Systems, devices and methods for isotachophoresis - Patents.com

Abstract

Sample preparation is the first step to genomic and transcriptomic analysis and, nevertheless, can be a major source of analytical variation. Sample preparation can also be manually intensive. The present disclosure relates to fluidic systems and devices for processing, extracting, or purifying one or more analytes. These systems and devices can be used to process samples and extract nucleic acids, for example, by isotachophoresis. In particular, the systems and related methods can enable the extraction of nucleic acids, including uncrosslinked nucleic acids, from samples, such as tissues or cells. The systems and devices can also be used for multiplexed parallel sample processing.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 540,515, entitled "Isotachophoresis for Purification of Nucleic Acids," filed August 2, 2017 [Docket No. 43647-718.101]; U.S. Provisional Patent Application No. 62 / 541,086, entitled "Isotachophoresis Devices for Purification of Nucleic Acids," filed August 3, 2017 [Docket No. 43647-719.101]; and U.S. Provisional Patent Application No. 62 / 541,089, entitled "Nucleic Acid Analysis Using Isotachophoresis and Intercalating Dye," filed August 3, 2017 [Docket No. 43647-720.101], the entire contents of which are incorporated herein by reference.

[0002] This application is related to co-pending PCT Application No. PCT / US2017 / 015519, entitled "Isotachophoresis for Purification of Nucleic Acids," filed January 28, 2017 [Docket No. 43647-712.601], the entire contents of which are incorporated herein by reference.

[0003] Statement Regarding Federally Sponsored Research This invention was made with United States Government support under Contract No. 1R43HG007620-01 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0004] background For over a century, formalin-fixed, paraffin-embedded (FFPE) samples have been collected, prepared, preserved, and stored in large tissue banks. As of 2008, over 400 million FFPE samples were stored in biobanks worldwide, and the number continues to grow. These samples are often accompanied by clinical information, such as primary diagnosis, treatment regimen, and follow-up data, making such samples an important resource for therapeutic drug development and genomic and transcriptomic biomarker discovery.

[0005] Sample preparation methods for extracting and purifying nucleic acids from FFPE samples remain laborious and manual. While FFPE extraction and purification techniques vary widely, they often involve steps that are difficult to automate or accelerate, such as dewaxing, centrifugation, buffer exchange, temperature control, cross-linking reduction, and enzymatic treatment. FFPE generally refers to the use of formalin to cross-link proteins in a sample and embedding the sample in paraffin (wax). FFPE processing of samples often allows for, and is particularly useful for, long-term storage. Cross-linking proteins can bind DNA and RNA in a sample, generally rendering the sample unusable for downstream applications, such as amplification, library preparation, or sequencing.

[0006] Removal of paraffin and protein crosslinks in FFPE samples can be a very difficult process. Deparaffinization is traditionally performed using highly flammable xylenes. Samples may be treated alternately or sequentially with other solvents, mineral oil, alkaline chemistry, and / or high temperatures. After deparaffinization, proteins in the sample may be treated with various agents or subjected to conditions that may require additional time and effort.

[0007] When digestion and denaturation are complete, a mixture of crosslinked and non-crosslinked nucleic acids may remain. Removing the non-crosslinked material may be important for obtaining high-quality results from assays such as amplification or sequencing; in some cases, if the proportion of non-crosslinked material is too low, downstream assays may fail to run, resulting in the loss of the sample itself as well as labor, time, and resources. Summary of the Invention

[0008] overview Isotachophoresis (ITP) is an electrophoretic technique that uses discontinuous buffers containing a leading electrolyte (LE) with a higher effective mobility magnitude and a trailing electrolyte (TE) with a lower effective mobility magnitude (e.g., relative to the LE) to focus sample species with an effective mobility magnitude greater than that of the trailing electrolyte but less than that of the leading electrolyte. ITP can selectively focus nucleic acids from a sample by more than 10,000-fold in less than 5 minutes. The present disclosure provides methods and devices that use and automate ITP for sample preparation, including extraction, purification, concentration, and sensitive quantification, and is particularly useful for preparing and purifying nucleic acids from FFPE samples and other biological samples.

[0009] Although sample preparation is important for genome analysis, it remains a major source of analytical variability and can require a lot of manual labor.The present disclosure includes techniques and devices to address this challenge, for example, by using on-chip isotachophoresis (ITP) for nucleic acid extraction and purification.These techniques enable higher yield and higher quality nucleic acid sample preparation from FFPE and other archived samples or fresh samples, and include methods for concentrating uncrosslinked nucleic acids to produce more usable samples (for example, fewer samples that fail quality checks).

[0010] The present disclosure includes techniques and devices for automating nucleic acid sample preparation from samples including solid tissue, lysed solid tissue, preserved or fixed tissue samples (e.g., FFPE), whole blood, plasma and serum, buccal swabs, dried bloodstains and other forensic samples, fresh or fresh-frozen (FF) tissue, tissue biopsies, organ tissue, solid organ tissue, samples containing intercellular junctions (e.g., gap junctions, tight junctions, adherens junctions), cells cultured or harvested from blood or tissue, stool, and bodily fluids (e.g., saliva, urine), or any combination thereof. Samples can contain both eukaryotic and prokaryotic cellular and cell-free nucleic acids, or any combination thereof. Compared to existing methods, the disclosed techniques are faster, less labor-intensive, and more suitable for both small and large tissue starting volumes, achieving higher sample yields and higher-quality sample analysis.

[0011] An aspect of the present disclosure provides a fluidic device including an isotachophoresis (ITP) circuit including: (a) a first flow path including first and second spaced-apart capillary barriers; and (b) a first loading reservoir in fluid communication with the first flow path via a first opening in the first flow path, the first opening being disposed between the first and second capillary barriers to allow liquid entering the first flow path via the first opening to flow in one direction along the first flow path, terminate at the first capillary barrier, and flow in another direction along the first flow path, terminate at the second capillary barrier.

[0012] In some embodiments of the aspects provided herein, the liquid entering the first flow path through the first opening flows to the first capillary barrier along a path longer than the width of the first flow path. In some embodiments of the aspects provided herein, the liquid entering the first flow path through the first opening flows to the second capillary barrier along a path longer than the width of the first flow path. In some embodiments of the aspects provided herein, the liquid entering the first flow path through the first opening flows such that a meniscus of the first liquid stops at the first capillary barrier or the second capillary barrier. In some embodiments of the aspects provided herein, the first capillary barrier is configured and arranged to be breached by a liquid when a first burst pressure is applied to the one or more branched fluid circuits, and the second capillary barrier is configured and arranged to be breached by a liquid when a second burst pressure is applied to the one or more branched fluid circuits. In some embodiments of the aspects provided herein, the first and second burst pressures are approximately equal. In some embodiments of the aspects provided herein, the first burst pressure is greater than the second burst pressure. In some embodiments of the aspects provided herein, one or both of the first and second capillary barriers are cliff-type capillary barriers. In some embodiments of the aspects provided herein, the ITP circuit includes a second flow path in fluid communication with the first flow path, and the first capillary barrier is configured and arranged to stop the flow of a second liquid as the second liquid flows along the second flow path, forming a liquid-liquid interface between the first and second liquids at the first capillary barrier. In some embodiments of the aspects provided herein, one or both of the first and second capillary barriers are plateau-type capillary barriers. In some embodiments of the aspects provided herein, the plateau capillary barrier is constructed and arranged to form an air gap between the first liquid after it stops at the plateau capillary barrier and a second liquid that flows in another direction toward the plateau capillary barrier and stops at the plateau capillary barrier on the opposite side from the first liquid.In some embodiments of the aspects provided herein, one or both of the first and second capillary barriers include a plateau portion. In some embodiments of the aspects provided herein, one or both of the first and second capillary barriers include a ramp portion without a plateau portion. In some embodiments of the aspects provided herein, the first capillary barrier is a cliff-type capillary barrier and the second capillary barrier is a plateau-type capillary barrier. In some embodiments of the aspects provided herein, the at least one ITP branch further includes a third capillary barrier that is a plateau-type capillary barrier. In some embodiments of the aspects provided herein, the first minimum pressure is at least twice as high as the second minimum pressure. In some embodiments of the aspects provided herein, the fluidic device further includes a substrate having a first surface and a second surface, the first surface including a plurality of reservoirs including the first loading reservoir, the second surface including a plurality of flow paths including the first flow path, the plurality of reservoirs communicating with the plurality of flow paths via through holes in the substrate. In some embodiments of the aspects provided herein, the ITP circuit further includes a second loading reservoir and a second flow path, the second loading reservoir fluidly communicating with the second flow path via a second opening, the second flow path including a third capillary barrier configured and arranged to arrest a meniscus of liquid flowing along the second flow path at the third capillary barrier using capillary force. In some embodiments of the aspects provided herein, the second flow path is adjacent to the second capillary barrier within the first flow path, and the second capillary barrier is configured and arranged to arrest a meniscus of the liquid flowing along the second flow path at the second capillary barrier using capillary forces. In some embodiments of the aspects provided herein, the ITP circuit further includes a third loading reservoir fluidly connected to the third flow path through a third opening, the third flow path being fluidly connected to the second reservoir, and the third flow path including a fourth capillary barrier disposed between the second opening and the third opening. In some embodiments of the aspects provided herein, the first flow path or the first loading reservoir contains a sample buffer.In some embodiments of the aspects provided herein, the second flow path or second loading reservoir comprises a first leading electrolyte buffer. In some embodiments of the aspects provided herein, the third flow path or third loading reservoir comprises a second leading electrolyte buffer. In some embodiments of the aspects provided herein, the fluidic device further comprises a fourth flow path or reservoir in fluid communication with the first flow path and adjacent to the first capillary barrier. In some embodiments of the aspects provided herein, the fourth flow path or loading reservoir comprises a trailing electrolyte buffer. In some embodiments of the aspects provided herein, the ITP circuit comprises an elution flow path connected to a first elution reservoir at an elution junction. In some embodiments of the aspects provided herein, the elution flow path, the first elution reservoir, or both comprise a first elution buffer. In some embodiments of the aspects provided herein, the first opening, the second opening, the third opening, the elution junction, or a combination thereof is a through-hole. In some embodiments of the aspects provided herein, the ITP circuit includes a second elution reservoir separated from the first elution reservoir by the elution flow path, the elution flow path including a fifth capillary barrier. In some embodiments of the aspects provided herein, the third, fourth, or fifth capillary barrier, in any combination, is a plateau-type capillary barrier. In some embodiments of the aspects provided herein, the second elution reservoir includes a second elution buffer having a higher ion concentration than the first elution buffer. In some embodiments of the aspects provided herein, the ITP circuit includes a first leading electrolyte buffer reservoir connected to a second leading electrolyte buffer reservoir by a buffer flow path, the buffer flow path including first and second leading electrolyte buffers that meet at an interface where the plateau-type capillary barrier is located. In some embodiments of the aspects provided herein, the first capillary barrier, the second capillary barrier, the third capillary barrier, the fourth capillary barrier, or the fifth capillary barrier, in any combination, are adjacent to an air flow path that includes a constriction.In some embodiments of the aspects provided herein, the fluidic device further includes at least two additional ITP circuits each including a first loading reservoir and a first flow path, wherein the first loading reservoir is in fluid communication with the first flow path through a first opening, and the first flow path includes first and second spaced apart capillary barriers disposed on either side of the first opening, allowing liquid entering the first flow path through the first opening to flow in one direction along the first flow path and be terminated at the first capillary barrier, and to flow in another direction along the first flow path and be terminated at the second capillary barrier. In some embodiments of the aspects provided herein, the fluidic device further includes at least five additional ITP circuits, each including a first loading reservoir and a first flow path, wherein the first loading reservoir is in fluid communication with the first flow path through a first opening, and the first flow path includes first and second spaced apart capillary barriers disposed on either side of the first opening, allowing liquid entering the first flow path through the first opening to flow in one direction along the first flow path, be stopped at the first capillary barrier, and flow in another direction along the first flow path, be stopped at the second capillary barrier. In some embodiments of the aspects provided herein, the sample reservoir is connected to the sample flow path through a through-hole. In some embodiments of the aspects provided herein, the sample reservoir is closed by a removable material. In some embodiments of the aspects provided herein, the removable material is a film. In some embodiments of the aspects provided herein, the removable material is a heat-sealing material or an adhesive material. In some embodiments of the aspects provided herein, the removable material is a film comprising a plastic or a polymer. In some embodiments of the aspects provided herein, the fluidic device further comprises one or more air channels that open to one or more air ports and communicate with each of the capillary barriers.In some embodiments of the aspects provided herein, the fluidic device further includes: (a) a substrate having a first surface and a second surface, the first surface including a plurality of reservoirs including the first loading reservoir, the second surface including a plurality of channels including the first channel, the plurality of reservoirs communicating with the plurality of channels via through-holes in the substrate; (b) a layer of material covering the second surface, thereby forming a closed channel; and (c) a cover covering at least a portion of the first surface, the cover including a through-hole communicating with a port in the first surface through a gasket. In some embodiments of the aspects provided herein, the first surface further includes the one or more air ports. In some embodiments of the aspects provided herein, the one or more air ports have a head height shorter than that of the first loading reservoir. In some embodiments of the aspects provided herein, the one or more air ports have a head height shorter than that of at least one of the plurality of reservoirs. In some embodiments of the aspects provided herein, the cover layer is attached to the second surface by solvent heat bonding, pressure, adhesive, laser welding, or a combination thereof. In some embodiments of the aspects provided herein, the cover further comprises a porous, breathable, hydrophobic material disposed between the through-holes in the port. In some embodiments of the aspects provided herein, the first flow path is a sample flow path less than 2 mm deep. In some embodiments of the aspects provided herein, the first flow path is a sample flow path having a depth greater than about 10 μm. In some embodiments of the aspects provided herein, the sample flow path has a depth between about 400 μm and about 1.2 mm. In some embodiments of the aspects provided herein, the second flow path is a leading electrolyte buffer flow path less than about 1 mm deep. In some embodiments of the aspects provided herein, the leading electrolyte buffer flow path has a depth between about 10 μm and about 600 μm. In some embodiments of the aspects provided herein, the elution flow path has a depth less than about 1 mm. In some embodiments of the aspects provided herein, the elution channel has a depth that is from about 10 μm to about 600 μm.In some embodiments of the aspects provided herein, the first, second, or elution flow path, or a combination thereof, has a depth greater than about 40 μm or a depth greater than about 10 μm. In some embodiments of the aspects provided herein, the sample flow path has a volume of about 10 μL to about 1 mL. In some embodiments of the aspects provided herein, the sample flow path, the lead electrolyte buffer flow path, the elution flow path, or a combination thereof, has a volume of less than about 1 mL. In some embodiments of the aspects provided herein, at least one loading reservoir includes (a) a conical section in a region of the at least one reservoir that contacts the substrate, and (b) a cylindrical through-hole or opening through the substrate. In some embodiments of the aspects provided herein, the fluidic device includes at least one fluidic device including (a) an inlet for ambient air at one end, and (b) an opening through the substrate at the other end of the loading reservoir. The substrate includes at least one loading reservoir, the at least one loading reservoir having a frustoconical shape, with a wider region of the frustoconical shape disposed in the access path for ambient air and a narrower region disposed in the opening through the substrate. In some embodiments of the aspects provided herein, the frustoconical shape includes a guide wall disposed at an angle within a range of about 60° to about 90° relative to the surface of the substrate. In some embodiments of the aspects provided herein, the substrate includes an air port configured with a height or depth to minimize sample loss. In some embodiments of the aspects provided herein, the air port has a height relative to the surface of the substrate that is less than the height of the sample loading reservoir. In some embodiments of the aspects provided herein, the air port on the substrate is recessed into the surface of the first side of the substrate at a depth of about 1 μm to about 1 mm, or protrudes from the surface of the first side of the substrate at a height of about 0 μm to about 2 mm. In some embodiments of the aspects provided herein, the air port on the substrate is recessed into the surface of the first side of the substrate to a depth of about 1 μm to about 500 μm or protrudes from the surface of the first side of the substrate to a height of about 0 μm to about 1 mm. In some embodiments of the aspects provided herein, the first loading reservoir is a sample loading reservoir, the second loading reservoir is a leading electrolyte buffer reservoir, the third loading reservoir is a second leading electrolyte buffer reservoir, the fourth loading reservoir is a trailing electrolyte buffer reservoir, the fifth loading reservoir is an elution buffer reservoir, and the sixth loading reservoir is an elution buffer high reservoir.

[0013] An aspect of the present disclosure provides a method of loading the fluidic device, comprising loading the first, second, third, fourth, fifth, or sixth loading reservoir with a buffer solution.

[0014] An aspect of the present disclosure provides a method of loading the fluidic device, comprising loading a buffer into the first, second, third, fourth, fifth, or sixth flow path.

[0015] In some embodiments of the aspects provided herein, the fluidic device includes a first flow path including a plateau-shaped capillary barrier adjacent to a second flow path, and the loading step includes loading a first buffer into the first flow path or reservoir and loading a second buffer into the second flow path or reservoir. In some embodiments of the aspects provided herein, the method further includes applying a first positive or negative air pressure to the fluidic device to cause the first and second buffer solutions to stop at bases of ramps within the plateau-shaped capillary barrier. In some embodiments of the aspects provided herein, the applying the first positive or negative air pressure includes increasing or decreasing the first positive or negative pressure in increments. In some embodiments of the aspects provided herein, the method further includes applying a second positive or negative air pressure to the fluidic device to cause the first and second buffer solutions to flow along ramps on both sides of the plateau-shaped capillary barrier. In some embodiments of the aspects provided herein, applying the second positive or negative air pressure comprises increasing or decreasing the second positive or negative pressure in increments. In some embodiments of the aspects provided herein, the first and second buffer solutions rest at a plateau portion of the plateau-shaped capillary barrier, creating an air gap between them, the air gap being located above or below the plateau portion of the plateau-shaped capillary barrier. In some embodiments of the aspects provided herein, the method further comprises applying a third positive or negative air pressure to the fluidic device, causing the first and second liquids to enter the air gap, thereby forming a liquid-liquid interface between the first and second buffer solutions above or below the plateau portion of the plateau-shaped capillary barrier.

[0016] An aspect of the present disclosure provides a fluidic device including a fluid flow path and a capillary barrier disposed in the fluid flow path that restricts the flow of liquid in the fluid flow path, the capillary barrier including: (a) a ramp portion that protrudes from a surface of the fluid flow path at a first angle; (b) a plateau region; and (c) a cliff region that extends from the plateau region to the surface of the fluid flow path, the cliff region intersecting the surface at a second angle that is substantially steeper than the first angle.

[0017] In some embodiments of the aspects provided herein, the second angle is at least about 10°, at least about 15°, or at least about 20° steeper than the first angle. In some embodiments of the aspects provided herein, the ramp varies in slope along the length of the fluid flow path. In some embodiments of the aspects provided herein, the first angle is less than 60°. In some embodiments of the aspects provided herein, the second angle is greater than 60°. In some embodiments of the aspects provided herein, the plateau region is substantially parallel to the surface of the fluid flow path. In some embodiments of the aspects provided herein, the plateau region is inclined by about 10° or less relative to the surface of the fluid flow path. In some embodiments of the aspects provided herein, the ramp, plateau region, or cliff region, in any combination, has a substantially flat surface. In some embodiments of the aspects provided herein, the ramp, plateau region, or cliff region, in any combination, has a curved surface. In some embodiments of the aspects provided herein, the ramp, plateau, or cliff region has a surface that includes one or more grooves, ridges, depressions, steps, etchings, or protrusions, in any combination. In some embodiments of the aspects provided herein, the ramp, plateau, or cliff region has a surface that includes regions with faces of various angles, in any combination. In some embodiments of the aspects provided herein, the width of the ramp, plateau, or cliff region substantially occupies the width of the fluid flow path.

[0018] An aspect of the present disclosure provides a fluidic device including a fluid flow path and a capillary barrier disposed in the fluid flow path that restricts the flow of liquid in the fluid flow path, the capillary barrier including: (a) a first ramp portion that protrudes from a surface of the fluid flow path at a first angle that is less than 80°; (b) a plateau region; and (c) a second ramp portion that extends from the plateau region to the surface of the fluid flow path, the second ramp portion intersecting the surface at a second angle that is less than 80°.

[0019] In some embodiments of the aspects provided herein, the first and second angles are the same or substantially the same. In some embodiments of the aspects provided herein, the first and second angles are different. In some embodiments of the aspects provided herein, the first ramp, the second ramp, or the plateau region has a surface including one or more grooves, ridges, depressions, steps, etchings, or protrusions, in any combination.

[0020] Aspects of the present disclosure provide a fluidic device including a capillary barrier that (a) includes a trapezoidal cross-sectional area; (b) projects from an inner surface of the fluid flow path; (c) has a plateau surface that is substantially parallel to the inner surface of the fluid flow path; (d) has a ramp surface that connects the plateau surface to the inner surface of the fluid flow path and that varies in slope along the length of the fluid flow path; and (e) is constructed and arranged to stop and position a meniscus of liquid flowing along the length of the fluid flow path.

[0021] In some embodiments of the aspects provided herein, the capillary barrier extends substantially across the width of the fluid flow path. In some embodiments of the aspects provided herein, the capillary barrier is further configured and arranged to create a liquid-liquid interface. In some embodiments of the aspects provided herein, the trapezoid is an isosceles trapezoid. In some embodiments of the aspects provided herein, the trapezoid is a right-angled trapezoid that includes two angles that are substantially right angles. In some embodiments of the aspects provided herein, the trapezoid is a scalene trapezoid.

[0022] In some embodiments of the aspects provided herein, the capillary barrier is a "plateau-type capillary barrier." In some embodiments of the aspects provided herein, the capillary barrier is a "cliff-type capillary barrier." In some embodiments of the aspects provided herein, the fluidic device includes both a cliff-type capillary barrier and a plateau-type capillary barrier in the same fluidic circuit. In some embodiments of the aspects provided herein, the fluidic device further includes a sample flow path including a cliff-type capillary barrier. In some embodiments of the aspects provided herein, the fluidic device further includes a plateau-type capillary barrier located between buffer flow paths.

[0023] An aspect of the present disclosure provides an isotachophoresis (ITP) system including: (a) an interface configured to engage with a fluidic device including one or more branched fluidic circuits each including a plurality of loading reservoirs including a trailing electrolyte reservoir, a first leading electrolyte reservoir, and a first elution buffer reservoir, wherein: (i) an air manifold including a plurality of manifold air flow paths communicating with a source of positive or negative air pressure, each opening into one or more manifold ports configured to engage with one or more air ports of the fluidic device when the fluidic device is engaged with the interface; and (ii) a plurality of electrodes including first, second, and third electrodes each communicating with a voltage or current source, the plurality of electrodes configured to be disposed in the trailing electrolyte reservoir, the first leading electrolyte reservoir, and the first elution buffer reservoir, respectively, when the fluidic device is engaged with the interface; (b) a source of positive or negative air pressure in communication with the air manifold; and (c) a voltage or current source in communication with the electrodes.

[0024] In some embodiments of the aspects provided herein, the isotachophoresis system includes a motor for actuating an interface with an engaged fluidic device. In some embodiments of the aspects provided herein, the fluidic device engages with the interface. In some embodiments of the aspects provided herein, the air manifold further includes a valve for controlling air pressure to an air flow path in at least one of the branched fluidic circuits. In some embodiments of the aspects provided herein, the isotachophoresis system further includes: (d) a ridge having a long, thin tip, a heating element configured to heat the tip, and an actuator configured to press the ridge tip against the fluidic device engaged with the interface to close multiple fluid flow paths in the microfluidic device. In some embodiments of the aspects provided herein, the system is configured so that multiple fluid flow paths can be closed with a heat-sealable material, PCR film, parafilm, plastic wrap, an adhesive layer, or a material not secured by a seal.

[0025] In some embodiments of the aspects provided herein, the system is configured such that a plurality of fluid flow paths within the fluidic device can be closed by a load-bearing block. In some embodiments of the aspects provided herein, the system is configured such that a plurality of fluid flow paths within the fluidic device can be closed by a mechanical actuator block having a rubber sealing member.

[0026] In some embodiments of the aspects provided herein, the system further includes a temperature measuring device. In some embodiments of the aspects provided herein, the system further includes a display device for displaying an operating parameter of the system. In some embodiments of the aspects provided herein, the display device displays the temperature.

[0027] In some embodiments of the aspects provided herein, the display device displays a measure of the light detected by the light sensor. In some embodiments of the aspects provided herein, the display device displays a voltage or current across the fluid circuit. In some embodiments of the aspects provided herein, the system further includes a voltage or current measuring device. In some embodiments of the aspects provided herein, the system further includes an optical assembly including one or more light sources configured to send light into the fluid flow paths of the fluid circuit and one or more light sensors for detecting light emitted from the fluid flow paths of the fluid circuit. In some embodiments of the aspects provided herein, the interface further includes one or more alignment marks for aligning the fluidic device in a particular orientation. In some embodiments of the aspects provided herein, the system further includes software that adjusts electrodes in response to temperature, current, or voltage. In some embodiments of the aspects provided herein, the fluidic device further includes multiple branched fluidic circuits, each including an independent electrical circuit. In some embodiments of the aspects provided herein, each of the branched fluidic circuits is coupled to the same voltage or current source or to different voltage or current sources.

[0028] An aspect of the present disclosure is a method for detecting a sample loading reservoir comprising: (a) providing a fluidic device comprising at least one branched fluidic circuit including a trailing electrolyte buffer reservoir, a first flow path, a first leading electrolyte buffer reservoir, a sample loading reservoir, a second leading electrolyte buffer reservoir, and a first elution buffer reservoir, all in fluid communication with each other, wherein (i) the trailing electrolyte buffer reservoir contains a trailing electrolyte buffer; (ii) the first leading electrolyte buffer reservoir contains a first leading electrolyte buffer; (iii) the second leading electrolyte buffer reservoir contains a second electrolyte buffer different from the first electrolyte buffer; and (iv) the first elution buffer reservoir contains a first elution buffer; (b) disposing the trailing electrolyte buffer reservoir in the first elution buffer reservoir; (c) loading a sample into the air gap between the trailing electrolyte buffer and the leading electrolyte buffer in the first flow path; and (d) applying air pressure to the second leading electrolyte buffer reservoir and the first elution buffer reservoir, such that the second leading electrolyte buffer and the first elution buffer each enter the fluid circuit substantially simultaneously.

[0029] In some embodiments of the aspects provided herein, the air pressure is positive or negative air pressure. In some embodiments of the aspects provided herein, applying the air pressure in step (b) results in the trailing electrolyte buffer being stopped at a first capillary barrier in the first flow path and the leading electrolyte buffer being stopped at a second capillary barrier in the first flow path. In some embodiments of the aspects provided herein, applying the air pressure in step (d) results in the second leading electrolyte buffer being stopped at a third capillary barrier in the fluid circuit and the first elution buffer being stopped at a fourth capillary barrier in the fluid circuit.

[0030] In some embodiments of the aspects provided herein, the first and second capillary barriers are cliff-type capillary barriers or ramp-type capillary barriers. In some embodiments of the aspects provided herein, the third and fourth capillary barriers are plateau-type capillary barriers. In some embodiments of the aspects provided herein, the third and fourth capillary barriers each have a lower burst pressure than the burst pressure of the first capillary barrier or the second capillary barrier. In some embodiments of the aspects provided herein, the sample comprises a wetting agent.

[0031] An aspect of the disclosure is a fluidic device including one or more branched fluidic circuits, each of which includes an isotachophoresis ("ITP") branch and an elution branch in communication with the ITP branch, (a) the ITP branches include a trailing electrolyte buffer reservoir, a sample flow path, a leading electrolyte buffer flow path, a first leading buffer electrolyte reservoir, and a second leading electrolyte buffer reservoir, all in communication with each other, (i) the sample flow path is separated from the trailing electrolyte reservoir by a first cliff-type capillary barrier and the leading electrolyte buffer reservoir is separated from the second cliff-type capillary barrier by a second cliff-type capillary barrier. (i) the first elution buffer reservoir is separated from the second elution buffer reservoir by a second plateau-type capillary barrier; and (ii) the leading electrolyte buffer reservoir is separated from the second leading electrolyte reservoir by a first plateau-type capillary barrier; and (b) the elution branch comprises an elution flow path, a first elution buffer reservoir, and a second elution buffer reservoir, all in communication with each other, and (i) the first elution buffer reservoir is separated from the second elution buffer reservoir by a second plateau-type capillary barrier; and (ii) the leading electrolyte buffer flow path is separated from at least a portion of the elution flow path by a third plateau-type capillary barrier.

[0032] An aspect of the present disclosure is a method for producing a fluidic device comprising: (a) providing a fluidic device of any aspect provided herein, wherein (i) the trailing electrolyte buffer reservoir comprises a trailing electrolyte buffer; (ii) the first leading electrolyte buffer reservoir comprises a first leading electrolyte buffer; (iii) the second leading electrolyte buffer reservoir comprises a second leading electrolyte buffer; (iv) the first elution buffer reservoir comprises a first elution buffer; and (v) the second elution buffer reservoir comprises a second elution buffer; (b) providing a fluidic device of any aspect provided herein, wherein (i) the trailing electrolyte buffer reservoir comprises a trailing electrolyte buffer; (ii) the first leading electrolyte buffer reservoir comprises a first leading electrolyte buffer; (iii) the second leading electrolyte buffer reservoir comprises a second leading electrolyte buffer; (iv) the first elution buffer reservoir comprises a first elution buffer; and (v) the second elution buffer reservoir comprises a second elution buffer; (c) applying negative air pressure to the first, second, and third plateau-type capillary barriers to prime a trailing electrolyte buffer and a first leading electrolyte buffer at the cliff-type capillary barrier; (c) loading a sample containing a wetting agent into the sample flow path sufficient to create a fluid connection across the first and second cliff-type capillary barriers; and (d) applying negative air pressure to the first, second, and third plateau-type capillary barriers to create a fluid connection across the first, second, and third plateau-type capillary barriers.

[0033] In some embodiments of the aspects provided herein, the method further includes: (e) inserting a first electrode into the subsequent electrolyte in the subsequent electrolyte buffer reservoir; (f) inserting a second electrode into the second leading electrolyte buffer in the second leading electrolyte buffer reservoir; and (g) applying a voltage or current across the first electrode and the second electrode.

[0034] In some embodiments of the aspects provided herein, the method further includes (h) inserting a third electrode into the second elution buffer in the second elution buffer reservoir; and (i) after step (g), applying a voltage or current across the first and third electrodes and optionally reducing the current at the second electrode.

[0035] In some embodiments of the aspects provided herein, the method further comprises adding a topper solution to the sample reservoir. In some embodiments of the aspects provided herein, the method further comprises spiking the sample with a subsequent electrolyte buffer. In some embodiments of the aspects provided herein, the method further comprises applying a voltage or current in response to a triggering event. In some embodiments of the aspects provided herein, the voltage is in the range of about 0 V to about 1500 V. In some embodiments of the aspects provided herein, the method further comprises applying a negative air pressure of about 0 mpsi to about 200 mpsi. In some embodiments of the aspects provided herein, the applied negative air pressure is about 10 mpsi to about 80 mpsi.

[0036] An aspect of the present disclosure provides a fluidic device including a fluid flow path, the fluid flow path including: (a) a first wall substantially parallel to a third wall and a second wall substantially parallel to a fourth wall; and (b) a capillary barrier, the capillary barrier including: (i) a side disposed on or incorporated within an interior surface of the second wall and extending substantially between the first wall and the third wall; (ii) first and second outer walls each including a trapezoidal cross-sectional area connected to, incorporated within, or adjacent to the first and third walls, respectively; (iii) a plateau surface substantially parallel to the second wall and located between the second and fourth walls; and (iv) a ramp portion connecting the second wall to the plateau surface, the ramp portion having a varying slope along the length of the fluid flow path.

[0037] In some embodiments of the aspects provided herein, the trapezoid is an isosceles trapezoid. In some embodiments of the aspects provided herein, the trapezoid is a right-angled trapezoid containing two angles that are substantially right angles. In some embodiments of the aspects provided herein, the trapezoid is a scalene trapezoid. In some embodiments of the aspects provided herein, the capillary barrier is a "plateau-type capillary barrier." In some embodiments of the aspects provided herein, the capillary barrier is a "cliff-type capillary barrier." In some embodiments of the aspects provided herein, the fluidic device includes both a cliff-type capillary barrier and a plateau-type capillary barrier in the same fluidic circuit. In some embodiments of the aspects provided herein, the fluidic device further includes a sample flow path including a cliff-type capillary barrier. In some embodiments of the aspects provided herein, the fluidic device further includes a plateau-type capillary barrier located between buffer flow paths.

[0038] An aspect of the present disclosure provides a fluidic device including a fluid flow path, the fluid flow path including a capillary barrier protruding into the fluid flow path from a first wall of the fluid flow path, the capillary barrier including: (i) two outer surfaces each having a trapezoidal cross-sectional area; (ii) a plateau side substantially parallel to the first wall of the fluid flow path; and (iii) a ramp portion having one edge intersecting the plateau side to form an internal obtuse angle of the capillary barrier and an opposite edge intersecting the first wall of the fluid flow path to form an internal acute angle of the capillary barrier.

[0039] In some embodiments of the aspects provided herein, the capillary barrier further includes a side connecting the plateau side to the first wall. In some embodiments of the aspects provided herein, the side connecting the plateau side to the first wall is approximately perpendicular to the first wall. In some embodiments of the aspects provided herein, the side connecting the plateau side to the first wall intersects the first wall at an acute angle. In some embodiments of the aspects provided herein, at least one of the outer surfaces is substantially parallel to or integrated into a second wall of the fluid flow path.

[0040] An aspect of the present disclosure provides a fluidic system including: (a) a first isotachophoresis circuit in a microfluidic chip, the first isotachophoresis circuit including: (i) a first sample reservoir; (ii) a trailing electrolyte buffer reservoir in fluid communication with the sample reservoir, the trailing electrolyte buffer reservoir containing a leading electrolyte buffer; and (iii) a leading electrolyte buffer flow path in fluid communication with the sample reservoir, the leading electrolyte buffer flow path containing a leading electrolyte buffer; (b) a sensor configured to detect a temperature change in the leading electrolyte buffer flow path; and (c) a device configured to monitor a voltage or current in the first isotachophoresis circuit and to provide a constant current within the first isotachophoresis circuit.

[0041] In some embodiments of the aspects provided herein, the leading electrolyte flow path comprises an elution flow path. In some embodiments of the aspects provided herein, the sensor is constructed and arranged to detect a temperature change in the elution flow path. In some embodiments of the aspects provided herein, the fluidic system further comprises an elution well. In some embodiments of the aspects provided herein, the first isotachophoresis circuit further comprises an elution flow path comprising an elution buffer.

[0042] An aspect of the present disclosure provides a fluidic system including: (a) a first isotachophoresis circuit in a microfluidic chip, the first isotachophoresis circuit including: (i) a first sample reservoir in fluid communication with a first fluid flow path; (ii) first, second, and third buffer reservoirs in fluid communication with the first fluid flow path, the first and second buffer reservoirs being separated by a first capillary barrier; and (iii) an elution reservoir in fluid communication with the first fluid flow path; (b) a sensor configured to detect a temperature change in the first fluid flow path within the first isotachophoresis region; and (c) a device configured to monitor voltage or current and provide a constant current in the first isotachophoresis circuit.

[0043] In some embodiments of the aspects provided herein, the first fluid flow path includes a second capillary barrier adjacent to the first sample reservoir. In some embodiments of the aspects provided herein, the first capillary barrier is a plateau-type capillary barrier and the second capillary barrier is a cliff-type capillary barrier. In some embodiments of the aspects provided herein, the first capillary barrier is a cliff-type capillary barrier, a plateau-type capillary barrier, or a ramp-type capillary barrier. In some embodiments of the aspects provided herein, the first fluid flow path includes a cliff-type capillary barrier and a constriction downstream from the cliff-type capillary barrier. In some embodiments of the aspects provided herein, the system further includes a temperature sensor configured downstream from the constriction.

[0044] An aspect of the present disclosure provides a fluidic system including: a fluidic chip including a plurality of circuits, each including an elution flow path in fluid communication with an elution reservoir; and a mechanical member including a ridge portion configured to simultaneously apply mechanical pressure to the plurality of elution flow paths via the ridge portion to at least partially close the elution flow paths by plastic deformation of at least one wall of the elution flow path.

[0045] In some embodiments of the aspects provided herein, the system further includes a bottom film adhered to the substrate layer, the bottom layer forming a wall of each of the elution channels, and the bottom film and the substrate layer each include a material having the same melting point. In some embodiments of the aspects provided herein, each elution channel includes a bend, and a ridge at least partially closes each elution channel at two locations on either side of the bend. In some embodiments of the aspects provided herein, the ridge completely closes the channel.

[0046] Aspects of the present disclosure provide a method of recovering an analyte from an assay, comprising: introducing an analyte into one of the circuits in the fluid system of aspects provided herein; allowing the analyte to migrate to the elution channel in the one of the circuits; and actuating the mechanical member to apply mechanical pressure to the plurality of elution channels via the ridges to at least partially close the elution channel by plastic deformation of at least one wall of the elution channel.

[0047] An aspect of the present disclosure provides a fluidic device including a first liquid flow path; a gas flow path in fluid communication with the first liquid flow path; an air port in fluid communication with the gas flow path; and a breathable hydrophobic membrane disposed in the air port, the hydrophobic membrane being non-liquid permeable and configured to prevent liquid from exiting the air port when negative pressure is applied to the gas flow path via the air port.

[0048] In some embodiments of the aspects provided herein, the device further includes a gasket disposed over the air port. In some embodiments of the aspects provided herein, the device further includes a constriction between the liquid flow path and the gas flow path to prevent liquid from exiting the gas flow path. In some embodiments of the aspects provided herein, the gasket is secured in place by a cover layer including a flow path that communicates with the gas flow path through the port. In some embodiments of the aspects provided herein, the cover layer includes an interference fit configured to maintain a compressive force on the gasket.

[0049] An aspect of the present disclosure provides a method comprising: (a) providing a fluid circuit including: (i) an elution well adjacent to an elution flow path containing an elution buffer, the elution flow path being connected to a leading electrolyte flow path containing a leading electrolyte buffer; and (ii) a capillary barrier located at an interface between the elution buffer and the leading electrolyte buffer; (b) flowing the interface between the leading electrolyte buffer and the elution buffer toward the elution well; and (c) stopping the flow at the interface between the leading electrolyte buffer and the elution buffer so that the capillary barrier is completely engulfed by the leading electrolyte buffer.

[0050] In some embodiments of the aspects provided herein, the fluidic circuit further includes a sample well in fluid communication with the elution well. In some embodiments of the aspects provided herein, the method further includes introducing a nucleic acid sample into the sample well and applying an electric current to the fluidic circuit to move the nucleic acid sample across the capillary barrier.

[0051] An aspect of the disclosure provides a fluidic device that (a) includes a fluidic circuit having a trailing electrolyte buffer reservoir, a sample flow path, a leading electrolyte buffer flow path, and an elution reservoir, all in communication with one another, where (i) the leading electrolyte buffer flow path is fluidly connected to the elution reservoir through an opening in the leading electrolyte buffer flow path located below the elution reservoir; (ii) the trailing electrolyte buffer reservoir contains a trailing electrolyte buffer, (ii) the sample flow path contains an analyte, (iii) the leading electrolyte buffer flow path contains a leading electrolyte buffer, and (iv) the elution reservoir contains an elution buffer. and (b) applying an electric current to the fluid circuit to move the analyte to the elution reservoir, the electric current constructed and arranged to generate a first temperature at an interface between the analyte and the trailing electrolyte buffer and a second temperature at an interface between the sample and the leading electrolyte buffer, wherein a temperature difference exists between the first temperature and the second temperature; and when the analyte reaches the opening in the leading electrolyte buffer flow path located below the elution reservoir, the analyte is driven by the temperature difference into the elution reservoir.

[0052] In some embodiments of the aspects provided herein, the method further comprises pipetting the analyte from the elution reservoir.

[0053] An aspect of the present disclosure provides a method for quantifying a nucleic acid sample, comprising: providing a fluidic device including an isotachophoresis (ITP) flow path containing a nucleic acid sample comprising nucleic acids complexed with an intercalating dye; performing ITP in the fluidic flow path to focus the nucleic acids complexed with the intercalating dye; and quantifying the nucleic acids complexed with the intercalating dye in the flow path by measuring the intensity of the intercalating dye after ITP has been performed, wherein the intercalating dye comprises one or more of SYTO™ 13, PicoGreen®, EvaGreen®, or Quantifluor®.

[0054] In some embodiments of the aspects provided herein, the dye comprises SYTO™ 13. In some embodiments of the aspects provided herein, the dye comprises PicoGreen®. In some embodiments of the aspects provided herein, the dye comprises EvaGreen®. In some embodiments of the aspects provided herein, the dye comprises Quantifluor®. In some embodiments of the aspects provided herein, the nucleic acid complexed with the intercalating dye is located in a region of the ITP flow path that comprises a leading electrolyte buffer or an elution buffer. In some embodiments of the aspects provided herein, the nucleic acid complexed with the intercalating dye comprises RNA or DNA, or a combination thereof. In some embodiments of the aspects provided herein, the nucleic acid sample further comprises a contaminant. In some embodiments of the aspects provided herein, the contaminant comprises a protein, cellular debris, lipids, plasma membranes, small molecules, or a combination thereof. In some embodiments of the aspects provided herein, performing ITP in the fluid flow path separates the nucleic acid complexed with the intercalating dye from the contaminant.

[0055] An aspect of the present disclosure provides a fluidic device including one or more branched fluidic circuits, each of the branched fluidic circuits including an isotachophoresis ("ITP") branch and an elution branch in communication with the ITP branch, the ITP branches including a trailing electrolyte buffer reservoir, a sample flow path, a leading electrolyte buffer flow path, a first leading buffer electrolyte reservoir, and a second leading electrolyte buffer reservoir, all in communication with each other; the elution branch including an elution flow path and an elution well, the elution well including first and second through-holes in communication with the elution flow path.

[0056] In some embodiments of the aspects provided herein, the first and second through-holes are circular. In some embodiments of the aspects provided herein, the first through-hole has an oval shape. In some embodiments of the aspects provided herein, the first through-hole has a maximum transverse dimension of less than 1.5 mm or less than 1 mm. In some embodiments of the aspects provided herein, the second through-hole has a maximum transverse dimension of less than 1.5 mm. In some embodiments of the aspects provided herein, the second through-hole has a maximum transverse dimension of less than 1 mm. In some embodiments of the aspects provided herein, the first through-hole and the second through-hole are on the same vertical plane within the elution well. In some embodiments of the aspects provided herein, the first through-hole and the second through-hole are on different vertical planes within the elution well. In some embodiments of the aspects provided herein, the first through-hole and the second through-hole are aligned with a longitudinal axis of the elution channel. In some embodiments of the aspects provided herein, the first through-hole is configured to restrain a pipette tip in a predetermined binding position. In some embodiments of the aspects provided herein, the first through-hole comprises a circular cross-section. In some embodiments of the aspects provided herein, the first through-hole comprises an oval cross-section. In some embodiments of the aspects provided herein, the first through-hole comprises a D-shaped cross-section. In some embodiments of the aspects provided herein, the first through-hole comprises a guide wall disposed at an angle within a range of about 60° to about 90° relative to the flow path. In some embodiments of the aspects provided herein, the elution well comprises one or more vertical gates separating the first through-hole and the second through-hole. In some embodiments of the aspects provided herein, the elution well comprises a circular cross-section. In some embodiments of the aspects provided herein, the elution well comprises an elongated cross-section.

[0057] An aspect of the present disclosure provides a fluidic device comprising: a first flow path terminating at an end in a first through hole; a second flow path terminating at an end in a second through hole; and a fluid reservoir defined by a wall having a height of 25 mm or less, 15 mm or less, 10 mm or less, or greater than 10 mm; the reservoir is in fluid communication with each of the two fluid flow paths through the first and second through holes, and the first through hole enters the reservoir at a location in the reservoir that is lower than the second through hole.

[0058] In some embodiments of the aspects provided herein, the first flow path communicates with a first electrode; the second flow path communicates with a second electrode; the flow path and reservoir contain a conductive fluid, and application of a voltage across the first and second electrodes generates a current that travels through the reservoir. In some embodiments of the aspects provided herein, the wall has a height within a range of about 8 mm to about 10 mm. In some embodiments of the aspects provided herein, the first and second through-holes each have a height within a range of about 0.2 mm. 2 ~7mm 2 and approximately 0.2 mm 2 ~7mm 2 In some embodiments of the aspects provided herein, the first and second through holes each have an area of ​​about 0.8 mm 2 ~1.5mm 2 and approximately 1 mm 2 ~2.75mm 2 In some embodiments of the aspects provided herein, the second through-hole enters the reservoir through a platform in the reservoir that is positioned about 1 mm to about 6 mm above the entry point of the first through-hole into the reservoir. In some embodiments of the aspects provided herein, the volume of the reservoir between the first and second through-holes is about 2.5 mL, 1 mL, or 0.5 mL or less. In some embodiments of the aspects provided herein, the volume of the reservoir between the first and second through-holes is 0.1 mL.

[0059] An aspect of the present disclosure provides a method comprising providing any of the fluidic devices described herein, wherein the flow paths and reservoirs contain a conductive fluid and the first flow path further contains an ionic analyte; applying a voltage across the first and second electrodes to generate a current that moves through the reservoir; and moving the analyte through the first through-hole and into the reservoir.

[0060] In some embodiments of the aspects provided herein, the analyte comprises a nucleic acid, e.g., RNA or DNA. In some embodiments of the aspects provided herein, the method further comprises performing isotachophoresis in the first flow path, whereby the analyte migrates into the reservoir.

[0061] An aspect of the present disclosure provides a fluidic device comprising: a fluid flow path having a length and a width; and a reservoir disposed above the fluid flow path and fluidly connected to the fluid flow path through one or more through holes; at least a portion of each through hole is substantially coextensive with the fluid flow path across the width of the fluid flow path, the fluid flow path and the reservoir contain a conductive fluid, and are shaped such that when an electric current is passed through the fluid flow path, at least 5%, at least 6%, at least 7%, at least 10%, or at least 20% of the electric current passes through the reservoir.

[0062] In some embodiments of the aspects provided herein, the reservoir includes one through-hole. In some embodiments of the aspects provided herein, the reservoir includes two through-holes arranged along the longitudinal axis of the flow channel. In some embodiments of the aspects provided herein, the one or two through-holes have a square, rectangular, oval, or elongated dimension in the width direction. In some embodiments of the aspects provided herein, the one or two through-holes have one or two sides that span the width of the fluid flow channel or the width of the reservoir, and each of the sides is at least 75% straight.

[0063] An aspect of the present disclosure provides a method comprising providing any of the devices described herein, wherein the flow path and reservoir contain a conductive fluid, and the reservoir contains an ionic analyte; and passing an electric current through the flow path; wherein the electric current moves at least some of the analyte from the reservoir into the flow path.

[0064] An aspect of the present disclosure provides a fluidic device including one or more branched fluidic circuits, each of the branched fluidic circuits including an isotachophoresis ("ITP") branch including a trailing electrolyte buffer reservoir, a sample flow path, a leading electrolyte buffer flow path, a first leading buffer electrolyte reservoir, and a second leading electrolyte buffer reservoir, all in communication with each other, and a sample reservoir including a first through-hole in communication with the sample flow path.

[0065] In some embodiments of the aspects provided herein, the first through-hole has a square or rectangular shape. In some embodiments of the aspects provided herein, the first through-hole has a maximum dimension in a range of about 0.5 mm to about 5 mm. In some embodiments of the aspects provided herein, the first through-hole has a maximum dimension of about 1.5 mm. In some embodiments of the aspects provided herein, the first through-hole has a maximum dimension of about 1 mm. In some embodiments of the aspects provided herein, the first through-hole has a volume of less than about 15 μL. In some embodiments of the aspects provided herein, the first through-hole has a volume of about 7 μL. In some embodiments of the aspects provided herein, the first through-hole has a width in a range of about 80% to about 120% of the width of the sample channel. In some embodiments of the aspects provided herein, the first through-hole has a width of about 100% of the width of the sample channel. In some embodiments of the aspects provided herein, the sample reservoir further comprises a second through-hole in communication with the sample channel. In some embodiments of the aspects provided herein, the first and second through-holes are separated by a filler block. In some embodiments of the aspects provided herein, the filler block has an in-channel height in the range of about 0.2 mm to about 2 mm. In some embodiments of the aspects provided herein, the filler block has an in-channel height of about 1.2 mm. In some embodiments of the aspects provided herein, the second through-hole has a square or rectangular shape. In some embodiments of the aspects provided herein, the second through-hole has a maximum dimension in the range of about 0.5 mm to about 5 mm. In some embodiments of the aspects provided herein, the second through-hole has a maximum dimension of about 1.5 mm. In some embodiments of the aspects provided herein, the second through-hole has a maximum dimension of about 1 mm. In some embodiments of the aspects provided herein, the second through-hole has a volume of less than about 15 μL. In some embodiments of the aspects provided herein, the second through-hole has a volume of about 7 μL.In some embodiments of the aspects provided herein, the second through-hole has a width in the range of about 80% to about 120% of the width of the sample channel. In some embodiments of the aspects provided herein, the second through-hole has a width in the range of about 80% to about 120% of the width of the sample channel. In some embodiments of the aspects provided herein, when an electric field is applied to the ITP branch, more than 10% of the applied current travels above the top surface of the sample channel along the length of the sample reservoir. In some embodiments of the aspects provided herein, the sample reservoir has a conical shape, the narrower portion of which has a diameter in the range of about 0.1 mm to about 4 mm. In some embodiments of the aspects provided herein, the sample reservoir has a conical shape, the narrower portion of which has a diameter in the range of about 1 mm to about 4 mm. In some embodiments of the aspects provided herein, the sample reservoir has an oval shape.

[0066] An aspect of the present disclosure provides an apparatus for performing vertical isotachophoresis, comprising one or more cylindrical columns including an internal flow path defined by the interior walls of the cylindrical columns, each cylindrical column comprising: a first stage including a first gel plug disposed at a first position within the internal flow path and a first space within the internal flow path disposed between the first gel plug and an upper end of the cylindrical column; a second stage including a second gel plug disposed at a second position within the internal flow path located below the first position and oriented in alignment with gravity, and a second space within the internal flow path disposed between the first gel plug and the second gel plug; and a third stage including a third gel plug disposed at a third position within the internal flow path located below the second position and oriented in alignment with gravity, and a third space within the internal flow path disposed between the second gel plug and the third gel plug.

[0067] In some embodiments of the aspects provided herein, the one or more cylindrical columns comprise a plurality of cylindrical columns, the plurality of cylindrical columns being arranged to fit standard microtiter plate dimensions. In some embodiments of the aspects provided herein, the one or more cylindrical columns have a cross-sectional column area of ​​approximately 9 mm x 9 mm. In some embodiments of the aspects provided herein, the first space comprises a trailing electrolyte buffer, the second space comprises the analyte, and the third space comprises a first leading electrolyte buffer. In some embodiments of the aspects provided herein, the device further comprises a fourth stage comprising a fourth gel plug disposed in a fourth position within the internal flow path that is lower than the third position and oriented in alignment with gravity, and a fourth space disposed within the internal flow path between the third and fourth gel plugs. In some embodiments of the aspects provided herein, the fourth space comprises an elution buffer. In some embodiments of the aspects provided herein, the device further includes a fifth stage including a fifth gel plug disposed in the internal flow channel at a fifth position that is lower than the fourth position and oriented in alignment with gravity, and a fifth space disposed in the internal flow channel between the fourth and fifth gel plugs. In some embodiments of the aspects provided herein, the fifth space contains a second lead electrolyte buffer.

[0068] An aspect of the present disclosure provides a method of focusing an analyte, comprising: introducing an analyte into the second space of the second stage above the second gel plug of any of the vertical or column-type ITP devices described herein; and applying an electric current to the device to move the analyte from the second space, through the second gel plug, and into the third space in the direction of gravity.

[0069] 256. The method of claim 255, further comprising applying said current to said device to cause said analyte to migrate in the direction of gravity from the third space, through a third gel plug, and into said fourth space.

[0070] In some embodiments of the aspects provided herein, the method further includes removing the analyte from the fourth space.

[0071] An aspect of the present disclosure provides a sample purification method comprising: (a) loading into a fluidic device (i) a tissue sample comprising dissolved solid tissue containing nucleic acids and contaminants, (ii) a trailing electrolyte buffer comprising first trailing electrolyte ions of an effective mobility having a magnitude lower than the magnitude of the effective mobility of the nucleic acid, and (iii) a leading electrolyte buffer comprising first leading electrolyte ions of a second effective mobility having a magnitude higher than the magnitude of the effective mobility of the nucleic acid; and (b) applying an electric field within the fluidic device to perform isotachophoresis on the first trailing electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby purifying the nucleic acid from the contaminants in the tissue sample.

[0072] In some embodiments of the aspects provided herein, the effective mobility of the first subsequent electrolyte ion is greater than the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the fluidic device is a microfluidic chip, and the tissue sample, the subsequent electrolyte buffer, and the leading electrolyte buffer are loaded into a first zone of the microfluidic chip. Some embodiments of the aspects provided herein may further include performing at least one sample preparation procedure on the tissue sample in the first zone of the microfluidic chip, selected from the group consisting of: (1) removal of embedding material, (2) tissue disruption, (3) cell lysis, (4) decrosslinking of the nucleic acid, (5) protein digestion, and (6) digestion of the nucleic acid. In some embodiments of the aspects provided herein, the isotachophoresis is performed in a second zone of the microfluidic chip, and the second zone is separated from and fluidly connected to the first zone. In some embodiments of the aspects provided herein, the solid tissue is derived from a solid organ. In some embodiments of the aspects provided herein, the lysed solid tissue comprises a chemical fixative. In some embodiments of the aspects provided herein, the chemical fixative is formalin. In some embodiments of the aspects provided herein, the solid tissue is formalin-fixed, paraffin-embedded (FFPE) tissue. In some embodiments of the aspects provided herein, the lysed solid tissue comprises urea or thiourea. Some embodiments of the aspects provided herein further comprise disrupting intercellular junctions, extracellular matrix, or connective tissue to obtain the lysed solid tissue. In some embodiments of the aspects provided herein, the lysed solid tissue comprises solid particles. In some embodiments of the aspects provided herein, the nucleic acid comprises dispersed or solvated nucleic acid. In some embodiments of the aspects provided herein, the contaminant is selected from the group consisting of crosslinked nucleic acid, embedding material, tissue debris, fixative, protein, inhibitor, and combinations thereof. In some embodiments of the aspects provided herein, the contaminant comprises crosslinked nucleic acid. In some embodiments of the aspects provided herein, the tissue sample is combined with the subsequent electrolyte buffer prior to the loading.In some embodiments of the aspects provided herein, the tissue sample is combined with the leading electrolyte buffer prior to the loading. In some embodiments of the aspects provided herein, the loading of the leading electrolyte buffer is performed prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the solid tissue is dissolved in the leading electrolyte buffer prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the solid tissue is dissolved in the trailing electrolyte buffer prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure comprises removing embedding material by incubating the tissue sample in the fluidic device at a temperature of at least about 37°C for a period of at least about 1 minute prior to the application of the electric field. In some embodiments of the aspects provided herein, the temperature is from about 40°C to about 80°C. In some embodiments of the aspects provided herein, the period is from about 1 minute to about 120 minutes. In some embodiments of the aspects provided herein, the sample preparation procedure comprises disrupting tissue or lysing cells by applying mechanical stress to the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure comprises disrupting tissue or lysing cells by applying heat to the tissue sample. In some embodiments of the aspects provided herein, the application of heat results in a temperature of the tissue sample of about 30°C to about 80°C. In some embodiments of the aspects provided herein, the sample preparation procedure comprises disrupting tissue or lysing cells by contacting the tissue sample with a solution having a pH of at least 10 or proteolytically digesting the tissue sample. In some embodiments of the aspects provided herein, the proteolytic digestion is performed at a temperature greater than about 25°C. In some embodiments of the aspects provided herein, the sample preparation procedure comprises disrupting tissue or lysing cells by adding at least one detergent to the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure comprises disrupting tissue or lysing cells by adding a solution comprising urea to the tissue or cell sample.In some embodiments of the aspects provided herein, the solution further comprises thiourea. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is in the range of about 4 M to about 9 M, and the concentration of the thiourea in the solution is in the range of about 0.5 M to about 3.5 M. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is in the range of about 6.5 M to about 7.5 M, and the concentration of the thiourea in the solution is in the range of about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the sample preparation procedure comprises decrosslinking the nucleic acid by digesting crosslinked proteins with proteinase K. In some embodiments of the aspects provided herein, the sample preparation procedure comprises digesting the nucleic acid with DNase or RNase. Some embodiments of the aspects provided herein further comprise eluting an output solution comprising the purified nucleic acid from an outlet reservoir of the fluidic device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the tissue sample and the purified nucleic acid in the output solution comprise crosslinked nucleic acids, and the concentration of the crosslinked nucleic acid in the output solution is at least about two-fold lower than the concentration of the crosslinked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the contaminant is present in the output solution at a concentration that is at least two-fold lower than the concentration of the contaminant in the tissue sample. In some embodiments of the aspects provided herein, the first subsequent electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the first leading electrolyte ion comprises chloride. In some embodiments of the aspects provided herein, the subsequent electrolyte buffer comprises a second subsequent electrolyte ion having a different effective mobility than the first subsequent electrolyte ion. In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or MOPS (3-(N-morpholino)propanesulfonic acid).In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and the first subsequent electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid) and the first subsequent electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and the first subsequent electrolyte ion comprises MOPS. In some embodiments of the aspects provided herein, the subsequent electrolyte buffer comprises a second subsequent electrolyte ion having a second effective mobility, the second effective mobility having an order of magnitude that is about the same as or smaller than the order of magnitude of the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the tissue sample loaded into the fluidic device has a volume of at least 50 μL. Some embodiments of the aspects provided herein further include performing a first sample processing procedure on the tissue sample in the first zone of the microfluidic chip and performing an enzymatic reaction on the tissue sample in the second zone of the microfluidic chip. In some embodiments of the aspects provided herein, the first sample processing procedure includes removal of embedding material, tissue disruption, or cell lysis, and the enzymatic reaction includes decrosslinking of the nucleic acid, digestion of proteins, or digestion of nucleic acids. In some embodiments of the aspects provided herein, the first zone and the second zone are each heated to a temperature greater than 37° C. In some embodiments of the aspects provided herein, the first zone is heated to a temperature of about 60° C. to 100° C. during the first sample processing procedure, and the second zone is heated to a temperature of 40° C. to 60° C.

[0073] An aspect of the present disclosure provides a method for detecting nucleic acids, the method comprising the steps of: (a) loading, into a first flow path of a microfluidic chip, (i) a first sample comprising a first nucleic acid and a first contaminant, (ii) a first subsequent electrolyte buffer comprising a first subsequent ion, the magnitude of the effective mobility of which is smaller than the magnitude of the effective mobility of the first nucleic acid, and (iii) a first leading electrolyte buffer comprising a first leading ion, the magnitude of the effective mobility of which is larger than the magnitude of the effective mobility of the first nucleic acid; (b) loading, into a second flow path of the microfluidic chip, (i) a second sample comprising a second nucleic acid and a second contaminant, (ii) a second subsequent electrolyte buffer comprising a second subsequent ion, the magnitude of the effective mobility of which is larger than the magnitude of the effective mobility of the second nucleic acid. and (iii) a second leading electrolyte buffer containing second leading ions, the magnitude of the effective mobility of which is smaller than the magnitude of the effective mobility of the second nucleic acid; and (c) applying a first electric field within the microfluidic chip to perform isotachophoresis with the first trailing ions, the first nucleic acid, and the first leading ions in the first flow path, and applying a second electric field to perform isotachophoresis with the second trailing ions, the second nucleic acid, and the second leading ions in the second flow path, thereby purifying the first nucleic acid from the first contaminant and simultaneously purifying the second nucleic acid from the second contaminant.

[0074] In some embodiments of the aspects provided herein, the first sample and the second sample are different sample types. In some embodiments of the aspects provided herein, the first nucleic acid and the second nucleic acid are nucleic acids of different types or lengths. In some embodiments of the aspects provided herein, the first subsequent electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or tissue disruption agent. In some embodiments of the aspects provided herein, the lysing agent or tissue disruption agent comprises one or more agents selected from the group consisting of a solution having a pH greater than about 12, proteinase, urea, thiourea, and a surfactant. In some embodiments of the aspects provided herein, the first sample comprises lysed solid tissue. In some embodiments of the aspects provided herein, the second sample comprises lysed cells. In some embodiments of the aspects provided herein, the first sample does not contact the second sample during isotachophoresis. Some embodiments of the aspects provided herein further include loading a third flow path of the microfluidic chip with (i) a third sample containing a third nucleic acid and a third contaminant, (ii) a third subsequent electrolyte buffer containing a third subsequent ion, the magnitude of the effective mobility of which is smaller than that of the third nucleic acid, and (iii) a third leading electrolyte buffer containing a third leading ion, the magnitude of the effective mobility of which is larger than that of the third nucleic acid, and applying the electric field within the microfluidic chip to perform the isotachophoresis with the third subsequent ion, the third nucleic acid, and the third leading ion in the third flow path, thereby simultaneously purifying the first nucleic acid from the first contaminant, purifying the second nucleic acid from the second contaminant, and purifying the third nucleic acid from the third contaminant. In some embodiments of the aspects provided herein, the first and second electric fields are generated from a single electrode pair. In some embodiments of the aspects provided herein, the first and second electric fields are generated from different electrode pairs. In some embodiments of the aspects provided herein, the first and second flow paths are coupled to independent sensors.In some embodiments of the aspects provided herein, the first and second electric fields are independently controlled using feedback from the independent sensors. In some embodiments of the aspects provided herein, the independent sensors sense voltage, and the feedback is used to control current (or resistance) in the first and second flow paths. In some embodiments of the aspects provided herein, the nucleic acid comprises DNA. In some embodiments of the aspects provided herein, the nucleic acid comprises RNA.

[0075] An aspect of the present disclosure provides a method for sample purification, comprising: (a) loading into a fluidic device (i) a sample containing nucleic acid, the sample comprising fixed cells, fixed tissue, or embedded tissue; (ii) a trailing electrolyte buffer solution containing a trailing electrolyte having an effective mobility lower than that of the nucleic acid; and (iii) a leading electrolyte buffer solution containing a leading electrolyte having an effective mobility higher than that of the nucleic acid; and (b) applying an electric field to the fluidic device to perform isotachophoresis on the trailing electrolyte ions, the nucleic acid, and the leading electrolyte, thereby purifying the nucleic acid from contaminants in the sample.

[0076] In some embodiments of the aspects provided herein, the contaminant is selected from the group consisting of cross-linked nucleic acids, embedding materials, fixatives, enzymes, and inhibitors. In some embodiments of the aspects provided herein, the sample comprises the fixed cells, the fixed tissue, or both the fixed cells and the fixed tissue. In some embodiments of the aspects provided herein, the sample is formalin-fixed. In some embodiments of the aspects provided herein, the sample comprises the embedded tissue. In some embodiments of the aspects provided herein, the sample comprises the paraffin-embedded tissue. In some embodiments of the aspects provided herein, the sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. In some embodiments of the aspects provided herein, the sample comprises a tissue biopsy. In some embodiments of the aspects provided herein, the sample is an autopsy formalin-fixed, paraffin-embedded (FFPE) sample. Some embodiments of the aspects provided herein further include comparing a characteristic of the nucleic acid with nucleic acid from another sample, the characteristic being expression level, nucleic acid sequence, molecular weight, nucleic acid integrity, strandedness (e.g., double-stranded or single-stranded) of the nucleic acid, or nucleic acid purity. In some embodiments of the aspects provided herein, the sample is a tumor sample. In some embodiments of the aspects provided herein, the subsequent electrolyte buffer has a pH greater than about 7. Some embodiments of the aspects provided herein further include incubating the tissue sample in the fluidic device at a temperature of at least about 37°C for a period of at least about 1 minute prior to applying the electric field. In some embodiments of the aspects provided herein, the temperature is from about 40°C to about 80°C. In some embodiments of the aspects provided herein, the period is from about 1 minute to about 120 minutes. In some embodiments of the aspects provided herein, the initial electrolyte buffer comprises proteinase K. Some embodiments of the aspects provided herein further include using proteinase K to remove protein crosslinks from the nucleic acid. Some embodiments of the aspects provided herein further include, after the step of applying an electric field, using heat to remove protein crosslinks from the nucleic acid.Some embodiments of the aspects provided herein further include eluting an output solution containing the purified nucleic acid from the outlet reservoir of the fluidic device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the concentration of the cross-linked nucleic acid in the output solution is at least about two-fold lower than the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the output solution has a volume of about 50 μL or less. In some embodiments of the aspects provided herein, the tissue sample has a mass of at least about 1 ng. In some embodiments of the aspects provided herein, the tissue sample has a volume of more than 25 μL. In some embodiments of the aspects provided herein, the subsequent electrolyte has a higher effective mobility than the contaminant. In some embodiments of the aspects provided herein, the subsequent electrolyte comprises (i) a first ion having an effective mobility magnitude greater than that of the contaminant, and (ii) a second ion having an effective mobility magnitude approximately the same as or smaller than that of the contaminant. In some embodiments of the aspects provided herein, the step of performing isotachophoresis quenches the pH of the tissue sample to about 7.5. Some embodiments of the aspects provided herein further comprise deparaffinizing the sample prior to the loading step. Some embodiments of the aspects provided herein further comprise detecting the concentration of the nucleic acid. In some embodiments of the aspects provided herein, the concentration is about 1 picogram / microliter (pg / μL) or less.

[0077] An aspect of the disclosure is a method for detecting a nucleic acid fragment from a tissue sample, the method comprising: (a) providing a fluidic device with: (i) a tissue sample comprising lysed solid tissue and nucleic acids; (ii) a trailing electrolyte buffer comprising trailing electrolyte ions having a first effective mobility having a magnitude smaller than the magnitude of the effective mobility of the nucleic acid; (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir comprising first leading electrolyte ions having a second effective mobility having a magnitude larger than the magnitude of the effective mobility of the nucleic acid; and (iv) a third effective mobility buffer in a first leading electrolyte reservoir having a magnitude larger than the magnitude of the effective mobility of the nucleic acid. and (c) performing a second isotachophoresis run with the trailing electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby purifying the nucleic acid from the contaminants in the tissue sample.

[0078] In some embodiments of the aspects provided herein, performing the second isotachophoresis includes varying the applied current from the first flow path to the second flow path. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the second effective mobility has a magnitude greater than the magnitude of the third effective mobility. In some embodiments of the aspects provided herein, the first leading electrolyte ion is different from the second leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer contains a third leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer contains a third leading electrolyte ion. Some embodiments of the aspects provided herein further include collecting the nucleic acid in the second leading electrolyte reservoir and retrieving the nucleic acid from the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the steps of performing the first isotachophoresis and performing the second isotachophoresis are performed by applying a single electric field. In some embodiments of the aspects provided herein, the steps of performing the first isotachophoresis and performing the second isotachophoresis are performed by applying more than one electric field. In some embodiments of the aspects provided herein, the concentration of the second leading electrolyte ion in the second leading electrolyte buffer is less than 50 mM. In some embodiments of the aspects provided herein, the second leading electrolyte buffer comprises 50 mM Tris-HCl.

[0079] An aspect of the present disclosure provides a microfluidic device comprising: (a) a first isotachophoresis region in a microfluidic chip, the first isotachophoresis region comprising (i) a first sample reservoir in fluid communication with a first fluid flow path, (ii) a first buffer reservoir in fluid communication with the first fluid flow path, and (iii) a second buffer reservoir in fluid communication with the first fluid flow path; and (b) a second isotachophoresis region in the microfluidic chip, the second isotachophoresis region comprising (i) a second sample reservoir in fluid communication with a second fluid flow path, (ii) a third buffer reservoir in fluid communication with the second fluid flow path, and (iii) a fourth buffer reservoir in fluid communication with the second fluid flow path, wherein the first isotachophoresis region is not in fluid communication with the second isotachophoresis region; and the microfluidic device is configured to independently control a first electrical circuit that applies current to the first isotachophoresis region and a second electrical circuit that applies current to the second isotachophoresis region.

[0080] In some embodiments of the aspects provided herein, the leakage rate between the first and second isotachophoresis regions is less than 1 μL per hour. In some embodiments of the aspects provided herein, the leakage rate between the first and second isotachophoresis regions is less than 1 μA. In some embodiments of the aspects provided herein, the impedance is greater than 1 megaohm. In some embodiments of the aspects provided herein, the first fluid flow path holds a volume of greater than 100 μL. In some embodiments of the aspects provided herein, the first fluid flow path is separated from the second fluid flow path by a distance that is at least 5 times smaller than the width of the first fluid flow path. In some embodiments of the aspects provided herein, the microfluidic device is configured to control the first electrical circuit simultaneously with the second electrical circuit. Some embodiments of the aspects provided herein further include an elution reservoir in fluid communication with the first flow path, and a temperature sensor is located within 5 mm of the elution reservoir.

[0081] An aspect of the present disclosure provides a method comprising: (a) providing an electrokinetic fluid device including a sample input reservoir in fluid communication with a flow path; (b) loading a sample volume into the sample input reservoir; (c) transferring at least 50% of the sample volume from the sample input reservoir to the flow path without adding an additional volume to the sample input reservoir; and (d) applying an ionic current to the flow path.

[0082] In some embodiments of the aspects provided herein, the moving step is performed with the assistance of gravity. In some embodiments of the aspects provided herein, the ionic current does not substantially pass through the flow path. In some embodiments of the aspects provided herein, the at least 50% of the sample volume comprises at least 80% of the sample volume. In some embodiments of the aspects provided herein, the sample volume comprises nucleic acids. In some embodiments of the aspects provided herein, the sample volume comprises a tissue sample or a formalin-fixed, paraffin-embedded (FFPE) sample. In some embodiments of the aspects provided herein, the applying the ionic current comprises performing isotachophoresis. In some embodiments of the aspects provided herein, the total sample volume loaded into the sample input reservoir is equal to or less than the volume of the input reservoir. In some embodiments of the aspects provided herein, the sample input reservoir includes an upper region connected to a bottom region via a tapered region, the upper region having a first diameter and the bottom region having a second diameter, the first diameter being at least twice as long as the second diameter, facilitating the transfer of at least 50% of the sample volume from the sample input reservoir to the flow path. In some embodiments of the aspects provided herein, the sample volume is at least 25 μL. In some embodiments of the aspects provided herein, the sample volume is at least 50 μL. In some embodiments of the aspects provided herein, the sample volume is at least 100 μL.

[0083] An aspect of the present disclosure provides a microfluidic chip including: a first sample input reservoir, the first sample input reservoir including a top region connected to a bottom region via a tapered region, the top region having a first hydraulic inner diameter and the bottom region having a second hydraulic inner diameter, the first hydraulic inner diameter being at least twice as long as the second hydraulic inner diameter, the first sample input reservoir in fluid communication with a first flow path; a first buffer reservoir in fluid communication with the first flow path, the first sample reservoir configured such that a free surface of liquid in the first sample reservoir has a negligible buffer head height difference with respect to liquid in the first buffer reservoir; and a second buffer reservoir in fluid communication with the first flow path.

[0084] In some embodiments of the aspects provided herein, the first hydraulic inner diameter ranges from about 1 mm to about 15 mm. In some embodiments of the aspects provided herein, the second hydraulic inner diameter ranges from about 0.5 mm to about 5 mm. In some embodiments of the aspects provided herein, the first sample reservoir is configured to hold a sample volume of at least 100 μL. In some embodiments of the aspects provided herein, the microfluidic chip is configured to transfer at least 50% of the sample volume from the first sample reservoir to the first flow path upon application of a vacuum. In some embodiments of the aspects provided herein, the microfluidic chip is configured to perform isotachophoresis on a sample entering the first flow path.

[0085] An aspect of the present disclosure provides a method for extracting nucleic acids, comprising the steps of: (a) exposing a biological sample containing cells or tissue to a solution containing urea or thiourea, thereby lysing the cells or tissue within the biological sample and producing a cell lysate; (b) introducing the cell lysate into an apparatus; and (c) performing isotachophoresis using the apparatus to isolate nucleic acids from the cell lysate.

[0086] Some embodiments of the aspects provided herein further include digesting the sample with proteinase K. In some embodiments of the aspects provided herein, the solution includes urea and thiourea. In some embodiments of the aspects provided herein, the solution includes a ratio of urea:thiourea of ​​about 2:1. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is about 4 M to about 9 M, and the concentration of the thiourea in the solution is about 0.5 M to about 3.5 M. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is about 6.5 M to about 7.5 M, and the concentration of the thiourea in the solution is about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the solution includes trailing electrolyte ions, leading electrolyte ions, or both trailing and leading electrolyte ions.

[0087] An aspect of the present disclosure is a method for purifying high molecular weight nucleic acids from a tissue sample, comprising: (a) loading into a fluidic device: (i) a cell sample containing genomic DNA and contaminants, the cell sample being contacted with a lysis buffer before or after being loaded into the fluidic device; (ii) a subsequent electrolyte buffer containing subsequent electrolyte ions having a first effective mobility that is smaller in magnitude than the effective mobility of the high molecular weight nucleic acid and larger in magnitude than the contaminant; and (iii) a subsequent electrolyte buffer containing subsequent electrolyte ions having a first effective mobility that is larger in magnitude than the effective mobility of the high molecular weight nucleic acid. (b) performing isotachophoresis with the trailing electrolyte ions, the high molecular weight nucleic acids, and the first leading electrolyte ions, thereby separating the high molecular weight nucleic acids from the contaminants and concentrating the high molecular weight nucleic acids in the isotachophoresis zone; and (c) eluting the genomic DNA into a solution in an output reservoir, wherein more than 50% of the mass of the nucleic acids in the solution is greater than 30 kilobases.

[0088] In some embodiments of the aspects provided herein, the lysis buffer does not comprise an alkaline buffer. In some embodiments of the aspects provided herein, the lysis buffer comprises octylphenol ethoxylate. In some embodiments of the aspects provided herein, more than 50% of the masses of nucleic acids in the solution are greater than 50 kilobases.

[0089] An aspect of the present disclosure provides a method of performing isotachophoresis, comprising: (a) providing a fluidic device including a first flow path in fluid communication with a sample input reservoir containing a tissue sample comprising lysed solid tissue, a first buffer reservoir containing a first leading electrolyte buffer, and a second buffer reservoir containing a trailing electrolyte buffer; (b) contacting a first electrode with the first leading electrolyte buffer in the first buffer reservoir; (c) contacting a second electrode with the trailing electrolyte buffer in the second buffer reservoir; and (d) applying an electric field within the fluidic device to perform isotachophoresis, wherein the isotachophoresis occurs without direct contact between the tissue sample and the first and second electrodes.

[0090] In some embodiments of the aspects provided herein, the fluidic device further includes a third buffer reservoir in fluid communication with the first flow path and the first buffer reservoir, the third buffer reservoir containing the first lead electrolyte buffer at a lower concentration than the first buffer reservoir. In some embodiments of the aspects provided herein, the third buffer reservoir and the first buffer reservoir are connected by a second flow path comprising one or more capillary barriers to restrict pressure-driven flow between the third buffer reservoir and the first buffer reservoir within the second flow path. In some embodiments of the aspects provided herein, the fluidic device further includes an elution reservoir. In some embodiments of the aspects provided herein, the elution reservoir is in fluid communication with a fourth buffer reservoir.

[0091] An aspect of the present disclosure provides a microfluidic system including: (a) a microfluidic chip including a first flow path and a first reservoir in fluid communication with the first flow path, the first flow path and the first reservoir abutting at a first junction; and (b) a mechanical member including first teeth and configured to apply mechanical pressure to the first flow path via the first teeth to at least partially close the first flow path by plastic deformation of at least one wall of the first flow path and increase fluid resistance between the first flow path and the first reservoir.

[0092] In some embodiments of the aspects provided herein, the microfluidic chip further includes a second reservoir in fluid communication with the first reservoir and a second flow path connecting the first reservoir and the second reservoir, and the mechanical member further includes a second tooth configured to apply mechanical pressure to the second flow path to plastically close the second flow path and prevent fluid communication between the first reservoir and the second reservoir. In some embodiments of the aspects provided herein, the first tooth is configured to apply mechanical pressure to the first junction to close the first flow path by plastic deformation of at least one wall of the first flow path. In some embodiments of the aspects provided herein, the first tooth is configured to heat the first flow path. In some embodiments of the aspects provided herein, the mechanical member includes a material having a Young's modulus higher than that of the first flow path. In some embodiments of the aspects provided herein, the microfluidic system is configured to perform isotachophoresis. In some embodiments of the aspects provided herein, the first tooth is thermally coupled to a heating element. In some embodiments of the aspects provided herein, the first tooth is heated to a temperature higher than the glass transition temperature of the at least one wall of the first flow path. Some embodiments of the aspects provided herein include a method of using the microfluidic system to complete a process in a fluid system, the method including at least partially closing the first flow path by plastic deformation, thereby increasing resistance to fluid flow between the first flow path and the first reservoir. In some embodiments of the aspects provided herein, the first tooth of the mechanical member applies a force of at least 0.25 lb to the first flow path. In some embodiments of the aspects provided herein, the process in the fluid system is isotachophoresis.

[0093] An aspect of the present disclosure provides a method for performing isotachophoresis on a sample containing nucleic acid, the method comprising: (a) loading a sample containing nucleic acid into a first reservoir of a microfluidic chip; (b) loading a second reservoir of the microfluidic chip with a trailing electrolyte buffer containing first trailing electrolyte ions having an effective mobility that is smaller than the magnitude of the effective mobility of the nucleic acid; (c) loading a third reservoir of the microfluidic chip with a leading electrolyte buffer containing first leading electrolyte ions having a second effective mobility that is larger than the magnitude of the effective mobility of the nucleic acid; (d) applying an electric field within the microfluidic chip to perform isotachophoresis on the first trailing electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby confining the nucleic acid or a portion thereof in an isotachophoresis zone; and (e) using a temperature sensor to sense temperature changes in or near the isotachophoresis zone, wherein the electric field is controlled using feedback from the temperature sensor.

[0094] In some embodiments of the aspects provided herein, the control of the electric field causes the nucleic acid, or a portion thereof, to be disposed in an elution reservoir or region of the microfluidic chip. In some embodiments of the aspects provided herein, the temperature sensor is disposed within at most 8 mm of the elution reservoir. In some embodiments of the aspects provided herein, the temperature change is within a range of about 0.2°C to 5°C. In some embodiments of the aspects provided herein, the applied electric field brings the leading electrolyte and the trailing electrolyte together at an isotachophoresis interface, and the temperature sensor senses the isotachophoresis interface.

[0095] An aspect of the present disclosure provides a microfluidic device including: (a) a first isotachophoresis region in a microfluidic chip, the first isotachophoresis region including: (i) a first sample reservoir in fluid communication with a first fluid flow path; (ii) first, second, and third buffer reservoirs in fluid communication with the first fluid flow path, the first and second buffer reservoirs being separated by a capillary barrier; and (iii) an elution reservoir in fluid communication with the first fluid flow path; (b) a sensor configured to detect a temperature change in the first fluid flow path within the first isotachophoresis region; and (c) an apparatus arranged to provide an electric current in the first flow path within the first isotachophoresis region.

[0096] Some embodiments of the aspects provided herein further include a controller configured to trigger the reduction or elimination of the current when the sensor receives a thermal signal. In some embodiments of the aspects provided herein, the temperature change is an increase in temperature in a range of about 0.2°C to 5°C. In some embodiments of the aspects provided herein, the microfluidic device is further configured to isolate a sample of nucleic acid in the elution reservoir after the sensor detects the temperature change. In some embodiments of the aspects provided herein, the sensing of the nucleic acid is performed by a sensor located within at most 8 mm of the elution reservoir. In some embodiments of the aspects provided herein, the first flow path includes a single sensor.

[0097] Aspects of the present disclosure provide a kit comprising: (a) the microfluidic device of claim 111, the microfluidic device of claim 165, or the microfluidic chip of claim 128; (b) a subsequent electrolyte buffer comprising a subsequent electrolyte; and (c) a leading electrolyte buffer comprising a leading electrolyte.

[0098] In some embodiments of the aspects provided herein, the subsequent electrolyte buffer comprises a mixture of at least two electrolytes having different effective mobilities. In some embodiments of the aspects provided herein, the mixture comprises (i) a first electrolyte having an effective mobility magnitude lower than the nucleic acid and higher than the contaminant, and (ii) a second electrolyte having an effective mobility magnitude lower than the contaminant. In some embodiments of the aspects provided herein, the first electrolyte comprises caproic acid. In some embodiments of the aspects provided herein, the second electrolyte comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments of the aspects provided herein, the kit further comprises a sample buffer, wherein the sample buffer comprises a leading electrolyte buffer, a subsequent electrolyte buffer, or urea, in any combination. In some embodiments of the aspects provided herein, the kit further comprises a sample buffer comprising urea and thiourea.

[0099] An aspect of the present disclosure provides a sample purification method comprising: (a) loading into a fluidic device (i) a tissue sample containing nucleic acids and contaminants, the tissue sample being other than an unlysed whole blood sample; (ii) a trailing electrolyte buffer containing trailing electrolyte ions having effective mobilities greater than the magnitude of the effective mobility of the contaminants and less than the magnitude of the effective mobility of the nucleic acids; and (iii) a leading electrolyte buffer containing leading electrolyte ions having a second effective mobility greater than the magnitude of the effective mobility of the nucleic acids; and (b) applying an electric field within the fluidic device to perform isotachophoresis on the trailing electrolyte ions, the nucleic acids, and the leading electrolyte ions, thereby purifying the nucleic acids from the contaminants in the tissue sample.

[0100] In some embodiments of the aspects provided herein, the tissue sample is not a whole blood sample. In some embodiments of the aspects provided herein, the subsequent electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the leading electrolyte ion comprises chloride. In some embodiments of the aspects provided herein, the subsequent electrolyte buffer comprises a second subsequent electrolyte ion having a different effective mobility than the first subsequent electrolyte ion. In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and the first subsequent electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid) and the first subsequent electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the second subsequent electrolyte ion comprises HEPES and the first subsequent electrolyte ion comprises MOPS. In some embodiments of the aspects provided herein, the subsequent electrolyte buffer comprises a second subsequent electrolyte ion having a second effective mobility, the second effective mobility having a magnitude that is about the same as or lower than the magnitude of the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the contaminant is selected from the group consisting of crosslinked nucleic acids, embedding materials, fixatives, proteins, inhibitors, and combinations thereof. In some embodiments of the aspects provided herein, the contaminant comprises crosslinked nucleic acids. In some embodiments of the aspects provided herein, the tissue sample is combined with the subsequent electrolyte buffer prior to the loading. In some embodiments of the aspects provided herein, the tissue sample is combined with the lead electrolyte buffer prior to the loading.In some embodiments of the aspects provided herein, the loading of the lead electrolyte buffer is performed before the loading of the tissue sample. In some embodiments of the aspects provided herein, the method further includes eluting an output solution containing the purified nucleic acid from the outlet reservoir of the fluidic device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the concentration of the cross-linked nucleic acid in the output solution is at least about two-fold lower than the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the output solution is free of the contaminant. In some embodiments of the aspects provided herein, the tissue sample is fresh tissue. In some embodiments of the aspects provided herein, the tissue sample is fresh-frozen (FF) tissue. In some embodiments of the aspects provided herein, the tissue sample is formalin-fixed, paraffin-embedded (FFPE) tissue. In some embodiments of the aspects provided herein, the method further comprises lysing or disrupting the tissue sample prior to the loading. In some embodiments of the aspects provided herein, the lysis or disruption is performed using urea or thiourea.

[0101] An aspect of the present disclosure provides a method for detecting a nucleic acid fragment comprising the steps of: (a) loading into a first flow path of a fluidic device (i) a first tissue sample comprising a first nucleic acid and a first contaminant, (ii) a first subsequent electrolyte buffer comprising a first subsequent ion, the magnitude of the effective mobility of which is smaller than the magnitude of the effective mobility of the first nucleic acid; and (iii) a first leading electrolyte buffer comprising a first leading ion, the magnitude of the effective mobility of which is larger than the magnitude of the effective mobility of the first nucleic acid; (b) loading into a second flow path of the fluidic device (iv) a second tissue sample comprising a second nucleic acid and a second contaminant, and (v) a second subsequent electrolyte buffer comprising a second subsequent ion, the magnitude of the effective mobility of which is larger than the magnitude of the effective mobility of the first nucleic acid. (vi) loading a second leading electrolyte buffer solution containing second leading ions, the magnitude of which is smaller than the magnitude of the effective mobility of the second nucleic acid; and (c) applying an electric field within the fluidic device to perform isotachophoresis with the first trailing ions, the first nucleic acid, and the first leading ions in the first flow path, and to perform isotachophoresis with the second trailing ions, the second nucleic acid, and the second leading ions in the second flow path, thereby purifying the first nucleic acid from the first contaminant and purifying the second nucleic acid from the second contaminant.

[0102] In some embodiments of the aspects provided herein, the first subsequent electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or tissue disruption agent. In some embodiments of the aspects provided herein, the second subsequent electrolyte buffer or the second leading electrolyte buffer further comprises a lysing agent or tissue disruption agent. In some embodiments of the aspects provided herein, the lysing agent or tissue disruption agent comprises one or more agents selected from the group consisting of a solution having a pH greater than about 12, a proteinase, urea, thiourea, and a surfactant.

[0103] An aspect of the present disclosure provides a sample purification method comprising: (a) loading into a first zone of a fluidic device (i) a tissue sample containing nucleic acids and contaminants, (ii) a trailing electrolyte buffer containing trailing ions, the trailing ions having an effective mobility magnitude lower than that of the nucleic acid; and (iii) a leading electrolyte buffer containing leading ions, the leading ions having an effective mobility magnitude greater than that of the nucleic acid; and (b) applying an electric field to the fluidic device to perform isotachophoresis on the trailing ions, the nucleic acids, and the leading ions in a second zone of the fluidic device, thereby purifying the nucleic acids from the contaminants, wherein during the application of the electric field, the first zone is maintained at a first temperature and the second zone is maintained at a second temperature different from the first temperature.

[0104] In some embodiments of the aspects provided herein, the trailing electrolyte buffer or the leading electrolyte buffer further comprises a lysing agent or tissue disruption agent. In some embodiments of the aspects provided herein, the lysing agent or the tissue disruption agent comprises one or more agents selected from the group consisting of a solution having a pH greater than about 12, a proteinase, urea, thiourea, and a surfactant. In some embodiments of the aspects provided herein, the first temperature is from about 4°C to about 40°C. In some embodiments of the aspects provided herein, the first temperature is from about 40°C to about 80°C.

[0105] An aspect of the present disclosure provides a sample purification method comprising: (a) loading into a first zone of a fluidic device (i) a tissue sample containing nucleic acids, (ii) a trailing electrolyte buffer solution containing trailing ions, the trailing ions having an effective mobility magnitude lower than that of the nucleic acid, and (iii) a leading electrolyte buffer solution containing leading ions, the leading ions having an effective mobility magnitude greater than that of the nucleic acid; (b) performing at least one sample preparation step on the tissue sample in the first zone selected from the group consisting of: (1) removal of embedding material, (2) tissue disruption, (3) cell lysis, (4) decrosslinking of nucleic acids, (5) protein digestion, and (6) digestion of nucleic acids; and (c) applying an electric field within the fluidic device to perform isotachophoresis on the trailing ions, the nucleic acids, and the leading ions in a second zone of the fluidic device, thereby purifying the nucleic acids from contaminants in the tissue sample.

[0106] In some embodiments of the aspects provided herein, removing the embedding material or lysing the cells comprises incubating the tissue sample in the fluidic device at a temperature of at least about 37°C for a period of at least about 1 minute prior to applying the electric field. In some embodiments of the aspects provided herein, the temperature is from about 40°C to about 80°C. In some embodiments of the aspects provided herein, the period is from about 1 minute to about 60 minutes. In some embodiments of the aspects provided herein, disrupting the tissue or lysing the cells comprises applying mechanical stress to the sample. In some embodiments of the aspects provided herein, disrupting the tissue or lysing the cells comprises applying heat to the sample. In some embodiments of the aspects provided herein, the heating results in a temperature of the tissue sample of from about 30°C to about 65°C. In some embodiments of the aspects provided herein, disrupting the tissue or lysing the cells comprises a solution pH of at least 12. In some embodiments of the aspects provided herein, disrupting the tissue or lysing the cells comprises proteolytic digestion. In some embodiments of the aspects provided herein, the proteolytic digestion is carried out at a temperature greater than about 25°C. In some embodiments of the aspects provided herein, the temperature is about 30°C to about 65°C. In some embodiments of the aspects provided herein, the tissue disruption or cell lysis comprises applying at least one detergent to the tissue or cells. In some embodiments of the aspects provided herein, the tissue disruption or cell lysis comprises applying a solution comprising urea to the tissue or cells. In some embodiments of the aspects provided herein, the solution further comprises thiourea. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is about 4M to about 9M, and the concentration of the thiourea in the solution is about 0.5M to about 3.5M. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is about 6.5M to about 7.5M, and the concentration of the thiourea in the solution is about 1.5M to about 2.5M. In some embodiments of the aspects provided herein, decrosslinking the nucleic acid comprises digesting crosslinked proteins with proteinase K.In some embodiments of the aspects provided herein, digestion of the nucleic acid is carried out with DNase or RNase.

[0107] An aspect of the present disclosure provides a sample purification method comprising: (a) loading into a fluidic device (i) a tissue sample containing embedded or fixed nucleic acids, (ii) a trailing electrolyte buffer containing a trailing electrolyte having an effective mobility lower than that of the nucleic acids, and (iii) a leading electrolyte buffer containing a leading electrolyte having an effective mobility higher than that of the nucleic acids; and (b) applying an electric field to the fluidic device to perform isotachophoresis on the trailing electrolyte, the nucleic acids, and the leading electrolyte, thereby purifying the nucleic acids from contaminants in the tissue sample.

[0108] In some embodiments of the aspects provided herein, the contaminant is selected from the group consisting of cross-linked nucleic acids, embedding materials, fixatives, enzymes, and inhibitors. In some embodiments of the aspects provided herein, the embedding material comprises paraffin. In some embodiments of the aspects provided herein, the tissue sample is formalin-fixed. In some embodiments of the aspects provided herein, the tissue sample is embedded and fixed. In some embodiments of the aspects provided herein, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. In some embodiments of the aspects provided herein, the tissue sample is an autopsy tissue sample. In some embodiments of the aspects provided herein, the autopsy tissue sample is an autopsy FFPE sample. In some embodiments of the aspects provided herein, the method further comprises comparing the nucleic acid feature with nucleic acids from other samples. In some embodiments of the aspects provided herein, the feature is an expression level. In some embodiments of the aspects provided herein, the feature is a nucleic acid sequence. In some embodiments of the aspects provided herein, the feature is a molecular weight. In some embodiments of the aspects provided herein, the feature is nucleic acid integrity. In some embodiments of the aspects provided herein, the characteristic is nucleic acid purity. In some embodiments of the aspects provided herein, the method further comprises administering a drug based on the characteristic of the nucleic acid. In some embodiments of the aspects provided herein, the tissue sample is a tumor sample. In some embodiments of the aspects provided herein, the subsequent electrolyte buffer has a pH of about 7. In some embodiments of the aspects provided herein, the subsequent electrolyte buffer has a pH greater than about 7. In some embodiments of the aspects provided herein, the method further comprises incubating the tissue sample in the fluidic device at a temperature of at least about 37°C for a period of at least about 1 minute prior to applying the electric field. In some embodiments of the aspects provided herein, the temperature is about 40°C to about 80°C. In some embodiments of the aspects provided herein, the period is about 1 minute to about 60 minutes.In some embodiments of the aspects provided herein, the leading electrolyte buffer comprises proteinase K. In some embodiments of the aspects provided herein, the method further comprises removing protein crosslinks from the nucleic acid using proteinase K. In some embodiments of the aspects provided herein, the method further comprises removing protein crosslinks from the nucleic acid using heat after applying the electric field. In some embodiments of the aspects provided herein, the method further comprises eluting an output solution comprising the purified nucleic acid from an outlet reservoir of the fluidic device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the concentration of the crosslinked nucleic acid in the output solution is at least about two-fold lower than the concentration of the crosslinked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the output solution is free of the contaminant. In some embodiments of the aspects provided herein, the output solution has a volume of about 50 μL or less. In some embodiments of the aspects provided herein, the tissue sample has a mass of at least about 1 ng. In some embodiments of the aspects provided herein, the tissue sample has a volume of less than about 500 μL. In some embodiments of the aspects provided herein, the subsequent electrolyte has a higher effective mobility than the contaminant. In some embodiments of the aspects provided herein, the subsequent electrolyte includes (i) a first ion having an effective mobility magnitude greater than the contaminant, and (ii) a second ion having an effective mobility magnitude approximately the same as or lower than the contaminant. In some embodiments of the aspects provided herein, the isotachophoresis is performed by quenching the pH of the tissue sample to about 7. In some embodiments of the aspects provided herein, the method further includes deparaffinizing the tissue sample prior to the loading step. In some embodiments of the aspects provided herein, the tissue sample is a historical formalin-fixed, paraffin-embedded (FFPE) sample, and the method further includes comparing the nucleic acid signature with different nucleic acid signatures from different tissue samples.In some embodiments of the aspects provided herein, the method further comprises detecting the concentration of the nucleic acid. In some embodiments of the aspects provided herein, the concentration is about 1 picogram / microliter (pg / μL) or less. In some embodiments of the aspects provided herein, the concentration is about 0.5 pg / μL or less. In some embodiments of the aspects provided herein, the concentration is at least about 1 picogram / microliter (pg / μL).

[0109] An aspect of the present disclosure provides a fluidic device comprising: (a) a first zone; (b) a sample inlet located in the first zone; (c) a trailing electrolyte reservoir in fluid communication with the first zone; (d) a second zone in fluid communication with the first zone; (e) a leading electrolyte reservoir in fluid communication with the second zone; (f) a sample outlet in fluid communication with the second zone; (g) a first heater in thermal communication with the first zone; and (h) a second heater configured to transfer heat to the second zone, wherein the first zone is substantially thermally isolated from the second zone.

[0110] An aspect of the present disclosure provides a fluidic device including: (a) a first zone; (b) a sample inlet located in the first zone; (c) a trailing electrolyte reservoir in fluid communication with the first zone; (d) a second zone in fluid communication with the first zone; (e) a leading electrolyte reservoir in fluid communication with the second zone; (f) a sample outlet in fluid communication with the second zone; and (g) a heater in thermal communication with the first zone and the second zone.

[0111] In some embodiments of the aspects provided herein, the device further includes a second sample purification region. In some embodiments of the aspects provided herein, the first zone is a deparaffinization zone. In some embodiments of the aspects provided herein, the first zone is a disruption zone. In some embodiments of the aspects provided herein, the second zone is an isotachophoresis zone. In some embodiments of the aspects provided herein, the first zone or the second zone has a width of less than about 1 mm. In some embodiments of the aspects provided herein, the first zone or the second zone has a width of less than about 0.5 mm.

[0112] An aspect of the present disclosure provides a kit that includes a device provided herein, a trailing electrolyte buffer solution that includes a trailing electrolyte, and a leading electrolyte buffer solution that includes a leading electrolyte.

[0113] In some embodiments of the aspects provided herein, the subsequent electrolyte buffer contains a mixture of at least two electrolytes with different effective mobilities. In some embodiments of the aspects provided herein, the mixture includes (i) a first electrolyte having an effective mobility magnitude lower than that of the nucleic acid and higher than that of the contaminant, and (ii) a second electrolyte having an effective mobility magnitude lower than that of the contaminant. In some embodiments of the aspects provided herein, the contaminant includes cross-linked nucleic acid. In some embodiments of the aspects provided herein, the first electrolyte includes caproic acid. In some embodiments of the aspects provided herein, the second electrolyte includes HEPES.

[0114] An aspect of the present disclosure is a method for detecting a nucleic acid in a fluidic device, the method comprising: (a) providing a fluidic device with: (i) a tissue sample containing nucleic acid; (ii) a trailing electrolyte buffer containing trailing electrolyte ions having a first effective mobility that is less than the magnitude of the effective mobility of the nucleic acid; (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir containing first leading electrolyte ions having a second effective mobility that is greater than the magnitude of the effective mobility of the nucleic acid; and (iv) a second leading electrolyte buffer containing first leading electrolyte ions having a third effective mobility that is greater than the magnitude of the effective mobility of the nucleic acid. A method for purifying a sample is provided, comprising the steps of: (a) loading a second leading electrolyte buffer in a second leading electrolyte reservoir (the first leading electrolyte buffer being different from the second leading electrolyte buffer) containing two leading electrolyte ions; (b) performing isotachophoresis a first time with the trailing electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby purifying the nucleic acid from the contaminants in the tissue sample; and (c) performing isotachophoresis a second time with the trailing electrolyte ions, the nucleic acid, and the second leading electrolyte ions.

[0115] In some embodiments of the aspects provided herein, performing the isotachophoresis a second time includes varying the applied current from the first flow path to the second flow path. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is at least 1.5-fold different from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the first leading electrolyte ion is different from the second leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer includes a third leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer includes a third leading electrolyte ion. In some embodiments of the aspects provided herein, the method further includes collecting the nucleic acid in the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the method further includes removing the nucleic acid from the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the subsequent electrolyte buffer is loaded into a subsequent electrolyte reservoir that is separate from the first leading electrolyte reservoir and the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the steps of performing isotachophoresis a first time and performing isotachophoresis a second time are performed by applying an electric field.In some embodiments of the aspects provided herein, the steps of performing isotachophoresis a first time and performing isotachophoresis a second time are performed by applying more than one electric field.

[0116] An aspect of the present disclosure provides a fluidic device comprising: (a) a flow path including a first zone and a second zone in fluid communication with the first zone; (b) a sample inlet, a trailing electrolyte reservoir containing a trailing electrolyte buffer, and a first leading electrolyte reservoir containing a first leading electrolyte buffer, each in fluid communication with the first zone; and (c) a second leading electrolyte reservoir containing a second leading electrolyte buffer, wherein the second leading electrolyte buffer is in fluid communication with the second zone and the second leading electrolyte buffer is different from the first leading electrolyte buffer.

[0117] In some embodiments of the aspects provided herein, the sample inlet can receive a sample containing at least some non-liquid biological material. In some embodiments of the aspects provided herein, the second leading electrolyte buffer contains a different leading electrolyte co-ion than the first leading electrolyte buffer. In some embodiments of the aspects provided herein, the first leading electrolyte buffer contains a first leading electrolyte ion and the second leading electrolyte buffer contains a second leading electrolyte ion that is the same as the first leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer differs from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the first leading electrolyte buffer contains a first leading electrolyte ion and the second leading electrolyte buffer contains a second leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer differs from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer by at least 1.5-fold. In some embodiments of the aspects provided herein, the first leading electrolyte buffer comprises a first leading electrolyte ion and the second leading electrolyte buffer comprises a second leading electrolyte ion that is different from the first leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte buffer comprises a first leading electrolyte ion and the second leading electrolyte buffer comprises a second leading electrolyte ion that is the same as the first leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer comprises a third leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte buffer comprises a first leading electrolyte ion, the second leading electrolyte buffer comprises a second leading electrolyte ion that is the same as the first leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer comprises a third leading electrolyte ion.

[0118] An aspect of the present disclosure provides a method comprising: (a) providing an electrokinetic fluid device including a reservoir in fluid communication with a flow path; (b) loading a sample volume into the reservoir; (c) transferring at least 50% of the sample volume from the reservoir to the flow path; and (d) applying an ionic current to the flow path.

[0119] In some embodiments of the aspects provided herein, the transferring step is performed with the assistance of gravity. In some embodiments of the aspects provided herein, the ionic current does not substantially pass through the reservoir. In some embodiments of the aspects provided herein, the at least 50% of the sample volume comprises at least 80% of the sample volume. In some embodiments of the aspects provided herein, the sample volume comprises nucleic acid. In some embodiments of the aspects provided herein, the sample volume comprises a tissue sample. In some embodiments of the aspects provided herein, the sample volume comprises a formalin-fixed, paraffin-embedded (FFPE) sample. In some embodiments of the aspects provided herein, the applying the ionic current step comprises performing isotachophoresis (ITP). [The present invention 1001] a first flow path including spaced apart first and second capillary barriers; a first loading reservoir in fluid communication with the first flow path through a first opening in the first flow path; Including, the first opening is disposed between the first and second capillary barriers to allow liquid entering the first flow path through the first opening to flow in one direction along the first flow path, terminate at the first capillary barrier, and flow in another direction along the first flow path, terminate at the second capillary barrier; Isotachophoresis (ITP) circuit A fluidic device comprising: [The present invention 1002] 1001. A fluidic device according to claim 1001, wherein liquid entering the first flow path through said opening flows to a first or second capillary barrier along a path longer than the width of said first flow path. [The present invention 1003] A fluidic device of the present invention 1001, wherein liquid entering the first flow path through the opening flows so that the meniscus of the first liquid stops at the first capillary barrier or the second capillary barrier or at both the first and second capillary barriers. [The present invention 1004] the first capillary barrier is constructed and arranged to be breached by the liquid when a first burst pressure is applied to the one or more branched fluid circuits; a second capillary barrier constructed and arranged to be breached by the liquid when a second burst pressure is applied to the one or more branched fluid circuits; the first and second burst pressures are approximately equal; A fluidic device according to the present invention. [The present invention 1005] the first capillary barrier is constructed and arranged to be breached by the liquid when a first burst pressure is applied to the one or more branched fluid circuits; a second capillary barrier constructed and arranged to be breached by the liquid when a second burst pressure is applied to the one or more branched fluid circuits; the first burst pressure is greater than the second burst pressure; A fluidic device according to the present invention. [The present invention 1006] The fluidic device of the present invention 1001, wherein one or both of the first and second capillary barriers is a cliff-type capillary barrier. [The present invention 1007] The fluidic device of the present invention 1001, wherein one or both of the first and second capillary barriers is a plateau-type capillary barrier. [The present invention 1008] Any of the above fluidic devices of the present invention, wherein the plateau capillary barrier is constructed and arranged so that an air gap is formed between a first liquid after it stops at the plateau capillary barrier and a second liquid that flows in another direction towards the plateau capillary barrier and stops at the plateau capillary barrier on the opposite side to the first liquid. [The present invention 1009] the ITP circuit includes a second flow path in fluid communication with the first flow path; a first capillary barrier constructed and arranged to stop the flow of a second liquid as the second liquid flows along the second flow path, such that a liquid-liquid interface forms between the first and second liquids at the first capillary barrier; A fluidic device according to the present invention. [The present invention 1010] further comprising a substrate having a first side and a second side; the first surface includes a plurality of reservoirs including a first loading reservoir; the second surface includes a plurality of channels including the first channel; the plurality of reservoirs communicate with the plurality of channels via through holes in the substrate; A fluidic device according to the present invention. [The present invention 1011] the ITP circuit further includes a second loading reservoir and a second flow path; the second loading reservoir is in fluid communication with the second flow path through a second opening; the second flow path includes a third capillary barrier; the third capillary barrier is constructed and arranged to use capillary forces to arrest a meniscus of liquid flowing along the second flow path at the third capillary barrier; A fluidic device according to the present invention. [The present invention 1012] the ITP circuit further includes a third loading reservoir fluidly connected to the third flow path through a third opening; the third flow path is fluidly connected to a second reservoir; the third flow path includes a fourth capillary barrier disposed between the second opening and the third opening. A fluidic device according to the present invention. [The present invention 1013] the first flow path or the first loading reservoir contains a sample buffer; the second flow path or the second loading reservoir contains a first lead electrolyte buffer; or the third flow path or the third loading reservoir contains a second lead electrolyte buffer; A fluidic device according to the present invention. [The present invention 1014] a fourth channel or reservoir in fluid communication with the first channel and adjacent to the first capillary barrier; further comprising the fourth flow path or loading reservoir contains a subsequent electrolyte buffer; A fluidic device according to the present invention. [The present invention 1015] 1001. A fluidic device according to claim 10, wherein the ITP circuit comprises an elution flow path connected to a first elution reservoir at an elution junction. [The present invention 1016] A fluidic device of the present invention 1001, wherein the ITP circuit includes a first leading electrolyte buffer reservoir connected to a second leading electrolyte buffer reservoir by a buffer flow path, the buffer flow path including first and second leading electrolyte buffers that meet at an interface where a plateau-type capillary barrier is located. [The present invention 1017] 1001. The fluidic device of the present invention, wherein the first capillary barrier or the second capillary barrier or both are adjacent to an air flow path that includes a constriction. [The present invention 1018] further comprising at least two additional isotachophoresis (ITP) circuits, each including an ITP branch including a first loading reservoir and a first flow path; the first loading reservoir in fluid communication with the first flow path through a first junction; the first flow path includes first and second capillary barriers disposed on opposite sides of the first junction; the first capillary barrier is constructed and arranged to use capillary forces to arrest a meniscus of liquid flowing along the first flow path at the first capillary barrier; the second capillary barrier is constructed and arranged to stop the flow of another meniscus of the liquid at the second capillary barrier using capillary forces; A fluidic device according to the present invention. [The present invention 1019] further comprising at least five additional fluid circuits, each including an isotachophoresis (ITP) branch including a first loading reservoir and a first flow path; the first loading reservoir in fluid communication with the first flow path through a first junction; the first flow path includes first and second capillary barriers disposed on opposite sides of the first junction; the first capillary barrier is constructed and arranged to use capillary forces to arrest a meniscus of liquid flowing along the first flow path at the first capillary barrier; the second capillary barrier is constructed and arranged to stop the flow of another meniscus of the liquid at the second capillary barrier using capillary forces; A fluidic device according to the present invention. [The present invention 1020] 10. The fluidic device of the present invention 1001, wherein the first reservoir is a sample reservoir closed by a removable material. [The present invention 1021] one or more air channels opening to one or more air ports and communicating with each of the capillary barriers; The fluidic device of the present invention 1001 further comprises: [The present invention 1022] (a) a substrate having a first surface and a second surface, the first surface including a plurality of reservoirs including a first loading reservoir and one or more air ports, the second surface including a plurality of flow channels including a first flow channel, the plurality of reservoirs communicating with the plurality of flow channels via through holes in the substrate; (b) a layer of material covering the second surface, thereby forming a closed flow path; and (c) a cover covering at least a portion of the first surface, the cover including a through hole communicating with the port in the first surface through a gasket; Any of the fluidic devices of the present invention further comprising: [The present invention 1023] A fluidic device of the present invention 1022, wherein the one or more air ports have a head height relative to the substrate that is shorter than the first loading reservoir. [The present invention 1024] A fluidic device according to claim 1022, wherein the cover further comprises a porous, breathable, hydrophobic material disposed between the through holes in the port. [The present invention 1025] 1001. A fluidic device according to the present invention, wherein the first channel is a sample channel having a depth of less than 2 mm or a volume of about 10 μL to about 1 mL. [The present invention 1026] The first reservoir comprises: (a) a conical section in the region of the first reservoir that contacts the substrate; and (b) a cylindrical through-hole or opening extending through the substrate; The fluidic device of the present invention 1001, comprising: [The present invention 1027] The first reservoir comprises: (a) an inlet for ambient air at one end; and (b) An opening through the substrate at the other end of the loading reservoir. Including, the first reservoir has a frustoconical shape, a wider region of the frustoconical shape disposed at the inlet path for ambient air and a narrower region of the frustoconical shape disposed at the first opening through the substrate; A fluidic device according to the present invention. [The present invention 1028] A fluidic device of the present invention 1022, wherein the air port on the substrate is embedded into the surface of the first side of the substrate to a depth of about 1 μm to about 1 mm, or protrudes from the surface of the first side of the substrate to a height of about 0 μm to about 2 mm. [The present invention 1029] A method of loading a fluidic device of the present invention 1001 further comprising a second, third, fourth, fifth or sixth loading reservoir or flow path, the method comprising the step of loading different buffer solutions into the first, second, third, fourth, fifth or sixth flow path or reservoir. [The present invention 1030] the fluidic device includes a first flow path including a plateau-type capillary barrier adjacent to a second flow path; the step of loading buffers includes loading a first buffer into the first channel or reservoir and loading a second buffer into the second channel or reservoir; applying a first positive or negative air pressure to the fluidic device so that the first and second buffers stop at the base of a ramp within the plateau-shaped capillary barrier; the step of applying the first positive or negative air pressure includes increasing or decreasing the first positive or negative pressure in increments; The method of the present invention 1029. [The present invention 1031] The method of claim 1029, further comprising applying a second positive or negative air pressure to the fluidic device to cause the first and second buffer solutions to flow along ramp portions on both sides of the plateau-shaped capillary barrier, wherein applying the second positive or negative air pressure comprises increasing or decreasing the second positive or negative pressure in fixed increments. [The present invention 1032] the first and second buffer solutions stop at a plateau portion of the plateau-type capillary barrier, forming an air gap between them, the air gap being located above or below the plateau portion of the plateau-type capillary barrier; the method further comprising applying a third positive or negative air pressure to the fluidic device to cause the first and second liquids to enter the air gap, thereby forming a liquid-liquid interface between the first and second buffer solutions above or below the plateau portion of the plateau-shaped capillary barrier; Any of the methods of the present invention. [The present invention 1033] a fluid flow path; and a capillary barrier disposed in the fluid flow path to restrict the flow of liquid through the fluid flow path; The capillary barrier is a ramp portion projecting at a first angle from a surface of the fluid flow path; Plateau region; and a cliff region extending from the plateau region to the surface of the fluid flow path; Including, the cliff section intersects the surface at a second angle that is substantially steeper than the first angle; Fluidic devices. [The present invention 1034] The fluidic device of the present invention 1033, wherein the second angle is at least about 10°, at least about 15°, or at least about 20° steeper than the first angle. [This invention 1035] A fluidic device according to the present invention 1033, wherein the plateau region is approximately parallel to the surface of the fluid flow path. [The present invention 1036] a fluid flow path; and a capillary barrier disposed in the fluid flow path to restrict the flow of liquid through the fluid flow path; The capillary barrier is (a) a first ramp portion projecting from a surface of the fluid flow path at a first angle less than 80°; (b) Plateau region; and (c) a second ramp portion extending from the plateau region to the surface of the fluid flow path; Including, the second ramp portion intersects the surface at a second angle less than 80°; Fluidic devices. [This invention 1037] The fluidic device of the present invention 1036, wherein the first and second angles are the same or substantially the same. [The present invention 1038] The fluidic device of the present invention 1036, wherein the first and second angles are different. [This invention 1039] (a) Contains a trapezoidal cross-sectional area; (b) protruding from the inner surface of the fluid flow path; (c) having a plateau surface that is substantially parallel to the interior surface of the fluid flow path; (d) a ramp surface connecting the plateau surface to the inner surface of the fluid flow path, the ramp surface having a slope that varies along the length of the fluid flow path; (e) constructed and arranged to arrest and position a meniscus of liquid flowing along the length of the fluid flow path; Capillary Barrier A fluidic device comprising a fluid flow path comprising: [The present invention 1040] 1039. The fluidic device of claim 1039, wherein the capillary barrier is further constructed and arranged to create a liquid-liquid interface. [The present invention 1041] (a) A fluidic device comprising at least one branched fluidic circuit including a trailing electrolyte buffer reservoir, a first flow path, a first leading electrolyte buffer reservoir, a sample loading reservoir, a second leading electrolyte buffer reservoir, and a first elution buffer reservoir, all in fluid communication with each other; (i) the subsequent electrolyte buffer reservoir contains a subsequent electrolyte buffer; (ii) the first leading electrolyte buffer reservoir contains a first leading electrolyte buffer; (iii) the second leading electrolyte buffer reservoir contains a second electrolyte buffer different from the first electrolyte buffer; (iv) the first elution buffer reservoir contains a first elution buffer; providing a fluidic device; (b) applying air pressure to the trailing electrolyte buffer reservoir and the leading electrolyte buffer reservoir such that the trailing electrolyte buffer and the leading electrolyte buffer, respectively, enter the first flow path and remain within the first flow path, creating an air gap between the trailing electrolyte buffer and the leading electrolyte buffer; (c) loading a sample into the air gap between the trailing electrolyte buffer and the leading electrolyte buffer in the first flow path; and (d) applying air pressure to the second leading electrolyte buffer reservoir and the first elution buffer reservoir so that the second leading electrolyte buffer and the first elution buffer, respectively, enter the fluid circuit substantially simultaneously. A method for making a fluid circuit, comprising: [The present invention 1042] 1. A fluidic device comprising one or more branched fluidic circuits, each of the branched fluid circuits includes an isotachophoresis ("ITP") branch and an elution branch in communication with the ITP branch; (a) the ITP branch includes a trailing electrolyte buffer reservoir, a sample flow path, a leading electrolyte buffer flow path, a first leading buffer electrolyte reservoir, and a second leading electrolyte buffer reservoir, all in communication with each other; (i) the sample flow path is separated from the trailing electrolyte reservoir by a first cliff-type capillary barrier and from the leading electrolyte buffer flow path by a second cliff-type capillary barrier; (ii) the leading electrolyte reservoir is separated from the second leading electrolyte reservoir by a first plateau-type capillary barrier; (b) the elution branch comprises an elution flow path, a first elution buffer reservoir, and a second elution buffer reservoir, all in communication with each other; (i) the first elution buffer reservoir is separated from the second elution buffer reservoir by a second plateau-type capillary barrier; (ii) the leading electrolyte buffer flow path is separated from at least a portion of the elution flow path by a third plateau-shaped capillary barrier; Fluidic devices. [This invention 1043] (a)(i) the trailing electrolyte buffer reservoir contains a trailing electrolyte buffer; (ii) the first leading electrolyte buffer reservoir contains a first leading electrolyte buffer; (iii) the second leading electrolyte buffer reservoir contains a second leading electrolyte buffer; (iv) the first elution buffer reservoir contains a first elution buffer; (v) the second elution buffer reservoir contains a second elution buffer; providing any one of the fluidic devices of the present invention; (b) applying negative air pressure to the first and second cliff-type capillary barriers to prime the trailing electrolyte buffer and the first leading electrolyte buffer at the cliff-type capillary barriers; (c) loading a sample containing sufficient wetting agent into the sample flow path to create a fluid connection across the first and second cliff-type capillary barriers; and (d) applying negative air pressure to the first, second, and third plateau-type capillary barriers to create fluid connections across the first, second, and third plateau-type capillary barriers. A method for making a fluid circuit, comprising: [This invention 1044] (e) inserting the first electrode into the subsequent electrolyte buffer in the subsequent electrolyte buffer reservoir; (f) inserting a second electrode into a second leading electrolyte buffer in a second leading electrolyte buffer reservoir; and (g) applying a voltage or current across the first and second electrodes. The method of the present invention 1043 further comprising: [This invention 1045] (h) inserting a third electrode into the second elution buffer in the second elution buffer reservoir; and (i) after step (g), applying a voltage or current across the first and third electrodes and, optionally, reducing the current at the second electrode. The method of the present invention 1044 further comprises: [The present invention 1046] 1043. The method of claim 1043, further comprising the step of adding a topper fluid to the sample reservoir. [This invention 1047] 1. A fluidic device including a fluid flow path, The fluid flow path is (a) a first wall substantially parallel to a third wall and a second wall substantially parallel to a fourth wall; and (b) Capillary barrier Including, The capillary barrier is (i) a side surface disposed on or incorporated within the interior surface of the second wall and extending substantially between the first wall and the third wall; (ii) first and second outer walls each including a trapezoidal cross-sectional area connected to, embedded within, or adjacent to said first and third walls, respectively; (iii) a plateau surface substantially parallel to the second wall and located between the second and fourth walls; and (iv) a ramp section connecting the second wall to the plateau surface, the ramp section having a varying slope along the length of the fluid flow path; Including, Fluidic devices. [This invention 1048] 1. A fluidic device including a fluid flow path, The fluid flow path is a capillary barrier protruding from a first wall of the fluid flow path into the fluid flow path; Including, The capillary barrier is two outer surfaces each having a trapezoidal cross-sectional area; a plateau side of the channel substantially parallel to the first wall; and (c) a ramp portion having one edge intersecting the plateau side to form an internal obtuse angle of the capillary barrier and an opposite edge intersecting the first wall of the channel to form an internal acute angle of the capillary barrier; Including, Fluidic devices. [This invention 1049] (a)(i) a first sample reservoir; (ii) a subsequent electrolyte buffer reservoir in fluid communication with the sample reservoir, the subsequent electrolyte buffer reservoir containing the subsequent electrolyte buffer; and (iii) a leading electrolyte buffer flow path in fluid communication with the sample reservoir, the leading electrolyte buffer flow path containing the leading electrolyte buffer; a first isotachophoresis circuit in a microfluidic chip; (b) a sensor configured to detect a temperature change in the leading electrolyte buffer flow path; and (c) a device configured to monitor the voltage or current in the first isotachophoresis circuit and to provide a constant current in the first isotachophoresis circuit. 1. A fluid system comprising: [The present invention 1050] (a)(i) a first sample reservoir in fluid communication with the first fluid flow path; (ii) first, second, and third buffer reservoirs in fluid communication with the first fluid-flow path, the first and second buffer reservoirs being separated by a first capillary barrier; and (iii) an elution reservoir in fluid communication with the first fluid flow path. a first isotachophoresis circuit in a microfluidic chip; (b) a sensor configured to detect a temperature change in the first fluid flow path within the first isotachophoresis region; and (c) a device configured to monitor the voltage or current and to provide a constant current within the first isotachophoresis circuit; 1. A fluid system comprising: [This invention 1051] a first liquid flow path; a gas flow path in fluid communication with the first liquid flow path; an air port in fluid communication with the gas flow path; and a breathable hydrophobic membrane disposed across the air port; Including, the hydrophobic membrane is not liquid permeable and is configured to prevent liquid from exiting the air port when negative pressure is applied to the gas flow path through the air port. Fluidic devices. [This invention 1052] The fluidic device of the present invention 1051 further comprising a gasket disposed over the air port. [This invention 1053] (a)(i) an elution well adjacent to an elution flow path containing an elution buffer, the elution flow path being connected to a leading electrolyte flow path containing a leading electrolyte buffer; and (ii) a capillary barrier located at the interface between the elution buffer and the leading electrolyte buffer; providing a fluid circuit comprising: (b) flowing the interface between the leading electrolyte buffer and the elution buffer toward the elution well; and (c) stopping the flow of the interface between the leading electrolyte buffer and the elution buffer so that the capillary barrier is completely engulfed by the leading electrolyte buffer. A method comprising: [This invention 1054] (a) a fluid circuit having a trailing electrolyte buffer reservoir, a sample flow path, a leading electrolyte buffer flow path, and an elution reservoir, all in communication with each other; (i) the leading electrolyte buffer flow path is fluidly connected to the elution reservoir through an opening in the leading electrolyte buffer flow path located below the elution reservoir; (ii) the subsequent electrolyte buffer reservoir contains a subsequent electrolyte buffer; (ii) the sample flow path contains an analyte; (iii) the leading electrolyte buffer flow path comprises a leading electrolyte buffer; (iv) the elution reservoir comprises an elution buffer; Providing a fluidic device; and (b) applying an electric current to the fluid circuit to move the analyte to the elution reservoir. Including, the current is constructed and arranged to generate a first temperature at an interface between the analyte and the trailing electrolyte buffer and a second temperature at an interface between the sample and the leading electrolyte buffer, wherein a temperature difference exists between the first temperature and the second temperature; when the analyte reaches the opening in the leading electrolyte buffer flow path located below the elution reservoir, the analyte is driven by the temperature difference to enter the elution reservoir. method. [This invention 1055] providing a fluidic device comprising an isotachophoresis (ITP) flow path containing a nucleic acid sample comprising nucleic acids complexed with an intercalating dye; performing ITP in the fluid flow path to focus the nucleic acid complexed with the intercalating dye; and quantitating the nucleic acid complexed with the intercalating dye in the flow path by measuring the intensity of the intercalating dye after ITP has been performed. Including, the intercalating dye comprises one or more of SYTO™ 13, PicoGreen®, EvaGreen® or Quantifluor®; A method for quantifying a nucleic acid sample. [The present invention 1056] 1. A fluidic device comprising one or more branched fluidic circuits, each of the branched fluid circuits includes an isotachophoresis ("ITP") branch and an elution branch in communication with the ITP branch; the ITP branch includes a trailing electrolyte buffer reservoir, a sample flow path, a leading electrolyte buffer flow path, a first leading buffer electrolyte reservoir, and a second leading electrolyte buffer reservoir, all in communication with each other; the elution branch includes an elution flow path and an elution well, the elution well including first and second through holes in communication with the elution flow path; Fluidic devices. [This invention 1057] The fluidic device of the present invention 1056, wherein the first and second through-holes are circular, curved, or elliptical. [This invention 1058] The fluidic device of the present invention 1056, wherein the first through-hole and the second through-hole are on the same or different vertical planes within the elution well. [This invention 1059] a first flow path terminating at an end in the first through hole; a second flow path terminating at an end in the second through hole; and a fluid reservoir defined by a wall having a height of 25 mm or less, 15 mm or less, 10 mm or less, or greater than 10 mm; Including, the reservoir is in fluid communication with two fluid flow paths through the first and second through-holes, respectively, and the first through-hole enters the reservoir at a location in the reservoir that is lower than the second through-hole; Fluidic devices. [The present invention 1060] a first flow path in communication with the first electrode; a second flow path communicating with the second electrode; the channel and reservoir contain a conductive fluid, and application of a voltage across the first and second electrodes generates a current that travels through the reservoir; A fluidic device according to the present invention. [This invention 1061] Providing a fluidic device of the present invention 1059, wherein the flow path and the reservoir contain a conductive fluid, and the first flow path further contains an ionic analyte; applying a voltage across the first and second electrodes to generate a current that travels through the reservoir; and transferring the analyte through a first through-hole into the reservoir. A method comprising: [This invention 1062] a fluid flow path having a length and a width; a reservoir disposed above the fluid flow path and fluidly connected to the fluid flow path through one or more through holes; Including, At least a portion of each through hole is substantially coextensive with the fluid flow path across the width of the fluid flow path; the fluid flow path and the reservoir contain a conductive fluid and are shaped such that when a current is passed through the fluid flow path, at least 5%, at least 6%, at least 7%, at least 10%, or at least 20% of the current passes through the reservoir; Fluidic devices. [This invention 1063] providing a device of the invention 1062, wherein the flow path and reservoir contain a conductive fluid, and the reservoir contains an ionic analyte; and passing an electric current through the flow path; Including, the current moves at least some of the analyte from the reservoir into the flow path; method. [This invention 1064] 1. A fluidic device comprising one or more branched fluidic circuits, Each of the branched fluid circuits comprises: an isotachophoresis ("ITP") branch including a trailing electrolyte buffer reservoir, a sample flow path, a leading electrolyte buffer flow path, a first leading electrolyte buffer reservoir, and a second leading electrolyte buffer reservoir, all in communication with each other; a sample reservoir including a first through-hole in communication with the sample flow path; Including, Fluidic devices. [This invention 1065] The fluidic device of the present invention 1064, wherein the first through-hole has a square or rectangular shape. [The present invention 1066] one or more cylindrical columns including an internal flow path defined by an interior wall of the cylindrical columns; Each cylindrical column is a first stage including a first gel plug disposed at a first position within the internal flow channel and a first space disposed within the internal flow channel between the first gel plug and an upper end of the cylindrical column; a second stage including a second gel plug disposed in the internal flow passage at a second position located below the first position and oriented in alignment with gravity, and a second space disposed in the internal flow passage between the first gel plug and the second gel plug; and a third stage including a third gel plug disposed in the internal flow passage at a third position located below the second position and oriented in alignment with gravity, and a third space disposed in the internal flow passage between the second gel plug and the third gel plug. Including, An apparatus for performing vertical isotachophoresis. [This invention 1067] introducing the analyte into the second space of the second stage above the second gel plug of the device of the present invention 1066; and applying an electric current to the device to move the analyte from the second space through the second gel plug in the direction of gravity and into a third space. 1. A method for focusing an analyte, comprising:

[0120] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0121] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0122] [Figure 1A] 1 provides exemplary protocols for sample processing and nucleic acid extraction or purification. [Figure 1B] 1 provides an exemplary protocol for automated sample processing and nucleic acid extraction or purification. [Figure 2A] FIG. 1 shows an exemplary schematic diagram of isotachophoretic separation and purification of DNA and RNA from contaminants. [Figure 2B] FIG. 1 shows an exemplary schematic of isotachophoretic separation and purification of nucleic acids from paraffin and other possible sample contaminants, in parallel with proteinase-mediated tissue disruption and nucleic acid decrosslinking. [Figure 3] Exemplary results of DNA extraction and purification by automated isotachophoresis in a fluidic device are shown in comparison with exemplary results from a common solid-phase column extraction kit. [Figure 4] Figure 4A shows an example result of unbiased (e.g., sequence-wise) extraction of GC-rich and AT-rich synthetic DNA oligonucleotides mixed at a sample concentration ratio using isotachophoresis. Figure 4B shows an example result of unbiased (e.g., size or molecular weight) pre- and post-purification of a DNA molecular weight ladder using isotachophoresis. A comparison with two solid-phase column-based nucleic acid purification methods is provided. [Figure 5A] 1 shows an exemplary schematic of a flow path having a sample preparation zone and an isotachophoretic purification zone. [Figure 5B] Shown in Figure 5A is an exemplary fluidic device cartridge containing eight parallel fluidic flow paths and reservoirs for the simultaneous processing of up to eight samples. [Figure 5C] An exemplary top view of one flow path and its connected reservoir for the fluidic device cartridge shown in Figure 5B is shown, further illustrating the use of a gas port for applying external pressure or vacuum to the flow path within the fluidic device cartridge. [Figure 5D] 5C shows an exemplary side view of the fluidic device cartridge shown in FIG. 5B. [Figure 5E] 5C shows an exemplary end view of the fluidic device cartridge shown in FIG. 5B. [Figure 6A] 1 shows an exemplary top view of a fluidic device cartridge. [Figure 6B] 1 illustrates an exemplary side view of a fluidic device cartridge. [Figure 6C] 1 shows an exemplary bottom view of a fluidic device cartridge. [Figure 6D] 1 shows an exemplary three-dimensional top view of a fluidic device cartridge. [Figure 7A] 1 shows an exemplary top view of a fluidic device cartridge. [Figure 7B] 1 illustrates an exemplary side view of a fluidic device cartridge. [Figure 7C] 1 shows an exemplary bottom view of a fluidic device cartridge. [Figure 7D] 1 illustrates an exemplary three-dimensional overall bottom view of a fluidic device cartridge. [Figure 8A] 1 shows an exemplary top view of a fluidic device cartridge. [Figure 8B] 1 illustrates an exemplary side view of a fluidic device cartridge. [Figure 8C] 1 shows an exemplary bottom view of a fluidic device cartridge. [Figure 8D] 1 illustrates an exemplary three-dimensional overall bottom view of a fluidic device cartridge. [Figure 9]Figure 9A shows an exemplary schematic of a fluidic device cartridge including the eight parallel flow paths shown in Figure 5B. Figure 9B shows an exemplary schematic of two temperature control devices, each aligned with a zone of the eight parallel flow paths shown in Figure 9A. [Figure 10] Figure 10A shows an exemplary gas flow path that may include a capillary barrier, and Figure 10B is an enlarged view of the gas flow path of Figure 10A. [Figure 11] 1 illustrates an exemplary low-loss sample reservoir. [Figure 12] Figure 12A shows an exemplary mechanical element that can be used to apply pressure to close a flow path of a fluidic device, Figure 12B shows an exemplary comb-like mechanical element, and Figure 12C shows the alignment of the comb-like mechanical element with a flow path of a fluidic device. [Figure 13A] 1 shows an exemplary benchtop apparatus for performing automated sample preparation and isotachophoresis on a fluidic device cartridge. [Figure 13B] 1 illustrates an exemplary computerized system that is programmed or otherwise configured to implement the methods provided herein. [Figure 14] 1 shows exemplary results of fluorescence-based measurement and quantification for a titration series of nucleic acids using isotachophoresis. [Figure 15] Schematic diagram of an exemplary design of a fluid flow channel with connected reservoirs, non-contact electrodes (which can be used as conductivity sensors), and gas ports for automated fluid loading into the channel / device and for performing automated isotachophoresis. [Figure 16] 1 shows a graph of voltage measurements over time in a running ITP flow path. [Figure 17] 17 shows two graphs of the differential analysis of the voltage measurements from FIG. [Figure 18] An example of conductivity measurement over time in an ITP flow path near the elution reservoir is shown. [Figure 19] 1 shows an exemplary schematic diagram of a C4D sensor implementation. [Figure 20A] 1 shows an exemplary temperature map of an ITP flow channel taken using a thermal imaging camera. [Figure 20B] A plot of temperature over time at the position of cursor 1 in FIG. 20A is shown. [Figure 21] Graphs of temperature measurements and temperature derivatives over time during ITP are shown. [Figure 22] Figure 22A shows an exemplary schematic of a vertical (or column) ITP setup, and Figure 22B shows an exemplary image of the vertical ITP setup with DNA ITP bands. [Figure 23] 1 shows exemplary images and corresponding fluorescence intensity traces of the extraction and separation of amplifiable (e.g., decrosslinked) DNA from crosslinked DNA from FFPE samples using isotachophoresis. [Figure 24] Figure 24A shows an exemplary image of DNA extraction and purification from FFPE samples using isotachophoresis. Figure 24B shows an exemplary DNA yield measured by quantitative PCR for DNA extraction and purification from FFPE samples using isotachophoresis, compared to exemplary results from a common solid-phase column extraction kit. [Figure 25] Figure 25A shows an image of a single-channel ITP chip loaded with nucleic acids (RNA extraction and digests from human cells) stained with dyes for visualization. Figure 25B shows an image of a single-channel ITP chip loaded with nucleic acids (RNA extraction and digests from human cells) stained with dyes for visualization. [Figure 26A] Shown is an image of the RNA ITP band in the chip channel during purification. [Figure 26B] 1 shows an image of the total nucleic acid ITP band in the chip channel during purification. [Figure 26C] 26B shows a graph of the RNA quality electropherogram of the sample shown in FIG. 26A. [Figure 26D] 26B shows a graph of the RNA quality electropherogram of the sample shown in FIG. 26B. [Figure 27A] Results of DNA yield (ng) for ITP (squares) of mouse whole blood compared to column extraction (diamonds, Qiagen QiaAmp) are shown as a function of the volume percent of whole blood in the starting sample. [Figure 27B] Shown is an image of total nucleic acid in the ITP band during ITP purification of lysed whole mouse blood on a chip. [Figure 27C] Figure 27 shows white light and fluorescent overlay images of the ITP chip channels, showing the physical separation of heme in the elution channel and the sample / leading electrolyte channel from the reservoir, before and after ITP purification of 50% whole blood lysate by volume. Nucleic acids are stained with green dye in the elution well for visualization. Figure 27C shows the chip before ITP (blood lysate and ITP buffer loaded onto the chip; buffer only in the elution well). Figure 27D shows the chip after ITP (blood lysate and ITP buffer loaded onto the chip; purified DNA in the elution well). [Figure 27D] See legend to Figure 27C. [Figure 27E] The chip (50% blood by volume) after ITP purification is shown. [Figure 27F] The chip (25% blood by volume) after ITP purification is shown. [Figure 28] The results of high molecular weight DNA purification using ITP are shown in comparison with solid phase extraction. [Figure 29A] 1 shows a fluidic device containing eight closed channels. [Figure 29B] 10 shows a magnified microscope image of a second channel closure position adjacent to the elution reservoir of each channel. [Figure 29C] 1 shows the calculated percent closure as a function of the force applied to the fluidic device. [Figure 29D] The results of conductivity measurements of channel closure are shown. [Figure 30] Graphs of voltage measurements and voltage derivatives over time during ITP are shown. [Figure 31] Figure 31A shows a micrograph of ITP bands with focused DNA in each of the eight samples in the sample channel region of the device. Figure 31B shows independent voltage signal data over time at a fixed current for each of the eight channels. Figure 31C shows a micrograph of the same eight ITP bands with focused DNA from a sample eluted in the elution reservoir. Figure 31D is a magnified section of the voltage tracing (monitoring) used for triggering shown in Figure 31B. [Figure 32A-1] An exemplary method of ITP for sample preparation and DNA purification from cultured mammalian cells is shown. [Figure 32A-2] This is a figure showing a continuation of Figure 32A-1. [Figure 32B-1] 1 shows an exemplary method of ITP for sample preparation and DNA purification from tissue samples. [Figure 32B-2] This is a figure showing a continuation of Figure 32B-1. [Figure 33-1] 1 illustrates an exemplary process workflow for automated ITP. [Figure 33-2] This is a figure showing a continuation of Figure 33-1. [Figure 33-3] This is a continuation of Figure 33-2. [Figure 34] 1 shows a circuit configured to detect and prevent electrical faults in an ITP. [Figure 35A] 1 illustrates an exemplary fluidic device that includes two connecting components. [Figure 35B] See legend to Figure 35A. [Figure 36A] 1 illustrates an example of a fluidic device that includes multiple components. [Figure 36B] See legend to Figure 36A. [Figure 37A] 1 shows an example of a fluidic device that includes three parts. [Figure 37B] See legend to Figure 37A. [Figure 38] FIG. 1 shows an exemplary channel schematic of eight parallel channels on the underside of a chip of a three-part fluidic device. [Figure 39] 1 illustrates an exemplary multi-component fluidic device. [Figure 40] 1 illustrates an exemplary fluid circuit including voltage and temperature sensing. [Figure 41] 41A-41B show an exemplary "cliff-type capillary barrier." [Figure 42] 42A-42B show an exemplary "plateau capillary barrier." [Figure 43] 1 shows an exemplary flow path or fluid circuit highlighting the initial fluid interface location after loading. [Figure 44] 1 shows an exemplary flow path or fluid circuit highlighting the final fluid interface location after loading. [Figure 45] Figure 45A shows an example of the fluidic layer of the chip, Figure 45B shows a cutaway view of the air ports, and Figure 45C shows a realization of this design. [Figure 46A] 1 shows an exemplary microfluidic channel capable of detecting an empty sample channel. [Figure 46B] 1 shows the sequence of events for detecting whether a flow path is empty or full. [Figure 46C] 10 shows pressure traces and control signal traces from an implementation of this technology. [Figure 47A] 1 illustrates an exemplary pneumatic control scheme for staged liquid loading. [Figure 47B] See legend to Figure 47A. [Figure 48A] 1 shows an exemplary sample inlet reservoir designed for direct injection. [Figure 48B] See legend to Figure 48A. [Figure 48C] See legend to Figure 48A. [Figure 48D] See legend to Figure 48A. [Figure 48E] See legend to Figure 48A. [Figure 48F] See legend to Figure 48A. [Figure 48G] See legend to Figure 48A. [Figure 48H] See legend to Figure 48A. [Figure 49] 49A-49B show an exemplary low dispersion elution flow path. [Figure 50A1] The fluid surface of the substrate is shown with the channels terminating in through-holes that communicate with reservoirs on the opposing reservoir side of the substrate. [Figure 50A] 1 shows a technical drawing of an exemplary elution reservoir on a chip device. [Figure 50B] See legend to Figure 50A. [Figure 50C]50B shows background-subtracted fluorescence images of the fluidic device at various elution steps for the design in FIG. 50A. [Figure 50D] See legend to Figure 50C. [Figure 50E] See legend to Figure 50C. [Figure 50F] FIG. 1 is a block diagram representing steps in an elution workflow. [Figure 51A] 1 shows an exemplary fluid reservoir that can be used for nucleic acid elution. [Figure 51B] 10 shows a second embodiment of the reservoir with further modifications for injection molding compatibility. [Figure 51C] Two views of this third embodiment of the reservoir design are shown. [Figure 51D] See legend to Figure 51C. [Figure 51E] 1 shows a comparison of nucleic acid residence times between the disclosed design and a reference design. [Figure 51F] 1 shows a cross section of the reference design, which is a stylized vertical cylinder without internal structure. [Figure 52A] 1 shows a fluid reservoir consisting of an oval through-hole sized to allow easy placement of a pipette tip for reliable fluid withdrawal from the device. [Figure 52B] See legend to Figure 52A. [Figure 52C] See legend to Figure 52A. [Figure 52D] See legend to Figure 52A. [Figure 53A] 1 illustrates another exemplary fluid reservoir. [Figure 53B] See legend to Figure 53A. [Figure 53C] See legend to Figure 53A. [Figure 53D] 1 shows a top view of an exemplary elution reservoir. [Figure 53E] FIG. 53D shows a cross-sectional view of the elution reservoir of FIG. [Figure 54A] 1 illustrates an exemplary mechanical member that can be used to apply pressure to close a flow path in a fluidic device. [Figure 54B] 1 illustrates an exemplary ridge-like mechanical member. [Figure 54C] 1 illustrates the alignment of a ridge-like mechanical member with a flow channel of a fluidic device. [Figure 54D] FIG. 10 shows an exploded component assembly view of a mechanical actuator coupled to a ridge-like structure for closing a flow channel. [Figure 55] FIG. 1 shows the pneumatic control block diagram of the beta prototype and production equipment. [Figure 56] 56A-56B show an alternative mechanism for closing a flow path without using heat or pressure induced plastic deformation of the fluidic device. [Figure 57] 10 illustrates an alternative mechanism for closing a flow channel without using heat or pressure induced plastic deformation of a fluidic device. [Figure 58] 58A-58C show an alternative mechanism for closing a flow channel without using heat or pressure induced plastic deformation of the fluidic device. [Figure 59A] 1 shows an exemplary benchtop apparatus for performing automated isotachophoresis and / or sample preparation on a fluidic device cartridge. [Figure 59B] See legend to Figure 59A. [Figure 59C] See legend to Figure 59A. [Figure 60] 10 shows exemplary images that may be displayed to a user to instruct and guide the user through the reservoir loading process. [Figure 61] 1 shows an air manifold that can facilitate mating of the microfluidic chip with the instrument. [Figure 62] A cross section of the chip in the device is shown. [Figure 63] 1 shows a vertical manifold motion mechanism including a mechanical assembly design for alignment and motion with automatic retraction. [Figure 64] 1 shows a horizontal manifold motion mechanism design including a mechanical assembly design for horizontal motion using rack and pinion. [Figure 65] 1 shows a heat pipe with a thermoelectric cooler design for maintaining a zone of predetermined temperature away from the location of the thermoelectric cooler. [Figure 66] 1 shows another exemplary benchtop apparatus for performing automated isotachophoresis and / or sample preparation on a fluidic device cartridge. [Figure 67] 1 shows a plot of peak area response versus nucleic acid mass for various nucleic acid binding dyes. [Figure 68] 1 shows a plot of peak width response versus nucleic acid mass for various nucleic acid binding dyes. [Figure 69] Figures 69A-69C show Quantifluor® incompatibility with PCR. [Figure 70] 1 shows PicoGreen compatibility with Qubit dsDNA assays. [Figure 71] Demonstrates SYTO 13 compatibility with Qubit dsDNA assays. [Figure 72] Figures 72A-72B show PicoGreen compatibility with PCR. [Figure 73] Figures 73A-73C show SYTO 13 compatibility with PCR. gDNA was purified using isotachophoresis in the presence and absence of SYTO 13. [Figure 74] 1 shows PicoGreen compatibility with amplicon-based sequencing library preparation. [Figure 75] Demonstrating SYTO 13 compatibility with amplicon-based sequencing library preparation. [Figure 76] Figures 76A-76B show PicoGreen incompatibility with whole genome sequencing library preparation. [Figure 77] Figures 77A-77B show SYTO 13 compatibility with next-generation sequencing library preparation. [Figure 78] 1 shows PicoGreen incompatibility with whole genome sequencing library preparation. [Figure 79] Demonstrates SYTO 13 compatibility with next-generation sequencing library preparation. [Figure 80A]Demonstrates SYTO 13 compatibility with whole-exome next-generation sequencing library preparation. [Figure 80B] See legend to Figure 80A. [Figure 80C] See legend to Figure 80A. [Figure 80D] See legend to Figure 80A. [Figure 81] FIG. 1 shows a block diagram illustrating an optical signal processing algorithm. [Figure 82] 1 shows a diagram of a mechanical optical assembly design for illumination and detection of fluorescence of dye-bound samples. [Figure 83] The optical paths that achieve excitation of sample-bound dye fluorescence and capture of emitted light from sample-bound dye fluorescence are shown. [Figure 84A] 1 shows a control scheme for a method for establishing an electrical circuit created by electrodes in a channel. [Figure 84B] See legend to Figure 84A. [Figure 84C] See legend to Figure 84A. [Figure 84D] See legend to Figure 84A. [Figure 84E] See legend to Figure 84A. [Figure 84F] See legend to Figure 84A. [Figure 85] 1 shows a flow path closer, i.e. a tooth-like member with a mechanical actuator. [Figure 86] 1 shows a contrast image of the stained analyte in the flow channel. [Figure 87A] Conductivity data is shown, which was obtained by measuring the conductivity of the eluate using a conductivity meter. [Figure 87B] 1 shows a contrast image of the stained analyte in the flow channel. [Figure 88A] 1 shows a typical trace of the temperature signal captured by the IR sensor during a run. [Figure 88B] The first derivative of the temperature data in Figure A is shown. [Figure 88C]The likelihood ratio is calculated by comparing the residuals of the data-goodness-of-fit (0016) with the residuals of the data-null hypothesis (0015). [Figure 88D] 1 shows a block diagram of the triggering process. [Figure 88E] 1 shows the successful triggering of the nucleic acid extraction process. [Figure 88F] Indicates a failed triggering execution. [Figure 89A] A capillary barrier between the sample (in this case prepared in the leading electrolyte) and the leading electrolyte buffer is shown. [Figure 89B] The passage of nucleic acids with the addition of a slow ion (3-(N-morpholino)propanesulfonic acid) and a control is shown. [Figure 89C] The nucleic acid forms at different times during the extraction process. [Figure 89D] A capillary barrier between the leading electrolyte buffer and the elution buffer is shown. [Figure 89E] An example of buffer interface modulation is shown. [Figure 89F] 1 shows the enhancement of nucleic acid passage by modulating the interface position. [Figure 90] Figures 90A-90C show the lysis efficiency of three different cell lines. [Figure 91] 91A-91B show exemplary temperature measurements using an infrared temperature sensor to trigger a reduction or removal of current in one of the flow paths. [Figure 92A] A block diagram of the sample flow path to LE flow path triggering process used is shown. [Figure 92B] 10 shows exemplary traces of the voltage used for triggering from the sample channel to the LE channel, the voltage derivative, and the measurement error. [Figure 92C] FIG. 1 shows a block diagram of the LE flow channel triggering process used. [Figure 92D] 10 shows exemplary traces of the voltage, voltage derivative, and measurement error used for triggering at the constriction after the capillary barrier in the LE flow channel. [Figure 92E]FIG. 1 shows a block diagram of the elution triggering process used. [Figure 92F] 1 shows exemplary traces of the voltage used for elution triggering, the voltage derivative, and the measurement error. DETAILED DESCRIPTION OF THE INVENTION

[0123] Detailed Description Overview Sample preparation is the first step in nearly all genomic and transcriptomic analyses and can nevertheless be a major source of analytical variability. Sample preparation can also be labor-intensive, especially when the samples are formalin-fixed, paraffin-embedded (FFPE) samples, which contain cross-linked proteins.

[0124] The present disclosure provides processes and devices for improving the efficiency of nucleic acid extraction and purification from tissue and cell samples, including samples that have been processed in some way, such as paraffin-embedded or chemically fixed samples (e.g., FFPE samples, samples containing solid tissue). The methods provided herein include methods for on-chip or off-chip preparation of such processed samples prior to performing isotachophoresis using methods incorporating leading and trailing electrolyte ions. In some examples, the methods include treating fixed solid tissue in a trailing or leading electrolyte buffer (e.g., by removing, lysing, or enzymatically disrupting the tissue) prior to performing isotachophoresis on the sample. The methods can also include the use of a second, lower ionic strength leading electrolyte buffer to generate a sample compatible with downstream processes, such as amplification or other enzymatic assays. The devices and systems provided herein include apparatus suitable for performing isotachophoresis on tissue-derived samples, including microfluidic devices with parallel processing capabilities and automated feedback control mechanisms, which may include temperature sensors that detect temperature changes within the sample processing channels.

[0125] The disclosed processes and devices can provide improved nucleic acid recovery from samples, particularly low-abundance samples (e.g., less than 100 ng of nucleic acid), samples with relatively large volumes (e.g., greater than 25 μL total volume, greater than 50 μL total volume, greater than 100 μL total volume, or more total volume), or liquid samples containing solid particles. The disclosed processes and devices can also provide high repeatability and reduced bias for short-chain nucleic acids. The devices provided herein can integrate sample preparation (e.g., removal of crosslinking or embedding material) and nucleic acid extraction operations within a single device. The disclosed devices and processes can also provide compatibility with process automation, integration with downstream processes, integration with in-line quantification (e.g., at single picogram resolution), and / or integration with nucleic acid length and sequence distribution analysis.

[0126] The method provided herein is often a method for carrying out isotachophoresis under conditions suitable for extracting nucleic acid from specific sample, particularly FFPE sample.In some examples, the disclosed method comprises carrying out isotachophoresis using a trailing electrolyte buffer solution containing at least two ions with different effective mobility magnitudes.The method can also comprise carrying out isotachophoresis using two different leading electrolyte buffer solutions, one of which can act as sample elution buffer solution.The method can comprise process automation and parallel processing of multiple samples.

[0127] The present disclosure also includes protocols using buffer and spacer chemistries. These buffer and spacer chemistries can include the use of multiple electrolytes to perform ITP. For example, the subsequent electrolyte can include a mixture of electrolyte species that can separate either the non-crosslinked nucleic acids or both the crosslinked and non-crosslinked nucleic acids from contaminants in the sample while separating the non-crosslinked nucleic acids from the crosslinked nucleic acids.

[0128] The devices provided herein include injection-molded fluidic devices with parallel sample processing channels capable of performing ITP in multiplexed fashion, and ITP devices with two or more regions connected to thermal devices. The disclosed technology can use ITP to simultaneously capture, purify, and focus extracted RNA and DNA, quantify extracted total nucleic acids on-chip (e.g., via in-line ITP-assisted concentration to very small volumes or labeling with intercalating fluorescent dyes), and deliver nucleic acids downstream to parallel output reservoirs compatible with robotic pipetting.

[0129] The techniques of the present disclosure can enable the purification of sample materials (e.g., nucleic acids) without binding the sample materials to a solid support. The techniques of the present disclosure can enable the purification of sample materials (e.g., nucleic acids) without using liquid phase extraction. This can enable purification that does not rely on solubility differences.

[0130] The operation of the disclosed devices can be automated, largely automated, or partially automated. In some cases, the disclosed methods involve only a single off-chip mixing step of dispensing a sample (e.g., an FFPE section) into a solution (e.g., an alkaline solution, lysis solution, or buffer solution containing urea and / or thiourea), followed by sample loading into a reservoir of a fluidic device for further on-device sample preparation (e.g., deparaffinization, tissue disruption and cell lysis, protease digestion, proteolytic digestion, or other treatments including protein denaturation or nuclease digestion) and nucleic acid extraction, purification, concentration, in-line quantification, and sizing or fractionation (e.g., size selection). In some cases, the disclosed methods include dispensing a sample (e.g., an FFPE section or other tissue sample) into a reservoir or channel of a fluidic device (e.g., a cartridge) pre-filled with a solution (e.g., an alkaline solution containing urea and / or thiourea, a lysis solution, or a buffer solution) for on-device sample preparation (e.g., deparaffinization, tissue disruption and cell lysis, protease digestion, or other processing involving protein denaturation or nuclease digestion) and nucleic acid extraction, purification, concentration, in-line quantification, and sizing or fractionation (e.g., size sorting). In some cases, the disclosed methods include off-chip tissue disruption and / or cell lysis of the sample, followed by loading the sample, which may be a homogenous or heterogeneous mixture of lysed solid tissue and nucleic acids, into a reservoir of the fluidic device for further on-device sample preparation (e.g., deparaffinization, protease digestion, or other processing involving protein denaturation or nuclease digestion) and nucleic acid extraction, purification, concentration, in-line quantification, and sizing or fractionation (e.g., size sorting). Nuclease digestion can include DNA removal for DNA-free RNA extraction or RNA removal for RNA-free DNA extraction. The fluidic devices provided herein can be used with benchtop systems to automate electric field-based methods for DNA and RNA extraction from samples.

[0131] The disclosed devices include systems that can automate and integrate on-chip heating (e.g., to temperatures between 37°C and 80°C), sample preparation (e.g., deparaffinization, tissue disruption, and cell lysis), buffer exchange, nucleic acid extraction and purification, concentration of uncrosslinked or amplifiable nucleic acids (e.g., by cleaving and delivering them separately from crosslinked nucleic acids), and delivery of purified nucleic acids to output reservoirs, such as arrays compatible with manual or robotic pipetting. For example, the disclosure includes an integrated benchtop controller prototype that can provide automated control of the loading of eight-channel cartridges and buffers and other fluids in a standard robotic automation-compatible microtiter plate format, application of temperature and electric fields to the device, and automated start-stop parallel processing of samples. This system can be easily modified in the future to provide higher throughput for use in larger diagnostic or clinical laboratories (e.g., 96-well sample formats), if desired.

[0132] For example, FIG. 1A shows an exemplary process diagram for sample processing and nucleic acid extraction using the disclosed technology. A sample is provided (101) and can be subjected to optional pretreatment steps 102, such as mixing with a buffer, dissolution, or removal of embedding material (if present). The sample (and, e.g., buffer) can then be loaded into a fluidic device 103. Sample preparation steps 104, such as removal of embedding material (if present and if not removed during pretreatment), tissue disruption, cell lysis, protein or proteolytic digestion, and (e.g.,) nuclease digestion, can then be performed on the fluidic device. Isotachophoresis 105 can then be performed to separate and purify the nucleic acids from contaminants within the sample (e.g., cellular debris, plasma membranes, small molecules, embedding material, crosslinked nucleic acids, fixatives such as formalin, inhibitors, and enzymes such as digestion or restriction enzymes). Other steps, such as decrosslinking of crosslinked nucleic acids (e.g., by heat or protease digestion), can be performed simultaneously with isotachophoresis. During or after isotachophoresis, the nucleic acids can be detected and quantified (106). Once extracted or purified, the nucleic acids can then be eluted and recovered (107) from the device.

[0133] Figure 32A shows a non-limiting exemplary method of ITP for sample preparation and DNA purification from cultured mammalian cells utilizing the methods and apparatus provided herein. Generally, the steps may include cell isolation (step 151) and washing (step 152), cell lysis (steps 153-155) and proteolysis (steps 156-157), and homogenization of free DNA in the lysate (steps 158-159), all while maintaining the appropriate ionic content for downstream ITP.

[0134] In step 151, live, healthy (e.g., >90% viability) log-phase cell cultures may be pelleted by centrifugation (e.g., 250 g for 5 minutes). In at least some instances, when using adherent or semi-adherent cells, trypsinization may be performed prior to pelleting. After discarding the spent medium, the cells may be washed in fresh medium, pelleted, resuspended in fresh medium, and counted. A cell suspension of appropriate density (e.g., 100,000 live cells per lane of ITP extraction) may be placed in a microcentrifuge tube (e.g., a 2 mL Eppendorf Lo-Bind microcentrifuge tube) for downstream processing (e.g., steps 152-163 below). In some instances, as will be apparent to one of skill in the art, the cells of interest may be isolated using fluorescence-activated cell sorting (FACS) of the resuspension, counted, and placed in a recipient tube, which may then be prepared as described herein for ITP.

[0135] In step 152, the cells may be washed with a buffer, such as phosphate-buffered saline (PBS). The cells may be pelleted by centrifugation (e.g., 250 g x 5 minutes). The cell culture medium may then be discarded and the pellet resuspended in PBS (e.g., 190 μL of 1×PBS (without Ca2+ or Mg2+)). The resuspended cells may be centrifuged to form a pellet, and the PBS supernatant may be removed from the pelleted cells.

[0136] In step 153, the cell pellet may be lysed in a lysis buffer, such as proprietary alkaline CCD Lysis Buffer ("L1"), by pipetting and resuspension (e.g., by pipetting five times using a P1000 pipette). Lysis may be performed in place of or in combination with other lysis techniques known to those skilled in the art, such as sonication, manual trituration, Beise method, homogenization, freezing, enzymatic digestion, and / or chemical disruption. The lysed cells may then be vortexed to mix (e.g., for 3 seconds). Generally, in step 153, the lysis buffer is highly alkaline. Optionally, the alkaline solution may include 30-120 mM NaOH (optionally 40-80 mM NaOH) and have a pH of about 10-13. An exemplary alkaline solution may include 80 mM NaOH, 11 mM DTT, and 0.5% by volume Igepal CA-630.

[0137] In step 154, the cells may be incubated in the lysis buffer at room temperature (eg, for 2 minutes) to allow the lysis process to lyse the cells.

[0138] In step 155, lysis may be stopped and the sample pH may be neutralized. For example, a proprietary acidic Quench buffer ("Quench") may be applied to the lysed cell sample. In some cases, the Quench buffer may contain only LE, both LE and TE, or only TE. In some cases, the Quench buffer may contain both LE and TE or only TE to reduce retention of nucleic acids at the capillary barrier, particularly the capillary barrier between the sample and LE. In some cases, the quenching described in step 155 is not performed in the methods disclosed herein (e.g., for high molecular weight DNA applications). For example, it may be unnecessary when the lysis solution is in a neutral or non-alkaline pH range. In such cases, LE, LE and TE, or TE may be included in the lysis solution. Quenching is generally useful when an alkaline lysis solution is used during the lysis step (e.g., for cultured cell applications or primary human cells).

[0139] The sample may be mixed by pipetting (eg, by pipetting up and down 5 times) and then vortexed (eg, for 3 seconds).

[0140] In step 156, the lysed cell suspension may be treated to denature proteins in the cell lysate. Optionally, RNA (or DNA, if the sample molecule of interest in the ITP is RNA) may be degraded. For example, proteinase K (e.g., 20 mg / mL) and optionally RNase A (e.g., 10 mg / mL) may be added. The sample may be vortexed to mix (e.g., for 3 seconds), then pulse-spun, and then incubated at room temperature (e.g., for 2 minutes).

[0141] In step 157, the sample may be incubated at 56° C. for 10 minutes.

[0142] In step 158, the sample may be homogenized by vortexing (eg, for 3 seconds) and pulse spinning.

[0143] The sample can then be allowed to cool to room temperature (e.g., 5 minutes) in step 159. Optionally, if the sample is prepared in LE, TE can be added after cooling to reduce or minimize retention of nucleic acids in the capillary barrier, particularly the capillary barrier between the sample and LE.

[0144] In step 160, optionally, a sample surfactant (eg, MOPS) may be added to the sample.

[0145] At step 161, the sample may optionally be frozen for later use.

[0146] In step 162, the sample may be warmed to 20°C.

[0147] In step 163, optionally, a nucleic acid dye or stain may be added to the sample lysate for visualization and detection of nucleic acids (e.g., for quantification of nucleic acid mass). For example, if in-line quantification of nucleic acid mass is performed during the ITP process, the addition of a nucleic acid dye, such as a nucleic acid binding or intercalating dye, may be used.

[0148] In step 164, the sample may be loaded into one or more reservoirs. Prior to loading, the sample may be pulse vortexed (e.g., twice) and pulse spun to agitate and mix the sample. The ITP flow path may be pre-primed with a leading electrolyte buffer, a trailing electrolyte buffer, and / or other buffers described herein. Alternatively, the sample may be loaded simultaneously with the rest of the liquid in the flow path.

[0149] In step 165, ITP can be performed. During the ITP process, the DNA in the sample can be purified (i.e., concentrated) as it travels through the flow path and reaches the elution reservoir as described herein. The sample DNA can be quantified as described herein.

[0150] In step 166, the eluted sample may be collected from the elution reservoir by elution (eg, by pipetting).

[0151] 32B shows a non-limiting exemplary method of ITP for sample preparation and DNA purification from a tissue sample (e.g., a fresh or FFPE tissue sample) using the methods and apparatus provided herein. Generally, the steps can include trimming excess paraffin from the tissue (e.g., when using slide-mounted FFPE tissue sections) (step 171), tissue lysis (steps 172-175), proteolysis (steps 176-177), and decrosslinking free DNA in the lysate (steps 178-179), all while maintaining the appropriate ionic content for downstream ITP.

[0152] In step 171, excess paraffin may optionally be trimmed or removed from the FFPE tissue sample (e.g., before further sample preparation steps). The FFPE tissue sample may also be obtained directly from a paraffin block or a section mounted on a slide.

[0153] At step 172, the tissue may be collected in a microcentrifuge tube (e.g., an Eppendorf Lo-Bind microcentrifuge tube). Collection may optionally include scraping the FFPE tissue sample from a slide or otherwise placing the fresh or FFPE tissue sample into the tube.

[0154] At step 173, the tissue may be lysed in a lysis buffer, such as proprietary alkaline CCD Lysis ("L1"), by pipetting and resuspension (e.g., by pipetting five times using a P1000 pipette). Lysis may be performed alternatively or in combination with other lysis techniques known to those skilled in the art, such as sonication, manual grinding, bead processing, homogenization, freezing, enzymatic digestion, and / or chemical disruption. The lysed cells may then be vortexed to mix (e.g., for 5 seconds). Generally, at step 173, the lysis buffer is highly alkaline. Optionally, the alkaline solution may include 30-120 mM NaOH (optionally 40-80 mM NaOH) and have a pH of about 10-13. An exemplary alkaline solution may include 80 mM NaOH, 11 mM DTT, and 0.5% by volume Igepal CA-630.

[0155] At step 174, the tissue may be incubated in the lysis buffer at 80° C. (e.g., for 3 minutes) to allow the lysis process to lyse the cells. The sample may then be mixed by pipetting (e.g., by pipetting up and down 5 times) and then vortexed (e.g., for 3 seconds).

[0156] In step 175, the lysed sample may be allowed to cool by incubating at room temperature (eg, for 3 minutes).

[0157] In step 176, the lysed tissue suspension may be treated to denature proteins in the tissue lysate. Optionally, RNA (or DNA, if the sample molecule of interest in the ITP is DNA) may be degraded. For example, proteinase K (e.g., 20 mg / mL) may be added.

[0158] In step 177, the sample may be incubated for 1 hour at 56° C. After incubation, the sample may be vortexed and pulse spun.

[0159] In step 178, the sample may be incubated at 90° C. for 1 hour.

[0160] The sample may then be allowed to cool to room temperature (eg, 5 minutes) in step 179.

[0161] In step 180, optionally, RNase A (eg, 10 mg / mL) can be added.

[0162] In step 181, the sample may be incubated for 5 minutes at 25° C. After incubation, the sample may be vortexed and pulse spun.

[0163] In step 182, optionally, a sample lubricant (eg, MOPS) may be added to the sample.

[0164] In step 183, the sample may optionally be frozen for later use.

[0165] In step 184, the sample may be warmed to 20°C.

[0166] In step 185, optionally, a nucleic acid dye or stain may be added to the sample lysate for visualization and detection of nucleic acids (e.g., for quantification of nucleic acid mass). For example, if in-line quantification of nucleic acid mass is performed during the ITP process, the addition of a nucleic acid dye, such as a nucleic acid binding or intercalating dye, may be used.

[0167] In step 186, the sample may be loaded into one or more reservoirs. Prior to loading, the sample may be pulse vortexed (e.g., twice) and pulse spun to agitate and mix the sample. The ITP flow path may be pre-primed with a leading electrolyte buffer, a trailing electrolyte buffer, and / or other buffers described herein. Alternatively, the sample may be loaded simultaneously with the rest of the liquid in the flow path.

[0168] In step 187, ITP may be performed. During the ITP process, the DNA in the sample may be purified (i.e., concentrated) as it travels through the flow path and reaches the elution reservoir as described herein. The sample DNA may be quantified as described herein.

[0169] Also in step 187, the eluted sample can be collected from the elution reservoir by elution (eg, by pipetting).

[0170] FIG. 1B shows an exemplary process workflow for automated ITP. In step 110, a protocol can be selected on the benchtop instrument, e.g., by using a graphical user interface. User interface software can enable easy-to-use or hands-free operation. For example, the user can select from a menu (e.g., a drop-down menu). Alternatively, the instrument can scan a barcode (e.g., an optical barcode, an RFID chip) associated with the sample or fluidic device chip, which can indicate the protocol to be performed. In step 111, the instrument lid can be opened (e.g., manually or automatically by a motor). Motorized lid opening is compatible with robotic laboratory automation. In step 112, a user can load a chip (e.g., a fluidic device) into the benchtop instrument. The chip can include a monolithic multichannel SLAS standard microtiter plate (MTP) footprint for automated ITP. In step 113, ITP fluids can be loaded into the chip wells. The reservoirs for ITP fluids and user samples can be designed for easy loading, for example, via a multi-channel pipette (e.g., a 9 mm pitch SLAS standard microtiter plate format). The geometric design of the channels connecting the reservoirs to the ITP channels (e.g., capillary barriers) can resist gravity-driven flow or wetting of liquids into the channels prior to operation. These structures can stop fluids at predetermined locations within the ITP channels, including the establishment of a leading electrolyte / trailing electrolyte interface, and can enable bubble-free loading. In some cases, pneumatic actuation can be applied to prime the channels prior to operation. The chip material can be selected to prevent or resist wetting or wicking of fluids into the channels (e.g., hydrophobic or high-contact-angle plastics). The user can load the chip with ITP reagents and buffers (e.g., five different fluids). Alternatively, the chip can be provided with pre-loaded reagents. In step 114, the user or the device can close the lid of the device.Sample loading can be actuated through gas or air ports on the chip. For example, the channels can be filled with liquid using wet and / or gravity-driven flow without active pressure application.

[0171] In step 115, the instrument can apply pressure to load fluid into the chip to prime the flow paths. In step 116, the device can check that the flow paths were properly primed. For example, optical (e.g., reflectance), electrical, pressure, and / or flow sensors can be used to check that the fluid was loaded into the correct location within the chip. Sensors and device software can enable real-time monitoring and control of fluid loading. Loading of ITP reagents and buffers can be performed before loading the sample into the chip so that sample material is not wasted in the event of a misloading. If the flow paths are not properly primed, the device can execute an error report 130. In step 117, the device lid can be opened. In step 118, the sample can be loaded into the device. Sample loading can be performed manually by a user or in an automated manner, for example, by laboratory automation robotics. Other sample preparation steps can also be performed. For example, a paraffin-embedded sample (e.g., FFPE) can be loaded, in which case the device can control the temperature in the sample reservoir to deparaffinize the sample. In step 119, the lid of the device can be closed. In step 120, the device can perform a self-test. For example, electrical feedback from device electrodes interfacing with the on-chip reservoirs can be used to self-test the success of liquid priming (e.g., air bubble detection). Optical sensors can be used to provide feedback on the liquid priming status (e.g., whether the liquid has reached a designated capillary barrier). Other sensing mechanisms, such as those disclosed herein, can also be used. If the cell test determines that the device is not properly primed, the device can perform an error report 131.

[0172] In step 121, an ITP-based purification can be performed. Sensor-based feedback control and process timing (e.g., triggering) as described herein can be used to control and / or automate ITP purification. The device determines whether the purification was successful and, if not, can perform error reporting 132. In step 122, a sensor (e.g., an optical sensor) on the device can be used to quantify the sample, e.g., by fluorescence, UV, or other optical detection. Sample sizing can also be performed. If the device determines that the sample was not properly quantified or discovers other problems, the device can perform error reporting 133. In step 123, a change in conductivity can be detected and used to indicate when to terminate the ITP run (e.g., when the nucleic acid reaches a designated elution position or reservoir). Other detection methods described herein, e.g., temperature or driving voltage, can also be used to determine the timing of the end of the run or other trigger. For example, to automate the ITP process, a temperature or voltage sensor can be used to control the electric field applied to a flow path within the device. As an example, an electric field can be applied to a flow path to initiate ITP purification. The sensed voltage change can be used to trigger the initiation of temperature or other sensing at a fixed location within the flow path, such as at or near an elution reservoir. As the ITP zone containing the trapped nucleic acid moves, the voltage can change. A voltage sensor can sense a change indicating that the ITP zone is passing through a feature of the flow path, such as a section of a reduced cross-sectional area, and feedback can be used to modify the electric field, for example, by reducing the applied current. A temperature sensor can detect a temperature change as the ITP zone passes the elution reservoir or near it, and feedback from the sensor can be used to control the electric field, for example, by removing the electric field, to terminate the ITP run. In step 124, the device can terminate the run, for example, based on a trigger signal. When the ITP run is terminated, the nucleic acid can be positioned or sequestered in an elution reservoir or region.At step 125, the device can close the flow path, which can fix the elution volume to maintain a constant volume for elution (e.g., by resisting or preventing flow into the elution or output reservoir while pipetting out the elution volume). Fixing the elution volume can aid in ease of use and can aid in reporting the concentration of eluted sample material. At step 126, the device can be opened (e.g., by a user or automatically).

[0173] In step 127, the purified sample can be extracted from the device. The chip and / or device can be designed for a given elution volume, as detailed herein. Recovering the purified material from the device can be performed by pipetting or otherwise removing the material from the chip. Alternatively, sample extraction can be performed by interfacing the ITP chip with another fluidic chip or system (e.g., in the absence of an elution reservoir). Other fluidic systems can then be used to perform other operations on the purified sample material, such as next-generation sequencing (NGS) library preparation, sample analysis, e.g., PCR, ddPCR, other sequencing operations, or other downstream processes. In step 128, the device can report quantitative data about the sample, such as sample amount and / or sample concentration. The device can include algorithms or other software for converting measurements (e.g., fluorescent signals) into sample quantification values ​​and can report that data to the user. The process ends in step 129.

[0174] FIG. 33 shows a non-limiting, exemplary detailed process workflow for automated ITP using the methods, apparatus, and systems described herein.

[0175] At step 250, the user may turn on the device.

[0176] In step 251, an optional instrument initialization may be performed. Initialization may include, for example, one or more of the following checks / steps: (a) checking pressure control tolerances at 0 psi and -0.1 psi; (b) verifying negligible flow in the flow path with the vacuum valve closed; (c) observing the proportional valve value; (d) checking the HVS temperature; (e) checking the HVS voltage rails; (f) verifying no voltage with the HVS disabled; (g) checking the optics temperature; (h) checking the optics voltage rails; (i) verifying the pickoff value increase with each LED turned on; (j) verifying the detection value increase with each LED turned on; and / or (k) verifying the infrared sensor at or near room temperature.

[0177] At step 252, the device may optionally display a "Start" or "Home" menu to the user.

[0178] At step 253, the user may optionally key in "Start New Run" on the device display.

[0179] At step 254, the instrument door may be opened.

[0180] In step 255, the user may place a new microfluidic chip on the instrument stage.

[0181] In step 256, a "chip-in-place" sensor may optionally be used to detect whether the chip has been placed on the instrument stage and / or whether the chip is in the correct position or orientation on the stage.

[0182] In step 257, the device may optionally read the barcode identifier on the chip. For example, a user may use a handheld scanner.

[0183] In step 258, the device may optionally select a protocol to execute based on the scanned barcode identifier.

[0184] In step 259, the instrument may optionally check the temperature of the instrument (e.g., of the heater). For example, the temperature of the heating / cooling element may be read by the instrument. The temperature detected by an optional temperature sensor may be read by the instrument. For example, the instrument's temperature range may be confirmed by checking that the instrument temperature and the temperature detected by the temperature sensor differ by no more than about 5°C.

[0185] At step 260, the instrument may optionally display instructions on how to load buffers (e.g., one or more trailing electrolyte buffers, one or more leading electrolyte buffers, one or more elution buffers, etc.) into the chip. For example, the instrument may display visual and / or color-coded instructions (e.g., as shown in FIG. 60) to the user.

[0186] In step 261, the user may pipette a buffer onto the tip.

[0187] At step 262, the user may close the appliance door, for example, by pressing a button on the display device.

[0188] At step 263, the instrument may load one or more of the buffer solutions into the tip. The display device may optionally provide an indication to the user that loading is occurring, for example, by displaying a "priming in progress" message.

[0189] In step 264, the instrument may optionally check to confirm that the buffer reagent has been loaded and that the flow paths have been properly primed with buffer. Loading may be confirmed, for example, by testing the conductivity between two high voltage electrodes as described herein. For example, 10 μA may be sourced from one electrode and 10 μA may be sinked from the other electrode. The voltage difference between the two electrodes may be measured as described herein. Priming of the flow paths may be confirmed before, during, or after confirming buffer loading. Flow path priming may be confirmed, for example, by ensuring conductivity between the source and ground electrodes.

[0190] In step 265, the instrument may open the door / lid to allow the user access to the chip after verifying that one or more buffers have been loaded correctly.

[0191] In step 266, the instrument may optionally display instructions to the user on how to load the sample onto the tip.

[0192] At step 267, the user may pipette a sample onto the chip. The user may optionally remove the seal from the sample well of the chip to allow for sample loading. The user may optionally add an additional amount of "topper" to the sample reservoir after loading the sample as described herein.

[0193] At step 268, the user may close the appliance door, for example, by pressing a button on the display device.

[0194] In step 269, the instrument may optionally confirm that the sample has been loaded correctly. Sample loading may be confirmed by checking the conductivity from each electrode to ground as described herein. If one or more buffers remain unloaded after sample loading, the instrument may load the remaining buffers into the tip.

[0195] At step 270, the instrument may begin an ITP run. The instrument may optionally wait a predetermined period after loading the chip to allow the fluid in the chip to equilibrate. Initiating an ITP run may require activating a high voltage ("HV") power supply, adjusting the temperature of the chip to a run temperature ("T_run"), and / or turning on an optical detection system (e.g., a light-emitting diode). Run temperatures for a typical ITP procedure may be, for example, within a range of about 15°C to about 23°C.

[0196] In step 271, the instrument may optionally record and process the detected voltage signal as described herein.

[0197] In step 272, the device may optionally detect a voltage change to act as a trigger to initiate ITP as described herein. The voltage change may optionally act as a trigger to change drive electrode polarity and / or drive voltage as described herein.

[0198] In step 273, the instrument may optionally perform optical detection.

[0199] In step 274, the instrument may optionally process the detected optical signal.

[0200] In step 275, the instrument may optionally sense a temperature change at a predetermined location within the chip, for example, using an infrared sensor as described herein, which may optionally act as a trigger to terminate the ITP as described herein.

[0201] In step 276, the device may optionally detect a voltage change to act as a trigger to terminate the ITP as described herein.

[0202] In step 277, the instrument may optionally be triggered to shut off the high voltage power supply, thereby terminating the ITP run. The instrument may also shut down the optical system.

[0203] In step 278, the device may optionally close the flow path using a flow path closer as described herein.

[0204] At step 279, the device may optionally display an indicator or message to the user to alert the user that the ITP is complete.

[0205] At step 280, the device may optionally display to the user any quantitative data collected during the ITP run.

[0206] At step 281, the user may return or remain at the machine.

[0207] In step 282, the instrument may optionally hold the tip at a constant temperature until the user returns to the instrument as in step 281. The constant temperature may be, for example, in the range of about 4°C to about 20°C.

[0208] At step 283, the user may optionally open the appliance door / lid, for example, by pressing a button on the display device.

[0209] At step 284, the user may optionally retrieve the sample from the elution reservoir. Alternatively, or in combination, the instrument may optionally automatically retrieve the sample from the elution reservoir. The sample may then optionally be used in further downstream assays as desired by the user.

[0210] At step 285, the user may remove the used tip from the instrument.

[0211] A chip-in-place sensor may optionally detect removal of the chip by the user in step 286. Barcode information stored in the device may be erased.

[0212] In step 287, the user may optionally prepare reagents and samples for further ITP runs using new chips if desired.

[0213] At step 288, the user may close the door.

[0214] In step 289, the user may optionally repeat steps 253-288 as many times as desired with new chips, buffers, samples, etc. The instrument may optionally adjust the temperature of the instrument while idling between runs, for example, to a temperature in the range of about 20°C to about 25°C.

[0215] At step 290, the user may optionally transfer data collected during the ITP run, for example, via a USB port on the device or via a wireless connection.

[0216] The user may optionally turn off the device in step 291. In some examples, the device may be programmed to turn off after a predetermined idle period when the chip-in-place sensor confirms that there are no more chips in the device.

[0217] Table 1 shows typical operating times for various manual (user-performed) or automated (instrument-performed) steps of the ITP process using the exemplary method described in FIG.

[0218] [Table 1]

[0219] These issues can be particularly significant when dealing with precious, difficult-to-collect, or low-abundance samples (e.g., samples containing less than 100 ng of nucleic acid or low abundance intact or uncrosslinked nucleic acids). For such samples, current protocols can lack repeatability, result in sample loss, introduce bias for short or long nucleic acid targets, introduce bias for the sequence of nucleic acid targets, and / or lack repeatability. Such protocols may also lack compatibility with process automation or downstream analysis. Current protocols for nucleic acid preparation include liquid-phase extraction (LPE), such as phenol-chloroform or Trizol extraction, and solid-phase extraction (SPE). SPE-type techniques can use packed beads, monolithic porous structures, and / or structures containing magnetic beads. In some cases, LPE and SPE-type techniques can generate mechanical shear during processing, which can result in fragmentation of long or high-molecular-weight nucleic acids and / or reduce their yield.

[0220] The isotachophoresis methods and devices provided herein are particularly suitable for performing nucleic acid extraction from solid or semi-solid tissue lysates. Solid-phase extraction (SPE) techniques typically process lysates by pumping the entire lysate sample through a column to selectively adsorb nucleic acids to the column surface. Pumping such complex lysates, which may contain a liquid-particle mixture, through a porous column can lead to clogging or fouling of the column, which can reduce the efficiency of nucleic acid extraction. In contrast, the isotachophoresis methods and devices described herein often do not involve pumping or "filtering" the entire lysate sample through a column. Instead, an electric field is applied to the lysate to migrate charged, solvated nucleic acids dispersed throughout the complex sample lysate through and out of the sample's continuous liquid phase. Nucleic acids may contain a relatively high electrophoretic mobility magnitude compared to other solutes, debris, or contaminants in the sample lysate. Solutes in a sample may have relatively low electrophoretic mobilities, too low to be focused into the isotachophoresis zone located at the interface between the leading and trailing electrolytes. Application of an electric field can migrate nucleic acids, but particles and / or other tissue debris (e.g., including cellular debris, unlysed cells, or tissue that may bind cells to other cells) remain behind. Thus, the isotachophoresis methods and devices provided herein can be suitable for extracting charged, solvated nucleic acids from complex, lysed solid tissue samples without the need to process the entire mixture through a column, as in SPE.

[0221] As used herein, "particle" may refer to a component of a sample mixture or sample dissolution mixture that is a phase different from the continuous liquid phase (e.g., aqueous solution) of the sample. A particle may also be a non-liquid component of a sample mixture. A particle can be, for example, a suspended solid particle or a colloidal body suspended within a sample. Such particles can have a variety of characteristic length scales ranging from about 1 nanometer (nm) to about 1 millimeter (mm). In some instances, a particle may not be a single-cell organism or a cell.

[0222] The isotachophoresis methods and devices provided herein can provide a reduced strain rate as the sample passes through the flow path compared to typical SPE methods. In some cases, the methods and devices provided herein can provide a reduced strain rate as the sample passes through the flow path, typically about 250 s -1 , 500s -1 , 750s -1 , 1000s -1 , 2000s -1 , 3000s -1 , 4000s -1 , 5000s -1 , 6000s -1 , 7000s -1 , 8000s -1 , 9000s -1 or 10,000s -1 In some cases, the methods and apparatus provided herein have a strain rate of less than about 250 s -1 , 500s -1 , 750s -1 , 1000s -1 , 2000s -1 , 3000s -1 , 4000s -1 , 5000s -1 , 6000s -1 , 7000s -1 , 8000s -1 , 9000s -1 or 10,000s -1 In some cases, the methods provided herein can be performed without centrifugation.

[0223] Isotachophoresis Chemistry and Operation 2A shows an exemplary schematic diagram of an isotachophoresis (ITP) process for purifying nucleic acids. A sample 201, e.g., a dissolved solid tissue sample, containing nucleic acids (DNA and RNA) 202 and contaminants 203 is loaded into an isotachophoresis channel 200 containing a leading electrolyte (LE) 205 along with a trailing electrolyte (TE) 204. Under the influence of an electric field 220 applied to the isotachophoresis channel 210, the nucleic acids 212 can migrate away from the contaminants 213. The electric field also migrates the trailing electrolyte 214 to a position in the channel generally behind the nucleic acids, and the leading electrolyte 215 to a position in the channel generally ahead of the nucleic acids. The leading electrolyte has an effective mobility greater than that of the nucleic acids, which in turn is greater than that of the trailing electrolyte, which in turn is greater than that of the contaminants.

[0224] FIG. 2B shows an exemplary schematic diagram of a process using isotachophoresis (ITP) on a fluidic device to simultaneously decrosslink nucleic acids and separate the decrosslinked nucleic acids from crosslinked nucleic acids and contaminants (e.g., paraffin). In some instances, the contaminants may include crosslinked nucleic acids. For tissue lysis and initial deparaffinization, a paraffin-embedded sample may be loaded into the fluidic device in an alkaline buffer and incubated at a pH of about 10 and a temperature of about 50°C to about 80°C for 10 to 30 minutes. Incubation may be performed before or during application of an electric field to perform isotachophoresis. Alternatively, the sample may be loaded into a leading electrolyte buffer. After incubation, at a first time point 240, the sample containing crosslinked nucleic acids 236 and paraffin 237 may be positioned in the ITP flow path along with the trailing electrolyte 232. At the front of the ITP flow path, in the leading electrolyte (LE) zone 238, are leading electrolyte 231 and proteinase K enzyme 233. At a second time point 250, at 50°C, ITP-driven pH quenching lowers the pH, and Proteinase K enzyme contacts and decrosslinks the crosslinked nucleic acid, generating uncrosslinked nucleic acid 235, which is focused between the trailing and leading electrolytes in the ITP zone 239. Lowering the pH (e.g., from a range of about 10-12 to about 7 (or about 6.5 to about 8.5)) can provide a suitable environment for improved enzyme activity and chemical stability of the nucleic acid. At a third time point 260, Proteinase K decrosslinks more nucleic acid, generating free protein 234, which migrates further upstream away from paraffin, free protein, and other contaminants. This process can be performed automatically by a fluidic device or benchtop system.

[0225] In some cases, the sample may be loaded into a sample buffer containing a concentration of leading electrolyte 205, 231 that is different from the concentration of the leading electrolyte 205, 231 used to perform the isotachophoresis. In some cases, the sample may be loaded into a sample buffer containing a second leading electrolyte that is different from the leading electrolyte 215. The second leading electrolyte can have an effective mobility magnitude that is greater than the effective mobility magnitude of the nucleic acid. The second leading electrolyte can have an effective mobility magnitude that is less than the effective mobility magnitude of the leading electrolyte 215.

[0226] In some cases, the pH of the sample may be quenched by performing isotachophoresis. In some examples, the pH of the sample may be quenched to a range of about 6.5 to about 8.5, for example, about 7 or 7.5.

[0227] ITP can be performed using a variety of leading and trailing electrolytes. The leading electrolyte can be selected to have an effective mobility magnitude greater than that of the extraction target (e.g., nucleic acid), and the trailing electrolyte can be selected to have an effective mobility magnitude less than that of the extraction target. The leading and / or trailing electrolytes can be present at a concentration of about 10 mM to about 200 mM. The leading and / or trailing electrolytes can be present at a concentration of about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The leading and / or trailing electrolytes can be present at a concentration of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM. The leading and / or trailing electrolytes can be present at a concentration of at most about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The leading electrolyte used in the ITP embodiment can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different ionic species. The trailing electrolyte used in the ITP embodiment can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different ionic species. The different ionic species in the leading and / or trailing electrolytes can be present at different concentrations. For example, different ion concentrations in the trailing or leading electrolytes can be selected to manipulate the size of spacing zones, which can be used to further separate one type of target from another type of target, e.g., to separate decrosslinked nucleic acids from protein-crosslinked nucleic acids.

[0228] The subsequent electrolyte can contain a mixture of ions with different effective mobility magnitudes. Using a first subsequent electrolyte ion with a first effective mobility magnitude and a second subsequent electrolyte ion with a second effective mobility magnitude lower than the effective mobility magnitude of the first ion can separate non-crosslinked nucleic acids from protein-crosslinked nucleic acids while simultaneously separating both (or at least the non-crosslinked nucleic acids) from contaminants. In such cases, the non-crosslinked nucleic acids can have an effective mobility magnitude greater than the first subsequent electrolyte ion, which can have an effective mobility magnitude greater than the crosslinked nucleic acids; on the other hand, the crosslinked nucleic acids can have an effective mobility magnitude greater than the second subsequent electrolyte ion, which can have an effective mobility magnitude greater than the contaminants. For example, crosslinked and non-crosslinked nucleic acids can be separately concentrated by performing isotachophoresis using a leading electrolyte and two subsequent electrolytes, e.g., caproic acid as the first ion and HEPES as the second ion.

[0229] Electrolyte ions can also be selected based on acidity (e.g., pKa). Ions with specific pKa can be selected, for example, to cause pH changes along the ITP flow path. Ions can also be selected for non-electrophoretic reasons, such as compatibility with downstream processes (e.g., enzymatic processes, such as PCR or next-generation sequencing library preparation). For example, caproic acid, MOPS, and HEPES can be selected for good downstream enzymatic compatibility.

[0230] Exemplary leading electrolyte ions include, but are not limited to, hydrochloric acid, acetic acid, 2-chloroisocrotonic acid, salicylic acid, chlorocrotonic acid, nicotinic acid, gallic acid, trichlorolactic acid, butyric acid, sulfanilic acid, benzoic acid, crotonic acid, trichloroacrylic acid, propionic acid, levulinic acid, sorbic acid, orotic acid, valeric acid, picric acid, 2-naphthalenesulfonic acid, saccharin, dinitrophenol, p-toluenesulfonic acid, aspartic acid, trimethylacrylic acid, isocaproic acid, caproic acid, octylsulfonic acid, nitrophenol, GABA, cacodylic acid, trimethylpyruvic acid, ethylmaleate, ethylfumarate, toluic acid, enanthic acid, mandelic acid, cinnamic acid, cresol, glutamic acid, MES, isomers thereof, and combinations thereof.

[0231] Exemplary subsequent electrolyte ions include, but are not limited to, caprylic acid, gluconic acid, vanillic acid, decyl sulfonic acid, aspirin, glucuronic acid, pelargonic acid, benzylaspartic acid, ascorbic acid, dodecyl sulfonic acid, MOPS (3-(N-morpholino)propanesulfonic acid), dichlorophenol, caproic acid, capric acid, tyrosine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), isomers thereof, and combinations thereof.

[0232] Mixtures of different subsequent electrolyte ions can be used to achieve mobility-graded separations (e.g., separation of non-crosslinked nucleic acids from crosslinked nucleic acids from contaminants), compatibility with downstream assays, favorable surface energy or contact angle between the fluid and the fluidic device material, buffering capacity, and total ion solubility.

[0233] The leading electrolyte can be loaded into a leading electrolyte buffer. The leading electrolyte buffer can include one or more leading electrolytes to compact the target nucleic acid behind the leading electrolyte in the flow path during ITP. The leading electrolyte can also separate the target nucleic acid from any contaminants or inhibitors with an effective mobility magnitude greater than that of the target nucleic acid. The leading electrolyte buffer can include, for example, chloride. The leading electrolyte buffer can include a sufficient concentration of chloride such that the separation capacity of the leading electrolyte buffer is greater than the ionic strength of the sample nucleic acid. The leading electrolyte buffer can have a pH compatible with nucleic acid stability. The leading electrolyte buffer can include a surfactant (e.g., Tween) to reduce or minimize electroosmotic flow. The leading electrolyte buffer can include a surfactant (e.g., Brij-35) to reduce or minimize surface adsorption. The leading electrolyte buffer can include one or more surfactants described herein. The concentration of the surfactant(s) may be adjusted to ensure that the LE does not uncontrollably wet past the capillary barriers described herein (eg, plateau-type capillary barriers).

[0234] In some embodiments, the leading electrolyte is loaded into a high-concentration leading electrolyte buffer, which can act to buffer the lower-concentration leading electrolyte buffer. The high-concentration electrolyte buffer can have sufficient buffering capacity to prevent pH changes during the electrolysis process occurring during ITP. The high-concentration leading electrolyte buffer can include one or more leading electrolytes, which may be the same as the leading electrolytes in the leading electrolyte buffer but at higher concentrations. The high-concentration leading electrolyte buffer can include Tris and chloride. The high-concentration leading electrolyte buffer can include a Tris:chloride ratio configured to maximize buffering capacity, for example, a high Tris:chloride ratio to provide a source of Tris during ITP. The high-concentration leading electrolyte buffer can include one or more surfactants to ensure that the high-concentration leading electrolyte buffer wets the walls of the high-concentration leading electrolyte buffer reservoir and loads the capillary barrier upon application of negative pressure. The concentration of the one or more surfactants can be adjusted to ensure that the LE does not uncontrollably wet past the capillary barriers (e.g., ramp-type barriers) described herein.

[0235] In some embodiments, the leading electrolyte may be loaded into the elution buffer. The elution buffer may contain one or more leading electrolytes. The one or more leading electrolytes of the elution buffer may have a lower ionic strength than the leading electrolyte of the leading electrolyte buffer. The one or more leading electrolytes may allow for the transfer of ITP bands from a higher ionic strength electrolyte buffer to a lower ionic strength elution buffer. The elution buffer may provide compatibility with one or more downstream assays described herein (e.g., NGS library preparation, PCR, etc.). The elution buffer may contain Tris and chloride, e.g., 10 mM Tris-HCl. The elution buffer may have a pH compatible with nucleic acid stability. The leading electrolyte buffer may contain a surfactant (e.g., Tween) to reduce or minimize electroosmotic flow. The leading electrolyte buffer may contain a surfactant to reduce or minimize bubble growth in the fluid flow path (e.g., during temperature measurement as described herein). The leading electrolyte buffer may contain a surfactant to reduce or minimize surface adsorption. The elution buffer may include one or more surfactants described herein. The concentration of the surfactant(s) may be adjusted to ensure that the elution buffer does not uncontrollably wet past the capillary barriers (e.g., ramp-type barriers) described herein.

[0236] In some embodiments, the leading electrolyte is loaded into a high-concentration elution buffer, which can act to buffer a lower-concentration elution buffer (e.g., the lower concentration is suitable for use in extraction and downstream assays described herein). The high-concentration elution buffer can have sufficient buffering capacity so as not to change pH during the electrolysis process that occurs during an ITP run. The high-concentration elution buffer can have minimal carryover (e.g., less than 1 μL) between the high-concentration elution buffer and the elution buffer. The high-concentration elution buffer can contain ionic concentrations low enough that carryover does not affect downstream compatibility. The high-concentration elution buffer can contain Tris and chloride. The high-concentration elution buffer can contain a Tris:chloride ratio configured to maximize buffering capacity, e.g., a high Tris:chloride ratio to provide a source of Tris during an ITP run. The high-concentration elution buffer can contain Tris and chloride at ionic concentrations high enough to minimize the effects of carryover while achieving robust buffering. The concentrated elution buffer may include one or more surfactants to ensure that the concentrated elution buffer wets the walls of the concentrated elution buffer reservoir and loads the capillary barrier upon application of negative pressure. The concentration of the one or more surfactants may be adjusted to ensure that the concentrated elution buffer does not uncontrollably wet past the capillary barriers described herein (e.g., ramp-type barriers).

[0237] The subsequent electrolyte may be in a subsequent electrolyte buffer. The subsequent electrolyte buffer may contain one or more subsequent electrolytes to pack the target nucleic acid into the flow path before the subsequent electrolyte during ITP. The subsequent electrolyte may also separate the target nucleic acid from any contaminants or inhibitors whose effective mobility magnitude is smaller than that of the target nucleic acid. The subsequent electrolyte buffer may have sufficient buffering capacity to prevent pH changes during the electrolysis process occurring during ITP. The subsequent electrolyte buffer may contain, for example, caproic acid. The subsequent electrolyte buffer may contain MOPS. The subsequent electrolyte buffer may contain caproic acid and MOPS. The subsequent electrolyte buffer may contain a high concentration of caproic acid. A high concentration of caproic acid may result in an overly wetting fluid. Adding MOPS to the caproic acid in the subsequent electrolyte buffer may provide the necessary buffering capacity in the subsequent electrolyte reservoir without increasing the wetting properties of a high concentration of caproic acid. The subsequent electrolyte buffer may have a pH compatible with nucleic acid stability.

[0238] The lead electrolyte buffer can include one or more surfactants (e.g., Tween) as described herein. The concentration of the surfactant(s) can be adjusted to ensure that the TE does not uncontrollably wet past the capillary barrier (e.g., a plateau-type capillary barrier) as described herein. The concentration of the surfactant(s) can be adjusted to produce only small or no bubbles during the electrolysis process, as opposed to large bubbles or very large bubbles that can cause fluid fluctuations that can cause disturbances in the ITP band, voltage trace, and / or temperature trace, and that can move and negatively affect triggering.

[0239] Isotachophoresis can quench the pH of the sample to neutral or near neutral. Ions that affect the local pH (e.g., sodium ions (Na+)) are displaced from the sample zone during isotachophoresis, thereby shifting the pH in the sample zone toward neutral.

[0240] Isotachophoresis can be performed over a range of voltages, currents, and electric field strengths. For example, isotachophoresis can be performed at a voltage of about 100 V to about 1500 V. Isotachophoresis can be performed at a voltage of about 100 V, 200 V, 300 V, 400 V, 500 V, 600 V, 700 V, 800 V, 900 V, 1000 V, 1100 V, 1200 V, 1300 V, 1400 V, or 15000 V. Isotachophoresis can be performed at a voltage of at least about 100 V, 200 V, 300 V, 400 V, 500 V, 600 V, 700 V, 800 V, 900 V, 1000 V, 1100 V, 1200 V, 1300 V, 1400 V, or 15000 V. Isotachophoresis can be performed at a voltage of at most about 100 V, 200 V, 300 V, 400 V, 500 V, 600 V, 700 V, 800 V, 900 V, 1000 V, 1100 V, 1200 V, 1300 V, 1400 V, or 15000 V. Isotachophoresis can be performed at a current of about 10 nA to about 10 mA. Isotachophoresis can be performed at a current of about 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA or 10 mA. Isotachophoresis can be performed at a current of at least about 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA or 10 mA. Isotachophoresis can be performed at a current of at most about 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA, or 10 mA. Isotachophoresis can be performed at a field strength of about 10 V / cm to about 100 V / cm.Isotachophoresis can be performed at a field strength of about 10V / cm, 15V / cm, 20V / cm, 25V / cm, 30V / cm, 35V / cm, 40V / cm, 45V / cm, 50V / cm, 55V / cm, 60V / cm, 65V / cm, 70V / cm, 75V / cm, 80V / cm, 85V / cm, 90V / cm, 95V / cm or 100V / cm. Isotachophoresis can be carried out at a field strength of at least about 10V / cm, 15V / cm, 20V / cm, 25V / cm, 30V / cm, 35V / cm, 40V / cm, 45V / cm, 50V / cm, 55V / cm, 60V / cm, 65V / cm, 70V / cm, 75V / cm, 80V / cm, 85V / cm, 90V / cm, 95V / cm or 100V / cm. Isotachophoresis can be carried out at a field strength of at most about 10V / cm, 15V / cm, 20V / cm, 25V / cm, 30V / cm, 35V / cm, 40V / cm, 45V / cm, 50V / cm, 55V / cm, 60V / cm, 65V / cm, 70V / cm, 75V / cm, 80V / cm, 85V / cm, 90V / cm, 95V / cm or 100V / cm.

[0241] Isotachophoresis can be used to concentrate nucleic acids in a sample. The concentration of nucleic acids in a sample can be increased by at least about 2-fold, 5-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, 100,000,000-fold, or 1,000,000,000-fold. The operating time for concentrating nucleic acids by isotachophoresis can be about 5 hours, 4.5 hours, 4 hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 10 seconds, or 1 second or less. In some cases, isotachophoresis can be used to increase the concentration of nucleic acids in a sample by 1,000,000-fold in about 2 minutes or less. In some cases (e.g., from a 25 μL sample of blood lysate), isotachophoresis can be used to increase the concentration of nucleic acids in a sample by 100,000-fold in about 5 minutes or less.

[0242] The technique of the present disclosure can be used to reduce the concentration of crosslinked nucleic acids in a sample.The concentration of crosslinked nucleic acids in a sample can be reduced by at least about 2 times, 5 times, 10 times, 100 times, 1,000 times, 10,000 times, 100,000 times, 1,000,000 times, 10,000,000 times, 100,000,000 times or 1,000,000,000 times after isotachophoresis.Isotachophoresis can be used to reduce the concentration of contaminants in a sample. The concentration of a contaminant in a sample can be reduced by at least about 2-fold, 5-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, 100,000,000-fold, or 1,000,000,000-fold after isotachophoresis.

[0243] A nucleic acid sample can contain from about 0.1 picograms (pg) to about 25 micrograms (μg). For example, a nucleic acid sample can contain from about 5 pg to about 5 μg. The nucleic acid samples were prepared in the following concentrations: approximately 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 nanogram (ng), 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng The amount of sucrose in the solution may be 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, or 25 μg.

[0244] The nucleic acid sample can include deoxyribonucleic acid (DNA), single-stranded DNA, double-stranded DNA, genomic DNA, complementary DNA, ribonucleic acid (RNA), ribosomal RNA, transfer RNA, messenger RNA, microRNA, etc., or any combination thereof. The nucleic acid sample can include a length of at least about 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kb or greater. Different sample types can be extracted using the techniques of the present disclosure in different flow paths of a fluidic device. For example, different lengths and / or types of nucleic acids can be extracted using different flow paths.

[0245] In some examples, a feature of a nucleic acid sample can be compared to one or more nucleic acids from another sample. The feature can be, for example, expression level, nucleic acid sequence, molecular weight, nucleic acid integrity, strandedness of the nucleic acid, or nucleic acid purity.

[0246] Nucleic acid samples can be of a particular quality before and / or after extraction or other processing. Nucleic acid quality can be assessed by various metrics, including RIN value (RNA integrity number), DIN value (DNA integrity number), size distribution (e.g., using electrophoresis), and the ability to be amplified (e.g., by PCR) or otherwise enzymatically processed (e.g., fragmentation, ligation, a-tailing, or hybridization for next-generation sequencing library preparation). Nucleic acids are extracted or processed using the disclosed techniques to obtain a nucleic acid sequence of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.10, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17, 8.18, 8.19, 8.20, 8.21, 8.22, 8.23, 8.24, 8.25, 8.26, 8.27, 8.28, 8.29, 9.20, 9.21, 9.22, Extracted or processed nucleic acid can be provided having a RIN value of 0.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0.The disclosed techniques are used to extract or process nucleic acids and provide at most about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 1 Extracted or processed nucleic acid can be provided having an RIN value of 6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. Nucleic acids are extracted or processed using the disclosed techniques to obtain a nucleic acid sequence of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.10, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17, 8.18, 8.19, 8.20, 8.21, 8.22, 8.23, 8.24, 8.25, 8.26, 8.27, 8.28, 8.29, 9.20, 9.21, 9.22, Extracted or processed nucleic acid can be provided having a DIN value of 0.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0.Nucleic acids extracted or processed using the disclosed techniques are at most about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 1 In some embodiments, the extracted or processed nucleic acid may have a DIN value of 0.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. The disclosed techniques can be used to extract or process nucleic acids to provide extracted or processed nucleic acids in which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the mass of nucleic acids in a sample has a molecular weight of at least about 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kB or greater. In some cases, about 90% to about 100% of the mass of the processed nucleic acids is about 10 to about 1,000 bp, about 200 to about 2,000 bp, or about 200 to 5,000 bp.

[0247] Isotachophoresis can be used to extract nucleic acids from a given starting amount of nucleic acid with an extraction efficiency or yield characterized as a percentage yield of nucleic acid. The disclosed techniques can provide extracted nucleic acids with a yield of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%. The disclosed techniques can provide extracted nucleic acids with a yield of at least about 10%. 4nanogram (ng), 10 3 ng, 10 2 ng, 10 1 ng, 10 0 ng, 10 -1 ng or 10 -2 Even low nucleic acid inputs, including ng or less, can provide high yields. Figure 3, for example, shows exemplary nucleic acid yields from a range of different nucleic acid inputs and sources. High nucleic acid yields and / or low loss can be important for next-generation sequencing library preparation. Nucleic acid recovery can be at or near 100%.

[0248] The disclosed techniques can extract nucleic acids with low or no sequence bias. That is, the sequence composition (e.g., the ratio of GC-rich nucleic acids to AT-rich nucleic acids) of the extracted and purified nucleic acids can be similar to or the same as the sequence composition of the input nucleic acids (see, e.g., Figure 4A). The difference between the sequence composition of the extracted nucleic acids and the sequence composition of the input nucleic acids can be less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0249] The disclosed techniques can extract nucleic acids with low or no length bias. That is, the length distribution of extracted nucleic acids (e.g., the proportion of nucleic acids of various sizes) can be similar to or the same as the length distribution of input nucleic acids (see, e.g., Figure 4B). The difference between the length distribution of extracted nucleic acids and the length distribution of input nucleic acids can be about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or less. For example, short-chain nucleic acids (e.g., about 10 to about 300 bp), long-chain nucleic acids (e.g., about 10 kB, 20 kB, 30 kB, 40 kB, 50 kB, 60 kB, 70 kB, 80 kB, 90 kB, 100 kB, or larger), or both short-chain and long-chain nucleic acids can be extracted with reduced loss or bias. Solid-phase columns can, in some cases, lose up to 100% of short-chain and / or long-chain nucleic acid material. The disclosed techniques can recover nucleotides ranging in size from one base to hundreds of kilobases, and can recover at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the short and / or long nucleic acids present in a sample.

[0250] The disclosed techniques can result in the removal of contaminants from a sample. Contaminants include, but are not limited to, embedding materials, cellular debris, extracellular matrix components, tissue debris, embedding debris, lipids, carbohydrates, enzymes, ligation by-products, primers, unbound probes or ligators, divalent metals, detergents, preservatives, fixatives, anticoagulants, collagen fibers, and PCR inhibitors. Contaminants can originate from the tissues or cells of the sample, preservatives or embedding materials used on the sample, or previous preparations, reactions, or assays performed on the sample. For example, enzymes such as restriction nucleases can be used to prepare DNA for fingerprinting assays, and the DNA can be released from the enzymes after digestion (e.g., DNase digestion).

[0251] Sample The disclosed technology can be used to process a variety of sample types, including biological samples, solid tissues, biopsies, tissue biopsies, liquid biopsies, organs, tumors, fresh tissues, solid organs, preserved tissues (e.g., FFPE), dissected FFPE, fresh-frozen tissues, fixed samples, fixed tissues, embedded samples, lysed samples, unlysed samples, samples containing intercellular junctions (e.g., gap junctions, tight junctions, adherens junctions), lysed solid tissues and samples containing nucleic acids, multiphasic samples, heterogeneous liquids or solutions (e.g., tissue, whole blood, or unlysed cell suspensions), biological samples containing genomic DNA, lysed and unlysed whole blood, plasma and serum, buccal swabs, dried bloodstains and other forensic samples, fresh or fresh-frozen (FF) tissues, cells (lysed and unlysed) cultured or harvested from blood or tissue, fixed cells, stool, and body fluids (e.g., saliva, urine), or any combination thereof. Non-limiting examples of solid organs include the liver, pancreas, brain, heart, gallbladder, colon, lung, and reproductive organs. Samples can contain cellular and cell-free nucleic acids from both eukaryotic and prokaryotic organisms. Fixed samples can be chemically fixed or physically fixed (e.g., by heating or freezing). For example, samples can be chemically fixed with chemical fixatives such as formalin, neutral buffered formalin (NBF), formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, mercuric chloride, zinc salts, Bouin's solution, alcohol-formalin-acetic acid (AFA or FAA), citrate-acetone-formalin (CAF), acetone, methanol, ethanol, Clarke's solution, Carnoy's solution, or Puchtler's methacin. Embedded samples can be embedded in substances including wax (e.g., paraffin), agar, gelatin, or plastic resins. Formalin-fixed, paraffin-embedded (FFPE) samples can be processed using the techniques of the present disclosure. Samples can include buccal swabs, blood stains, and other forensic samples.Samples can include clinical samples, fine needle aspirates, biopsies, whole blood, lysed blood, serum, plasma, urine, cell culture lysates or freshly harvested cell (e.g., blood cells, dissociated fresh tissue, stem cell) lysates, blood cells, circulating cells (e.g., circulating tumor cells (CTCs)), nucleic acids from blood or other bodily fluids, and other sample categories. Cell-free nucleic acids (e.g., cfDNA or cfRNA) can be recovered, for example, from unlysed whole blood using the techniques disclosed herein. In many cases, the cell-free nucleic acids are circulating cell-free nucleic acids. Samples can be derived from a variety of sources, including normal tissue, benign neoplasms, malignant neoplasms, stem cells, human tissue, animal tissue, plant tissue, bacteria, viruses, and environmental sources (e.g., water). Human or animal tissues include, but are not limited to, epithelial tissue, connective tissue (e.g., blood, bone), muscle tissue (e.g., smooth muscle, musculoskeletal, cardiac muscle), and nervous tissue (e.g., brain, spinal cord).

[0252] The sample can include one or more particles in suspension. The one or more particles can range from colloidal size to visible. The one or more particles can be at least about 1 nanometer (nm), 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 950 nm, 1 micrometer (μm), 10 μm, 20 μm, 30 μm, or 400 nm. The size may be 1 mm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm or 1 millimeter (mm). The one or more particles may be at most about 1 nanometer (nm), 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 950 nm, 1 micrometer (μm), 10 μm, 20 μm, 30 μm The particle size may be 1 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, or 1 millimeter (mm). One or more particles may be the same size or different sizes. A sample may include, for example, multiple particles ranging in size from 1 nm to 500 μm.

[0253] Various amounts of sample can be processed on the fluidic device (eg, to extract and purify nucleic acids). For example, the sample volume (with or without buffer) can be at least about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 175 μL, 200 μL, 225 μL, 250 μL, 275 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. The sample volume (with or without buffer) can be at most about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. In some cases, the sample volume can be from about 1 nL to about 10 nL. Sample volumes (with or without buffer) can be at least about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. In some cases, sample volumes can be from about 1 nL to about 10 nL.

[0254] Samples containing various numbers of cells can be processed on the fluidic device (e.g., to extract and purify nucleic acids). For example, samples can be about 20,000 or less, 15,000 or less, 10,000 or less, 9,000 or less, 8,000 or less, 7,000 or less, 6,000 or less, 5,000 or less, 4,500 or less, 4,000 or less, 3,500 or less, 3,000 or less, 2,500 or less, 2,000 or less, 1,500 or less, 1,000 or less, or 2,500 or less. The cell may contain up to 00, up to 900, up to 800, up to 700, up to 600, up to 500, up to 400, up to 300, up to 200, up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, up to 10, up to 5, up to 2, or 1 cell. In some cases, the sample contains at least about 10,000,000, 5,000,000, 1,000,000, 500,000, 100,000, 50,000, 20,000, 15,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,500, 4,000, 3,500, 3,000, 2,500, 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 cells.

[0255] Samples of various masses can be processed on the fluidic device (e.g., to extract and purify nucleic acids). For example, a sample can contain from about 0.001 milligrams (mg) to about 10 mg of tissue. A sample can contain at most about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue. The sample can contain at least about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue. The sample can contain about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg of tissue.

[0256] Samples containing various amounts of nucleic acids can be processed on a fluidic device (e.g., to extract and purify nucleic acids). For example, a sample can contain about 1 microgram (1 μg) or less, 100 nanograms (ng) or less, 10 ng or less, 1 ng or less, 100 picograms (pg) or less, 10 pg or less, or 1 pg or less of nucleic acid. In some cases, a sample can contain about 1 microgram (1 μg) or more, 100 nanograms (ng) or more, 10 ng or more, 1 ng or more, 100 picograms (pg) or more, 10 pg or more, or 1 pg or more of nucleic acid.

[0257] The sample can be loaded into a sample buffer. The sample buffer may contain a lysing agent or surfactant to dissolve the input sample during off-chip processing to provide access to the target nucleic acid. The sample buffer may contain one or more leading electrolytes. The sample buffer may have sufficient wettability to self-load into the sample flow channel by gravity and / or surface tension. The sample buffer may contain one or more surfactants to reduce or minimize adsorption of the target nucleic acid to the walls of the fluid flow channel. The sample buffer may have an ionic content optimized to accommodate nucleic acid separation while having sufficient salt for lysis and / or nucleic acid preservation. One skilled in the art will understand that the higher the ionic content of the sample buffer (or any of the buffers described herein), the more current is required to transfer the charge of the sample buffer.

[0258] The sample can be loaded into a buffer containing a trailing or leading electrolyte. The sample can be loaded into a buffer containing a second leading electrolyte that is different from the leading electrolyte used to perform ITP. The sample can be loaded into a buffer, such as an aqueous alkaline or aqueous neutral buffer. An exemplary alkaline solution or buffer (e.g., for DNA extraction) can contain 30-120 mM NaOH (optionally 40-80 mM NaOH) and have a pH of about 10-13 (optionally containing at least one additional component). In some examples, if the sample is lysed by treatment with an alkaline solution or buffer before loading onto the chip, the lysed sample can then be quenched by adding an acidic solution or buffer to bring the pH of the lysed sample to a range of about 7.5 to about 8.5 before performing isotachophoresis. Exemplary aqueous buffers (e.g., for DNA or RNA extraction) can include 2-150 mM Tris-HCl (pH about 7 to about 8) or BisTris-HCl (pH about 5.8 to about 7.3) along with at least one additional component. Additional components used in buffers can include non-ionic surfactants or detergents, ionic or zwitterionic surfactants or detergents, chaotropic agents, disulfide bond reducing agents, proteases, nucleases, and other additives or components that digest, denature, disrupt, or degrade nucleic acids in order to extract, purify, concentrate, or otherwise isolate nucleic acids.

[0259] The sample can be loaded into a buffer containing a trailing or leading electrolyte added to reduce or minimize retention of nucleic acids at capillary barriers, particularly those between the sample and the LE. For example, a small amount of trailing electrolyte (e.g., MOPS and / or caproic acid) can be added to the lysate sample to intentionally slow down the compaction of the DNA ITP band. While not wishing to be bound by any particular theory, it is believed that this may help maintain the DNA in a more dispersed state as it passes through the constricted space of the capillary barrier (e.g., a cliff-type capillary barrier at the junction of the sample and the LE), which could otherwise impair the passage of the more compact ITP band. Because the spiked-in TE has a slower mobility magnitude than both the DNA and the LE, as the ITP band enters the LE buffer, the TE may lag behind, allowing the ITP band to fully compact before it reaches the elution reservoir.

[0260] Nonionic surfactants or detergents include, but are not limited to, surfactants from the following classes: octylphenol ethoxylate, polysorbate, poloxamer, or polyoxyethylene. Octylphenol ethoxylate surfactants include, but are not limited to, branched octylphenoxypolyethoxyethanol (IGEPAL CA-630), t-octylphenoxypolyethoxyethanol (Triton™ X-100), or other polyethylene oxide chains with aromatic hydrocarbon lipophilic or hydrophobic groups. Polysorbate surfactants include, but are not limited to, polyethylene glycol sorbitan monolaurate (Tween® 20), polyethylene glycol sorbitan monooleate (Tween® 80), or sorbitan monooleate (Span® 80). Poloxamer surfactants (i.e., block copolymers based on ethylene oxide and propylene oxide) include, but are not limited to, polyoxyethylene-polyoxypropylene block copolymer (Pluronic® F-68) or polyethylene-polypropylene glycol block copolymer (Pluronic® F-127). Polyoxyethylene surfactants include, but are not limited to, nonylphenoxypolyethoxylethanol (NP-40).

[0261] Nonionic surfactants or detergents include, but are not limited to, IGEPAL® (e.g., IGEPAL® CA-630), Triton™ X-100, Tween® 20, Tween® 80, NP-40, other block copolymers such as Pluronic® (e.g., F-68 or F-127), Span® 80, and PEGylated polymers or copolymers. Nonionic surfactants or detergents can be used to reduce or prevent biomolecular adsorption to channel walls or to control fluid wettability and / or surface tension to control sample loading into fluidic devices. Nonionic surfactants or detergents can be present at a concentration of about 0.0005-5% (v / v or w / v). For example, IGEPAL CA-630 can be used at about 0.05-0.5% v / v. Ionic surfactants or detergents include, but are not limited to, sodium dodecyl sulfate (e.g., 0.01-2% w / v), sodium dodecylbenzenesulfonate (e.g., 0.01-2% w / v), sodium cholesteryl sulfate (e.g., 0.01%-2% w / v), and sodium deoxycholate (e.g., about 10-1000 mM). Chaotropic agents include, but are not limited to, urea (e.g., about 0.5-9.5 M or, in some cases, 5-9.5 M), thiourea, butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, lithium chloride, magnesium chloride, phenol, and propanol. For example, for either RNA or DNA extraction or total nucleic acid extraction, 7.0 M urea and 2.0 M thiourea can be used in a 5-50 mM Tris-HCl (in some cases, 10-20 mM Tris-HCl) buffer solution. The ratio of urea:thiourea can be at least about 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 6.5:1, 7:1, 7.5:1 or 8:1.Disulfide bond reducing agents include, but are not limited to, DTT (e.g., about 0.1-40 mM, or optionally about 10 mM) and beta-mercaptoethanol (e.g., about 0.5-2%, or optionally about 1%). Proteases include, but are not limited to, proteinase K, proteases, endoproteinases (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Nucleases include, but are not limited to, non-specific nucleic acid digestion enzymes, such as DNases, e.g., DNase I (e.g., for preparing DNA-free RNA extracts), and RNases, e.g., RNase A, RNase T, or combinations thereof (e.g., for preparing RNA-free DNA extracts). Nucleases also include specific nucleic acid digestion enzymes (e.g., restriction enzymes) that can cleave at specific nucleic acid sequences and generate predictable fragment sizes and fragment size distributions. In some cases, one or more of the methods or processes provided herein are performed without the use of nucleases, without the use of DNases, or without the use of RNAases. For example, the methods provided herein include RNA extraction without the use of DNAases.

[0262] Restriction enzymes include, but are not limited to, type I to type V restriction enzymes, BamHI, EcoP15I, EcoRI, EcoRII, EcoRV, HaeIII, HgaI, HindIII, HinFI, KpnI, NotI, PstI, PvuII, SacI, SalI, SmaI, SpeI, SphI, XbaI, and StuI. Nucleases can be used at concentrations ranging from 50 to 400 μg / mL. Nuclease digestion can be performed at temperatures ranging from about 20°C to about 37°C. Other nucleic acid-modifying enzymes, such as transposases, ligases, polymerases, and phosphatases, can also be used. Other protein or polynucleotide digestion or degradation agents, such as lysozyme, can also be used.

[0263] Prior to loading into the fluidic device, the sample can be subjected to various degrees of pre-processing. In some cases, the sample can simply be loaded into a buffer solution before being loaded into the fluidic device, and any other necessary or desired sample preparation steps can be performed on the device. In other cases, the sample can be added to a sample reservoir that is pre-filled with a processing fluid, such as a solution or buffer. In other cases, the sample can be subjected to embedding material removal, tissue disruption, cell lysis, or digestion before being loaded into the fluidic device. In one example, the sample is deparaffinized before being loaded into the fluidic device, and nucleic acid decrosslinking is performed on the fluidic device. In another example, the sample is deparaffinized, disrupted, and lysed before being loaded into the fluidic device, and optionally nucleic acid decrosslinking is performed on the fluidic device. In another example, the sample is deparaffinized before being loaded into the fluidic device, and tissue disruption and cell lysis are performed on the fluidic device. In another example, the sample is loaded into a fluidic device, and deparaffinization, tissue disruption, cell lysis, and nucleic acid decrosslinking are all performed on the fluidic device. The sample preparation steps are described in further detail in this disclosure.

[0264] Sample preparation Samples can be prepared before isotachophoresis. Sample preparation can include steps such as removal of embedding material, tissue disruption, cell lysis, protein digestion, nucleic acid cross-linking, isothermal enzymatic processes, enzymatic amplification, enzymatic digestion, disruption of intercellular junctions, disruption of the extracellular matrix, disruption of connective tissue, and combinations thereof. Sample preparation can include techniques such as polymerase chain reaction (PCR) or other nucleic acid amplification, isolation or purification of the material of interest (e.g., cells, nucleic acids), probe hybridization, and antibody hybridization (e.g., hybridization of antibodies to nucleosomes). In some cases, samples can be prepared for further analysis by isolating a portion of material from cells from the sample. For example, circulating tumor cells can be isolated from a heterogeneous cell population using a cell sorting device such as a flow cytometer or a magnetic column. In another example, peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs) can be isolated from a blood sample. Sample preparation can be performed on-device or off-device. In some cases, some sample preparation steps are performed off-device before the sample is loaded into a fluidic device where further sample preparation steps are performed.

[0265] Biological materials (e.g., cells, tissues, nucleic acids) in embedded samples can be removed from the embedding material. For example, paraffin-embedded samples can be deparaffinized. Removal of the embedding material can be achieved using techniques including heat treatment, chemical treatment (e.g., acid or base), enzymatic treatment, and combinations thereof. Deparaffinization can be achieved by chemical treatment of the sample, heat treatment of the sample, enzymatic treatment of the sample, or other methods. For example, deparaffinization can be achieved at elevated temperatures (e.g., about 50°C to about 80°C) in the presence of a neutral buffer or a slightly acidic buffer (e.g., up to about pH 5.5), or a slightly basic (up to about pH 9) or alkaline solution (e.g., about pH 12 to about 13). Removal of the embedding material can be achieved off-device or on-device. In one example, the embedded sample can be incubated in a container at elevated temperatures before being loaded into a fluidic device. In another example, the embedded sample can be loaded into a fluidic device and incubated at elevated temperature on the device, for example in a channel or reservoir.

[0266] The embedding material can be removed by heat treatment. Incubation for removing the embedding material can be carried out at a temperature of at least about 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 96°C, 97°C, 98°C, 99°C, 99.5°C, or 100°C. Incubation for removing the embedding material can be carried out at a temperature of about 40°C to about 80°C, about 50°C to about 80°C, about 50°C to about 99.9°C, or about 95 to about 99.5°C. Incubation to remove the embedding material can be carried out for a period of at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, or 120 minutes. Incubation to remove the embedding material can be carried out for a period of about 1 minute to about 20 minutes, about 1 minute to about 30 minutes, about 1 minute to about 60 minutes, about 1 minute to about 120 minutes, or about 5 minutes to about 20 minutes. Incubation to remove the embedding material can be carried out, for example, at a temperature of at least about 37°C for a period of at least about 1 minute. Incubation to remove the embedding material can be performed in the presence of an alkaline or neutral buffer (e.g., a lysis buffer). The alkaline buffer (e.g., a lysis buffer) can have a pH of at least about 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, or 13.5. The neutral buffer can have a pH of about 7.0 (e.g., about 7 to about 8).

[0267] Once tissue or cells are disrupted or lysed, they can release nucleic acids for separation, purification, or extraction. Tissue disruption or cell lysis can be achieved using techniques including mechanical stress, sonication, electroporation, osmotic pressure, chemical treatment (e.g., acid or base), enzymatic treatment, heat treatment, and combinations thereof. For example, pressure can be used to force tissue through a structure (e.g., a channel, a resin, e.g., a frit or porous resin, or a glass material) to mechanically disrupt the tissue or lyse the cells. In some cases, the subsequent electrolyte buffer can contain one or more tissue disruption agents and / or cell lysis agents. In some cases, the leading electrolyte buffer can contain one or more tissue disruption agents and / or cell lysis agents. In some cases, removal of the embedding material can be achieved by the same process as tissue disruption or cell lysis. For example, incubation at elevated temperatures (e.g., about 30°C to about 80°C, about 50°C to about 80°C, or about 30°C to about 65°C) can achieve embedding material removal, tissue disruption, and cell lysis. Tissue disruption or cell lysis can be performed off-device or on-device. In one example, the tissue sample is disrupted in a container before being loaded into the fluidic device, and in another example, the tissue sample is previously loaded into the fluidic device and is disrupted on the device.

[0268] Samples containing tissues or cells can be lysed using a lysis solution or buffer compatible with isotachophoresis before or after loading into a fluidic device. Lysis buffers compatible with isotachophoresis can contain non-ionic surfactants or detergents, ionic or zwitterionic surfactants or detergents, chaotropic agents, disulfide bond reducing agents, proteases, nucleases, and other additives or components that digest, denature, disrupt, or degrade nucleic acids for extraction, purification, enrichment, or other isolation. In some cases, the lysis buffer can include an alkaline buffer. In other cases, the lysis buffer can be free of an alkaline buffer. Exemplary lysis buffers can include 0.5M-9.5M, 4M-9M, or 6.5M-7M urea, as described herein. Exemplary lysis buffers can include 0.5M-3.5M, or 1.5M-2.5M thiourea, as described herein. An exemplary lysis buffer can include 0.5-9.5 M urea and thiourea, e.g., 7 M urea and 2 M thiourea, along with a non-ionic surfactant as described herein. Urea can be used alone or in combination with thiourea to lyse cells for nucleic acid purification. When combined, urea and thiourea can act synergistically to lyse cells and provide an uncharged, isotachophoresis-compatible buffer for nucleic acid purification.

[0269] An exemplary lysis buffer may include a non-ionic surfactant, such as 0.05-0.5% v / v IGEPAL CA-630, as described herein. Optionally, the lysis buffer may include one or more trailing electrolytes. Optionally, the lysis buffer may include a trailing electrolyte buffer along with additives for tissue disruption or cell lysis, as described herein. Optionally, the lysis buffer may include one or more leading electrolytes. Optionally, the lysis buffer may include a leading electrolyte buffer along with additives for tissue disruption or cell lysis, as described herein. Optionally, the lysis buffer may include one or more leading electrolytes and one or more trailing electrolytes. Optionally, the lysis buffer may include one or more leading electrolytes and one or more trailing electrolytes along with additives for tissue disruption or cell lysis, as described herein.

[0270] In some cases, the methods or processes herein may include lysing cell or tissue samples using a lysis buffer that minimizes mechanical disruption of DNA and / or RNA during the lysis reaction. For example, cells or tissues may be lysed in a buffer solution containing Tris (e.g., 5 mM, 10 mM, 20 mM, 30 mM Tris) together with HCl (e.g., 1 mM, 5 mM, 10 mM HCl) and a non-ionic detergent. The non-ionic detergent (e.g., IGEPAL CA-630) may be present in the lysis buffer at about 1%, about 2%, about 3%, about 4%, or more, or less than about 1%. Cells or tissues may be lysed in the lysis buffer by gentle mixing, such as by inversion and low speed (automated pipette). In some cases, enzymes such as proteinase K may be included in the lysate or lysis buffer. In some cases, lysis is performed without centrifugation. In some cases, centrifugation is used in the lysis method. The lysate can be introduced into an isotachophoresis apparatus to purify desired analytes, such as high molecular weight DNA fragments.

[0271] Proteins in a sample can be digested, for example, by enzymatic digestion with proteases. Proteases include, but are not limited to, proteinase K, proteases, endoproteinases (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Other protein or polynucleotide digesting or degrading agents, such as lysozyme, can also be used. Protein digestion can remove cross-linking proteins from cross-linked nucleic acids, converting the cross-linked nucleic acids to non-cross-linked nucleic acids. Protein digestion can be performed at room temperature or at elevated temperatures (e.g., greater than about 25°C) as described herein.

[0272] The sample can be processed on a device (e.g., an electrokinetic device or system having at least one reservoir connected to at least one flow path) in which a sample volume passes through a reservoir and enters a flow path, with less than 20% of the sample volume remaining in the reservoir, and then an ionic current can be applied to the sample volume in the flow path. The ionic current can be substantially absent from the flow path. In some cases, less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the sample volume remains in the reservoir.

[0273] The sample can be processed on an apparatus (e.g., an electrokinetic device or system having at least one reservoir connected to at least one flow path) such that the amount of sample passing through the reservoir and entering the flow path is at least 50% of the amount of sample loaded into the reservoir, and then an ionic current can be applied to the amount of sample in the flow path. In some cases, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the sample volume is transferred from the reservoir to the flow path. In some cases, the total amount loaded into the reservoir is equal to or less than the volume of the reservoir. Substantially no ionic current can pass through the flow path. In some cases, applying an ionic current includes performing isotachophoresis.

[0274] In some embodiments, after a sample volume is loaded into a reservoir and then into a flow path, a topper buffer may be added to the reservoir to facilitate movement of the sample volume into the flow path. An amount of topper buffer may be added to the reservoir to "push" additional sample volume into the flow path. For example, an amount of topper buffer equal to or greater than the amount of sample remaining in the reservoir may be added to the reservoir to move at least a portion of the remaining sample volume into the flow path. The topper buffer may, for example, comprise the same buffer as the sample buffer, but without the analyte therein.

[0275] isotachophoresis apparatus Isotachophoresis and / or sample preparation (e.g., deparaffinization, digestion, lysis) can be performed in a fluidic device, e.g., a microfluidic chip. For example, FIG. 5A shows a schematic diagram of a flow path having a sample preparation (e.g., deparaffinization) zone 500 with a sample inlet 501 and a trailing electrolyte reservoir 502, and a purification (e.g., isotachophoresis) zone 510 with a leading electrolyte reservoir 511 and an elution outlet 520. A capillary barrier can provide an interface between the sample fluid and the leading electrolyte buffer prior to application of a voltage. A capillary barrier can be provided between the sample preparation zone 500 and the trailing electrolyte reservoir 502 to limit, reduce, or prevent mixing or pressure-driven flow of the sample fluid and the trailing electrolyte buffer. A capillary barrier can be provided between the purification zone 510 and the leading electrolyte reservoir 511 to limit, reduce, or prevent mixing or pressure-driven flow of the contents of zone 510 and the leading electrolyte reservoir 511. In another example, deparaffinization can be performed initially off-chip or can be unnecessary due to the starting material, in which case the flow path can include a lysis and digestion zone (e.g., pH 7, 56°C) and a decrosslinking and purification (e.g., isotachophoresis) zone (e.g., pH 7, 80°C). In another example, deparaffinization can be performed initially off-chip or can be unnecessary due to the starting material, in which case the flow path can include a lysis and digestion zone (e.g., pH 7, temperature T1) and a decrosslinking and / or purification (e.g., isotachophoresis) zone (e.g., pH 7, temperature T2). In another example, deparaffinization can be performed initially off-chip or can be unnecessary due to the starting material, in which case the flow path can include a disruption and / or lysis zone (e.g., pH 7, temperature T1) and a digestion and / or purification (e.g., isotachophoresis) zone (e.g., pH 7, temperature T2).In another example, deparaffinization can be performed initially off-chip or may not be required due to the starting material, in which case the flow path can include a disruption and / or lysis zone (e.g., pH 7, temperature T1) and an isothermal enzymatic amplification zone (e.g., pH 7, temperature T2). In another example, deparaffinization can be performed initially off-chip or may not be required due to the starting material, in...

Claims

1. a fluid flow path; and a three-dimensional capillary barrier disposed in the fluid flow path to restrict flow of liquid through the fluid flow path; The three-dimensional capillary barrier comprises: (a) a ramp portion protruding from a surface of the fluid flow path at a first angle; (b) a plateau surface substantially parallel to the surface of the fluid flow path; and (c) a cliff surface extending from the plateau surface to the surface of the fluid flow path. Including, the ramp portion includes an edge that intersects with the plateau surface; the cliff face intersects the surface at a second angle that is at least 10° steeper than the first angle; the three-dimensional capillary barrier extends substantially across the width of the fluid flow path; A fluidic device, comprising: The fluidic device further comprising a gas flow path in fluid communication with the fluid flow path and an air port in fluid communication with the gas flow path, the gas flow path and the air port in fluid communication with the three-dimensional capillary barrier.

2. a fluid flow path; and a three-dimensional capillary barrier disposed in the fluid flow path to restrict flow of liquid through the fluid flow path; The three-dimensional capillary barrier comprises: (a) a first ramp portion projecting from a surface of the fluid flow path at a first angle less than 80°; (b) a plateau surface substantially parallel to the surface of the fluid flow path; and (c) a second ramp portion extending from the plateau surface to the surface of the fluid flow path. Including, the plateau surface includes first and second steps configured such that a height of the plateau surface decreases in a region between the first and second steps; the first ramp portion includes an edge that intersects with the plateau surface; the second ramp portion intersects the surface at a second angle less than 80°; the three-dimensional capillary barrier extends substantially across the width of the fluid flow path; Fluidic devices.

3. The fluidic device according to any one of claims 1 to 2, wherein the first angle is 60° or less.

4. The fluidic device according to any one of claims 1 to 2, wherein the first angle is 45° or less.

5. The fluidic device according to any one of claims 1 to 2, wherein the first angle is 30° or less.

6. 10. The fluidic device of claim 1, wherein the second angle is between about 80 degrees and about 100 degrees.

7. 10. The fluidic device of claim 1, wherein the second angle is about 90 degrees.

8. 10. The fluidic device of claim 1, wherein at least one of the ramp portion, the cliff surface, and the plateau surface has a surface that includes one or more grooves, ridges, depressions, steps, etchings, or protrusions.

9. 3. The fluidic device of claim 2, wherein at least one of the first ramp portion, the second ramp portion, and the plateau surface has a surface including one or more grooves, ridges, depressions, steps, etchings, or protrusions.

10. The fluidic device of any one of claims 1 to 2, wherein the fluid flow path further comprises a second three-dimensional capillary barrier spaced from the three-dimensional capillary barrier.

11. the fluid flow path further includes a first loading reservoir in fluid communication with the fluid flow path through an opening in the fluid flow path; The fluidic device of claim 10, wherein the opening is disposed between the three-dimensional capillary barrier and the second three-dimensional capillary barrier to allow the liquid entering the fluid flow path through the opening to flow in one direction along the fluid flow path, to be stopped at the three-dimensional capillary barrier, to flow in another direction along the fluid flow path, to be stopped at the second three-dimensional capillary barrier.

12. 12. The fluidic device of claim 11, wherein the liquid entering the fluid flow path through the opening flows along a path that is longer than a width of the fluid flow path to the three-dimensional capillary barrier or the second three-dimensional capillary barrier.

13. 12. The fluidic device of claim 11, wherein the liquid entering the fluid flow path through the opening flows such that a meniscus of the liquid terminates at the three-dimensional capillary barrier or the second three-dimensional capillary barrier or both.

14. The fluidic device of claim 10, further comprising one or more branched fluid circuits, each of the one or more branched fluid circuits comprising an isotachophoresis ("ITP") zone and an elution zone in communication with the ITP zone.

15. The fluidic device of claim 14, wherein the three-dimensional capillary barrier is configured and arranged to be breached by the liquid when a first burst pressure is applied to the one or more branch-type fluid circuits, and the second three-dimensional capillary barrier is configured and arranged to be breached by the liquid when a second burst pressure is applied to the one or more branch-type fluid circuits.

16. 16. The fluidic device of claim 15, wherein the first and second burst pressures are approximately equal.

17. 16. The fluidic device of claim 15, wherein the first burst pressure is greater than the second burst pressure.

18. The second three-dimensional capillary barrier is a cliff-type capillary barrier, the cliff-type capillary barrier comprising: a ramp portion of the cliff-type capillary barrier protruding from a surface of the fluid flow path at a third angle; a plateau surface of the cliff-shaped capillary barrier substantially parallel to the surface of the fluid flow path; and a cliff surface of the cliff-type capillary barrier extending from the plateau surface of the cliff-type capillary barrier to the surface of the fluid flow path. Including, the ramp portion of the cliff-type capillary barrier includes an edge that intersects with the plateau surface of the cliff-type capillary barrier; the cliff face of the cliff-type capillary barrier intersects the surface at a fourth angle that is 10° or more steeper than the third angle; The fluidic device of claim 10.

19. The second three-dimensional capillary barrier is a plateau-type capillary barrier, the plateau-type capillary barrier comprising: a first ramp portion of the plateau-shaped capillary barrier protruding from a surface of the fluid flow path at a third angle; a plateau surface of the plateau-shaped capillary barrier substantially parallel to the surface of the fluid flow path; and a second ramp portion of the plateau-type capillary barrier extending from the plateau surface of the plateau-type capillary barrier to the surface of the fluid flow path. Including, 11. The fluidic device of claim 10, wherein a fourth angle at which the second ramp portion of the plateau-shaped capillary barrier intersects the surface of the fluid flow path and the third angle are oblique with respect to each other.

20. 20. The fluidic device of claim 19, wherein the plateau-type capillary barrier is constructed and arranged to form an air gap between the liquid after it stops at the plateau-type capillary barrier and a second liquid flowing in another direction toward the plateau-type capillary barrier and stopping at the plateau-type capillary barrier on an opposite side to the liquid.

21. The fluidic device of claim 1 , wherein the three-dimensional capillary barrier is disposed adjacent to the gas flow path.

22. The fluidic device of any one of claims 1 to 2, wherein the three-dimensional capillary barrier spans the width of the fluid flow path.

23. The fluidic device of any one of claims 1 to 2, further comprising a plurality of fluid flow paths including capillary barriers.

24. 3. The fluidic device of claim 1, wherein the fluid flow path has a width in the range of about 1 mm to about 3.8 mm and a height in the range of about 0.1 mm to about 1.2 mm.

25. 2. The fluidic device of claim 1, wherein the ramp portion of the three-dimensional capillary barrier is configured to gradually reduce the height of the fluid flow path from a first height in a range of about 50 μm to about 2 mm to a second height in a range of about 10 μm to about 30 μm.

26. 3. The fluidic device of claim 2, wherein the first ramp portion and the second ramp portion of the three-dimensional capillary barrier are configured to gradually reduce the height of the fluid flow path from a first height in a range of about 50 μm to about 2 mm to a second height in a range of about 10 μm to about 30 μm.

27. 2. The fluidic device of claim 1, wherein the second angle is at least 15° steeper than the first angle.

28. 2. The fluidic device of claim 1, wherein the second angle is at least 20 degrees steeper than the first angle.

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