Device for separation of biological materials

Abstract

A device for capturing analytes is disclosed. The device includes a substrate, a conductive layer disposed over the substrate, the conductive layer configured to generate an electrokinetic field, and an insulating layer having at least one insulating portion substantially on an edge of the conductive layer. At least one insulating portion comprises an outer portion with a first height and an inner portion having a second height that is the same or different from the first height.

Description

Docket No.: XZOM-013 / 02WO 355524-2169DEVICE FOR SEPARATION OF BIOLOGICAL MATERIALSCROSS REFERENCE TO RELATEFD APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 750,690 filed on January 28, 2025, the content of which is incorporated herein by reference in its entirety for all purposes.BACKGROUND

[0002] Progress has been made in characterizing and isolating analytes from complex samples, including the isolation of biomolecules, such as nucleic acids or virus particles from biological samples, for next generation sequencing, immunoassays, or other diagnostic applications. These techniques and others are expected to transform fields as diverse as medicine, renewable energy, biosecurity and agriculture to name a few. However, devices, methods, compositions, and systems for efficiently isolating analytes suitable for analysis with these advanced methods have not kept pace. Accordingly, new and improved devices, methods, compositions, and systems for efficiently isolating analytes suitable for analysis are desired.SUMMARY

[0003] The present methods, devices, processes, and systems disclosed herein fulfill a need for improved methods of isolating and / or quantifying analytes from samples. Particular attributes of certain embodiments provided herein include devices comprising dielectric materials for isolation of analytes. In some embodiments, the dielectric materials are used with electrodes. In some embodiments, the present methods, devices, processes, and systems can be used to isolate and / or separate analytes such as cell-free nucleic acids, exosomes, high molecular weight (mw) nucleic acids, including high molecular weight DNA, oligonucleosome complexes, nucleosomes, aggregated proteins, vesicle bound DNA, cell membrane fragments and cellular debris, proteins, lipids, viruses, or other analyte from dilute and / or complex fluids such as blood, environmental samples, or any other sample source comprising analytes. In some embodiments, the disclosed technology uses small amounts of starting material, achieves isolation of highly pure analytes, and is amenable to multiplexed and high-throughput operation.Docket No.: XZOM-013 / 02WO 355524-2169

[0004] A hydrogel may be used to coat a surface of the electrode. The hydrogel may be a very thin polymer with a porous membrane. The hydrogel is non-biofouling, wherein analytes, such as blood, interact with the hydrogel in a manner which does not cause protein degradation. Hydrogel may inhibit the precipitation of blood proteins. In some embodiments, the pores of the hydrogel, over time, expand and result in a more porous membrane than was initially treated on the electrode. Such a reaction by analytes, such as blood, changes the analyte flow and dielectrophoretic effect on the analytes such that the analytes are captured by the hydrogel, on the side facing the electrode. The hydrogel may cause the analytes to be caught in the corner where the insulating layer (e.g., dielectric layer like silicon dioxide) and the hydrogel connect. While a hydrogel has many advantages as described herein, it can be difficult to create a layer of hydrogel over the electrode due to high costs and long times to form the hydrogel. Accordingly, there is a need for a different way to mimic the behavior of hydrogel without the hydrogel itself.

[0005] In some embodiments, disclosed herein are devices for capturing analytes comprising: a substrate; a conductive layer disposed over the substrate, where the conductive layer is configured to generate an electrokinetic field; and an insulating layer disposed partially over the conductive layer, the insulating layer having at least one insulating portion substantially on an edge of the conductive layer, wherein the at least one insulating portion comprises an outer portion with a first height and an inner portion having a second height that is the same or different from the first height.. In some embodiments, the insulating layer further comprises a peripheral insulating portion disposed over the substrate and adjacent to the at least one insulating portion and the conductive layer. In some embodiments, a distance of the top surface of the peripheral insulating portion from the substrate is less than a distance of the top surface of the at least one insulating portion from the substrate. In some embodiments, the second height is less than the first height. In some embodiments, the inner portions and outer portions of at least one insulating portion forms an opening that exposes the conductive layer. In some embodiments, the first length of the opening formed by inner portions is less than a second length of the opening formed by the outer portions. In some embodiments, the first length is within a range of about 5 pm to about 600 pm. In some embodiments, the second length is within a range of about 5 pm to about 700 pm. In some embodiments, the first height is within a range of about 100 nm to about 10 pm. In some embodiments, the second height is within a range of about 25 nm to about 7.5 pm. In some embodiments, each of the inner portions has a width that is within a range of about 500 nm to about 50 pm. In someDocket No.: XZOM-013 / 02WO 355524-2169embodiments, the conductive layer comprises platinum. In some embodiments, the conductive layer comprises a copper layer and a graphene layer. In some embodiments, the copper layer is disposed over the substrate and the graphene layer is disposed over the copper layer. In some embodiments, the length of the copper layer is greater than the length of the graphene layer. In some embodiments, the length of the copper layer is in the range of about 10 pm to about 1 nm. In some embodiments, the length of the graphene layer is in the range of about 6 pm to about 600 pm. In some embodiments, the thickness of the copper later is greater than the graphene layer. In some embodiments, the thickness of the copper layer is in the range of about 200 nm to about 20 pm. In some embodiments, the thickness of the graphene layer is in the range of about 2.5 nm to about 250 nm. In some embodiments, the insulating layer has a center insulating portion that is disposed over a center portion of the conductive layer. In some embodiments, the center insulating portion has a height that is greater than the first and second heights. In some embodiments, the device comprises a semipermeable membrane connected to at least one insulating portion. In some embodiments, the semipermeable membrane comprises an inorganic, organic, or composite material. In some embodiments, the inorganic semipermeable membrane comprises anodic Al2O3, anodic SiO2, ceramic Al2O3, ceramic SiO2, ZrO2, TiO2, Si, SiC, or other semiconductors. In some embodiments, the organic semipermeable membrane comprises polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), or polystyrene. In some embodiments, the composite semipermeable membrane comprises an anodic aluminum oxide (AAO) coated with carbon, metal, or metal oxide, an oxide / polymer composite, or a polymer coating on a support membrane. In some embodiments, the semipermeable membrane has a plurality of pores having the size in the range of 0.1 nm to 1000 nm. In some embodiments, the opening is substantially circular, and the first and second lengths include diameters.

[0006] In some embodiments, an electrode array is created, comprising a plurality of electrodes arranged in a plurality of rows, wherein each electrode comprises: a substrate; a conductive layer disposed over the substrate, wherein the conductive layer is configured to generate an electrokinetic field; and an insulating layer disposed partially over the conductive layer, the insulating layer having at least one insulating portion substantially on an edge of the conductive layer, wherein the at least one insulating portion comprises an outer portion with a first height and an inner portion having a second height that is the same or different from the first height.Docket No.: XZOM-013 / 02WO 355524-2169

[0007] In some embodiments, there is a method of fabricating a device for capturing analytes, comprising: a. depositing a conductive layer over a substrate, b. forming a first dielectric layer over the conductive layer and the substrate; and c. etching the first dielectric layer to create an opening that exposes the conductive layer, wherein the opening has a first length. In some embodiments, the fabrication method comprises etching the first dielectric layer to widen a top portion of the opening such that the opening has the first length closer to the conductive layer and a second length farther from the conductive layer, and wherein the second length is greater than the first length. In some embodiments, the fabrication method further comprises forming a second dielectric layer over the opening to create a pillar that does not connect to the first dielectric layer. In some embodiments, the fabrication method further comprises forming a semipermeable membrane over the conductive layer, wherein the semipermeable membrane is coupled to opposing portions of the first dielectric layer that form the opening.

[0008] Also provided herein are devices for capturing analytes comprising an electrode comprising platinum, wherein the electrode is configured to generate an AC dielectrophoretic field region; and a layer in contact with a portion of the electrode.INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0011] FIG. 1 shows an example electrode configuration in a device including a plurality of electrodes, in accordance with some embodiments.Docket No.: XZOM-013 / 02WO 355524-2169

[0012] FIG. 2 shows a cross-sectional view of an example electrode having a conductive layer and stepped insulating portions formed over the conductive layer, in accordance with some embodiments.

[0013] FIG. 3 shows a cross-sectional view of an example electrode including a conductive layer with an overlying graphene layer and stepped insulating portions, in accordance with some embodiments.

[0014] FIG. 4 shows a cross-sectional view of an example electrode including outer insulating portions and a center insulating portion formed over a conductive layer, in accordance with some embodiments.

[0015] FIG. 5 shows a cross-sectional view of an example electrode including outer insulating portions with notches and a center insulating portion formed over a conductive layer, in accordance with some embodiments.

[0016] FIG. 6 shows a cross-sectional view of an example electrode including a semi- permeable porous membrane disposed over a conductive layer and insulating portions formed thereon, in accordance with some embodiments.DETAILED DESCRIPTION

[0017] Described herein are methods, devices and systems suitable for isolating or separating nanoscale analytes from complex samples. In specific embodiments, provided herein are methods, devices and systems for isolating or separating a nanoscale analyte from a sample comprising other particulate material. In some embodiments, the methods, devices and systems may allow for rapid separation of particles and nanoscale analytes in a sample. In some embodiments, the methods, devices and systems may allow for rapid isolation of nanoscale analytes from particles in a sample. In some embodiments, the methods, devices and systems may allow for a rapid procedure that requires a minimal amount of material and / or results in a highly purified nanoscale analyte isolated from complex fluids such as blood or environmental samples.

[0018] The articles “a”, “an” and “the” are non-limiting. For example, “the method” includes the broadest definition of the meaning of the phrase, which can be more than one method.

[0019] The term “Vp-p” refers to peak-to-peak voltage.

[0020] The term “TBE” refers to a buffer solution containing a mixture of Tris base, boric acid and EDTA.Docket No.: XZOM-013 / 02WO 355524-2169

[0021] The term “TE” refers to a buffer solution containing a mixture of Tris base and EDTA.

[0022] The term “L-Histidine buffer” refers to a solution containing L-histidine.

[0023] The term “DEP” refers to an abbreviation for dielectrophoresis.

[0024] The term “ACE” refers to an abbreviation for Alternate Current Electrokinetics.

[0025] The term “ACET” refers to an abbreviation for AC electrothermal.

[0026] Provided in certain embodiments herein are methods, devices and systems for isolating or separating nanoscale analytes from a sample, the methods, devices, and systems comprising applying the fluid to a device comprising an array of electrodes as disclosed herein and being capable of generating AC electrokinetic forces (e.g., when the array of electrodes are energized). AC Electrokinetics (ACE) capture is a functional relationship between the dielectrophoretic force (FDEP), and the flow force (FFlow) derived from the combination of AC electrothermal (ACET) and AC electroosmotic (ACEO) flows. In some embodiments, the dielectrophoretic field generated is a component of AC electrokinetic force effects. In other embodiments, the component of AC electrokinetic force effects is AC electroosmosis or AC electrothermal effects. In some embodiments the AC electrokinetic force, including dielectrophoretic fields, comprises high-field regions (positive DEP, e.g., area where there is a strong concentration of electric field lines due to a non-uniform electric field) and / or low-field regions (negative DEP, e.g., area where there is a weak concentration of electric field lines due to a non-uniform electric field).

[0027] In some embodiments, the nanoscale analytes (e.g., nucleic acid) are isolated (e.g., isolated or separated from particulate material) in a field region (e.g., a high field region) of a dielectrophoretic field. In some embodiments, the method, device, or system includes isolating and concentrating nanoscale analytes in a high field DEP region. In some embodiments, the method, device, or system includes isolating and concentrating nanoscale analytes in a low field DEP region 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 or proteinaceous) material from the nanoscale analyte (e.g., rinsing the array with water or buffer while the nanoscale analyte is concentrated and maintained within a high field DEP region of the array), degrading residual proteins (e.g., degradation occurring according to any suitable mechanism, such as with heat, a protease, or a chemical), flushing degraded proteins from the nanoscale analyte, and collecting the nanoscale analyte. In some embodiments, the result of the methods, operation of the devices,Docket No.: XZOM-013 / 02WO 355524-2169and operation of the systems described herein is an isolated nanoscale analyte, optionally of suitable quantity and purity for further analysis or characterization in, for example, enzymatic assays (e.g, PCR assays).

[0028] In some embodiments, the methods, devices and compositions disclosed herein utilize electrode configurations and designs to improve separation and capture of the nanoscale analytes from particulate material. In some embodiments, the electrode arrays are configured such that fluid flow around or within the vicinity of the electrodes are disrupted or altered, allowing the localization and / or retention of nanoscale analytes around or within the electrode arrays. In other embodiments, the improvement in nanoscale analyte capture is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% or more nanoscale analyte captured than if using conventional electrode configuration or designs, which do not have a reduction in conductive material within the electrodes.

[0029] In some embodiments, the isolated nanoscale analyte comprises less than about 10% non-nanoscale analyte by mass. In some embodiments, the method is completed in less than 10 minutes.

[0030] In some embodiments, the method further comprises degrading residual proteins on the array. In some embodiments, the residual proteins are degraded by one or more of a chemical degradants or an enzymatic degradant. In some embodiments, the residual proteins are degraded by Proteinase K.

[0031] In some embodiments, the nanoscale analyte is a nucleic acid. In other embodiments, the nucleic acid is further amplified 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 cellular material and / or protein by mass. In some embodiments, 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. In some embodiments, the method is completed in less than about one hour. In some embodiments, centrifugation is not used. In some embodiments, the residual proteins are degraded by one or more of chemicalDocket No.: XZOM-013 / 02WO 355524-2169degradation 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. In some embodiments, the enzyme is inactivated by heat (e.g., 50 to 95 °C for 5 — 15 minutes). In some embodiments, the residual material and the degraded proteins are flushed in separate or concurrent steps. In some embodiments, the isolated nanoscale analyte is collected by (i) turning off the second AC electrokinetic field region, and (ii) eluting the nanoscale analyte from the array in an eluant. In some embodiments, a nanoscale analyte is isolated in a form suitable for sequencing. In some embodiments, the nanoscale analyte is isolated in a fragmented form suitable for shotgun-sequencing.

[0032] In some embodiments, the nucleic acid is sequenced by Sanger sequencing, pyrosequencing, ion semiconductor sequencing, polony sequencing, sequencing by ligation, DNA nanoball sequencing, sequencing by ligation, or single molecule sequencing. In some embodiments, the method further comprises performing a reaction on the DNA (e.g., fragmentation, restriction digestion, ligation) that is isolated and eluted from the devices disclosed herein. In some embodiments, the reaction occurs on or near the array or in the device. In some embodiments, the fluid or biological sample comprises no more than 10,000 cells.

[0033] In some embodiments, the sample is a biological sample and has a low conductivity or a high conductivity. In some embodiments, the sample 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, the cells comprise clonal cells, pathogen cells, bacteria cells, viruses, plant cells, animal cells, insect cells, and / or combinations thereof.

[0034] In some embodiments, it is advantageous that 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. In some embodiments, 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 nanoscale analyte. 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.

[0035] In some embodiments, the period of time is short with reference to the “hands-on time” measured as the cumulative amount of time that a person must attend to the procedureDocket No.: XZOM-013 / 02WO 355524-2169from the time between adding the fluid to the device and obtaining isolated nanoscale analyte. In some embodiments, the hands-on time is less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds.

[0036] In some embodiments, it is advantageous that the devices described herein comprise a single vessel, the systems described herein comprise a device comprising a single vessel and the methods described herein can be performed in a single vessel, e.g., in a dielectrophoretic device as described herein. In some embodiments, such a single-vessel embodiment minimizes the number of fluid handling steps and / or is performed in a short amount of time. In some embodiments, the present methods, devices and systems are contrasted with methods, devices and systems that use one or more centrifugation steps and / or medium exchanges. In some embodiments, centrifugation increases the amount of hands-on time required to isolate nanoscale analytes. In some embodiments, the single-vessel procedure or device isolates nanoscale analytes using a minimal amount of consumable reagents.Devices and Systems

[0037] In some embodiments, described herein are devices for isolating, purifying and collecting a nanoscale analyte from a sample. In some embodiments, described herein are devices for isolating, purifying and collecting or eluting a nanoscale from a complex sample other particulate material, including cells and the like. In some embodiments, the devices disclosed herein are capable of isolating, purifying, collecting and / or eluting nanoscale analytes from a sample comprising cellular or protein material. In some embodiments, the devices disclosed herein are capable of isolating, purifying, collecting and / or eluting nanoscale analytes from samples comprising a complex mixture of organic and inorganic materials. In some embodiments, the devices disclosed herein are capable of isolating, purifying, collecting and / or eluting nanoscale analytes from samples comprising organic materials. In some embodiments, the devices disclosed herein are capable of isolating, purifying, collecting and / or eluting nanoscale analytes from samples comprising inorganic materials.

[0038] In some embodiments, the device disclosed herein is a device for capturing analytes, the device comprising: a substrate, a conductive layer disposed over the substrate, wherein the conductive layer is configured to generate an electrokinetic field, and an insulating layer disposed partially over the conductive layer, the insulating layer having at least one insulating portion substantially on an edge of the conductive layer, wherein at least one insulatingDocket No.: XZOM-013 / 02WO 355524-2169portion comprises an outer portion with a first height and an inner portion having a second height that is the same or different from the first height.

[0039] In some embodiments, the insulating layer comprises a peripheral insulating portion disposed directly upon the substrate. In some embodiments, the peripheral insulating portion may be disposed adjacent to, but not upon, the conductive layer. In some embodiments, the peripheral portion may be disposed adjacent to, but not upon, at least one insulating portion. In some embodiments, the height of the peripheral insulating portion may be substantially uniform around the electrode. In some embodiments, the height of the peripheral insulating portion may not be uniform around the electrode. In some embodiments, the distance of the top surface of the peripheral insulating portion from the substrate may be less than the distance the top surface of the at least one insulating portion from the substrate. In some embodiments, the top surface of the peripheral and the at least one other insulating portions are equal.

[0040] In some embodiments, the substrate comprises a metalloid, such as silicon. In some embodiments, the substrate comprises a polymer. In some embodiments, the substrate comprises a metal. In some embodiments, the length of the substrate is greater than the length of the conductive layer. In some embodiments, the opening of the device is substantially circular. In some embodiments, the lengths represent diameters.

[0041] In some embodiments, the conductive layer is disposed over the substrate configured to generate an electrokinetic field. In some embodiments, the conductive layer comprises platinum.

[0042] In some embodiments, the conductive layer comprises a copper layer and a graphene layer, wherein the graphene layer is disposed over the copper layer. In some embodiments, the length of the copper layer is greater than the length of the graphene layer. In some embodiments, the length of the copper layer is about 10 nm to about 1 pm, such as about 100 pm to about 500 pm, or about 500 pm to about 1 nm. In some embodiments, the length of the copper layer is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1 pm. In some embodiments, the length of the graphene layer is about 6 pm to about 600 pm. In some embodiments, the length of the graphene layer is about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, aboutDocket No.: XZOM-013 / 02WO 355524-216980 pm, about 90 pm, or about 100 pm. In some embodiments, the length of the graphene layer is about 200 pm, about 300 pm, about 400 pm, about 500 pm, or about 600 pm. In some embodiments, the thickness of the copper layer is about 200 nm to about 20 pm. In some embodiments, the thickness of the copper layer is about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. In some embodiments, the thickness of the copper layer is about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, or about 20 pm. In some embodiments, the thickness of the graphene layer is about 2.5 nm to about 250 nm. In some embodiments, the thickness of the graphene layer is about 2.5 nm, about 3 nm, about 3.5 nm, about 4 nm, about 4.5 nm, or about 5 nm. In some embodiments, the thickness of the graphene layer is about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, or about 250 nm. In some embodiments, the thickness of the copper layer is greater than the thickness of the graphene layer. In some embodiments, the thickness of the copper layer is at least about 1.25 times, 1.5 times, 1.75 times, 2 times, 5 times, or at least about 10 times greater than the thickness of the graphene layer.

[0043] In some embodiments, the second height is less than the first height. In some embodiments, this inner portion with the second height forms an indentation. In some embodiments, the indentation is about 500 nm to about 50 pm. In some embodiments, the indentation is about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1 pm, about 2 pm about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, or about 50 pm.

[0044] In some embodiments, the at least one insulating portion forms an opening that exposes the conductive layer. In some embodiments, the first length of the exposed conductive layer form ed between the inner portions of the two i nsulating portions is less than the second length of the exposed conductive layer formed between the outer portions of the two insulating portions. In some embodiments, the first length of exposed conductive layer is about 5 pm to about 600 pm. In some embodiments, the first length of exposed conductiveDocket No.: XZOM-013 / 02WO 355524-2169layer is about 5 μm, about 10 μm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 200 pm, about 300 pm, about 400 pm, about 500 pm, or about 600 pm. In some embodiments, the second length of exposed conductive layer is about 5 pm to about 700 pm. In some embodiments, the second length of exposed conductive layer is about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 200 pm, about 300 pm, about 400 pm, about 500 pm, about 600 pm, or about 700 pm. In some embodiments, the first height, which is greater than the second height of the insulating portion is about 100 nm to about 10 pm. In some embodiments, the first height is about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, or about 10 pm. In some embodiments, the second height, which is less than the first height, is about 25 nm to about 7.5 pm. In some embodiments, the second height of the insulating portion is about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1 pm, about 1.5 pm, about 2 pm, about 2.5 pm, about 3 pm, about 3.5 pm, about 4 pm, about 4.5 pm, about 5 pm, about 5.5 pm, about 6 pm, about 6.5 pm, about 7 pm, or about 7.5 pm.

[0045] In some embodiments, a center insulating portion is disposed directly on the conductive layer. In some embodiments, the center insulating portion is disposed on the center of the conductive layer. In some embodiments, the center insulating portion is disposed between more than one insulating portion. In some embodiments, the center insulating portion has a height greater than the greatest height of at least one insulating portion. In some embodiments, the height of the center insulating portion is about 200 nm to about 20 pm. In some embodiments, the center insulating portion is about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm,Docket No.: XZOM-013 / 02WO 355524-2169about 8 pm, about 9 μm, about 10 μm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, or about 20 pm. In some embodiments, the center insulating portion has a height equal to that of at least one insulating portion.

[0046] In some embodiments, the conductive layer comprises a platinum layer and a nanoporous layer, wherein the nanoporous layer is disposed over the copper layer. In some embodiments, the nanoporous layer acts as a membrane. In some embodiments, the nanoporous membrane is semi -permeable. Further description of the semi-permeable nanoporous membrane is found in Shashishekar P. Adiga, et al., Nanoporous membranes for medical and biological applications, 1 Wiley Interdiscip, Rev. Nanomed. And Nanobiotechnol. 568 (2009), which is incorporated herein for such disclosure.

[0047] In some embodiments, the nanoporous layer is composed of an inorganic material, wherein said material provides physical and chemical stability and better membrane performance due to the ordered pores. Exemplary inorganic compositions may include aluminum oxide, silicon dioxide, zirconium dioxide, and / or titanium dioxide. In some embodiments, the inorganic material may be fabricated via an anodization process. In some embodiments, aluminum oxide is anodic. In some embodiments, silicon dioxide is anodic. In some embodiments, aluminum oxide is ceramic. In some embodiments, silicon dioxide is ceramic. In some embodiments, zirconium dioxide is ceramic. In some embodiments, titanium dioxide is ceramic. In some embodiments, ceramic inorganic materials may be fabricated by powder sintering. In some embodiments, ceramic inorganic materials may be fabricated by a sol-gel process. In some embodiments, the inorganic material may comprise a semiconductor, which may comprise silicon or silicon carbide. In some embodiments, the semiconductor is fabricated by micromachining.

[0048] In some embodiments, the inorganic nanoporous membrane comprises a plurality of pores having a size in range of about 0.1 nm to about 1000 nm. In some embodiments, the pores are about 0.1 nm, about 0.2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. In some embodiments, the pores are narrowly distributed. In some embodiments, the pores are widely distributed. InDocket No.: XZOM-013 / 02WO 355524-2169some embodiments, the plurality of pores has a density of about 108cm'2to about 1011cm'2. In some embodiments, the pores have a density of about 108cm'2, about 109cm'2, about 1010cm'2, or about 1011cm'2. In some embodiments, the pores are ordered. In some embodiments, the pores are thermoset. In some embodiments, the pores are tortuous.

[0049] In some embodiments, the nanoporous layer is composed of an organic material, wherein said material provides adequate biocompatibility. In some embodiments, the organic material comprises a polymer. Exemplary polymer compositions include polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), or polystyrene (PS). In some embodiments, polymer materials may be fabricated by ion-track etching. In some embodiments, polymer materials may be fabricated by lithography. In some embodiments, polymer materials may be fabricated by phase separation.

[0050] In some embodiments, the organic nanoporous membrane comprises a plurality of pores having a size in range of about 0.1 nm to about 1000 nm. In some embodiments, the pores are about 0.1 nm, about 0.2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. In some embodiments, the pores are narrowly distributed. In some embodiments, the pores are not narrowly distributed. In some embodiments, the plurality of pores has a density of about 107cm'2to about 1011cm'2. In some embodiments, the pores have a density of about 107cm'2, about 108cm'2, about 109cm'2, about 1010cm'2, or about 1011cm'2. In some embodiments, the pores are ordered. In some embodiments, the pores are tortuous.

[0051] In some embodiments, the nanoporous layer is composed of a composite material, wherein said material provides adequate biocompatibility and additional functionality. In some embodiments, the composite material comprises a coated anodic aluminum oxide. In some embodiments, the anodic aluminum oxide is coated with a metal. In some embodiments, the anodic aluminum oxide is coated with carbon. In some embodiments, the anodic aluminum oxide is coated with a metal oxide. In some embodiments, the coated anodic aluminum oxide is fabricated by chemical vapor or atomic layer deposition. In some embodiments, the composite material comprises a combination of an oxide and polymer. In some embodiments, the combination is of nation and titanium dioxide. In someDocket No.: XZOM-013 / 02WO 355524-2169embodiments, the oxide / polymer combination is fabricated by a sol-gel or solution casting. In some embodiments, the composite material comprises a support membrane with a polymer coating. In some embodiments, this polymer-coated support membrane is fabricated by layer- by-layer deposition. In some embodiments, the composite nanoporous membrane comprises a plurality of pores having a size in range of about 0.1 nm to about 1000 nm, such as about 5 nm to about 900 nm, about 10 nm to about 750 nm, about 25 nm to about 500 nm, about 75 nm to about 250 nm, or about 100 nm to about 200 nm. In some embodiments, the pores may be about 0.1 nm, about 0,2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. In some embodiments, the pore density and shape may be defined by the fabrication process. In some embodiments, the pore density and shape may be the same as organic or inorganic membranes.

[0052] In some embodiments, a plurality of electrodes is arranged in a plurality of rows, wherein each individual electrode comprises the device, wherein said device comprises a substrate, a conductive layer, and an insulating layer.

[0053] In some embodiments, the analyte capture device is contained in an electrode unit comprises a housing, a heater and / or a reservoir comprising a protein degradation agent, and a plurality of alternating current (AC) electrodes within the housing

[0054] In some embodiments, an AC electrokinetic field is generated to collect, separate or isolate nanoscale analytes. In some embodiments, the nanoscale analytes are biomolecules, such as nucleic acids. In some embodiments, the AC electrokinetic field is a di electrophoretic field. Accordingly, in some embodiments dielectrophoresis (DEP) is utilized in various steps of the methods and devices described herein.

[0055] Accordingly provided herein are systems and devices comprising a plurality of alternating current (AC) electrodes as disclosed herein, the AC electrodes configured to be selectively energized to establish a di electrophoretic (DEP) field region. In some embodiments, the AC electrodes may be configured to be selectively energized to establish multiple di electrophoretic (DEP) field regions, including di electrophoretic (DEP) high field and dielectrophoretic (DEP) low field regions. In some embodiments, AC electrokinetic effects provide for concentration of larger particulate material in low field regions and / orDocket No.: XZOM-013 / 02WO 355524-2169concentration (or collection or isolation) of nanoscale analytes (e g., macromolecules, such as nucleic acid) in high field regions of the DEP field. For example, further description of the electrodes and the concentration of cells in DEP fields may be found in Int’l Patent Publication No. WO 2009 / 146143 A2, the content of which is hereby incorporated herein in its entirety.

[0056] In specific embodiments, DEP is used to concentrate nanoscale analytes and larger particulate matter either concurrently or at different times. In certain embodiments, methods and devices described herein are capable of energizing the array of electrodes as disclosed herein so as to produce at least one DEP field. In other embodiments, the methods and devices described here further comprise energizing the array of electrodes so as to produce a first, second, and any further optional DEP fields. In some embodiments, the devices and systems described herein are capable of being energized so as to produce a first, second, and any further optional DEP fields.

[0057] 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, some embodiments of the devices and systems described herein, and the like, comprise a dielectric particle in various embodiments herein is a biological nanoscale analyte, such as a nucleic acid molecule. Different steps of the methods described herein or some embodiments of the devices or systems described herein may be utilized to isolate and separate different components, such as intact cells or other particular material. Further, different field regions of the DEP field may be used in different steps of the methods or embodiments of the devices and systems described herein. The di electrophoretic force generated in the device does not require the particle to be charged. In some embodiments, the strength of the force depends on the medium and the specific particles’ electrical properties, on the particles’ shape and size, as well as on the frequency of the electric field. In some embodiments, fields of a particular frequency selectively manipulate particles. In some embodiments described herein, these processes allow for the separation of nanoscale analytes, including nucleic acid molecules, from other components, such as cells and proteinaceous material.

[0058] 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 throughout the array. In some embodiments, the electrodes areDocket No.: XZOM-013 / 02WO 355524-2169located at the edges of the array. In some embodiments, DC electrodes are interspersed between AC electrodes.

[0059] In some embodiments, the aforementioned device for isolating a nanoscale analyte in a sample is present in an electrode unit comprising a housing and a plurality of alternating current (AC) electrodes as disclosed herein within the housing. The AC electrodes may be 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 the nanoscale analytes cells in an electrokinetic field region of the device. In some embodiments, the plurality of electrodes is configured to be selectively energized to establish a dielectrophoretic high field and di electrophoretic low field regions.

[0060] In some embodiments, the device disclosed herein is part of an electrode unit comprising: (1) a plurality of alternating current (AC) electrodes as disclosed herein, the AC electrodes configured to be selectively energized to establish AC electrokinetic high field and AC electrokinetic low field regions, and (2) a module capable of performing enzymatic reactions, such as polymerase chain reaction (PCR) or other enzymatic reaction. In some embodiments, the plurality of electrodes is configured to be selectively energized to establish a dielectrophoretic high field and dielectrophoretic low field regions. In some embodiments, the device is capable of isolating a nanoscale analyte from a sample, collecting or eluting the nanoscale analyte and further performing an enzymatic reaction on the nanoscale analyte. In some embodiments, the enzymatic reaction is performed in the same chamber as the isolation and elution stages. In other embodiments, the enzymatic reaction is performed in another chamber than the isolation and elution stages. In still other embodiments, a nanoscale analyte is isolated, and the enzymatic reaction is performed in multiple chambers.

[0061] In some embodiments, the electrode unit further comprises at least one of an elution tube, a chamber, and a reservoir to perform an enzymatic reaction. In some embodiments, the enzymatic reaction is performed in a serpentine microchannel comprising a plurality of temperature zones. In some embodiments, the enzymatic reaction is performed in aqueous droplets entrapped in immiscible fluids (e.g., digital PCR). In some embodiments, the thermal reaction comprises convection. In some embodiments, 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.

[0062] In some embodiments, the device described herein is part of an electrode unit comprising electrodes, wherein the electrodes are placed into separate chambers and DEPDocket No.: XZOM-013 / 02WO 355524-2169fields are created within an inner chamber by passage through pore structures. The exemplary' device includes a plurality of electrodes and electrode- containing chambers within a housing. A controller of the device independently controls the electrodes, as described further in Int’1 Patent Publication No. WO 2009 / 146143 A2, which was previously incorporated herein.

[0063] In some embodiments, chambered devices are created with a variety of pore and / or hole structures (nanoscale, microscale and even macroscale) and contain membranes, gels, or filtering materials which control, confine or prevent cells, nanoparticles or other entities from diffusing or being transported into the inner chambers while the AC / DC electric fields, solute molecules, buffer, and other small molecules can pass through the chambers.

[0064] Such devices may include, but are not limited to, multiplexed electrode and chambered devices, devices that allow reconfigurable electric field patterns to be created, devices that combine DC electrophoretic and fluidic processes, sample preparation devices, sample preparation, enzymatic manipulation of isolated nucleic acid molecules and diagnostic devices that include subsequent detection and analysis, lab-on-chip devices, point-of-care and other clinical diagnostic systems or versions.

[0065] In some embodiments, a planar 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 lateral analyte outlet. The exemplary' devices include multiple AC electrodes. In some embodiments, the sample comprises a combinati on of micron-sized entities or cells, larger nanoscale analytes and smaller nanoscale analytes or biomolecules.

[0066] In some embodiments, the smaller nanoscale analytes are proteins, smaller DNA, RNA, and cellular fragments. In some embodiments, the planar electrode array device is a 60x20 electrode array that is optionally sectioned into three 20x20 arrays that can be separately controlled but operated simultaneously. The optional auxiliary DC electrodes can be switched on to positive charge, while the optional DC electrodes are switched on to negative charge for electrophoretic purposes. In some embodiments, each of the controlled AC and DC systems is used in both a continuous and / or pulsed manner (e.g., each can be pulsed on and off at relatively short time intervals) in various embodiments. The optional planar electrode arrays along the sides of the sample flow are optionally used to generate DC electrophoretic forces as well as AC DEP,

[0067] In various embodiments these methods, devices and systems are operated in the AC frequency range of from 1,000 Hz to 100 MHz, at voltages which could range from approximately 1 volt to 2000 volts pk-pk, at DC voltages from 1 volt to 1000 volts, at flowDocket No.: XZOM-013 / 02WO 355524-2169rates of from 10 microliters per minute to 10 milliliter per minute, and in temperature ranges from 1 °C to 120 °C. In some embodiments, the methods, devices, and systems are operated in AC frequency ranges of from about 3 to about 15 kHz. In some embodiments, the methods, devices, and systems are operated at voltages of from 5-25 volts pk-pk. In some embodiments, the methods, devices, and systems are operated at voltages of from about 1 to about 50 volts / cm. In some embodiments, the methods, devices, and systems are operated at DC voltages of from about 1 to about 5 volts. In some embodiments, the methods, devices, and systems are operated at a flow rate of from about 10 microliters to about 500 mi croliters per minute. In some embodiments, the methods, devices, and systems are operated in temperature ranges of from about 20° C to about 60° C.

[0068] In some embodiments, the methods, devices and systems are operated in AC frequency ranges of from 1,000 Hz to 10 MHz. In some embodiments, the methods, devices and systems are operated in AC frequency ranges of from 1,000 Hz to 1 MHz. In some embodiments, the methods, devices and systems are operated in AC frequency ranges of from 1,000 Hz to 100 kHz. In some embodiments, the methods, devices and systems are operated in AC frequency ranges of from 1,000 Hz to 10 kHz. In some embodiments, the methods, devices and systems are operated in AC frequency ranges of from 10 kHz to 100 kHz. In some embodiments, the methods, devices and systems are operated in AC frequency ranges of from 100 kHz to 1 MHz.

[0069] In some embodiments, the methods, devices and systems are operated at voltages from approximately 1 volt to 1500 volts pk-pk. In some embodiments, the methods, devices and systems are operated at voltages from approximately 1 volt to 1500 volts pk-pk. In some embodiments, the methods, devices and systems are operated at voltages from approximately 1 volt to 1000 volts pk-pk. In some embodiments, the methods, devices and systems are operated at voltages from approximately 1 volt to 500 volts pk-pk. In some embodiments, the methods, devices and systems are operated at voltages from approximately 1 volt to 250 volts pk-pk. In some embodiments, the methods, devices and systems are operated at voltages from approximately 1 volt to 100 volts pk-pk. In some embodiments, the methods, devices and systems are operated at voltages from approximately 1 volt to 50 volts pk-pk.

[0070] In some embodiments, the methods, devices and systems are operated at DC voltages from 1 volt to 1000 volts. In some embodiments, the methods, devices and systems are operated at DC voltages from 1 volt to 500 volts. In some embodiments, the methods, devices and systems are operated at DC voltages from 1 volt to 250 volts. In some embodiments, theDocket No.: XZOM-013 / 02WO 355524-2169methods, devices and systems are operated at DC voltages from 1 volt to 100 volts. In some embodiments, the methods, devices and systems are operated at DC voltages from 1 volt to 50 volts.

[0071] In some embodiments, the AC electrokinetic field is produced using an alternating current having a voltage of 1 volt to 40 volts peak-peak, and / or a frequency of 5 Hz to 5,000,000 Hz and duty cycles from 5% to 50%.

[0072] In some embodiments, the methods, devices, and systems are operated at flow rates of from 10 microliters per minute to 1 ml per minute. In some embodiments, the methods, devices, and systems are operated at flow rates of from 10 microliters per minute to 500 microliters per minute. In some embodiments, the methods, devices, and systems are operated at flow rates of from 10 microliters per minute to 250 microliters per minute. In some embodiments, the methods, devices, and systems are operated at flow rates of from 10 microliters per minute to 100 microliters per minute.

[0073] In some embodiments, the methods, devices, and systems are operated in temperature ranges from 1 °C to 100 °C. In some embodiments, the methods, devices, and systems are operated in temperature ranges from 20 °C to 95°C. In some embodiments, the methods, devices, and systems are operated in temperature ranges from 25 °C to 100 °C. In some embodiments, the methods, devices, and systems are operated at room temperature.

[0074] In some embodiments, the controller independently controls each of the electrodes. In some embodiments, the controller is externally connected to the device such as by a socket and plug connection or is integrated with the device housing.

[0075] In some embodiments, a housing and a heater or thermal source and / or a reservoir comprises a protein degradation agent. In some embodiments, the heater or thermal source is capable of increasing the temperature of the fluid to a desired temperature (e.g., 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 thermocycler. In other embodiments, the heater or thermal source is used to maintain a constant temperature (isothermal conditions). In some embodiments, the protein degradation agent is a protease. In other embodiments, the protein degradation agent is Proteinase K and the heater or thermal source is used to inactivate the protein degradation agent.

[0076] In some embodiments, the device comprises an eluant. The eluant is any fluid suitable for eluting the isolated nanoscale analyte from the device. In some embodiments, the eluant isDocket No.: XZOM-013 / 02WO 355524-2169water or a buffer. In some embodiments, the eluant comprises reagents required for a DNA sequencing method.

[0077] In some embodiments, a system or device described herein is capable of maintaining a constant temperature. In some embodiments, a system or device described herein is capable of cooling the array or chamber. In some embodiments, a system or device described herein 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 disclosed herein comprise a localized temperature control element. In some embodiments, the devices disclosed herein are capable of both sensing and controlling temperature.

[0078] In some embodiments, the devices further comprise heating or thermal elements. In some embodiments, a heating or thermal element is localized underneath 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 achieved by a heating or thermal element is proportional to the current running through it. In some embodiments, the devices disclosed herein comprise localized cooling elements. In some embodiments, heat resistant elements are placed directly under the exposed electrode array. In some embodiments, the devices disclosed herein are capable of achieving and maintaining a temperature between about 20 °C and about 120 °C. In some embodiments, the devices disclosed herein are capable of achieving and maintaining a temperature between about 30 °C and about 100°C. In other embodiments, the devices disclosed herein are capable of achieving and maintaining a temperature between about 20°C and about 95°C. In some embodiments, the devices disclosed herein are capable of achieving and maintaining a temperature between about 25“C and about 90 C, between about 25 C and about 85 °C, between about 25 C and about 75 C, between about 25 C and about 65 C or between about 25 C and about 55 C. In some embodiments, the devices disclosed herein are capable of achieving 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

[0079] In some embodiments, the methods, devices and compositions disclosed herein utilize electrode configurations and designs to improve separation and capture of the nanoscale analytes from particulate material. In some embodiments, the electrode arrays are configuredDocket No.: XZOM-013 / 02WO 355524-2169such that fluid flow around or within the vicinity of the electrodes are disrupted or altered, allowing the localization and / or retention of nanoscale analytes around or within the electrode arrays. In other embodiments, the improvement in nanoscale analyte capture is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% or more nanoscale analyte captured than if using conventional electrode configuration or designs.

[0080] In some embodiments, the conductive material is in the shape of an open disk. In some embodiments, the electrode is configured in a hollow ring shape. In some embodiments, the electrode is configured in a hollow tube shape. In some embodiments, the array of electrodes as disclosed herein comprises non-conductive material. In some embodiments, the non-conductive material surrounds the conductive material within the electrodes and serves as a physical barrier to the conductive material. In some embodiments, the conductive material within the electrodes fills depressions in the non-conductive material of the array. In some embodiments, the array of electrodes as disclosed herein is configured in three-dimensions.

[0081] In some embodiments, the array of electrodes as disclosed herein comprises conductive material in only a fraction of the electrode array. In some embodiments, the conductive material is only present in less than about 10% of the electrode array. In some embodiments, the conductive material is only present in about 10% of the electrode array. In other embodiments, the conductive material is only present in about 20% of the electrode array. In still other embodiments, the conductive material is only present in about 30% of the electrode array. In yet other embodiments, the conductive material is only present in about 40% of the electrode array. In still other embodiments, the conductive material is only present in about 50% of the electrode array. In some embodiments, the conductive material is only present in about 60% of the electrode array. In some embodiments, the conductive material is only present in about 70% of the electrode array. In still other embodiments, the conductive material is only present in about 80% of the electrode array. In yet other embodiments, the conductive material is only present in about 90% of the electrode array.

[0082] In still other embodiments, the conductive material is only present in about 10%, in about 15%, in about 20%, in about 25%, in about 30%, in about 35%, in about 40%, in about 45%, in about 50%, in about 55%, in about 60%, in about 65%, in about 70%, in about 75%, in about 80%, in about 85% and in about 90% of the electrode array. In yet other embodiments, the conductive material is present in about 10-70% of the electrode array, inDocket No.: XZOM-013 / 02WO 355524-2169about 10-60% of the electrode array, in about 10-50% of the electrode array, in about 10-40% of the electrode or in about 10-30% of the electrode array. In other embodiments, the conductive material is present in about 30-90% of the electrode array, in about 30-80% of the electrode array, in about 30-70% of the electrode array, in about 30-60% of the electrode array or in about 30-50% of the electrode array. In some embodiments, the conductive material is present in about 8 to about 40% of the electrode array.

[0083] In yet other embodiments, the conductive material is substantially absent from the center of the individual electrodes in the electrode array. In other embodiments, the conductive material is only present at the edges of the individual electrodes in the electrode array. In still other embodiments, the conductive material is in the shape of an open disk, which comprises conductive material that is discontinuous in the open disk electrode. In some embodiments, the electrode is a hollow ring electrode shape, which comprises conductive material in the electrode array that is substantially absent from the center of the individual electrodes or is only at the edge of the individual electrodes. The hollow ring electrode shape, like the open disk shape, reduces the surface area of the conductive material in an electrode. The reduction in conductive material present on the electrode results in flow in and around the electrode surface, leading to increases in nanoscale analyte captured on the surface of the electrode.

[0084] In some embodiments, a layer of non-conductive material is present in certain areas of the electrode or in the proximal vicinity of the electrode array. In some embodiments, a layer of nonconductive material surrounds the electrode array, creating a physical barrier or wall surrounding the array. In some embodiments, the electrode array is depressed into the array material, creating a well or depression on the array surface wherein electrode material or substantially electrode material is present in the well or depression.

[0085] In some embodiments, the electrode configuration is in three-dimensions. In some embodiments, the electrode material is folded into an angle configuration. In other embodiments, the electrode material is formed into a triangular tube. In other embodiments, the electrode material is formed into a hollow triangular tube. In still other embodiments, the three dimensional electrode comprises angles between neighboring planar electrode surfaces of less than about 180 degrees, less than about 170 degrees, less than about 160 degrees, less than about 150 degrees, less than about 140 degrees, less than about 130 degrees, less than about 120 degrees, less than about 110 degrees, less than about 100 degrees, less than about 90 degrees, less than about 80 degrees, less than about 70 degrees, but not less than about 60Docket No.: XZOM-013 / 02WO 355524-2169degrees. In some embodiments, the conductive material configured into angles between neighboring planar electrode surfaces of equal to or less than 180 degrees. In some embodiments, the three dimensional electrode configuration comprises angles between neighboring planar electrode surfaces of more than about 60 degrees, more than about 70 degrees, more than about 80 degrees, more than about 90 degrees, more than about 100 degrees, more than about 110 degrees, more than about 120 degrees, more than about 130 degrees, more than about 140 degrees, more than about 150 degrees, more than about 160 degrees, more than about 170 degrees, but not more than about 180 degrees. In some embodiments, the conductive material configured into angles between neighboring planar electrode surfaces of equal to or more than 60 degrees. In some embodiments, the conductive material within the electrodes is configured into a depressed concave shape. In yet other embodiments, the electrode configuration is a depressed basket electrode. The three-dimensional structure of the electrode increases the total surface area of the electrode, allowing interrogation of more fluid in a defined unit of time.

[0086] In some embodiments, the individual electrodes are about 40 pm to about 100 pm in diameter. In still other embodiments, the individual electrodes are about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm or about 100 pm in diameter. In yet other embodiments, the individual electrodes are about 40 pm to about 50 pm, about 40 pm to about 60 pm or about 40 pm to about 70 pm. In still other embodiments, the individual electrodes are about 100 pm, about 200 pm, about 300 pm, about 400 pm, about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, or about 1000 pm in diameter.

[0087] The plurality of alternating current electrodes may optionally be configured in any manner suitable for the separation processes described herein. In other embodiments, the array of electrodes as disclosed herein comprises a pattern of electrode configurations, wherein the configuration comprises a repeating unit of electrode arrays. In some embodiments, the edge-to-edge distance between a parallel set of repeating units is equidistant, or roughly equidistant. Further description of the system or device including electrodes and / or concentration of cells in DEP fields is found in Int'l Patent Publication No. WO 2009 / 146143 A2, which was previously incorporated herein.

[0088] In some embodiments, the electrodes disclosed herein comprise any suitable metal. In other embodiments, the electrodes disclosed herein comprise a noble metal. In some embodiments, the electrodes can include but are not limited to: aluminum, copper, carbon,Docket No.: XZOM-013 / 02WO 355524-2169iron, 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 silicide materials such as platinum silicide, titanium silicide, gold silicide, or tungsten silicide. In some embodiments, the electrodes can comprise a conductive ink capable of being screen-printed. In some embodiments, the electrodes comprise a conductive polymer, such as polyacetylene or polythiophene. In some embodiments, the electrodes can include a metalloid, such as silicon.

[0089] In some embodiments, the electrode material is about 1 to about 20 pm thick. In some embodiments, the electrode material is about 200 to about 800 nm thick. In some other embodiments, the electrode material is about 1 to about 100 nm thick. In yet other embodiments, the electrode material is about 300 to about 500 nm thick. In still other embodiments, the electrode material is about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1000 nm thick, about 2 um thick, about 5 pm thick, about 10 pm thick, or about 20 pm thick.

[0090] In some embodiments, an adhesion layer is deposited or printed onto the array as a protective layer prior to deposition of the electrode material. In some embodiments, the adhesion layer comprises any suitable material. In some embodiments, the adhesion layer comprises titanium or tungsten. In other embodiments, the adhesion layer is between about 10 to about 50 nm thick. In some embodiments, the adhesion layer is between about 20 to about 40 nm thick. In yet other embodiments, the adhesion layer is between about 20 to about 30 nm thick. In still other embodiments, the adhesion layer is about 10 nm, about 20 nm, about 30 nm, about 40 nm or about 50 nm thick.

[0091] In some embodiments, the edge to edge (E2E) to diameter ratio of an individual electrode is about 10 pm to about 500 pm. In some embodiments, the E2E of an electrode is about 50 pm to about 300 pm. In yet other embodiments, the E2E of an electrode is about 100 pm to about 200 pm. In still other embodiments, the E2E of an electrode is about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pmm about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, aboutDocket No.: XZOM-013 / 02WO 355524-2169360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm or about 500 pm. In some embodiments, the E2E of an electrode is about 750 pm, about 1000 pm, about 1500 pm, or about 2000 pm.

[0092] In some embodiments, the electrodes disclosed herein are dry-etched. In some embodiments, the electrodes are wet etched. In some embodiments, the electrodes undergo a combination of dry etching and wet etching.

[0093] In some embodiments, each electrode is individually site-controlled.

[0094] In some embodiments, an array of electrodes as disclosed herein is controlled as a unit.

[0095] The array can be of any suitable material. In some embodiments, the array comprises plastic or silica. In some embodiments, the array comprises silicon dioxide. In some embodiments, the array comprises aluminum.

[0096] 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 from about 2.0 to about 8.0. In some embodiments, the passivation layer has a relative electrical permittivity of from 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 from 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.

[0097] In some embodiments, the passivation layer is between about 0.1 microns and about 10 microns in thickness. In some embodiments, the passivation layer is between about 0.5 microns and 8 microns in thickness. In some embodiments, the passivation layer is between about 1.0 micron and 5 microns in thickness. In some embodiments, the passivation layer is between about 1.0 micron and 4 microns in thickness. In some embodiments, the passivation layer is between about 1.0 micron and 3 microns in thickness. In some embodiments, the passivation layer is between about 0.25 microns and 2 microns in thickness. In some embodiments, the passivation layer is between about 0.25 microns and 1 micron in thickness.Docket No.: XZOM-013 / 02WO 355524-2169

[0098] In some embodiments, the passivation layer is comprised of any suitable insulative low k dielectric material, including but not limited to silicon nitride, silicon dioxide or titanium dioxide. In some embodiments, the passivation layer is chosen from the group comprising polyamides, carbon, doped silicon nitride, carbon doped silicon dioxide, fluorine doped silicon nitride, fluorine doped silicon dioxide, porous silicon dioxide, or any combinations thereof. In some embodiments, the passivation layer can comprise a dielectric ink capable of being screen-printed.Electrode Geometry

[0099] In some embodiments, the electrodes disclosed herein can be arranged in any manner suitable for practicing the methods disclosed herein.

[0100] In various embodiments, a variety of configurations for the devices are possible. For example, a device comprising a larger array of electrodes, for example in a square or rectangular pattern configured to create a repeating non-uniform electric field to enable 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, 20x100 electrode configuration, or a 20x80 electrode configuration.

[0101] In some embodiments, the electrodes are in a dot configuration, e.g. the electrodes comprise a generally circular or round configuration. In some embodiments, the electrodes are configured as disks. In some embodiments, the electrodes are configured as rings. In some embodiments, the angle of orientation between dots is from about 30° to about 90° degrees. In some embodiments, 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°. In some embodiments, the angle of orientation between dots is about 45°. In some embodiments, the angle of orientation between dots is about 50°. In some embodiments, the angle of orientation between dots is about 55°. In some embodiments, the angle of orientation between dots is about 60°. In some embodiments, the angle of orientation between dots is about 65°. In some embodiments, the angle of orientation between dots isDocket No.: XZOM-013 / 02WO 355524-2169about 70°, In some embodiments, the angle of orientation between dots is about 75°. In some embodiments, the angle of orientation between dots is about 80°. In some embodiments, the angle of orientation between dots is about 85°. In some embodiments, the angle of orientation between dots is about 90°.

[0102] In other embodiments, the electrodes are in a non-circular configuration. In some embodiments, the angle of orientation between non-circular configurations is between about 25 and 90 degrees. In some embodiments, the angle of orientation between non-circular configurations is from about 30° to about 90° degrees. In some embodiments, the angle of orientation between non-circular configurations is from about 25° to about 60°. In some embodiments, the angle of orientation between non-circular configurations is from about 30° to about 55°. In some embodiments, the angle of orientation between non-circular configurations is from about 30° to about 50°. In some embodiments, the angle of orientation between non-circular configurations is from about 35° to about 45°. In some embodiments, the angle of orientation between non-circular configurations is about 25°. In some embodiments, the angle of orientation between non-circular configurations is about 30°. In some embodiments, the angle of orientation between non-circular configurations is about 35°. In some embodiments, the angle of orientation between non-circular configurations is about 40°. In some embodiments, the angle of orientation between non-circular configurations is about 45°, In some embodiments, the angle of orientation between non-circular configurations is about 50°. In some embodiments, the angle of orientation between non- circular configurations is about 55°. In some embodiments, the angle of orientation between non-circular configurations is about 60°. In some embodiments, the angle of orientation between non-circular configurations is about 65°. In some embodiments, the angle of orientation between non-circular configurations is about 70°. In some embodiments, the angle of orientation between non-circular configurations is about 75°. In some embodiments, the angle of orientation between non-circular configurations is about 80°. In some embodiments, the angle of orientation between non-circular configurations is about 85°. In some embodiments, the angle of orientation between non-circular configurations is about 90°.

[0103] In some embodiments, the electrodes are in a substantially elongated configuration.

[0104] In some embodiments, the electrodes are in a configuration resembling wavy or nonlinear lines. In some embodiments, 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 theDocket No.: XZOM-013 / 02WO 355524-2169electrode, 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. In some embodiments, the electrodes are strips resembling wavy lines. In some embodiments, the edge-to-edge distance between the electrodes is equidistant, or roughly equidistant throughout the wavy line configuration. In some embodiments, the use of wavy line electrodes, as disclosed herein, lead to an enhanced DEP field gradient.

[0105] In some embodiments, the electrodes disclosed herein are in a planar configuration. In some embodiments, the electrodes disclosed herein are in a non-planar configuration.

[0106] In some embodiments, the devices disclosed herein surface selectively captures nanoscale biomolecules on its surface. For example, the devices disclosed herein may capture nanoscale analytes such as nucleic acids, by, for example, a) nucleic acid hybridization; b) antibody - antigen interactions; c) biotin - avidin interactions; d) ionic or electrostatic interactions; or e) any combination thereof. The devices disclosed herein, therefore, may incorporate a functionalized surface which 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 anchoring captures capable of capturing complementary target molecules such as avidin, capture molecules capable of capturing biomolecules (such as nucleic acids) by ionic or electrostatic interactions, or any combination thereof.

[0107] 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 interactions; c) surfactants; or d) any combination thereof. In some embodiments, the methods disclosed herein include use of additives which reduce non- specific binding interactions by interfering in such interactions, such as Tween 20 and the like, bovine serum albumin, nonspecific immunoglobulins, etc.

[0108] In some embodiments, the device comprises a plurality of microelectrode devices oriented (a) flat side by side, (b) facing vertically, and / or (c) facing horizontally. In other embodiments, the electrodes are in a sandwiched configuration, e.g., stacked on top of each other in a vertical format.Electrode DesignDocket No.: XZOM-013 / 02WO 355524-2169

[0109] In some embodiments, the conductive layer includes platinum. In some embodiments, the conductive layer is covered with an additional layer. In some embodiments, an additional layer is disposed over the conductive layer. In some embodiments, the conductive layer is covered with a semi-permeable porous (or nanoporous) membrane.

[0110] In some embodiments, the conductive layer is covered with a membrane that creates a similar effect to hydrogel. In some embodiments, the porous membrane comprises organic material. In some embodiments, the porous membrane comprises inorganic material. In some embodiments, the porous membrane comprises a composite material. In some embodiments. In some embodiments, the semi-permeable membrane is porous. In some embodiments, the pores are narrowly distributed. In some embodiments, the pores are widely distributed. In some embodiments, the pores are distributed in a medium distribution. In some embodiments, the pores are about 0.1 nanometers to about 500 nanometers in size. In some embodiments, the pores are more than 2 nanometers in size. In some embodiments, the pores are more than 10 nanometers in size. In some embodiments, the pores are more than 20 nanometers in size. In some embodiments, the pores are more than 100 nanometers in size. In some embodiments, the pores are about 1 nanometer to about 50 nanometers in size. In some embodiments, the pore size is determined by the fabrication method. In some embodiments, the pore density is about 107cm’2to about 1011cm'2. In some embodiments, the pore density is about 108cm’2. In some embodiments, the pore density is about 109cm’2. In some embodiments, the pore density is about 1010cm"2. In some embodiments, the pore density is high. In some embodiments, the pore morphonology is ordered. In some embodiments, the pore morphology is straight. In some embodiments, the pore morphology is tortuous.

[0111] In some embodiments, the conductive layer includes a metal. In some embodiments, the conductive layer includes copper. In some embodiments, the conductive layer includes or is covered with a graphene layer. In some embodiments, the conductive layer is coated with a material with a similar effect as hydrogel.

[0112] In some embodiments, two insulating portions are disposed over the conductive layer. In some embodiments, the two insulating portions form a ring around the conductive layer. In some embodiments, the two insulating portions are identical or substantially identical in size. In some embodiments, the two insulating portions are different sizes. In some embodiments, the insulating portions are dielectric and induce a DEP field. In some embodiments, the insulating portions are etched, wherein the insulating portion has both an inner and outer portion. This etch provides an improvement to analyte capture from insulating portionsDocket No.: XZOM-013 / 02WO 355524-2169without an etch, providing a device with properties comparable to those with a hydrogel. For example, the etch may provide a region (e g., the notches) in the insulation portions for the analytes to be captured and not be swept away by the AC electroosmosis or AC electrothermal effects. In some embodiments, the outer portion is where the insulating portion is at its maximum height. In some embodiments, the inner portion is where the insulating portion is at a lesser height, created by the etching. In some embodiments, the etching in each of the insulating portions are equal. In some embodiments, the inner portion height is less than 50% of the outer portion height. In some embodiments, the inner portion height is about 50% of the outer portion height. In some embodiments, the inner portion height is more than 50% of the outer portion height. In some embodiments, the distance between the inner portions is more than the distance between the outer portions. In some embodiments, the widths of the insulating portions are equal. In some embodiments, the widths of the insulating portions are not equal.

[0113] In some embodiments, a center insulating portion is disposed upon the center of the conductive layer. In some embodiments, the center insulating portion is dielectric and induces a DEP field. This insulating portion provides a similar flow around effect and generates the same fluidic activity as the electrodes coated with hydrogel. In some embodiments, the center insulating portion is a cube. In some embodiments, the center insulating portion is not a cube. In some embodiments, the center insulating portion is of equal height to at least one other insulating portion. In some embodiments, the center insulating portion height is greater than at least one other insulating portion height. In some embodiments, the center insulating portion height is double at least one other insulating portion height. In some embodiments, an etch is carved into the center insulating portion.

[0114] Referring to FIG. 1, an example electrode configuration is shown, in accordance with some embodiments. Each circle represents an electrode that comprises conductive material that is protected by an insulating layer such as a dielectric material. The shaded electrodes represent the anodes and the non-shaded electrodes represent the cathodes.

[0115] Referring to FIG. 2, a cross-sectional view of an example design of an electrode 200 is shown, in accordance with some embodiments. A silicon substrate may be used to form the electrode. A conductive layer may be disposed upon or over the silicon substrate. An insulating layer may be disposed upon or over the conductive layer, with two insulating portions disposed upon the conductive layer. The two insulating portions may extend around the conductive layer so as to form a ring or circular shape above and around the conductiveDocket No.: XZOM-013 / 02WO 355524-2169layer. The insulating portion that is formed over the conductive layer may be raised higher than the insulating portion that is not formed over the conductive layer (e.g., the portion of the insulating layer formed on the silicon substrate). The two insulating portions may have similar shapes about a center of ring formed by the two insulating portions. In some embodiments, the each of the two insulating portions may include an inner portion and an outer portion, where the inner portion is closer to the center of the ring and the outer portion is farther from the center of the ring. The inner portion may be shorter in height than the outer portion. The two insulating portions may be formed by first forming an insulating layer over the conductive layer, and then etching a portion of the insulating layer that overlaps the conductive layer. Each of the two insulating portions are additionally etched to create a smaller inner portion. The two insulating portions may be etched again to form a wider opening over the initial opening such that a staircase shape may be formed. In some embodiments, the etching of one insulating portion may vary in width and height from the etching of the other insulating portion such that the two insulating portions may not have the same widths or heights.

[0116] The numerical ranges provided in this disclosure as examples and not intended to be limiting. In some embodiments, the actual measurements of the distances may be more or less than the minimum and maximum of the ranges, respectively.

[0117] Referring to FIG. 2, the outer portion may have a height of about 100 nanometers to about 10 micrometers (2B) and the inner portion has a height of about 75 nanometers to about 7.5 micrometers (2B - 2C). In some embodiments, the outer portion height may be about 1 micrometer and the inner portion height may be about 750 nanometers. The width of the inner portion etched into the larger insulating portion may be about 500 nanometers to about 50 micrometers (2A). In some embodiments, the width of the etched portion may be about 5 micrometers. The length of the exposed conductive layer between the two insulating portions may be about 6 micrometers to about 600 micrometers (2D). In some embodiments, the length of exposed conductive layer may be about 60 micrometers. The distance from the outer portions of the two insulating portions may be about 7 micrometers to about 700 micrometers (2E). In some embodiments, the distance from the outer portion corner to the other outer portions corner may be about 70 micrometers.

[0118] Referring to FIG. 2, the conductive layer may be platinum which may be inert compared to other metals. While still biofouling, platinum is commonly used in such situations as it causes a lesser biofouling response when in contact with analytes, such asDocket No.: XZOM-013 / 02WO 355524-2169blood. In alternative embodiments, another metal may be used as a conductive layer, wherein said metal has a similar or superior voltage reactivity and biofouling properties as platinum.

[0119] Referring to FIG. 2, the corner further from the conductive layer but within the electrode unit itself, the highest DEP forces are present. Along this corner, the flow of analytes is also the highest, as analytes moving from one electrode unit to another follow the shortest path, coming in close contact with the insulating portion corner. Much like how the hydrogel interact with the analytes and helps capture analytes, the etch in the insulating portion produces a similar effect, wherein the notch acts as a capture point for the flowing analytes. In alternative embodiments, other insulating portion shapes may be applied, wherein similar or superior DEP is produced.

[0120] Referring to FIG. 3, a cross-sectional view of an example design of an electrode 300 is shown, in accordance with some embodiments. The design of the electrode 300 may be similar to that of electrode 200 except that the material for the conductive layer may be different.

[0121] In some embodiments, a silicon substrate may be used to form the electrode. A conductive layer may be disposed upon or over the silicon substrate. A graphene layer may be disposed upon or over the conductive layer. In some embodiments, the graphene layer may be part of the conductive layer. The center of the platinum layer may be aligned to the center of the graphene layer, but embodiments are not limited thereto. For example, the center of the platinum layer may not align with the center of the graphene layer, and the centers may be separated by a distance that is in the range of 50 nm to about 5 micrometers. The width of the graphene layer may be the same or substantially similar to the distance between the inner portions.

[0122] Referring to FIG. 3, the outer portion has a height from the conductive layer of about 100 nanometers to about 10 micrometers (3B) and the inner portion has a height from the conductive layer of about 25 nanometers to about 2.5 micrometers (3B - 3C). In some embodiments, the outer portion height may be about 1 micrometer and the inner portion height may be about 250 nanometers. The width of the inner portion etched into the larger insulating portion may be about 500 nanometers to about 50 micrometers (3 A). In some embodiments, the width of the etched portion m ay be about 5 micrometers. The length of the exposed conductive layer between the two insulating portions may be about 6 micrometers to about 600 micrometers (3D). In some embodiments, the exposed conductive layer may be about 60 micrometers.Docket No.: XZOM-013 / 02WO 355524-2169

[0123] Referring to FIG. 3, the conductive layer may include copper and graphene. A graphene layer may be disposed over the copper layer such that the copper layer is not exposed to the DEP. The graphene layer is more highly conductive than platinum and is non¬ biofouling. Therefore, graphene is able to produce a comparable effect to the hydrogel.

[0124] Referring to FIG. 4, a cross-sectional view of an example design of an electrode 400 is shown, in accordance with some embodiments. A silicon substrate may be used to form the electrode. A conductive layer may be disposed upon or over the silicon substrate. An insulating layer may be disposed upon or over the conductive layer, the insulating layer comprising at least one insulating portion. The at least one insulating portion may include an outer insulating portion and a center insulating portion. The outer and center insulating portions may be the same height and width as each other. In some embodiments, they may have different heights and widths. The two insulating portions may be formed by first forming an insulating layer over the conductive layer, and then etching a portion of the insulating layer that overlaps the conductive layer. The center insulating portion may be disposed upon the conductive layer, with the center of the center insulating portion aligned to the center of the conductive layer. However, embodiments are not limited thereto, and the center of the center insulating portion may not be aligned to the center of the conductive layer (e.g., off center). The center insulating portion may have the shape of a cylinder or pillar. In some embodiments, the center insulating layer may be formed over a center of the conductive layer. In some embodiments, the center insulating portion may have a height that is greater than the outer insulating portion formed over an edge of the conducti ve layer.

[0125] Referring to FIG 4, the outer insulating portion has a height from the conductive layer of about 100 nanometers to about 10 micrometers (4B). In some embodiments, the height of the of the outer insulating portion may be 1 micrometer. The center insulating portion has a height from the conductive layer of about 200 nanometers to about 20 micrometers (4C). In some embodiments, the center insulating portion may be about 2 micrometers. The distance between each outer insulating portions and the center insulating portion may be about 500 nanometers to about 50 micrometers (3 A). In some embodiments, the distance between the center and outer insulating portions may be about 5 micrometers. The distance between the two outer insulating portions may be about 6 micrometers to about 600 micrometers (4D). In some embodiments, the distance between the two outer insulating portions may be about 60 micrometers.Docket No.: XZOM-013 / 02WO 355524-2169

[0126] The conductive layer may include platinum as described in FIG. 2 or a copper and graphene as described in FIG. 3, but embodiments are not limited thereto. For example, the conductive layer may include any suitable conductor material.

[0127] Referring to FIG. 4, the center insulating portion may be disposed over the center of the conductive layer. This center insulating portion may increase the DEP force within the electrode and allow the analyte flow to remain close to the electrode for capturing the analytes. The analyte may flow around the center insulating portion, close to the outer edge with the strongest field strength. In some embodiments, the electrode 400 may provide a current that is less than that of electrodes 200 and 300, given the same length of the conductive layer, since the surface area of the conductive layer in electrode 400 will be less than that of the electrodes 200 and 300. Accordingly, the resulting DEP forces may be less but can be compensated by an increased voltage. The elevated dielectric insulating portion may also generate the same fluidic effect as the hydrogel, ensuring that the analyte flow will be comparable to that which is caused by the hydrogel. Therefore, this center electrode may produce a similar analyte flow.

[0128] Referring to FIG. 5, a cross-sectional view of an example design of an electrode 500 is shown, in accordance with some embodiments. The design of electrode 500 is similar to that of electrode 400 except that the insulating portion formed over the edges of the conductive layer may have a notch (e.g., an inner / outer portions) as described above.Accordingly, similar descriptions are omitted for clarity.

[0129] A silicon substrate may be used to form the electrode. A conductive layer may be disposed upon or over the silicon substrate. An insulating layer may be disposed upon or over the conductive layer, with two insulating portions disposed upon the conductive layer. The two insulating portions may be the same height and width as each other. The two insulating portions may be formed by first forming an insulating layer over the conductive layer, and then etching a portion of the insulating layer that overlaps the conductive layer to expose the conductive layer. The two insulating portions may be etched again to form a wider opening over the initial opening such that a staircase shape may be formed. In some embodiments, each of the two insulating portions may include an outer portion and an inner portion as described with respect FIG. 2. In alternative embodiments, the etching of one insulating portion may vary in width and height from the etching of the other insulating portion. A center insulating portion may be disposed upon the conductive layer, as described with respect to electrode 400.Docket No.: XZOM-013 / 02WO 355524-2169

[0130] Referring to FIG. 5, the outer insulating portions has a height from the conductive layer of about 100 nanometers to about 10 micrometer and the inner portion has a height from the conductive layer of about 25 nanometers to about 2.5 micrometers (Outer Portion Height - 5B ). In some embodiments, the outer portion height may be about 1 micrometer and the inner portion height may be about 250 nanometers. The width of the inner portion etched into the larger insulating portion may be about 500 nanometers to about 50 micrometers (5 A). In some embodiments, the width of the etched portion may be about 5 micrometers. The center insulating portions has a height from the conductive layer of about 200 nanometers to about 20 micrometers (5C) and a width of about 5 micrometers to about 500 micrometers (5F). In some embodiments, the center insulating portion height may be about 2 micrometers and the width may be about 50 micrometers. The distance between the outer insulating portion and the center insulating portion is about 500 nanometers to about 50 micrometers. In some embodiments, the distance between the two outer insulating portions and the center insulating portion may be 5 micrometers when the outer insulating portions are equidistant from the center insulating portion. The distance between the inner portions of the two insulating portions may be about 6 micrometers to about 600 micrometers (5E). In some embodiments, the distance between the inner portions of the two insulating portions may be about 60 micrometers. The distance between the outer portions of the two insulating portions may be about 7 micrometers to about 700 micrometers (5D). In some embodiments, the distance between the outer portions of the two insulating portions is about 70 micrometers.

[0131] Referring to FIG. 5, the conductive layer may be platinum or may be copper and graphene as described herein.

[0132] As described with respect to electrode 400, the center insulating portion may be formed so that the analyte may flow around the center insulating portion, above the conductive layer. Further, as described with respect to electrode 200, the notch (with the inner and outer portions) may allow the analyte to be better captured in the corners. Referring to FIG. 5, the combination of the center insulating portion formed over the insulating layer and the notches may help capture the analytes close to the electrode.

[0133] Referring to FIG. 6, a cross-sectional view of an example design of an electrode 600 is shown, in accordance with some embodiments. The electrode 600 may be similar to that of an electrode using a hydrogel, except that instead of hydrogel a different material may be used. For example, the material may include a semi-permeable porous membrane. The semi- permeable porous membrane may be used with the notches and / or the different conductiveDocket No.: XZOM-013 / 02WO 355524-2169materials (platinum or copper / graphene) and / or the center insulating portion over the conductive layer to maximize the effect of capturing the analytes.

[0134] Referring to FIG. 6, the outer portion has a height from the conductive layer of about 100 nanometers to about 10 micrometers (6B) and the inner portion has a height from the conductive layer of about 25 nanometers to about 2.5 micrometers (6B - 6C). In some embodiments, the outer portion height may be about 1 micrometer and the inner portion height may be about 250 nanometers. The width of the inner portion etched into the larger insulating portion may be about 500 nanometers to about 50 micrometers (6A). In some embodiments, the width of the etched portion may be about 5 micrometers. The length of the exposed semi-permeable porous membrane layer between the two insulating portions may be about 6 micrometers to about 600 micrometers (6D). In some embodiments, the exposed semi-permeable porous membrane layer may be about 60 micrometers.

[0135] Referring to FIG. 6, a semi-permeable porous membrane layer may be disposed over the conductive layer. This semi-permeable layer can include organic, inorganic, or composite material. In their construction, the pores size, distribution, density, and shape can differ as a result of manufacturing method and intention of the analyte capture method. The pores of a semi-permeable membrane may also be likely to remain constant, even after analyte capture has taken place. Additional descriptions of the semi-permeable porous membrane may be found in Adiga SP, Jin C, Curtiss LA, Monteiro- iviere NA, Narayan RJ. Nanoporous membranes for medical and biological applications. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2009 Sep-Oct;l(5):568-81. Doi: 10.1002 / wnan.50. PMID: 20049818;PMCID: PMC3684197, which is incorporated by reference in its entirety. Therefore, unlike hydrogel, where the pores may widen as the device is used, the pores of the semi -permeable membrane may stay constant or change very little over long periods of time and repeated use.

[0136] While certain embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, modification, combinations, and substitutions may occur by those skilled in the art without departing from the disclosure. As one example, a semi-permeable membrane may be disposed upon a platinum conductive layer or upon a graphene / copper conductive layer. For another example, a series of insulating portions may be disposed upon the conductive layer, as opposed to a single center insulating portion. Alternatively, multiple etchings may be carved into each of the insulating portions. It should be understood that various alternatives to the embodiments of the present disclosureDocket No.: XZOM-013 / 02WO 355524-2169may be employed in practicing the present disclosure. It is intended that the claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.Fabrication

[0137] In some embodiments, the analyte-capturing device is fabricated by a method comprising: a. depositing a conductive layer over a substrate, b. forming a first dielectric layer over the conductive layer and the substrate, and c. etching the first dielectric layer to create an opening that exposes the conductive layer, the opening having a first length. The first dielectric layer may be formed across the substrate to have a uniform thickness, with the top surface of the portion of the first dielectric layer formed over the conductive layer being higher than the top surface of the portion of the first dielectric layer formed over the substrate only. In some embodiments, the outer portion (or at least one portion) of the insulating layer may be formed.

[0138] In some embodiments, the method further comprises etching the first dielectric layer to widen a top portion of the opening such that the opening has the first length closer to the conductive layer and a second length farther from the conductive layer, the second length being greater than the first length. In some embodiments, the second etching here may provide an inner portion and an outer portion of the at least one insulating porti on, with the inner portion being closer to each other and having a shorter height than the outer portion which is farther apart (greater distance) and having a greater height.

[0139] In some embodiments, the method may further comprise forming a second dielectric layer over the opening to create the center insulating portion (or a pillar) over the conductive layer such that the center insulating portion does not contact the first dielectric layer. In some embodiments, the center insulating portion may be taller than the first and second heights of the outer and inner portions.

[0140] In some embodiments, the method may further comprise forming a semipermeable membrane over the conductive layer, where the semipermeable membrane is coupled to opposing portions of the first dielectric layer that form the opening.

[0141] In some embodiments, known semiconductor fabrication techniques may be used to fabricate the device.Docket No.: XZOM-013 / 02WO 355524-2169Hydrogels

[0142] Overlaying electrode structures with one or more layers of materials can reduce the deleterious electrochemistry' effects, including but not limited to electrolysis reactions, heating, and chaotic fluid movement that may occur on or near the electrodes, and still allow the effective separation of cells, bacteria, virus, nanoparticles, DNA, and other biomolecules to be carried out. In some embodiments, the materials layered over the electrode structures may be one or more porous layers. In other embodiments, the one or more porous layers may be a polymer layer. In other embodiments, the one or more porous layers may be a hydrogel.

[0143] In general, the hydrogel should have sufficient mechanical strength and be relatively chemically inert such that it will be able to endure the electrochemical effects at the electrode surface without disintegration or decomposition. In general, the hydrogel is sufficiently permeable to small aqueous ions, but keeps biomolecules away from the electrode surface.

[0144] In some embodiments, the hydrogel may be a single layer, or coating.

[0145] In some embodiments, the hydrogel comprises a gradient of porosity, wherein the bottom of the hydrogel layer has greater porosity than the top of the hydrogel layer.

[0146] In some embodiments, the hydrogel comprises multiple layers or coatings. In some embodiments, the hydrogel comprises two coats. 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 topcoat has less porosity than the first coating. In some embodiments, the topcoat has a mean pore diameter that functions as a size cut-off for particles of greater than 100 picometers in di ameter.

[0147] In some embodiments, the hydrogel has a conductivity from about 0.001 S / m to about 10 S / m. In some embodiments, the hydrogel has a conductivity of about 0.001 S / m, about 0.005 S / m, about 0.01 S / m, about 0.05 S / m, about 0.1 S / m, about 0.2 S / m, about 0.3 S / m, about 0.4 S / m, about 0.5 S / m, about 0.6 S / m, about 0.7 S / m, about 0.8 S / m, about 0.9 S / m, about 1.0 S / m, about 1.5 S / m, about 2 S / m, about 2.5 S / m, about 3 S / m, about 3.5 S / m, about 4 S / m, about 4.5 S / m, about 5 S / m, about 6 S / m, about 7 S / m, about 8 S / m, about 9 S / m, or about 10 S / m. In some embodiments, the hydrogel has a conductivity of about 0.1 S / m, about 0.2 S / m, about 0.3 S / m, about 0.4 S / m, about 0.5 S / m, about 0.6 S / m, about 0.7 S / m, about 0.8 S / m, about 0.9 S / m, or about 1,0 S / m. In some embodiments, the hydrogel has a thickness from about 0.1 microns to about 10 microns. In some embodiments, the hydrogel has a thickness of about 0.1 microns, about 0.2 microns, about 0.3 microns, about 0.4 microns, about 0.5 microns, about 0.6 microns, about 0.7 microns, about 0.8 microns, about 0.9Docket No.: XZOM-013 / 02WO 355524-2169microns, about 1 micron, about 1.5 microns, about 2 microns, about 2,5 microns, about 3 microns, about 3.5 microns, about 4 microns, about 4.5 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, or about 10 mi crons. In some embodiments, the viscosity of a hydrogel solution prior to spin-coating or deposition onto the array of electrodes ranges from about 0.5 cP to about 5 cP. In some embodiments, a single coating of hydrogel solution has a viscosity7of between about 0.75 cP and 5 cP prior to spincoating or deposition onto the array of electrodes. In some embodiments, in a multi -coat hydrogel, the first hydrogel solution has a viscosity from about 0.5 cP to about 1.5 cP prior to spin coating or deposition onto the array of electrodes. In some embodiments, 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 polymer’s concentration (0.1% -10%) and polymers molecular weight (10,000 to 300,000) in the solvent and the starting viscosity of the solvent.

[0148] In some embodiments, 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 embodiments, the first hydrogel coating has a thickness between about 0.75 and 1 micron. In some embodiments, 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.

[0149] In some embodiments, the hydrogel comprises any suitable synthetic polymer forming a hydrogel. In general, any sufficiently hydrophilic and polymerizable molecule may be utilized in the production of a synthetic polymer hydrogel for use as disclosed herein. Polymerizable moieties in the monomers may include alkenyl moieties including but not limited to substituted or unsubstituted a, P, unsaturated carbonyls wherein the double bond is directly attached to a carbon which is double bonded to an oxygen and single bonded to another oxygen, nitrogen, sulfur, halogen, or carbon; vinyl, wherein the double bond is singly- bonded to an oxygen, nitrogen, halogen, phosphorus or sulfur; allyl, wherein the double bond is singly bonded to a carbon which is bonded to an oxygen, nitrogen, halogen, phosphorus or sulfur; homoallyl, wherein the double bond is singly bonded to a carbon which is singly bonded to another carbon which is then singly bonded to an oxygen, nitrogen, halogen, phosphorus or sulfur; alkynyl moieties wherein a triple bond exists between two carbonDocket No.: XZOM-013 / 02WO 355524-2169atoms. In some embodiments, acryloyl or acryl amido monomers such as acrylates, methacrylates, acrylamides, methacrylamides, etc., are useful for formation of hydrogels as disclosed herein. More preferred acrylamido 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, ether-based polymers, alkylene-glycol based polymers (e.g., polypropylene glycol), and the like.

[0150] In some embodiments, the hydrogel comprises poly(2 -hydroxyethylmethacrylate) (pHEMA), cellulose acetate, cellulose acetate phthalate, cellulose acetate butyrate, or any appropriate acrylamide or vinyl-based polymer, or a derivative thereof.

[0151] In some embodiments, the hydrogel is applied by vapor deposition.

[0152] In some embodiments, the hydrogel is polymerized via atom-transfer radicalpolymerization (ATRP), Activators ReGenerated by Electron Transfer-polymerization (ARGET), Initiators for Continuous Activator Regeneration-polymerization (ICAR), Nitroxide-Mediated Radical Polymerization (NMP), Photoinitiated- ATRP, or reversible addition - fragmentation chain-transfer (RAFT) polymerization.

[0153] In some embodiments, additives may be added to a hydrogel to increase conductivity of the gel. In some embodiments, the hydrogel additives may be conductive polymers (e.g., PEDOT: PSS), salts (e.g., copper chloride), metals (e.g., gold), plasticizers (e.g., PEG200, PEG 400, or PEG 600), or co-solvents.

[0154] In some embodiments, the hydrogel may also comprise compounds or materials which help maintain the stability of the DNA hybrids, including, but not limited to histidine, histidine peptides, polyhistidine, lysine, lysine peptides, and other cationic compounds or substances.

[0155] In various embodiments provided herein, a method described herein may comprise producing a DEP field region and optionally a second DEP field region with the array. In various embodiments provided herein, a device or system described herein is capable of producing a DEP field region and optionally a second DEP field region with the array. In some instances, the first and second field regions are part of a single field (e.g., the first and second regions are present at the same time but are found at different locations within the device and / or upon the array). In some embodiments, the first and second field regions are different fields (e.g., the first region is created by energizing the electrodes at a first time, andDocket No.: XZOM-013 / 02WO 355524-2169the second region is created by energizing the electrodes a second time). In specific aspects, the DEP field region is suitable for concentrating or isolating cells (e.g., into a low field DEP region). In some embodiments, the optional second DEP field region is suitable for concentrating smaller particles, such as molecules (e.g., nucleic acid), for example into a high field DEP region. In some instances, a method described herein optionally excludes use of either the first or second DEP field region.

[0156] In some embodiments, the DEP field region is in the same chamber of a device as disclosed herein as the optional second DEP field region. In some embodiments, the DEP field region and the optional second DEP field region occupy the same area of the array of electrodes.

[0157] In some embodiments, the DEP field region is in a separate chamber of a device as disclosed herein, or a separate device entirely, from the second DEP field region,DEP Field Region

[0158] In some embodiments, e.g., high conductance buffers (>100 mS / m), the method described herein comprises applying a sample comprising nanoscale analytes and other particulate material to a device comprising an array of electrodes as disclosed herein, and, thereby, isolating and collecting the nanoscale analytes in a DEP field region. In some embodiments, the devices and systems described herein are capable of applying a sample comprising nanoscale analytes and other particulate material to the device comprising an array of electrodes as disclosed herein, and, thereby, isolating and collecting the nanoscale analytes in a DEP field region. Subsequent or concurrent second, or optional third and fourth DEP regions, may collect or isolate other sample components, including intact cells and other particulate material.

[0159] The DEP field region generated may be any field region suitable for isolating and collecting nanoscale analytes from a sample. For this application, the nanoscale analytes are generally concentrated near the array of electrodes as disclosed herein. In some embodiments, the DEP field region is a di electrophoretic low field region. In some embodiments, the DEP field region is a dielectrophoretic high field region. In some embodiments, e.g., low conductance buffers (<100 mS / m), the method described herein comprises applying a fluid comprising cells to a device comprising an array of electrodes as disclosed herein, and, thereby, concentrating the nanoscale analytes in a DEP field region.Docket No.: XZOM-013 / 02WO 355524-2169

[0160] In some embodiments, the devices and systems described herein are capable of applying a sample comprising nanoscale analytes and other particulate material to the device comprising an array of electrodes as disclosed herein, and concentrating the nanoscale analytes in a DEP field region. In some embodiments, the nanoscale analytes are captured in a dielectrophoretic high field region. In some embodiments, the nanoscale analytes are captured in a dielectrophoretic low-field region. High versus low field capture is generally dependent on the conductivity of the fluid, wherein generally, the crossover point between high and low conductivity fluid is between about 300-500 mS / m. In some embodiments, the DEP field region is a dielectrophoretic low field region performed in fluid conductivity of greater than about 300 mS / m. In some embodiments, the DEP field region is a dielectrophoretic low field region performed in fluid conductivity of less than about 300 mS / m, In some embodiments, the DEP field region is a dielectrophoretic high field region performed in fluid conductivity of greater than about 300 mS / m. In some embodiments, the DEP field region is a dielectrophoretic high field region performed in fluid conductivity of less than about 300 mS / m. In some embodiments, the DEP field region is a dielectrophoretic low field region performed in fluid conductivity of greater than about 500 mS / m. In some embodiments, the DEP field region is a dielectrophoretic low field region performed in fluid conductivity of less than about 500 mS / m. In some embodiments, the DEP field region is a dielectrophoretic high field region performed in fluid conductivity of greater than about 500 mS / m. In some embodiments, the DEP field region is a dielectrophoretic high field region performed in fluid conductivity of less than about 500 mS / m.

[0161] In some embodiments, the dielectrophoretic field region is produced by an alternating current. The alternating current has any amperage, voltage, frequency, and the like suitable for concentrating cells. In some embodiments, the 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. In some embodiments, the DEP field region is produced using an alternating current having a voltage of 5-25 volts peak to peak. In some embodiments, the DEP field region is produced using an alternating current having a frequency of from 3-15 kHz.

[0162] In some embodiments, the DEP field region is produced using an alternating current having an amperage of 100 milliamps to 5 amps. In some embodiments, the DEP field region is produced using an alternating current having an amperage of 0.5 Ampere — I Ampere. In some embodiments, the DEP field region is produced using an alternating current having anDocket No.: XZOM-013 / 02WO 355524-2169amperage of 0.5 Ampere — 5 Ampere. In some embodiments, the DEP field region is produced using an alternating current having an amperage of 100 milliamps ----- 1 Ampere. In some embodiments, the DEP field region is produced using an alternating current having an amperage of 500 milli Amperes - - - 2.5 Amperes.

[0163] In some embodiments, the DEP field region is produced using an alternating current having a voltage of 1-25 Volts peak to peak. In some embodiments, the DEP field region is produced using an alternating current having a voltage of 1-10 Volts peak to peak. In some embodiments, the DEP field region is produced using an alternating current having a voltage of 25-50 Volts peak to peak. In some embodiments, the DEP field region is produced using a frequency of from 10-1,000,000 Hz In some embodiments, the DEP field regi on is produced using a frequency of from 100-100,000 Hz. In some embodiments, the DEP field region is produced using a frequency of from 100-10,000 Hz. In some embodiments, the DEP field region is produced using a frequency of from 10,000-100,000 Hz. In some embodiments, the DEP field region is produced using a frequency of from 100,000-1,000,000 Hz.

[0164] In some embodiments, the first dielectrophoretic field region is produced by a direct current. The direct current has any amperage, voltage, frequency, and the like suitable for concentrating cells. In some embodiments, the first dielectrophoretic field region is produced using a direct current having an amperage of 0.1 micro Amperes - 1 Amperes, a voltage of 10 milli Volts – 10 Volts, and / or a pulse width of 1 millisecond - 1000 seconds and a pulse frequency of 0.001 Hz - 1000 Hz. In some embodiments, the DEP field region is produced using a direct current having an amperage of 1 micro Amperes -1 Amperes. In some embodiments, the DEP field region is produced using a direct current having an amperage of 100 micro Amperes -500 milli Amperes. In some embodiments, the DEP field region is produced using a direct current having an amperage of 1 milli Amperes - 1 Amperes. In some embodiments, the DEP field region is produced using a direct current having an amperage of 1 micro Amperes - 1 milli Amperes. In some embodiments, the DEP field region is produced using a direct current having a pulse width of 500 milliseconds-500 seconds. In some embodiments, the DEP field region is produced using a direct current having a pulse-width of 500 milliseconds- 100 seconds. In some embodiments, the DEP field region is produced using a direct current having a pulse width of 1 second - 1000 seconds. In some embodiments, the DEP field region is produced using a direct current having a pulse width of 500 milliseconds -1 second. In some embodiments, the DEP field region is produced using a pulse frequency of 0.01 Hz -1000 Hz. In some embodiments, the DEP fieldDocket No.: XZOM-013 / 02WO 355524-2169region is produced using a pulse frequency of 0.1 Hz -100 Hz. In some embodiments, the DEP field region is produced using a pulse frequency of 1 Hz -100 Hz. In some embodiments, the DEP field region is produced using a pulse frequency of 100 Hz -1000 Hz.

[0165] In some embodiments, the sample may comprise 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, one cell type (or any number of cell types less than the total number of cell types comprising the sample) may be preferentially concentrated in a DEP field region. In another non-limiting example, the 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).

[0166] Accordingly, in some embodiments, a method, device or system described herein is suitable for isolating or separating specific cell types in order to enable efficient isolation and collection of nanoscale analytes. In some embodiments, the DEP field of the method, device or system is specifically tuned to allow for the separation or concentration of a specific type of cell into a field region of the DEP field. In some embodiments, a method, device or system described herein provides more than one field region wherein more than one type of cell is isolated or concentrated. In some embodiments, a method, device, or system described herein is tunable so as to allow isolation or concentration of different types of cells within the DEP field regions thereof. In some embodiments, a method provided herein further comprises tuning the DEP field. In some embodiments, a device or system provided herein is capable of having the DEP field tuned. In some embodiments, such tuning may be in providing a DEP particularly suited for the desired purpose. For example, modifications in the array, the energy, or another parameter are optionally utilized to tune the DEP field. Tuning parameters for finer resolution include electrode diameter, edge to edge distance between electrodes, voltage, frequency, and fluid conductivity'.

[0167] In some embodiments, the DEP field region compri ses the entirety of an array of electrodes as disclosed herein. In some embodiments, the DEP field region comprises a portion of an array of electrodes as disclosed herein. In some embodiments, the 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 as disclosedDocket No.: XZOM-013 / 02WO 355524-2169herein. In some embodiments, the DEP field region comprises about a third of an array of electrodes as disclosed herein.

[0168] In some embodiments, the DEP field has an outer limit where the field does not have strong dielectric effect on analytes. In some embodiments, AC electrothermal flow creates vortices within the fluid due to the heating of the chip. In some embodiments, the AC electrothermal flow aids the DEP as the fluid motion created brings particles outside of the DEP field into the DEP field. In some embodiments, these particles brought into the DEP field are then in the capture region of the analyte capture device. In some embodiments, the fluid motion of the AC electrothermal flow can generate drag. In some embodiments, the DEP field voltage can be increased to compensate for this drag.

[0169] In some embodiments, the strongest point in the DEP field is located at the corner of the dielectric insulating portion. This strongest point is due to the converging field lines formed between multiple electrodes, wherein the particles traveling from one electrode to the other travel by the shortest possible pathway. In some embodiments, the dielectric portion has an etch, wherein the etch creates a wedge where particles are collected near the strongest portion of the DEP field.Cell Lysis

[0170] In some embodiments, following concentrating the cells in a first di electrophoretic field region, the method involves freeing nanoscale analytes from the cell. In some embodiments, the devices and systems described herein are capable of freeing nucleic acids from the cells. In some embodiments, the nucleic acids are freed from the cells in the first DEP field region.

[0171] In some embodiments, the methods described herein free nucleic acids from a plurality of cells by lysing the cells. In some embodiments, the devices and systems described herein are capable of freeing nucleic acids from a plurality of cells by lysing the cells. One method of cell lysis involves applying a direct current to the cells after isolation of the cells on the array. The direct current has any suitable amperage, voltage, and the like suitable for lysing cells. In some embodiments, the current has a voltage of 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 generally the driver of cell lysis, as high electric fields result in failed membrane integrity.Docket No.: XZOM-013 / 02WO 355524-2169

[0172] In some embodiments, the direct current used for lysis comprises one or more pulses having any duration, frequency, and the like suitable for lysing cells. In some embodiments, a voltage of about 100 volts is applied for about 1 millisecond to lyse cells. In some embodiments, the voltage of about 100 volts is applied 2 or 3 times over the course of a second.

[0173] In some embodiments, the frequency of the direct current depends on volts / cm, pulse width, and the fluid conductivity. In some embodiments, 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. In some embodiments, the pulse has a frequency of about 0.1 Hz. In other embodiments, the pulse has a frequency of about 1 Hz. In still other embodiments, 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.

[0174] In other embodiments, 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 1 s - 150 s. In some embodiments, 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 500 s or about 1000s. In some embodiments, the pulse has a frequency of 0.2 to 200 Hz with duty cycles from 10-50%.

[0175] In some embodiments, the direct current is applied once, or as multiple pulses. Any suitable number of pulses may be applied including about 1-20 pulses. There is any suitable amount of time between pulses including about 1 millisecond — 1000 second s. In some embodiments, the pulse duration is.01 to 10 seconds.

[0176] In some embodiments, the cells are lysed using other methods in combination with a direct current applied to the isolated cells. In yet other embodiments, the cells are lysed without use of direct current. In some embodiments, the devices and systems are capable of lysing cells with direct current in combination with other means, or may be capable of lysing cells without the use of direct current. Any method of cell lysis known to those skilled in theDocket No.: XZOM-013 / 02WO 355524-2169art may be suitable including, but not limited to application of a chemical lysing agent (e.g., an acid), an enzymatic lysing agent, heat, pressure, shear force, sonic energy, osmotic shock, or combinations thereof. Lysozyme is an example of an enzymatic-lysing agent.Nanoscale Analytes Isolation and Yields Thereof

[0177] In some embodiments, described herein are methods and devices for isolating a nanoscale analyte from a sample. In some embodiments, the nanoscale analyte is less than 1000 nm in diameter. In other embodiments, the nanoscale analyte is less than 500 nm in diameter. In some embodiments, the nanoscale analyte is less than 250 nm in diameter. In some embodiments, the nanoscale analyte is between about 100 nm to about 1000 nm in diameter. In other embodiments, the nanoscale analyte is between about 250 nm to about 800 nm in diameter. In still other embodiments, the nanoscale analyte is between about 300 nm to about 500 nm in diameter.

[0178] In some embodiments, the nanoscale analyte is less than 1000 pm in diameter. In other embodiments, the nanoscale analyte is less than 500 pm in diameter. In some embodiments, the nanoscale analyte is less than 250 pm in diameter. In some embodiments, the nanoscale analyte is between about 100 pm to about 1000 pm in diameter. In other embodiments, the nanoscale analyte is between about 250 pm to about 800 pm in diameter. In still other embodiments, the nanoscale analyte is between about 300 pm to about 500 pm in diameter.

[0179] In some embodiments, the method, device, or system described herein is optionally utilized to obtain, isolate, or separate any desired nanoscale analyte that may be obtained from such a method, device or system. In some embodiments, the nanoscale analyte is a nucleic acid. In other embodiments, the 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.

[0180] In various embodiments, an isolated or separated nanoscale analyte is a composition comprising nanoscale analyte that is free from at least 99% by mass of other materials, free from at least 99% by mass of residual cellular material, free from at least 98% by mass of other materials, free from at least 98% by mass of residual cellular material, free from at least 95% by mass of other materials, free from at least 95% by mass of residual cellular material,Docket No.: XZOM-013 / 02WO 355524-2169free 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 other materials, free from at least 80% by mass of residual cellular material, 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 residual cellular material, free from at least 50% 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.

[0181] In various embodiments, the nanoscale analyte has any suitable purity. For example, if an enzymatic assay requires nanoscale analyte samples having about 20% residual cellular material, then isolation of the nucleic acid to 80% is suitable. In some embodiments, the isolated nanoscale analyte 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-nanoscale analyte cellular material and / or protein by mass. In some embodiments, the isolated nanoscale analyte 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% nanoscale analyte by mass.

[0182] The nanoscale analytes may be isolated in any suitable form including unmodified, derivatized, fragmented, non -fragmented, and the like. In some embodiments, when the nanoscale analyte is a nucleic acid, the nucleic acid is collected in a form suitable for sequencing. In some embodiments, 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.

[0183] In some embodiments, the methods described herein result in an isolated nanoscale analyte sample that is approximately representative of the nanoscale analyte of the starting sample. In some embodiments, the devices and systems described herein are capable of isolating nanoscale analyte from a sample that is approximately representative of the nanoscale analyte of the starting sample. That is, the population of nanoscale analytesDocket No.: XZOM-013 / 02WO 355524-2169collected by the method, or capable of being collected by the device or system, may be substantially in proportion to the population of nanoscale analytes present in the cells in the fluid. In some embodiments, this aspect is advantageous in applications in which the fluid is a complex mixture of many cell types, and the practitioner desires a nanoscale analyte-based procedure for determining the relative populations of the various cell types.

[0184] In some embodiments, the nanoscale analyte 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 nanoscale analyte 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 nanoscale analyte 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 nanoscale analyte 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.

[0185] In some embodiments, about 50 pico-grams of nanoscale analyte is isolated from a sample comprising 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 nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 20 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 50 pico-grams of nanoscale analyte from about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 75 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 100 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 200 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 300 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 400 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 500 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devicesDocket No.: XZOM-013 / 02WO 355524-2169described herein yield at least 1,000 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 10,000 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 20,000 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 30,000 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 40,000 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells. In some embodiments, the methods, systems or devices described herein yield at least 50,000 pico-grams of nanoscale analyte from a sample comprising about 5,000 cells.

[0186] When the nanoscale analyte is a nucleic acid, the nucleic acid isolated using the methods described herein or capable of being isolated by the devices described herein may be high-quality and / or suitable for using directly in downstream procedures such as DNA sequencing, nucleic acid amplification, such as PCR, or other nucleic acid manipulation, such as ligation, cloning or further translation or transformation assays. In some embodiments, the collected nucleic acid comprises at most 0.01 % protein. In some embodiments, the collected nucleic acid comprises at most about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% protein.

[0187] In some embodiments, the methods, systems and devices described herein isolate nanoscale analytes from a sample. In some embodiments, the sample comprises a fluid. In some embodiments, the sample comprises cells or other particulate material and the nanoscale analytes. In some embodiments, the sample does not comprise cells.Samples

[0188] In some embodiments, the methods, systems and devices described herein isolate nanoscale analytes from a sample. In some embodiments, the sample comprises a fluid. In some embodiments, the sample comprises cells or other particulate material and the nanoscale analytes. In some embodiments, the sample does not comprise cells.

[0189] In some embodiments, the sample is a liquid, optionally water or an aqueous solution or dispersion. In some embodiments, the sample is a bodily fluid. Exemplary bodily fluids include blood, serum, plasma, bile, milk, cerebrospinal fluid, gastric juice, ejaculate, mucus,Docket No.: XZOM-013 / 02WO 355524-2169peritoneal fluid, saliva, sweat, tears, urine, synovial fluid and the like. In some embodiments, nanoscale analytes 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. In some embodiments, the sample is tissues and / or cells solubilized and / or dispersed in a fluid medium. For example, the tissue can be a cancerous tumor from which nanoscale analytes, such as nucleic acids, can be isolated using the methods, devices or systems described herein.

[0190] In some embodiments, the sample is an environmental sample. In some embodiments, 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. The 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. Exemplary environmental samples include municipal wastewater, industrial wastewater, water or fluid used in or produced as a result of various manufacturing processes, lakes, rivers, oceans, aquifers, ground water, storm water, plants or portions of plants, animals or portions of animals, insects, municipal water supplies, and the like.

[0191] In some embodiments, the sample is a food or beverage. The food or beverage can be assayed or monitored for the presence of a particular nanoscale analyte 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. In various embodiments, 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.

[0192] In some embodiments, the sample 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’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. In some embodiments, the medium comprises essentially of a plurality of clonal cells. In some embodiments, the medium comprises a mixture of at least two species.

[0193] In some embodiments, the sample is water. In some embodiments, the sample is salt water.

[0194] In some embodiments, the sample may also comprise other particulate material. Such particulate material may be, for example, inclusion bodies (e.g, ceroids or Mallory bodies),Docket No.: XZOM-013 / 02WO 355524-2169cellular 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. In some embodiments, particulate material is an aggregated protein (e.g., beta-amyloid).

[0195] The sample can have any conductivity including a 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 yet 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 to about 5 mS / m, between about 100 μS / m to about 4 mS / m, between about 100 μS / m to about 3 mS / m, between about 100 μS / m to about 2 mS / m, or between about 100 μS / m to about 1 mS / m.

[0196] 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 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.

[0197] In some embodiments, the conductivity is at least 1 μS / m. In yet 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 yet 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 at most 1 μS / m. In some embodiments, the conductivity is at most 10 μS / m. In other embodiments, the conductivity is at most 100 μS / m. 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.Docket No.: XZOM-013 / 02WO 355524-2169

[0198] In some embodiments, the sample is a small volume of liquid including less than 10 ml. In some embodiments, the sample is less than 8 ml. In some embodiments, the sample is less than 5 ml. In some embodiments, the sample is less than 2 ml. In some embodiments, the sample is less than 1 ml. In some embodiments, the sample is less than 500 μl. In some embodiments, the sample is less than 200 μl. In some embodiments, the sample is less than 100 μl. In some embodiments, the sample is less than 50 μl. In some embodiments, the sample is less than 10 μl. In some embodiments, the sample is less than 5 μl. In some embodiments, the sample is less than 1 μl.

[0199] In some embodiments, the quantity of sample applied to the device or used in the method comprises less than about 100,000,000 cells. In some embodiments, the sample comprises less than about 10,000,000 cells. In some embodiments, the sample comprises less than about 1,000,000 cells. In some embodiments, the sample comprises less than about 100,000 cells. In some embodiments, the sample comprises less than about 10,000 cells. In some embodiments, the sample comprises less than about 1,000 cells.

[0200] In some embodiments, isolation of a nanoscale analyte from a sample 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 a nanoscale analyte from a sample with the devices, systems and methods described herein takes not more than 30 minutes, not more than about 20 minutes, not more than about 15 minutes, not more than about 10 minutes, not more than about 5 minutes, not more than about 2 minutes or not more than about 1 minute. In additional embodiments, isolation of a nanoscale analyte from a sample 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.Removal of Residual Material

[0201] In some embodiments, following isolation of the nanoscale analytes in a DEP field region, the method includes optionally flushing residual material from the isolated nanoscale analytes. In some embodiments, the devices or systems described herein are capable of optionally and / or comprising a reservoir comprising a fluid suitable for flushing residual material from the nanoscale analytes. “Residual material” is anything originally present in the sample, originally present in the cells, added during the procedure, created through any step of the process including but not limited to ceils (e.g., intact cells or residual cellular material),Docket No.: XZOM-013 / 02WO 355524-2169and the like. For example, residual material may include intact cells, cell wall fragments, proteins, lipids, carbohydrates, minerals, salts, buffers, plasma, and the like. In some embodiments, a certain amount of nanoscale analyte is flushed with the residual material.

[0202] In some embodiments, the residual material is flushed in any suitable fluid, for example in water, TBE buffer, or the like. In some embodiments, the residual material is flushed with any suitable volume of fluid, flushed for any suitable period of time, flushed with more than one fluid, or any other variation. In some embodiments, the method of flushing residual material is related to the desired level of isolation of the nanoscale analyte, with higher purity nanoscale analyte requiring more stringent flushing and / or washing. In other embodiments, the method of flushing residual material is related to the particular starting material and its composition. In some embodiments, a starting material that is high in lipid requires a flushing procedure that involves a hydrophobic fluid suitable for solubilizing lipids.

[0203] In some embodiments, the method includes degrading residual material including residual protein. In some embodiments, the devices or systems are capable of degrading residual material including residual protein. For example, proteins may be degraded by one or more of chemical degradation (e.g, acid hydrolysis) and enzymatic degradation. In some embodiments, the enzymatic degradation agent is a protease. In other embodiments, the protein degradation agent is Proteinase K The optional step of degradation of residual material may be performed for any suitable time, temperature, and the like. In some embodiments, the degraded residual material (including degraded proteins) is flushed from the isolated nanoscale analytes.

[0204] In some embodiments, the agent used to degrade the residual material is inactivated or degraded. In some embodiments, the devices or systems are capable of degrading or inactivating the agent used to degrade the residual material. In some embodiments, an enzyme used to degrade the residual material is inactivated by heat (e.g., 50 to 95° C for 5-15 minutes). For example, enzymes including proteases (for example, Proteinase K) may be degraded and / or inactivated using heat (typically, 15 minutes, 70 °C). In some embodiments wherein the residual proteins are degraded by an enzyme, the method further comprises inactivating the degrading enzyme (e.g., Proteinase K) following degradation of the proteins. In some embodiments, heat is provided by a heating module in the device (temperature range, e.g., from 30 to 95 °C).Docket No.: XZOM-013 / 02WO 355524-2169

[0205] The order and / or combination of certain steps of the method can be varied. In some embodiments, the devices or methods are capable of performing certain steps in any order or combination. For example, in some embodiments, the residual material and the degraded proteins are flushed in separate or concurrent steps. That is, the residual material is flushed, followed by degradation of residual proteins, followed by flushing degraded proteins from the isolated nanoscale analytes. In some embodiments, one first degrades the residual proteins, and then flush both the residual material and degraded proteins from the nanoscale analytes in a combined step.

[0206] In some embodiments, the nanoscale analytes are retained in the device and optionally used in further procedures, such as PCR, enzymatic assays or other procedures that analyze, characterize or amplify the nanoscale analytes.

[0207] For example, in some embodiments, the isolated nanoscale analyte is a nucleic acid, and the devices and systems are capable of performing PCR or other optional procedures on the isolated nucleic acids. In other embodiments, the nucleic acids are collected and / or eluted from the device. In some embodiments, the devices and systems are capable of allowing collection and / or elution of nucleic acid from the device or system. In some embodiments, the isolated nucleic acid is collected by (i) turning off the second di electrophoretic field region, and (ii) eluting the nucleic acid from the array in an eluant. Exemplary' eluants include water, TE, TBE and L-Histidine buffer.Assays and Applications

[0208] In some embodiments, a system or device described herein includes a means of performing enzymatic reactions. In other embodiments, a system or device described herein includes a means of performing polymerase chain reaction (PCR), isothermal amplification, ligation reactions, restriction analysis, nucleic acid cloning, transcription or translation assays, or other enzymatic-based molecular biology assay.

[0209] In some embodiments, a system or device described herein comprises a nucleic acid sequencer. The sequencer is optionally any suitable DNA sequencing device including but not limited to a Sanger sequencer, pyro-sequencer, ion semiconductor sequencer, polony sequencer, sequencing by ligation device, DNA nanoball sequencing device, sequencing by ligation device, or single molecule sequencing device.

[0210] In some embodiments, the methods described herein further comprise optionally amplifying the isolated nucleic acid by polymerase chain reaction (PCR). In someDocket No.: XZOM-013 / 02WO 355524-2169embodiments, the PCR reaction is performed on or near the array of electrodes or in the device. In some embodiments, the device or system comprise a heater and / or temperature control mechanisms suitable for thermocycling.

[0211] PCR is optionally done using traditional thermocycling by placing the reaction chemistry analytes in between two efficient thermoconductive elements (e.g., aluminum or silver) and regulating the reaction temperatures using TECs. Additional designs optionally use infrared heating through optically transparent material like glass or thermoset polymers. In some embodiments, designs use smart polymers or smart glass that comprise conductive wiring networked through the substrate. This conductive wiring enables rapid thermal conductivity of the materials and (by applying appropriate DC voltage) provides the required temperature changes and gradients to sustain efficient PCR reactions. In certain embodiments, heating is applied using resistive chip heaters and other resistive elements that will change temperature rapidly and proportionally to the amount of current passing through them.

[0212] In some embodiments, used in conjunction with traditional fluorometry (ccd, pmt, other optical detector, and optical filters), fold amplification is monitored in real-time or on a timed interval. In certain embodiments, quantification of final fold amplification is reported via optical detection converted to AFU (arbitrary' fluorescence units correlated to analyze doubling) or translated to electrical signal via impedance measurement or other electrochemical sensing.

[0213] Given the small size of the micro electrode array, these elements may be optionally added around the micro electrode array and the PCR reaction will be performed in the main sample processing chamber (over the DEP array) or the analytes to be amplified may be optionally transported via fluidics to another chamber within the fluidic cartridge to enable on-cartridge Lab-On-Chip processing.

[0214] In some embodiments, light delivery schemes are utilized to provide the optical excitation and / or emission and / or detection of fold amplification. In certain embodiments, this includes using the flow cell materials (thermal polymers like acrylic (PMMA) cyclic olefin polymer (COP), cyclic olefin co-polymer, (COC), etc.) as optical wave guides to remove the need to use external components. In addition, in some embodiments light sources - light emitting diodes - LEDs, vertical-cavity surface-emitting lasers - VCSELs, and other lighting schemes are integrated directly inside the flow cell or built directly onto the micro electrode array surface to have internally controlled and powered light sources. MiniatureDocket No.: XZOM-013 / 02WO 355524-2169PMTs, CCDs, or CMOS detectors can also be built into the flow cell. This minimization and miniaturization may enable compact devices capable of rapid signal delivery and detection while reducing the footprint of similar traditional devices (e.g., a standard bench top PCR / QPCR / Fluorometer).Amplification on Chip

[0215] In some embodiments, silicon microelectrode arrays can withstand thermal cycling used in PCR. In some applications, on-chip PCR is advantageous because small amounts of target nucleic acids can be lost during transfer steps. In certain embodiments of devices, systems or processes described herein, any one or more of multiple PCR techniques are optionally used, such techniques optionally including any one or more of the following: thermal cycling in the flowcell directly, moving the material through microchannels with different temperature zones, and moving volume into a PCR tube that can be amplified on system or transferred to a PCR machine. In some embodiments, droplet PCR is performed if the outlet contains a T-junction that contains an immiscible fluid and interfacial stabilizers (surfactants, etc.). In certain embodiments, droplets are thermal cycled in by any suitable method.

[0216] In some embodiments, amplification is performed using an isothermal reaction, for example, transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification or circular helicase-dependent amplification.

[0217] In various embodiments, amplification is performed in homogenous solution or as heterogeneous system with anchored primer(s). In some embodiments of the latter, the resulting amplicons are directly linked to the surface for higher degree of multiplex. In some embodiments, the amplicon is denatured to render single stranded products on or near the electrodes. Hybridization reactions are then optionally performed to interrogate the 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 bisulfite treated and one is not. Bisulfite depurinates unmodified C becoming a U. Methylated C is unaffected in someDocket No.: XZOM-013 / 02WO 355524-2169embodiments. In some embodiments, allele specific base extension is used to report the base of interest.

[0218] Rather than specific interactions, the surface may be optionally modified with nonspecific moieties for capture. For example, surface could be modified with polycations, e.g., polylysine, to capture DNA molecules which can be released by reverse bias (-V). In some embodiments, 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.

[0219] In some applications, where multiple chip designs are employed, it is advantageous to have a chip sandwich where the two devices are facing each other, separated by a spacer, to form the flow cell. In various embodiments, devices are run sequentially or in parallel. For sequencing and next generation sequencing (NGS), size fragmentation and selection have ramifications on sequencing efficiency and quality'. In some embodiments, multiple chip designs are used to narrow the size range of material collected creating a band pass filter. In some embodiments, current chip geometry' (e.g., 80 um diameter electrodes on 200 um center-center pitch (80 / 200) acts as 500 bp cutoff filter (e.g., using voltage and frequency conditions around 10 Vpp and 10 kHz). In such embodiments, a nucleic acid of greater than 500 bp is captured, and a nucleic acid of less than 500 bp is not. Alternate electrode diameter and pitch geometries have different cutoff sizes such that a combination of chips should provide a desired fragment size. In some embodiments, a 40 pm diameter electrode on 100 pm center-center pitch (40 / 100) has a lower cutoff threshold, whereas a 160 pm diameter electrode on 400 pm center-center pitch (160 / 400) has a higher cutoff threshold relative to the 80 / 200 geometry, under similar conditions. In various embodiments, geometries on a single chip or multiple chips are combined to select for a specific sized fragments or particles. For example, 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. In some embodiments, size selection is accomplished using a single electrode geometry, wherein nucleic acid of >500 bp is isolated on the electrodes, followed by washing, followed by reduction of the ACEK high field strength (change voltage, frequency,Docket No.: XZOM-013 / 02WO 355524-2169conductivity) in order to release nucleic acids of <600 bp, resulting in a supernatant nucleic acid population between 500-600 bp.

[0220] In some embodiments, the chip device is oriented vertically with a heater at the bottom edge which creates a temperature gradient column. In some embodiments, the bottom is at denaturing temperature, the middle at annealing temperature, the top at extension temperature. In some embodiments, convection continually drives the process. In some embodiments, provided herein are methods or systems comprising an electrode design that specifically provides for electrothermal flows and acceleration of the process. In some embodiments, such design is optionally on the same device or on a separate device positioned appropriately. In some embodiments, active or passive cooling at the top, via fins or fans, or the like provides a steep temperature gradient. In some embodiments, 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. In some embodiments, such sensors are coupled with materials possessing different thermal transfer properties to create continuous and / or discontinuous gradient profiles.

[0221] In some embodiments, the amplification proceeds at a constant temperature (e.g., isothermal amplification).

[0222] In some embodiments, the methods disclosed herein further comprise sequencing the nucleic acid isolated as disclosed herein. In some embodiments, the nucleic acid is sequenced by Sanger sequencing or next generation sequencing (NGS). In some embodiments, the next generation sequencing methods include, but are not limited to, pyrosequencing, ion semiconductor sequencing, polony sequencing, sequencing by ligation, DNA nanoball sequencing, sequencing by ligation, or single molecule sequencing.

[0223] In some embodiments, the isolated nucleic acids disclosed herein are used in Sanger sequencing. In some embodiments, 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 may be performed asDocket No.: XZOM-013 / 02WO 355524-2169necessary. Reactions may be performed on a single chip with plurality of capture zones or on separate chips and / or reaction chambers.

[0224] In some embodiments, the method disclosed herein further comprise performing a reaction on the nucleic acids (e.g., fragmentation, restriction digestion, ligation of DNA or RNA) In some embodiments, the reaction occurs on or near the array or in a device, as disclosed herein.Other Assays

[0225] The isolated nucleic acids disclosed herein may be further utilized in a variety of assay formats. For instance, devices which are addressed with nucleic acid probes or amplicons may be utilized in dot blot or reverse dot blot analyses, base-stacking single nucleotide polymorphism (SNP) analysis, SNP analysis with electronic stringency, or in STR analysis. In addition, such devices disclosed herein may be utilized in formats for enzymatic nucleic acid modification, or protein-nucleic acid interaction, such as, e.g., gene expression analysis with enzymatic reporting, anchored nucleic acid amplification, or other nucleic acid modifications suitable for solid-phase formats including restriction endonuclease cleavage, endo- or exo-nuclease cleavage, minor groove binding protein assays, terminal transferase reactions, polynucleotide kinase or phosphatase reactions, ligase reactions, topoisomerase reactions, and other nucleic acid binding or modifying protein reactions.

[0226] In addition, the devices disclosed herein may be useful in immunoassays. For instance, in some embodiments, locations of the devices can be linked with antigens (e.g., peptides, proteins, carbohydrates, lipids, proteoglycans, glycoproteins, etc.) in order to assay for antibodies in a bodily fluid sample by sandwich assay, competitive assay, or other formats. Alternatively, the locations of the device may be addressed with antibodies, in order to detect antigens in a sample by sandwich assay, competitive assay, or other assay formats. As the isoelectric point of antibodies and proteins can be determined fairly easily by experimentation or pH / charge computations, the electronic addressing and electronic concentration advantages of the devices may be utilized by simply adjusting the pH of the buffer so that the addressed or analyte species will be charged.

[0227] In some embodiments, the isolated nucleic acids are useful for use in immunoassaytype arrays or nucleic acid arrays.Docket No.: XZOM-013 / 02WO 355524-2169

[0228] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Docket No.: XZOM-013 / 02WO 355524-2169CLAIMSWhat is claimed is:

1. A device for capturing analytes, comprising:a substrate;a conductive layer disposed over the substrate, the conductive layer configured to generate an electrokinetic field; andan insulating layer disposed partially over the conductive layer, the insulating layer having at least one insulating portion substantially on an edge of the conductive layer, wherein the at least one insulating portion comprises an outer portion with a first height and an inner portion having a second height that is the same or different from the first height,2. The device of claim 1, wherein the insulating layer further comprises a peripheral insulating portion disposed over the substrate and adjacent to the at least one insulating portion and the conductive layer.

3. The device of claim 2, wherein a distance of the top surface of the peripheral insulating portion from the substrate is less than a distance of the top surface of the at least one insulating portion from the substrate.

4. The device of claim 1, wherein the second height is less than the first height.

5. The device of claim 4, wherein the inner portion and outer portion of the at least one insulating portion form an opening that exposes the conductive layer.

6. The device of claim 5, wherein a first length of the opening formed by opposing ends of the inner portion is less than a second length of the opening formed by opposing ends of the outer portion.

7. The device of claim 6, wherein the first length is within a range of 5 μm to 600 μm.

8. The device of claim 6, wherein the second length is within a range of 5 μm to 700 μm.Docket No.: XZOM-013 / 02WO 355524-21699. The device of claim 4, wherein the first height is within a range of 100 nm to 10 μm.

10. The device of claim 4, wherein the second height is within a range of 25 nm to 7.5 μm.

11. The device of claim 4, wherein each of the inner portions has a width that is within a range of 500 nm to 50 μm.

12. The device of any one of claims 1-11, wherein the conductive layer comprises platinum.

13. The device of any one of claims 1-11, wherein the conductive layer comprises a copper layer and a graphene layer.

14. The device of claim 13, wherein the copper layer is disposed over the substrate, and the graphene layer is disposed over the copper layer.

15. The device of claim 13, wherein the length of the copper layer is greater than the length of the graphene layer.

16. The device of claim 15, wherein the length of the copper layer is in the range of 10 μm and 1 mm.

17. The device of claim 15, wherein the length of the graphene layer is in the range of 6 μm to 600 μm.

18. The device of claim 13, wherein the thickness of the copper layer is greater than the thickness of the graphene layer.

19. The device of claim 18, wherein the thickness of the copper layer is in the range of 200 nm and 20 μm.Docket No.: XZOM-013 / 02WO 355524-216920. The device of claim 18, wherein the thickness of the graphene layer is in the range of 2.5 nm and 250 nm.

21. The device of any one of claims 1-20, wherein the insulating layer has a center insulating portion that is disposed between opposing ends of the inner portion and over a center portion of the conductive layer.

22. The device of claim 21, wherein the center insulating portion has a height that is greater than the first and second heights.

23. The device of any one of claims 1-22, further comprising a semipermeable membrane connected to the inner portion or the outer portion,24. The device of claim 23, wherein the semipermeable membrane comprises an inorganic, organic, or composite material.

25. The device of claim 24, wherein the inorganic semipermeable membrane comprises anodic Al2O3, anodic SiO2, ceramic Al2O3, ceramic SiO2, ZrO2, TiO2, Si, SiC, or other semiconductors.

26. The device of claim 24, wherein the organic semipermeable membrane comprises polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), or polystyrene.

27. The device of claim 24, wherein the composite semipermeable membrane comprises an anodic aluminum oxide (AAO) coated with carbon, metal, or metal oxide, an oxide / polymer composite, or a polymer coating on a support membrane.

28. The device of claim 24, wherein the semipermeable membrane has a plurality of pores having the size in the range of 0.1 nm to 1000 nm.

29. The device of claim 1, wherein the opening is substantially circular, and the first and second lengths include diameters.Docket No.: XZOM-013 / 02WO 355524-216930. An electrode array, comprising:a substrate; anda plurality of electrodes arranged in a plurality of rows, wherein each electrode comprises:a conductive layer disposed over the substrate, the conductive layer configured to generate an electrokinetic field; andan insulating layer disposed partially over the conductive layer, the insulating layer having at least one insulating portion substantially on an edge of the conductive layer,wherein at least one insulating portion comprises an outer portion with a first height and an inner portion having a second height that is the same or different from the first height.

31. A method of fabricating a device for capturing analytes, comprising:depositing a conductive layer over a substrate;forming a first dielectric layer over the conductive layer and the substrate; and etching the first dielectric layer to create an opening that exposes the conductive layer, wherein the opening has a first length.

32. The method of claim 31, further comprising etching the first dielectric layer to widen a top portion of the opening such that the opening has the first length closer to the conductive layer and a second length farther from the conductive layer, and wherein the second length is greater than the first length.

33. The method of any one of claims 31-32, further comprising forming a second dielectric layer over the opening to create a pillar that does not connect to the first dielectric layer.

34. The method of claim 31, further comprising forming a semipermeable membrane over the conductive layer, wherein the semipermeable membrane is coupled to opposing portions of the first dielectric layer that form the opening.

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