Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

• the larger nanoparticulate molecular target is chosen from the group consisting of exosomes, high mw nucleic acids, including high mw DNA, oligo- nucleosome complexes, aggregated proteins, vesicle bound DNA, cell membrane fragments and cellular debris.
• the target circulating cell-free biomarker is chosen from the group consisting of mutations, deletions, rearrangements or methylated nucleic acid of circulating DNA, micro RNA, RNA from microvesicles or a combination thereof.
• the detection of the cell-free biomarker provides information useful for cancer diagnosis, cancer prognosis or treatment response in a patient.
• the tumor cell-free biomarker is associated with CNS tumors, neuroblastoma, gliomas, breast cancer, endometrial tumors, cervical tumors, ovarian tumors, hepatocellular carcinoma, pancreatic carcinoma, esophageal tumors, Stoch tumors, colorectal tumors, head and neck tumors, nasopharyngeal carcinoma, thyroid tumors, lymphoma, leukemia, lung cancer, non- small cell lung carcinoma, small cell lung carcinoma, testicular tumors, kidney tumors, prostate carcinoma, skin cancer, malignant melanoma, squamous cell carcinoma or a combination thereof.
• the tumor cell-free biomarker is GFAP, VEGF, EGFR, b-FGF, KRAS, YKL-40, MMP-9 or combinations thereof.
• the first AC electrokinetic high field capable of isolating larger nanoparticulate molecular targets
• a method for isolating a nucleic acid from a fluid comprising cells comprising: a. applying the fluid to a device, the device comprising an array of electrodes capable of establishing an AC electrokinetic field region; b. concentrating a plurality of cells in a first AC electrokinetic field region, wherein the first AC electrokinetic field region is a first dielectrophoretic low field region and the conductivity of the fluid is greater than 300 mS/m; c. isolating nucleic acid in a second AC electrokinetic field region, wherein the second AC electrokinetic field is a second eletrophoretic high field region; and d. flushing cells away from the array.
• the cells are lysed by applying a direct current to the cells.
• the direct current used to lyse the cells has a voltage of 1-500 volts; and a duration of .01 to 10 seconds applied once or as multiple pulses.
• the direct current used to lyse the cells is a direct current pulse or a plurality of direct current pulses applied at a frequency suitable for lysing the cells. In some embodiments, the pulse has a frequency of 0.2 to 200 Hz with duty cycles from 10-50%.
• the isolated nucleic acid comprises greater than about 99%, greater than about 98%, greater than about 95%, greater than about 90%, greater than about 80%), greater than about 70%, greater than about 60%, greater than about 50%, greater than about 40%), greater than about 30%, greater than about 20%), or greater than about 10% nucleic acid by mass.
• the method is completed in less than about one hour. In some embodiments, centrifugation is not used.
• the residual proteins are degraded by one or more of chemical degradation and enzymatic degradation. In some embodiments, the residual proteins are degraded by Proteinase K. In some embodiments, the residual proteins are degraded by an enzyme, the method further comprising inactivating the enzyme following degradation of the proteins.
• the second AC electrokinetic field region is produced using an alternating current having a voltage of 1 volt to 50 volts peak-peak; and/or a frequency of 5 Hz to 5,000,000 Hz, and duty cycles from 5% to 50%.
• the electrodes are selectively energized to provide the first AC electrokinetic field region and subsequently or continuously selectively energized to provide the second AC electrokinetic field region.
• the array of electrodes is coated with a hydrogel.
• the hydrogel comprises two or more layers of a synthetic polymer.
• the hydrogel is spin-coated onto the electrodes.
• 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 perform amplification. In some embodiments, amplification of the nucleic acid is polymerase chain reaction (PCR)-based. In some embodiments, amplification of the nucleic acid is performed in a serpentine microchannel comprising a plurality of temperature zones.
• PCR polymerase chain reaction
• the hydrogel has a thickness between about 0.1 microns and 1 micron. In some embodiments, the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m. In some embodiments, the array of electrodes comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0.
• the surface selectively captures biomolecules by: a.nucleic acid hybridization; b. antibody - antigen interactions; c. biotin - avidin interactions; d. ionic or electrostatic interactions; or e. any combination thereof. In some embodiments, the surface is functionalized to minimize and/or inhibit nonspecific binding interactions by: a. polymers (e.g., polyethylene glycol PEG); b. ionic or electrostatic
• a device comprising: a. a plurality of alternating current (AC) electrodes, the AC electrodes configured to be selectively energized to establish AC electrokmetic high field and AC electrokmetic low field regions, wherein the array of electrodes is in a wavy or nonlinear line configuration, wherein the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a linker, wherein the dots and linker define the boundaries of the electrode, wherein the linker tapers inward towards or at the midpoint between the pair of dots, wherein the diameters of the dots are the widest points along the length of the repeating unit, wherein the edge to edge distance between a parallel set of repeating units is equidistant, or roughly equidistant; and b.
• AC alternating current
• the hydrogel has a thickness between about 0.1 microns and 1 micron. In some embodiments, the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m. In some embodiments, the array of electrodes comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0.
• the surface selectively captures biomolecules by: a. nucleic acid hybridization; b. antibody - antigen interactions; c. biotin - avidin interactions; d. ionic or electrostatic interactions; or e. any combination thereof. In some embodiments, the surface is functionalized to minimize and/or inhibit nonspecific binding interactions by: a. polymers (e.g., polyethylene glycol PEG); b. ionic or electrostatic
• FIG. 8 exemplifies a wavy electrode configuration, as disclosed herein.
• the edge to edge distance between electrodes is generally equidistant throughout.
• a wavy electrode configuration maximizes electrode surface area while maintaining alternating non-uniform electric field to induce ACE gradient to enable DEP, ACEO, ACET, and other ACE forces.
• FIG. 9 exemplifies how the E-field gradient at a dielectric layer corner based on silicon nitride thickness. Lower K and lower thickness resulted in higher E-field gradient (bending) at a dielectric layer corner.
• FIG. 11 shows a picture of a two-chamber fluidic cartridge showing the layout for the unknown (U) and known (K) chambers.
• FIG. 12 shows a fluorescent image of YOYO ® - 1 labelled circulating cell-free DNA captured on electrodes.
• Region-of-Interest (ROI) segmentation enables rapid processing conversion from image to quantitative score.
• FIG. 14 shows the compatibility of the ACE fluidic wash solution with the downstream PCR mutation detection assays.
• DNA samples from the cell lines HI 75, RKO, OCI-AML3 and HEL 92.1.7 were diluted in either H 2 0 or ACE wash solution and used as positive controls for EGFR T790, BRAF V600, NPMla and JAK2 617V assays, respectively.
• Described herein are methods, devices and systems suitable for isolating or separating particles or molecules from a fluid composition.
• methods, devices and systems for isolating or separating a nucleic acid from a fluid comprising cells or other particulate material may allow for rapid separation of particles and molecules in a fluid composition.
• the methods, devices and systems may allow for rapid isolation of molecules from particles in a fluid composition.
• the methods, devices and systems may 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.
• the methods, devices, and systems comprising applying the fluid to a device comprising an array of electrodes and being capable of generating AC electrokinetic forces (e.g., when the array of electrodes are energized).
• the dielectrophoretic field is a component of AC electrokinetic force effects.
• the component of AC electrokinetic force effects is AC electroosmosis or AC electrothermal effects.
• the AC electrokinetic force, including dielectrophoretic fields comprises high- field regions (positive DEP, i.e.
• 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., rinsing the array with water or buffer while the nucleic acid is concentrated and maintained within a high field DEP region of the array), degrading residual proteins (e.g., residual proteins from lysed cells and/or other sources, such degradation occurring according to any suitable mechanism, such as with heat, a protease, or a chemical), flushing degraded proteins from the nucleic acid, and collecting the nucleic acid.
• residual e.g., cellular
• degrading residual proteins e.g., residual proteins from lysed cells and/or other sources, such degradation occurring according to any suitable mechanism, such as with heat, a protease, or a chemical
• flushing degraded proteins from the nucleic acid and collecting the nucleic acid.
• the methods described herein are performed in a short amount of time, the devices are operated in a short amount of time, and the systems are operated in a short amount of time.
• the period of time is short with reference to the "procedure time" measured from the time between adding the fluid to the device and obtaining isolated nucleic acid.
• 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.
• a device for isolating a nucleic acid from a fluid comprising cells or other particulate material comprising: a. a housing; b. a heater or thermal source and/or a reservoir comprising a protein degradation agent; and c. a plurality of alternating current (AC) electrodes within the housing, the AC electrodes configured to be selectively energized to establish AC electrokinetic high field and AC electrokinetic low field regions, whereby AC electrokinetic effects provide for concentration of cells in low field regions of the device.
• the plurality of electrodes is configured to be selectively energized to establish a dielectrophoretic high field and dielectrophoretic low field regions.
• the protein degradation agent is a protease.
• the device further comprises at least one of an elution tube, a chamber and a reservoir to perform PCR amplification or other enzymatic reaction.
• PCR amplification or other enzymatic reaction is performed in a serpentine microchannel comprising a plurality of temperature zones.
• PCR amplification or other enzymatic reaction is performed in aqueous droplets entrapped in immiscible fluids (i.e., digital PCR).
• the thermocycling comprises convection.
• the device comprises a surface contacting or proximal to the electrodes, wherein the surface is functionalized with biological ligands that are capable of selectively capturing biomolecules.
• pore or hole structures contain (or are filled with) porous material (hydrogels) or are covered with porous membrane structures.
• pore/hole structure DEP devices reduce electrochemistry effects, heating, or chaotic fluidic movement from occurring in the inner separation chamber during the DEP process.
• a planar platinum electrode array device comprises a housing through which a sample fluid flows.
• fluid flows from an inlet end to an outlet end, optionally comprising a lateral analyte outlet.
• the exemplary device includes multiple AC electrodes.
• the sample consists of a combination of micron-sized entities or cells, larger nanoparticulates and smaller nanoparticulates or biomolecules.
• the micron-sized entities may comprise blood cells, platelets, bacteria and the like.
• larger nanoparticulates comprise particulates in the range of about 10 nm and about 900 nm effective stokes diameter, and may comprise exosomes, high mw nucleic acids, including high mw DNA, oligo-nucleosome complexes, aggregated proteins, vesicle bound DNA, cell membrane fragments and cellular debris dispersed in the sample.
• smaller nanoparticulates ( ⁇ 1 Onm effective stokes diameter) comprise proteins such as immunoglobulins, human serum albumin, fibrinogen and other plasma proteins, smaller apoptotic DNA, and free ions.
• the AC electrokinetic field regions disclosed herein are capable of selectively isolating target particulates, including micron-sized entities, larger nanoparticulates and/or smaller nanoparticulates. In some embodiments, the AC electrokinetic field regions disclosed herein are capable of selectively isolating target particulates, including micron-sized entities, larger nanoparticulates and/or smaller nanoparticulates in complex biological or environmental samples. The target particulates are isolated in different field regions at or near the surface of the array, allowing non-target particulates or particulates that are not isolated at or near the surface of the array to be flushed from the array or cartridge.
• the electrodes are in a dot configuration, e.g. the electrodes comprises a generally circular or round configuration.
• the angle of orientation between dots is from about 25° to about 60°. In some embodiments, the angle of orientation between dots is from about 30° to about 55°. In some embodiments, the angle of orientation between dots is from about 30° to about 50°. In some embodiments, the angle of orientation between dots is from about 35° to about 45°. In some embodiments, the angle of orientation between dots is about 25°. In some embodiments, the angle of orientation between dots is about 30°. In some embodiments, the angle of orientation between dots is about 35°. In some embodiments, the angle of orientation between dots is about 40°.
• the viscosity of a hydrogel solution prior to spin-coating ranges from about 0.5 cP to about 5 cP.
• a single coating of hydrogel solution has a viscosity of between about 0.75 cP and 5 cP prior to spin-coating.
• the first hydrogel solution has a viscosity from about 0.5 cP to about 1.5 cP prior to spin coating.
• the second hydrogel solution has a viscosity from about 1 cP to about 3 cP.
• the viscosity of the hydrogel solution is based on the polymers concentration (0.1% -10%) and polymers molecular weight (10,000 to 300,000) in the solvent and the starting viscosity of the solvent.
• the first hydrogel coating has a thickness between about 0.5 microns and 1 micron. In some embodiments, the first hydrogel coating has a thickness between about 0.5 microns and 0.75 microns. In some embodioments, the first hydrogel coating has a thickness between about 0.75 and 1 micron.
• the second hydrogel coating has a thickness between about 0.2 microns and 0.5 microns. In some embodiments, the second hydrogel coating has a thickness between about 0.2 and 0.4 microns. In some embodiments, the second hydrogel coating has a thickness between about 0.2 and 0.3 microns. In some embodiments, the second hydrogel coating has a thickness between about 0.3 and 0.4 microns.
• 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, ether-based polymers, alkylene-glycol based polymers (e.g., polypropylene glycol), and the like.
• the method described herein comprises applying a fluid comprising cells or other particulate material to a device comprising an array of electrodes, and, thereby, concentrating the cells in a first DEP field region.
• the devices and systems described herein are capable of applying a fluid comprising cells or other particulate material to the device comprising an array of electrodes, and, thereby, concentrating the cells in a first DEP field region.
• Subsequent or concurrent second, or optional third and fourth DEP regions may collect or isolate other fluid components, including biomolecules, such as nucleic acids.
• the first DEP field region may be any field region suitable for concentrating cells from a fluid.
• the cells are generally concentrated near the array of electrodes.
• the first DEP field region is a dielectrophoretic low field region.
• the first DEP field region is a dielectrophoretic high field region.
• the method described herein 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.
• the first DEP field region is produced using a direct current having an amperage of 100 micro Amperes -500 milli Amperes. In some embodiments, the first DEP field region is produced using a direct current having an amperage of 1 milli Amperes - 1 Amperes. In some embodiments, the first DEP field region is produced using a direct current having an amperage of 1 micro Amperes - 1 milli Amperes. In some embodiments, the first DEP field region is produced using a direct current having a pulse width of 500 milliseconds-500 seconds. In some embodiments, the first DEP field region is produced using a direct current having a pulse width of 500 milliseconds- 100 seconds.
• the first DEP field region is produced using a direct current having a pulse width of 1 second - 1000 seconds. In some embodiments, the first DEP field region is produced using a direct current having a pulse width of 500 milliseconds- 1 second. In some embodiments, the first DEP field region is produced using a pulse frequency of 0.01 -1000 Hz. In some embodiments, the first DEP field region is produced using a pulse frequency of 0.1-100 Hz. In some embodiments, the first DEP field region is produced using a pulse frequency of 1-100 Hz. In some embodiments, the first DEP field region is produced using a pulse frequency of 100-1000 Hz.
• the first DEP field is operated in a manner that specifically concentrates viruses and not cells (e.g., in a fluid with conductivity of greater than 300 mS/m, viruses concentrate in a DEP high field region, while larger cells will concentrate in a DEP low field region).
• the first DEP field region comprises the entirety of an array of electrodes. In some embodiments, the first DEP field region comprises a portion of an array of electrodes. In some embodiments, 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 array of electrodes. In some embodiments, the first DEP field region comprises about a third of an array of electrodes. Second DEP Field Region
• the second dielectrophoretic field region is produced by an alternating current.
• the alternating current has any amperage, voltage, frequency, and the like suitable for concentrating nucleic acids.
• the second dielectrophoretic field region is produced using an alternating current having an amperage of 0.1 micro Amperes - 10 Amperes; a voltage of 1-50 Volts peak to peak; and/or a frequency of 1 - 10,000,000 Hz.
• the second DEP field region is produced using an alternating current having an amperage of 0.1 micro Amperes - 1 Ampere.
• the second DEP field region is produced using an alternating current having an amperage of 1 micro Amperes - 1 Ampere. In some embodiments, the second DEP field region is produced using an alternating current having an amperage of 100 micro Amperes - 1 Ampere. In some embodiments, the second DEP field region is produced using an alternating current having an amperage of 500 micro Amperes - 500 milli Amperes. In some embodiments, the second DEP field region is produced using an alternating current having a voltage of 1 -25 Volts peak to peak. In some embodiments, the second DEP field region is produced using an alternating current having a voltage of 1-10 Volts peak to peak.
• the second DEP field region is produced using an alternating current having a voltage of 25-50 Volts peak to peak. In some embodiments, the second DEP field region is produced using a frequency of from 10-1 ,000,000 Hz. In some embodiments, the second DEP field region is produced using a frequency of from 100-100,000 Hz. In some embodiments, the second DEP field region is produced using a frequency of from 100-10,000 Hz. In some embodiments, the second DEP field region is produced using a frequency of from 10,000-100,000 Hz. In some embodiments, the second DEP field region is produced using a frequency of from 100,000- 1,000,000 Hz.
• the second dielectrophoretic field region is produced by a direct current.
• the direct current has any amperage, voltage, frequency, and the like suitable for concentrating nucleic acids.
• the second dielectrophoretic field region is produced using a direct current having an amperage of O.lmicro Amperes - 1 Amperes; a voltage of 10 milli Volts - 10 Volts; and/or a pulse width of 1 milliseconds - 1000 seconds and a pulse frequency of 0.001 - 1000 Hz.
• the second DEP field region is produced using an alternating current having a voltage of 5-25 volts peak to peak.
• the second DEP field region is produced using a direct current having an amperage of 1 micro Amperes - 1 milli Amperes. In some embodiments, the second DEP field region is produced using a direct current having a pulse width of 500 milliseconds-500 seconds. In some embodiments, the second DEP field region is produced using a direct current having a pulse width of 500 milliseconds- 100 seconds. In some embodiments, the second DEP field region is produced using a direct current having a pulse width of 1 second - 1000 seconds. In some embodiments, the second DEP field region is produced using a direct current having a pulse width of 500 milliseconds -1 second.
• the second DEP field region is produced using a pulse frequency of 0.01- 1000 Hz. In some embodiments, the second DEP field region is produced using a pulse frequency of 0.1-100 Hz. In some embodiments, the second DEP field region is produced using a pulse frequency of 1-100 Hz. In some embodiments, the second DEP field region is produced using a pulse frequency of 100-1000 Hz.
• the second DEP field region comprises the entirety of an array of electrodes. In some embodiments, the second DEP field region comprises a portion of an array of electrodes. 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 array of electrodes. In some embodiments, the second DEP field region comprises about a third of an array of electrodes.
• a method for isolating a nucleic acid from a fluid comprising cells comprising: a. applying the fluid to a device, the device comprising an array of electrodes; b. concentrating a plurality of cells in a first AC electrokinetic (e.g., dielectrophoretic) field region; c.
• nucleic acid in a second AC electrokinetic (e.g., dielectrophoretic) field region; and d. flushing cells away.
• the cells are lysed in the high field region.
• the nucleic acids remain in the high field region and/or are concentrated in the high field region.
• residual cellular material is concentrated near the low field region.
• the residual material is washed from the device and/or washed from the nucleic acids.
• nucleic acid is concentrated in the second AC electrokinetic field region.
• the methods, systems and devices described herein isolate nucleic acid from a fluid comprising cells or other particulate material.
• dielectrophoresis is used to concentrate cells.
• the fluid is a liquid, optionally water or an aqueous solution or dispersion.
• the fluid is any suitable fluid including a bodily fluid.
• Exemplary bodily fluids include whole blood, serum, plasma, bile, milk, cerebrospinal fluid, gastric juice, ejaculate, mucus, peritoneal fluid, saliva, sweat, tears, urine, and other bodily fluids.
• nucleic acids are isolated from bodily fluids using the methods, systems or devices described herein as part of a medical therapeutic or diagnostic procedure, device or system.
• the fluid is tissues and/or cells solubilized and/or dispersed in a fluid.
• the tissue can be a cancerous tumor from which nucleic acid can be isolated using the methods, devices or systems described herein.
• the fluid is an environmental sample.
• the environmental sample is assayed or monitored for the presence of a particular nucleic acid sequence indicative of a certain contamination, infestation incidence or the like.
• environmental sample can also be used to determine the source of a certain contamination, infestation incidence or the like using the methods, devices or systems described herein.
• the fluid is a food or beverage.
• the food or beverage can be assayed or monitored for the presence of a particular nucleic acid sequence indicative of a certain contamination, infestation incidence or the like.
• the food or beverage can also be used to determine the source of a certain contamination, infestation incidence or the like using the methods, devices or systems described herein.
• the methods, devices and systems described herein can be used with one or more of bodily fluids, environmental samples, and foods and beverages to monitor public health or respond to adverse public health incidences.
• the fluid is a growth medium.
• the growth medium can be any medium suitable for culturmg cells, for example lysogeny broth (LB) for culturing E. coli, Ham's tissue culture medium for culturing mammalian cells, and the like.
• the medium can be a rich medium, minimal medium, selective medium, and the like.
• the medium comprises or consists essentially of a plurality of clonal cells.
• the medium comprises a mixture of at least two species.
• the fluid is water.
• the cells are any cell suitable for isolating nucleic acids from as described herein.
• the cells are eukaryotic or prokaryotic.
• the cells consist essentially of a plurality of clonal cells or may comprise at least two species and/or at least two strains.
• the fluid may also comprise other particulate material.
• particulate material may be, for example, inclusion bodies (e.g., ceroids or Mallory bodies), cellular casts (e.g., granular casts, hyaline casts, cellular casts, waxy casts and pseudo casts), Pick's bodies, Lewy bodies, fibrillary tangles, fibril formations, cellular debris and other particulate material.
• particulate material is an aggregated protein (e.g. , beta-amyloid).
• the fluid can have any conductivity including a high or low conductivity.
• the conductivity is between about 1 ⁇ / ⁇ to about 10 mS/m. In some embodiments, the conductivity is between about 10 ⁇ / ⁇ to about 10 mS/m. In other embodiments, the conductivity is between about 50 ⁇ / ⁇ to about 10 mS/m.
• the conductivity is between about 100 ⁇ 8/ ⁇ to about 10 mS/m, between about 100 ⁇ 8/ ⁇ to about 8 mS/m, between about 100 ⁇ / ⁇ to about 6 mS/m, between about 100 ⁇ / ⁇ to about 5 mS/m, between about 100 ⁇ / ⁇ to about 4 mS/m, between about 100 ⁇ / ⁇ to about 3 mS/m, between about 100 ⁇ 8/ ⁇ to about 2 mS/m, or between about 100 ⁇ 8/ ⁇ to about 1 mS/m.
• the conductivity is about 1 ⁇ / ⁇ . In some embodiments, the conductivity is about 10 ⁇ 8/ ⁇ . In some embodiments, the conductivity is about 100 ⁇ / ⁇ . 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 yet other embodiments, 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 embodiments, 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.
• the conductivity is at most 100 ⁇ / ⁇ . In some embodiments, the conductivity is at most 1 mS/m. In some embodiments, the conductivity is at most 10 mS/m. In some embodiments, the conductivity is at most 100 mS/m. In yet other embodiments, the conductivity is at most 1 S/m. In some embodiments, the conductivity is at most 10 S/m.
• the fluid is between about 50 ⁇ to about 500 ⁇ .
• the frequency of the direct current depends on volts/cm, pulse width, and the fluid conductivity.
• the pulse has a frequency of about 0.001 to about 1000 Hz. In some embodiments, the pulse has a frequency from about 10 to about 200 Hz. In other embodiments, the pulse has a frequency of about .01 Hz - 1000 Hz. In still other embodiments, the pulse has a frequency of about 0.1 Hz - 1000 Hz, about 1 Hz - 1000 Hz, about 1 Hz - 500 Hz, about 1 Hz - 400 Hz, about 1 Hz - 300 Hz, or about 1 Hz - about 250 Hz.
• the pulse has a duration of about 1 millisecond (ms) - 1000 seconds (s). In some embodiments, the pulse has a duration of about 10 ms - 1000 s. In still other embodiments, the pulse has a duration of about 100 ms - 1000 s, about I s - 1000 s, about I s - 500 s, about I s - 250 s or about I s - 150 s.
• the agent used to degrade the residual material is inactivated or degraded.
• the devices or systems are capable of degrading or inactivating the agent used to degrade the residual material.
• an enzyme used to degrade the residual material is inactivated by heat (e.g., 50 to 95° C for 5-15 minutes).
• enzymes including proteases, for example, Proteinase
• the method further comprises inactivating the degrading enzyme (e.g., Proteinase K) following degradation of the proteins.
• heat is provided by a heating module in the device (temperature range, e.g., from 30 to 95 °C).
• the isolated nucleic acid comprises greater than about 99%), greater than about 98%, greater than about 95%, greater than about 90%, greater than about 80%), greater than about 70%, greater than about 60%, greater than about 50%, greater than about 40%), greater than about 30%, greater than about 20%), or greater than about 10% nucleic acid by mass.
• the nucleic acids are isolated in any suitable form including unmodified, derivatized, fragmented, non-fragmented, and the like.
• the nucleic acid is collected in a form suitable for sequencing.
• the nucleic acid is collected in a fragmented form suitable for shotgun-sequencing, amplification or other manipulation.
• the nucleic acid may be collected from the device in a solution comprising reagents used in, for example, a DNA sequencing procedure, such as nucleotides as used in sequencing by synthesis methods.
• the 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, the 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, the 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, the 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.
• about 50 pico-grams of nucleic acid is isolated from about 5,000 cells using the methods, systems or devices described herein. In some embodiments, the methods, systems or devices described herein yield at least 10 pico-grams of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 20 pico-grams of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 50 pico-grams of nucleic acid from about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 75 pico-grams of nucleic acid from about 5,000 cells.
• Miniature PMTs, CCDs, or CMOS detectors can also be built into the flow cell. This minimization and miniaturization enables compact devices capable of rapid signal delivery and detection while reducing the footprint of similar traditional devices (i.e. a standard bench top PCPv/QPCR/Fluorometer).
• the surface is optionally modified with nonspecific moieties for capture.
• surface could be modified with polycations, i.e., polylysine, to capture DNA molecules which can be released by reverse bias (-V).
• modifications to the surface are uniform over the surface or patterned specifically for functionalizing the electrodes or non electrode regions. In certain embodiments, this is accomplished with photolithography, electrochemical activation, spotting, and the like.
• a 600 bp cutoff chip would leave a nucleic acid of less than 600 bp in solution, then that material is optionally recaptured with a 500 bp cutoff chip (which is opposing the 600 bp chip). This leaves a nucleic acid population comprising 500-600 bp in solution. This population is then optionally amplified in the same chamber, a side chamber, or any other configuration.
• the device or system described herein comprises, or a method described herein uses, temperature sensors on the device or in the reaction chamber monitor temperature and such sensors are optionally used to adjust temperature on a feedback basis.
• such sensors are coupled with materials possessing different thermal transfer properties to create continuous and/or discontinuous gradient profiles.
• the amplification proceeds at a constant temperature (i.e, isothermal amplification).
• the isolated nucleic acids disclosed herein are used in Sanger sequencing.
• Sanger sequencing is performed within the same device as the nucleic acid isolation (Lab-on-Chip).
• Lab-on-Chip workflow for sample prep and Sanger sequencing results would incorporate the following steps: a) sample extraction using ACE chips; b) performing amplification of target sequences on chip; c) capture PCR products by ACE; d) perform cycle sequencing to enrich target strand; e) capture enriched target strands; f) perform Sanger chain termination reactions; perform electrophoretic separation of target sequences by capillary electrophoresis with on chip multi-color fluorescence detection. Washing nucleic acids, adding reagent, and turning off voltage is performed as necessary. Reactions can be performed on a single chip with plurality of capture zones or on separate chips and/or reaction chambers.
• the isolated nucleic acids are useful for use in immunoassay- type arrays or nucleic acid arrays.
• E. coli genome is necessary for conventional manual methods, ⁇ e.g., 50 ng of input DNA is required for Nextera, assuming 50% recovery (Epicentre WaterMaster kit claims recovery about 30-60%) from DNA extraction purification). This is equivalent to about 20 million bacteria. In some embodiments of the present invention, less than 10,000 bacteria input is sufficient ⁇ e.g., since the chip is self contained and involves less transfers the efficiency is higher). In some embodiments, this is at least a 100 fold reduction in input, which can be important for applications where sample is limited, such as tumor biopsies.
• PCR amplification is accomplished in the device flow cell chamber, in a PCR tube that is on the cartridge, or though fluidic channels that possess heat zones for temperature cycling.
• the eluate from the device chamber is combined with side channel(s) primed with non aqueous miscible fluid, e.g., oil, and other droplet stabilizers to perform amplification in droplets.
• non aqueous miscible fluid e.g., oil
• the temperature cycling mechanics are as described above.
• the methods and devices disclosed herein may also be used in conjunction with DNA/RNA hybridization techniques to detect specific alleles implicated in cancer.
• specific electrodes and corresponding electrode trace lines can be designed to individually control separate electrode so as to achieve a unique electric field distribution. By designing nonuniform electric field distribution, specific DNA/RNA can be manipulated.
• pancreatitis Pancreas. 2004 Apr; 28(3):263-
• circulating DNA may depend 66(9):775-8.
• Lymphoma (DLBCL " ).
• APC are useful prognostic
• TBE is a buffer solution containing a mixture of Tris base, boric acid and EDTA.
• DEP dielectrophoresis
• hydrogel For a layer of hydrogel, approximately 70 microliters of hydrogel is used to coat a 10 x
• a low concentration ( ⁇ 1% solids by volume) cellulose acetate solution is dissolved into a solvent such as acetone, or an acetone and ethanol mixture and applied to an electrode array chip as disclosed herein.
• the chip is spun at a low rpm rate (1000-3000). The low rpm rate ensures that the height of the gel is in the range of 500nm or greater.
• the first (bottom) coating of cellulose acetate is dried at room temperature, in a convection oven, or a vacuum oven.
• the second layer of cellulose-acetate spin-coat is added immediately.
• the chip with two layers of cellulose acetate is then dried at room temperature, in a convection oven, or in a vacuum oven.
• a low concentration ( ⁇ 1% solids by volume) cellulose acetate solution is dissolved into a solvent such as acetone, or an acetone and ethanol mixture and applied to an electrode array chip as disclosed herein.
• the chip is spun at a low rpm rate (1000-3000).
• the low rpm rate ensures that the height of the gel is in the range of 500 nm or greater.
• the first (bottom) coating of cellulose acetate is dried at room temperature, in a convection oven, or a vacuum oven.
• the second layer of cellulose-acetate spin-coat is added immediately.
• the chip with two layers of cellulose acetate is then dried at room temperature, in a convection oven, or in a vacuum oven.
• hydrogel For a layer of hydrogel, approximately 70 microliters of hydrogel is used to coat a 10 x 12 mm chip.
• the first (bottom) coating of cellulose acetate is dried at room temperature, in a convection oven, or a vacuum oven.
• the second layer of cellulose-acetate spin-coat is added immediately.
• FIG. 2 & 3 A 45x20 custom 80 ⁇ diameter circular platinum microelectrode array on 200 um center-center pitch was fabricated based upon previous results (see references 1-3, below). All 900 microelectrodes are activated together and AC biased to form a
• the array is over-coated with a 200nm-500nm thick porous poly-Hema hydrogel layer (Procedure: 12% pHema in ethanol stock solution, purchased from PolySciences Inc., that is diluted to 5% using ethanol. 70uL of the 5% solution is spun on the above mentioned chip at a 6K RPM spin speed using a spin coater. The chip+hydrogel layer is then put in a 60 °C oven for 45 minutes) and enclosed in a microfluidic cartridge, forming a 50 ⁇ sample chamber covered with an acrylic window (FIG. 1).
• a 200nm-500nm thick porous poly-Hema hydrogel layer Providedure: 12% pHema in ethanol stock solution, purchased from PolySciences Inc., that is diluted to 5% using ethanol. 70uL of the 5% solution is spun on the above mentioned chip at a 6K RPM spin speed using a spin coater. The chip+hydrogel layer is then put in a 60 °C oven
• a function generator (HP 3245 A) provided sinusoidal electrical signal at lOKHz and 10 - 14V peak-peak, depending on solution conductivity. Images were captured with a fluorescent microscope (Leica) and an EGFP cube (485 nm emission and 525 nm excitation bandpass filters). The excitation source was a PhotoFluor II 200W Hg arc lamp.
• gDNA Human Genomic DNA
• Promega Promega, Madison, WI
• gDNA Human Genomic DNA
• the gDNA was diluted in DI water to the following concentrations: 50 nanograms, 5 nanograms, 1 nanogram, and 50 picograms.
• the gDNA was stained using lx SYBR Green I green fluorescent double stranded DNA dye purchased from Invitrogen (Life Technologies, Carlsbad, CA). This mixture was then inserted into the microelectrode arrays and run at 14 Volts peak to peak (Vp-p), at 10kHz sine wave for 1 minute.
• Vp-p Volts peak to peak
• each microelectrode had on average ⁇ 60 femtograms of DNA since there are 900 microelectrodes on the array.
• the low-level concentration ability of the ACE device is well within the range of 1 - lOng/mL needed to identify Cfc-DNA biomarkers in plasma and serum (see references 4-6 below).
• the process could also be optimized by changing the deposition rate or anchoring growth to the surface of the microelectrode array (i.e., to the passivation layer and exposed electrodes), using an adhesion promoter such as a silane derivative.
• QIAGEN® circulating nucleic acid Purification kit (cat# 5114) was used to purify 1 ml of plasma from chronic lymphocytic leukemia (CLL) patients, according to manufacturer's protocol. Briefly, incubation of 1 ml plasma with Proteinase K solution was performed for 30 minutes at 60 °C. The reaction was quenched on ice and the entire volume was applied to a QIAamp Mini column connected to a vacuum. The liquid was pulled through the column and washed with 3 different buffers (600-750 ul 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 ⁇ of elution buffer with 20,000 x g, 1 minute centrifugation. Total processing time was -2.5 hours.
• the chip die size was 10 x 12 mm, with 60 - 80 ⁇ diameter Pt electrodes on 180 - 200 ⁇ center-to-center pitch, respectively.
• the array was overcoated with a 5% pHEMA hydrogel layer (spun cast 6000 rpm from Ethanol solution, 12% pHEMA stock from
• the chip was pretreated using 0.5xPBS, 2V rms, 5 Hz, 15 seconds.
• the buffer was removed and 25 ⁇ of CLL patient plasma was added.
• DNA was isolated for 3 minutes at 11 V p-p, lOKhz, then washed with 500 ⁇ of TE buffer at a 100 ⁇ /min flow rate, with power ON. The voltage was turned off and the flow cell volume was eluted into a microcentrifuge tube. Total processing time was - 10 minutes.
• Values are in ng/ml and normalized to original plasma sample volume for comparison purposes.
• ACE microfluidic cartridges Using ACE microfluidic cartridges relative concentration of cell free biomarkers were determined in an unknown sample (sample can be whole blood, serum, plasma).
• the ACE microfluidic cartridge may be designed with one or more chambers for known standards and one or more chambers for the unknown sample as shown in FIG. 11.
• An ACE field at 10 Vp-p and 10 kHz is applied to select microfluidic cartridge chambers and cell-free target biomarkers of interest are captured on the electrodes as shown in FIG. 12.
• a fluidic wash solution water + osmolytes
• This fluidic wash is compatible with polymerase chain reaction (PCR) and next-generationsequencing thus allowing for secondary analysis post-elution.
• PCR polymerase chain reaction
• Target biomarkers include proteins, lipids, antibodies, high molecular weight DNA (greater than 300 bp), tumor cells, exosomes, nucleosomes, nanosomes.
• specific dyes use such as YOYO ® -l, SYBR ® Green, CBQCA protein quantitation kit, SYTO ® RNASelectTM.
• a CCD/CMOS/PMT detector is used in conjunction with fluorescence microscopy (with appropriate excitation / emission filters) to enable direct detection (binary) and / or quantification (concentration) of unknown analytes using a Region-of-Interest (ROI) image segmentation algorithm that compared pixel intensity between two regions in the electrodes (Fig. 12). Fluorescent quantification form both the known and unknown chambers is determined and the data is then compared using a linear fit calibration curve (FIG. 13) to create a relative ratio of intensity between the known chamber and the unknown chamber.
• ROI Region-of-Interest
• the analytes in the known chamber have known specific concentrations of the cell free target biomarkers and the fluorescence labels are specific for the target analytes, using an algebraic relationship between intensities of the known chamber and the unknown chamber enable and the linear calibration curve the determination of analyte concentration from the unknown chamber.
• EXAMPLE 10 Off-Chip Quantification using O-PCR, RT-PCR and Sequencing
• ACE microfluidic cartridges Using ACE microfluidic cartridges, relative concentrations of cell-free biomarkers were determined in an unknown sample.
• the sample may be whole blood, serum, plasma or other biological sample/fluid.
• the ACE microfluidic cartridge may be designed with one or more chambers for known standards and one or more chambers for the unknown sample.
• An ACE field at 10 Vp-p and 10 kHz is applied to the chamber and cell-free target biomarkers of interest are captured on the electrodes (FIG. 12).
• a fluidic wash (water + osmolytes) is applied using a peristaltic pump while the ACE field is still on in order to remove all unwanted sample. This fluidic wash is compatible with polymerase chain reaction (PCR) and next-generation sequencing thus allowing for secondary analysis post-elution.
• PCR polymerase chain reaction
• FIG. 14 illustrates the usage of PCR technique for detection of PCR mutations for DNA samples from the cell lines H1975, R O, OCI-AML3 and HEL 92.1.7 diluted in either H 2 0 or ACE fluidic wash solution and used as positive controls for EGFR T790, BRAF V600, NPMla and JAK2 617V assays.

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