA II Sequencer. E. Coli DNA was also isolated from 1x TBE buffer (10 million cells) using the Epicentre™ WaterMaster™ DNA purification procedure, to serve as a gold standard for comparison. The results are shown in Figure 4. EXAMPLE 7: HYDROGEL FORMATION WITH GVD
[0200] Hydrogel such as polyhydroxyethylmethacrylate (pHEMA) can also be layered onto the chip surface via vapor deposition using proprietary assays developed by GVD Corporation (Cambridge, MA) (see, www.gvdcorp.com). Hydrogels like pHEMA were deposited in various thicknesses (100, 200, 300, 400nm) and the crosslink density (5, 25, 40%) on electrode chips was performed using technology developed by GVD Corporation. The hydrogel films were tested using a standard ACE protocol (no pretreatment, 7Vp-p, 10KHz, 2 minutes, 0.5XPBS, 500ng/ml gDNA labeled with Sybr Green 1). Fluorescence on the electrodes was captured by imaging. Figure 10 shows that 100nm thick, 5% gel crosslinking device was identified as strong DNA capture. The process could also be optimized by altering the rate of deposition or growth of anchorage to the surface of the microelectrode array (ie, to the passivation layer and exposed electrodes) using an adhesion promoter such as a silane derivative. EXAMPLE 8: DESCRIBED DEVICE AND METHOD PERFORMANCE V. CONVENTIONAL METHOD
[0201] The QIAGEN® Circulating Nucleic Acid Purification Kit (cat#55114) was used to purify 1 ml of plasma from chronic lymphocytic leukemia (CLL) patients according to the manufacturer's protocol. Briefly, incubation of 1 ml of plasma with Proteinase K solution was carried out for 30 minutes at 60°C. The reaction was cooled on ice and the entire volume was applied to a QIAamp Mini column connected to a vacuum. The liquid was drawn through the column and washed with 3 different buffers (600 to 750 µl each). The column was centrifuged at 20,000 x g, 3 minutes and baked at 56°C for 10 minutes to remove excess liquid. The sample was eluted in 55 μl of elution buffer with 20,000 x g, 1 minute of centrifugation. Total processing time is ~2.5 hours.
[0202] The chip area is 10 x 12 mm, with Pt electrodes 60 to 80 µm in diameter in a center-to-center pitch of 180 to 200 µm, respectively. The array was coated with a 5% pHEMA hydrogel layer (deposited by 6000 rpm rotation of Ethanol solution, 12% pHEMA stock from Polysciences). The chip was pretreated using 0.5xPBS, 2V rms, 5Hz, 15 seconds. The buffer was removed and 25 µl of CLL patient plasma was added. DNA was isolated for 3 minutes at 11 V pp, 10Khz, then washed with 500 µl TE buffer at a flow rate of 100 µl/min, with power ON. The voltage was turned off and the flow cell volume was eluted in a microcentrifuge tube. Total processing time is ~10 minutes.
[0203] The same process can be applied to fresh whole blood without modification. The ability to extract and purify DNA from undiluted whole blood is uniquely enabled by the chip technology described here.
[0204] DNA quantification was performed on Qiagen eluates and chip using PicoGreen according to the manufacturer's protocol (Life Tech) (Table 2).
[0205] Subsequent gel electrophoresis, PCR and Sanger sequencing reactions showed performance similar to chip extraction techniques being able to process whole blood as well as plasma. A non-parametric Mann-Whitney U statistical test was also performed between the amounts of DNA isolated from plasma using Qiagen and chip techniques. There is no statistical difference (p<0.05 bilateral) using the DNA purification method. TABLE 2: DNA PURIFICATION, CHIP V. QIAGEN Values are in ng/ml and normalized to the original plasma sample volume for comparison purposes.

[0206] Although the preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Numerous variations, alterations, and substitutions will now occur for those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed to practice the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are covered.

权利要求:
Claims (24)
[0001]
1. Method for isolating a nucleic acid from a fluid comprising cells characterized in that the method comprises: a. applying the fluid to a device, the device comprising an array of electrodes capable of generating an AC electrokinetic field region with an alternating current; B. control the temperature of the device with a thermal element; ç. concentrating a plurality of cells or other particulate material in the fluid in a first AC electrokinetic field region, wherein the first AC electrokinetic region is a low-field dielectrophoretic region; d. isolating the nucleic acid in a second AC electrokinetic field region, wherein the second AC electrokinetic field is a second region of high dielectrophoretic field; and is. stain the concentrated cells and/or other particulate material from the first AC electrokinetic field region, where the fluid has a conductivity of at least 100 mS/m.
[0002]
2. Method according to claim 1, characterized in that the thermal element comprises tantalum, aluminum, tungsten or a combination thereof.
[0003]
3. Method according to claim 1 or claim 2, characterized in that the method further comprises the degradation of a residual protein and the release of degraded matrix proteins and isolated nucleic acid.
[0004]
4. Method according to any of the preceding claims, characterized in that the nanoparticulate comprises high molecular weight nucleic acids, high molecular weight DNA, smaller DNA, aggregated proteins, cell debris, RNA, viruses or combinations thereof.
[0005]
5. Method according to any of the preceding claims, characterized in that the detection of nanoparticulate provides useful information for the diagnosis of cancer, cancer prognosis or response to treatment in a patient.
[0006]
6. Method according to claim 5, characterized in that the detection of nanoparticulates is part of medical therapy or diagnostic procedure.
[0007]
7. Method according to any one of the preceding claims, characterized in that it further comprises the collection of nucleic acid by (i) turning off the second region of the AC electrokinetic field; and (ii) eluting the nanoparticulate nucleic acid from the matrix into an eluent.
[0008]
8. Method according to any one of the preceding claims, characterized in that the fluid comprises a bodily fluid, whole blood, serum, plasma, cerebrospinal fluid, body tissue, urine, saliva, a food, a drink, a growth medium, an environmental sample, a liquid, water, clonal cells, or a combination thereof.
[0009]
9. Method according to any of the preceding claims, characterized in that the sample is blood.
[0010]
10. Method according to any one of the preceding claims, characterized in that the first AC electrokinetic field region is produced using an alternating current that has a peak-to-peak voltage of 1 volt to 40 volts; and/or a frequency from 5 Hz to 5,000,000 Hz, and duty cycles from 5% to 50%.
[0011]
11. Method according to any of the preceding claims, characterized in that the second AC electrokinetic field region is a different region of the electrode array as the first AC electrokinetic field region.
[0012]
12. Method according to any one of the preceding claims, characterized in that the second AC electrokinetic field region is the same region of the electrode array as the first AC electrokinetic field region.
[0013]
13. Method according to any of the preceding claims, characterized in that the electrodes are selectively energized to provide the first region of AC electrokinetic field and subsequently or continuously selectively energized to provide the second region of AC electrokinetic field.
[0014]
14. Method according to any of the preceding claims, characterized in that the cells are lysed in the device using a direct current, a chemical lysing agent, an enzymatic lysing agent, heat, osmotic pressure, sonic energy or a combination of them.
[0015]
15. Method according to any one of the preceding claims, characterized in that the cells are lysed by applying a direct current to the cells, in which the direct current used to lyse the cells has a voltage of 1-500 volts and a duration of 0.1 to 10 seconds applied once or as multiple pulses, where the pulse has a frequency of 0.2 to 200 Hz with 10-50% duty cycles.
[0016]
16. Method according to any one of the preceding claims, characterized in that the fluid conductivity is 500 mS/m or greater.
[0017]
17. Method according to any one of the preceding claims, characterized in that the electrode array is coated by rotation with a hydrogel having a thickness between about 0.1 micron and 1 micron.
[0018]
18. Method according to claim 17, characterized in that the hydrogel comprises two or more layers of synthetic polymer.
[0019]
19. Method according to claim 17, characterized in that the hydrogel has a viscosity between about 0.5 cP to about 5 cP before rotational coating.
[0020]
20. Method according to claim 17, characterized in that the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m.
[0021]
21. Method according to any one of the preceding claims, characterized in that the electrode array is in a configuration of points and the orientation angle between the points is from about 25° to about 60°.
[0022]
22. Method according to any one of the preceding claims, characterized in that the electrode array is in a wavy or non-linear line configuration, wherein the configuration comprises a repeating unit comprising the shape of a pair of points connected by a ligand, where the points and the ligand define the boundaries of the electrode, where the ligand tapers inwards towards the midpoint between the pair of points, where the diameters of the points are the largest points along of repeat unit length, where the edge-to-edge distance between a parallel set of repeat units is equidistant, or nearly equidistant.
[0023]
23. Method according to any one of the preceding claims, characterized in that the electrode array comprises a passivation layer with a relative electrical permittivity of about 2.0 to about 4.0.
[0024]
24. Method according to any one of the preceding claims, characterized in that the method further comprises the amplification of isolated nucleic acid by polymerase chain reaction.

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-08-11| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-05-25| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-03| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 16/04/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261624897P| true| 2012-04-16|2012-04-16|

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US61/624,897|2012-04-16|

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PCT/US2013/036845|WO2013158686A1|2012-04-16|2013-04-16|Nucleic acid sample preparation|

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