Systems, devices, and methods for isoelectric focusing

Abstract

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 isoelectric focusing. In particular, the systems and related methods can allow for the extraction of nucleic acids, including non-crosslinked nucleic acids, from samples such as tissues or cells. The systems and devices can also be used for multiplexed parallel sample processing.

Description

[0001] This application is a divisional application of Chinese patent application No. 201880064555.9, entitled "System, apparatus and method for isokinetic electrophoresis" (the corresponding PCT application was filed on August 1, 2018, with application number PCT / US2018 / 044898). Cross-referencing

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 540,515, filed August 2, 2017, entitled “Isotachophoresis for Purification of Nucleic Acids” [Attorney’s File No. 43647-718.101]; U.S. Provisional Application No. 62 / 541,086, filed August 3, 2017, entitled “Isotachophoresis Devices for Purification of Nucleic Acids” [Attorney’s File No. 43647-719.101]; and U.S. Provisional Application No. 62 / 541,089, filed August 3, 2017, entitled “Nucleic Acid Analysis Using Isotachophoresis and Intercalating Dye” [Attorney’s File No. 43647-720.101], the entire contents of which are incorporated herein by reference.

[0003] This application relates to pending PCT application No. PCT / US2017 / 015519, filed on January 28, 2017, entitled “Isotachophoresis for Purification of Nucleic Acids” [Attorney’s Case No. 43647-712.601], the entire contents of which are incorporated herein by reference. Background Technology

[0004] Formalin-fixed paraffin-embedded (FFPE) samples have been collected, prepared, stored, and archived in large tissue libraries for over a century. As of 2008, more than 400 million FFPE samples were stored in biobanks worldwide, and this number continues to grow. These samples are often accompanied by clinical information such as preliminary diagnoses, treatment protocols, and follow-up data, making them an important resource for therapeutic development and the discovery of genomic and transcriptomic biomarkers.

[0005] Sample preparation methods for extracting and purifying nucleic acids from FFPE samples remain manual and labor-intensive. Methods for FFPE extraction and purification vary considerably, but typically involve steps that are difficult to automate and accelerate, such as dewaxing, centrifugation, buffer replacement, temperature control, cross-linking reduction, and enzymatic treatment. FFPE generally refers to the process of cross-linking proteins in a sample with formalin and embedding the sample in paraffin (wax). FFPE treatment of samples often allows for preservation over time and is particularly useful for long-term storage. Cross-linked proteins can bind to DNA and RNA in the sample, thus generally rendering them unusable for downstream applications such as amplification, library preparation, or sequencing.

[0006] Removing paraffin and protein crosslinks from FFPE samples can be a challenging process. Dewaxing is traditionally performed using highly flammable xylene. Alternatively, or continuously, samples can be treated with other solvents, mineral oils, and alkaline chemicals and / or elevated temperatures. After dewaxing, proteins in the sample can be treated with different reagents or subjected to conditions that may require additional time and effort.

[0007] At the end of digestion and denaturation, a mixture of cross-linked and non-cross-linked nucleic acids may still remain. Removing non-cross-linked substances can be important for high-quality results from assays such as amplification or sequencing; in some cases, if the fraction of non-cross-linked substances is too low, downstream assays may not be possible, resulting not only in the loss of the sample itself but also in the loss of labor, time, and resources. Summary of the Invention

[0008] Isotachyphoresis (ITP) is an electrophoresis technique that uses a discontinuous buffer containing a leading electrolyte (LE) with a high effective mobility and a trailing electrolyte (TE) with a lower effective mobility (e.g., relative to LE) to focus sample species with a higher effective mobility than the trailing electrolyte but a lower effective mobility than the leading electrolyte. ITP can selectively focus nucleic acids in a sample by more than 10,000 times in less than five minutes. This disclosure provides methods and apparatus for employing ITP and automating it for sample preparation, including extraction, purification, enrichment, and highly sensitive quantification, and is particularly suitable for the preparation and purification of nucleic acids from FFPE samples and other biological samples.

[0009] Sample preparation is crucial for genomic analysis, but it remains a major source of analytical variability and can often require significant manual labor. This disclosure includes techniques and equipment to overcome this challenge, such as the extraction and purification of nucleic acids using isotachypeptide-on-chip (ITP). These techniques include methods for enriching (concentrating) non-crosslinked nucleic acids to achieve higher yields and higher quality nucleic acid sample preparation and to generate more usable samples (e.g., fewer quality check failures) from FFPE and other preserved or fresh samples.

[0010] This disclosure includes techniques and apparatus for the automated preparation of nucleic acid samples from samples including solid tissues, lysed solid tissues, preserved or fixed tissue samples (e.g., FFPE), whole blood, plasma and serum, oral swabs, dried blood spots and other forensic samples, fresh or fresh-frozen (FF) tissues, biopsy tissues, organ tissues, solid organ tissues, samples containing intercellular junctions (e.g., gap junctions, tight junctions, adhesion junctions), cells cultured or harvested from blood or tissues, feces and body fluids (e.g., saliva, urine) or any combination thereof. For both eukaryotic and prokaryotic samples or any combination thereof, samples may include cellular nucleic acids and cell-free nucleic acids. Compared to existing methods, the techniques of this disclosure are faster, less manual, better suited for both small and large starting volumes of tissue, and can achieve higher yields and higher quality sample analysis.

[0011] One aspect of this disclosure provides a fluid apparatus including an isotachymeter electrophoresis (ITP) circuit, the ITP circuit comprising: (a) a first channel including a spaced-apart first capillary barrier and a second capillary barrier; and (b) a first loading reservoir in fluid communication with the first channel via a first orifice in the first channel, wherein the first orifice is positioned between the first capillary barrier and the second capillary barrier to allow liquid entering the first channel via the first orifice to flow along the first channel in one direction and stagnate at the first capillary barrier, and to flow along the first channel in another direction and stagnate at the second capillary barrier.

[0012] In some embodiments of the aspects provided herein, the liquid entering the first channel via the first orifice flows along a path to a first capillary barrier longer than the width of the first channel. In some embodiments of the aspects provided herein, the liquid entering the first channel via the first orifice flows along a path to a second capillary barrier longer than the width of the first channel. In some embodiments of the aspects provided herein, the liquid entering the first channel via the first orifice flows such that the meniscus of the first liquid stagnates at either the first or second capillary barrier. In some embodiments of the aspects provided herein, the first capillary barrier is configured and arranged to be breached by the liquid when a first burst pressure is applied to the one or more branched fluid loops, and the second capillary barrier is configured and arranged to be breached by the liquid when a second burst pressure is applied to the one or more branched fluid loops. In some embodiments of the aspects provided herein, the first burst pressure and the second burst pressure are substantially equal. In some embodiments of the aspects provided herein, the first burst pressure is higher 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 capillary barriers. In some embodiments provided herein, the ITP loop includes a second channel in fluid communication with the first channel, and the first capillary barrier is configured and arranged to impede the flow of the second liquid as it flows along the second channel, such that a liquid-liquid interface is formed at the first capillary barrier between the first and second liquids. In some embodiments provided herein, one or both of the first and second capillary barriers are plateau capillary barriers. In some embodiments provided herein, the plateau capillary barrier is configured and arranged such that an air gap is formed between the first and second liquids after the first liquid impedes at the plateau capillary barrier and after the second liquid flows in another direction toward the plateau capillary barrier and impedes relative to the first liquid at the plateau capillary barrier. In some embodiments provided herein, one or both of the first and second capillary barriers include a platform. In some embodiments provided herein, one or both of the first and second capillary barriers include a ramp without a platform. In some embodiments provided herein, the first capillary barrier is a cliff capillary barrier, and the second capillary barrier is a platform capillary barrier.In some embodiments provided herein, the at least one ITP branch further includes a third capillary barrier, which is a platform capillary barrier. In some embodiments provided herein, the first minimum pressure is at least twice the second minimum pressure. In some embodiments provided herein, the fluid device further includes a substrate having a first surface and a second surface, wherein the first surface includes a plurality of reservoirs containing the first loading reservoir, and the second surface includes a plurality of channels containing the first channel, wherein the plurality of reservoirs communicate with the plurality of channels via through-holes in the substrate. In some embodiments provided herein, the ITP loop further includes a second loading reservoir and a second channel, wherein the second loading reservoir is in fluid communication with the second channel via a second orifice, and the second channel includes a third capillary barrier, wherein the third capillary barrier is configured and arranged to allow flow along the second channel by capillary forces at which a meniscus of liquid stagnates at the third capillary barrier. In some embodiments provided herein, the second channel is adjacent to the second capillary barrier within the first channel, and the second capillary barrier is configured and arranged to allow flow along the second channel by capillary forces at which a meniscus of liquid stagnates at the second capillary barrier. In some embodiments provided herein, the ITP loop further includes a third loading reservoir fluidly connected to a third channel via a third orifice, wherein the third channel is fluidly connected to the second reservoir, and wherein the third channel includes a fourth capillary barrier positioned between the second orifice and the third orifice. In some embodiments provided herein, the first channel or the first loading reservoir contains a sample buffer. In some embodiments provided herein, the second channel or the second loading reservoir contains a first leading electrolyte buffer. In some embodiments provided herein, the third channel or the third loading reservoir contains a second leading electrolyte buffer. In some embodiments provided herein, the fluidic device further includes a fourth channel or reservoir fluidly in communication with the first channel and adjacent to the first capillary barrier. In some embodiments provided herein, the fourth channel or loading reservoir contains a trailing electrolyte buffer. In some embodiments provided herein, the ITP loop includes an elution channel connected to a first elution reservoir at an elution connection. In some embodiments provided herein, the elution channel, the first elution reservoir, or both contain a first elution buffer. In some embodiments provided herein, the first orifice, the second orifice, the third orifice, the elution connector, or combinations thereof are through holes.In some embodiments provided herein, the ITP loop includes a second elution reservoir separated from the first elution reservoir via the elution channel, wherein the elution channel includes a fifth capillary barrier. In some embodiments provided herein, any combination of the third, fourth, or fifth capillary barrier is a platform capillary barrier. In some embodiments provided herein, the second elution reservoir contains a second elution buffer with an ion concentration higher than the first elution buffer. In some embodiments provided herein, the ITP loop includes a first lead electrolyte buffer reservoir connected to a second lead electrolyte buffer reservoir via a buffer channel, wherein the buffer channel contains a first and a second lead electrolyte buffer that meet at the interface where the platform capillary barrier is located. In some embodiments provided herein, any combination of the first, second, third, fourth, or fifth capillary barriers is adjacent to an air channel including a constrictor. In some embodiments provided herein, the fluid device further includes at least two additional ITP loops, each ITP loop including a first loading reservoir and a first channel, wherein the first loading reservoir is in fluid communication with the first channel via a first orifice, and the first channel includes a first capillary barrier and a second capillary barrier spaced apart and positioned on either side of the first orifice to allow liquid to enter the first channel via the first orifice, to flow along the first channel in one direction and stagnate at the first capillary barrier, and to flow along the first channel in another direction and stagnate at the second capillary barrier. In some embodiments provided herein, the fluid device further includes at least five additional ITP loops, each ITP loop including a first loading reservoir and a first channel, wherein the first loading reservoir is in fluid communication with the first channel via a first orifice, and the first channel includes a first capillary barrier and a second capillary barrier spaced apart and positioned on either side of the first orifice to allow liquid to enter the first channel via the first orifice, to flow along the first channel in one direction and stagnate at the first capillary barrier, and to flow along the first channel in another direction and stagnate at the second capillary barrier. In some embodiments provided herein, the sample reservoir is connected to the sample channel via a through-hole. In some embodiments provided herein, the sample reservoir is closed by a removable material. In some embodiments provided herein, the removable material is a thin film. In some embodiments provided herein, the removable material is a heat-sealing material or an adhesive material. In some embodiments provided herein, the removable material is a film comprising a plastic or polymer.In some embodiments provided herein, the fluid device further includes one or more pneumatic channels that open at one or more pneumatic ports and communicate with each of the capillary barriers. In some embodiments provided herein, the fluid device further includes: (a) a substrate having a first surface and a second surface, wherein the first surface includes a plurality of reservoirs containing the first loading reservoir, and the second surface includes a plurality of channels containing the first channel, wherein the plurality of reservoirs communicate with the plurality of channels via through-holes in the substrate; (b) a material layer covering the second surface to form closed channels; and (c) a cap covering at least a portion of the first surface and including through-holes communicating with ports in the first surface via gaskets. In some embodiments provided herein, the first surface further includes the one or more pneumatic ports. In some embodiments provided herein, the one or more pneumatic ports have a head height shorter than the first loading reservoir. In some embodiments provided herein, the one or more pneumatic ports have a head height shorter than at least one of the plurality of reservoirs. In some embodiments provided herein, the capping layer is attached to the second surface by solvothermal bonding, pressure bonding, adhesive bonding, laser welding, or a combination thereof. In some embodiments provided herein, the cap further comprises a porous, breathable, and hydrophobic material positioned between through-holes in the port. In some embodiments provided herein, the first channel is a sample channel with a depth of less than 2 mm. In some embodiments provided herein, the first channel is a sample channel having a depth greater than about 10 μm. In some embodiments provided herein, the sample channel has a depth of about 400 μm to about 1.2 mm. In some embodiments provided herein, the second channel is a lead electrolyte buffer channel with a depth of less than about 1 mm. In some embodiments provided herein, the lead electrolyte buffer channel has a depth of about 10 μm to about 600 μm. In some embodiments provided herein, the elution channel has a depth of less than about 1 mm. In some embodiments provided herein, the elution channel has a depth of about 10 μm to about 600 μm. In some embodiments provided herein, the first channel, the second channel, or the elution channel, or a combination thereof, has a depth greater than about 40 μm or greater than about 10 μm. In some embodiments provided herein, the sample channel has a volume of about 10 μL to about 1 ml. In some embodiments provided herein, the sample channel, the lead electrolyte buffer channel, the elution channel, 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 cross-section in the region of the at least one reservoir adjacent to the substrate, and (b) a cylindrical through-hole or orifice penetrating the substrate. In some embodiments of the aspects provided herein, the fluid device includes at least one loading reservoir comprising: (a) an inlet channel for ambient air at one end and (b) an orifice penetrating the substrate at the other end of the loading reservoir, wherein the at least one loading reservoir has a frustoconical shape, a wider region of which is positioned at the inlet channel for ambient air, and a narrower region of which is positioned at the orifice penetrating the substrate. In some embodiments of the aspects provided herein, the frustoconical shape includes a guide wall positioned at an angle ranging from about 60 degrees to about 90 degrees relative to the surface of the substrate. In some embodiments of the aspects provided herein, the substrate includes a pneumatic port configured to have a height or depth that minimizes sample loss. In some embodiments of the aspects provided herein, the pneumatic port has a height relative to the surface of the substrate that is shorter than the height of the sample loading reservoir. In some embodiments provided herein, the pneumatic port on the substrate is inserted into the surface of the first surface of the substrate at a depth of about 1 μm to about 1 mm, or protrudes from the surface of the first surface of the substrate at a height of about 0 μm to about 2 mm. In some embodiments provided herein, the pneumatic port on the substrate is inserted into the surface of the first surface of the substrate at a depth of about 1 μm to about 500 μm, or protrudes from the surface of the first surface of the substrate at a height of about 0 μm to about 1 mm. In some embodiments provided herein, the first loading reservoir is a sample loading reservoir, the second loading reservoir is a lead electrolyte buffer reservoir, the third loading reservoir is a second lead electrolyte buffer reservoir, the fourth loading reservoir is a follower electrolyte buffer reservoir, the fifth loading reservoir is an elution reservoir buffer, and the sixth loading reservoir is an elution buffer high reservoir.

[0013] One aspect of this disclosure provides a method for loading the fluid device, including loading a buffer solution into a first loading reservoir, a second loading reservoir, a third loading reservoir, a fourth loading reservoir, a fifth loading reservoir, or a sixth loading reservoir.

[0014] One aspect of this disclosure provides a method for loading the fluid device, including loading a buffer solution into a first channel, a second channel, a third channel, a fourth channel, a fifth channel, or a sixth channel.

[0015] In some embodiments provided herein, the fluid device includes a first channel comprising a platform capillary barrier adjacent to a second channel, and the loading of the buffer solution includes loading a first buffer solution into the first channel or reservoir and loading a second buffer solution into the second channel or reservoir. In some embodiments provided herein, the method further includes applying a first positive pneumatic pressure or a first negative pneumatic pressure to the fluid device such that the first and second buffer solutions stagnate at the base of a ramp within the platform capillary barrier. In some embodiments provided herein, applying the first positive or first negative pneumatic pressure includes increasing or decreasing the first positive or first negative pressure in a fixed increment. In some embodiments provided herein, the method further includes applying a second positive or second negative pneumatic pressure to the fluid device such that the first and second buffer solutions flow along a ramp on either side of the platform capillary barrier. In some embodiments provided herein, applying the second positive or second negative pneumatic pressure includes increasing or decreasing the second positive or second negative pressure in a fixed increment. In some embodiments provided herein, the first and second buffer solutions stagnate at the platform of the platform capillary barrier and have an air gap between them, the air gap being located above or below the platform of the platform capillary barrier. In some embodiments provided herein, the method further includes applying a third positive or negative pneumatic pressure to the fluid 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 platform of the platform capillary barrier.

[0016] One aspect of this disclosure provides a fluid device comprising a fluid channel and a capillary barrier disposed in the fluid channel, the capillary barrier restricting the flow of liquid in the fluid channel, wherein the capillary barrier comprises: (a) a ramp projecting from a surface of the fluid channel at a first angle; (b) a plateau region; and (c) a cliff region extending from the plateau region to the surface of the fluid channel, wherein the cliff region intersects the surface at a second angle substantially steeper than the first angle.

[0017] In some embodiments provided herein, the second angle is steeper than the first angle by at least about 10 degrees, at least about 15 degrees, or at least about 20 degrees. In some embodiments provided herein, the ramp descends or slopes along the length of the fluid channel. In some embodiments provided herein, the first angle is less than 60 degrees. In some embodiments provided herein, the second angle is greater than 60 degrees. In some embodiments provided herein, the platform region is substantially parallel to the surface of the fluid channel. In some embodiments provided herein, the platform region is inclined relative to the surface of the fluid channel by no more than about 10 degrees. In some embodiments provided herein, any combination of the ramp, platform, or cliff regions has a substantially flat surface. In some embodiments provided herein, any combination of the ramp, platform, or cliff regions has a curved surface. In some embodiments provided herein, any combination of the ramp, platform, or cliff regions has a surface including one or more grooves, ridges, indentations, steps, etchings, or protrusions. In some embodiments provided herein, any combination of the ramp, platform, or cliff regions has a surface including areas with faces having different angles. In some embodiments provided herein, the width of the ramp, platform, or cliff region substantially occupies the width of the fluid channel.

[0018] One aspect of this disclosure provides a fluid device comprising a fluid channel and a capillary barrier disposed in the fluid channel, the capillary barrier restricting the flow of liquid in the fluid channel, wherein the capillary barrier includes: (a) a first ramp projecting from a surface of the fluid channel at a first angle of less than 80 degrees; (b) a plateau region; and (c) a second ramp extending from the plateau region to the surface of the fluid channel, wherein the second ramp intersects the surface at a second angle of less than 80 degrees.

[0019] In some embodiments provided herein, the first angle and the second angle are the same or substantially the same. In some embodiments provided herein, the first angle and the second angle are different. In some embodiments provided herein, any combination of the first ramp, the second ramp, or the platform region has a surface including one or more grooves, ridges, indentations, steps, etched surfaces, or protrusions.

[0020] One aspect of this disclosure provides a fluid device comprising a capillary barrier, the capillary barrier: (a) including a cross-section having a trapezoidal shape; (b) projecting from an inner surface of the fluid channel; (c) having a platform surface substantially parallel to the inner surface of the fluid channel; (d) having a ramp surface connecting the platform surface to the inner surface of the fluid channel, wherein the ramp surface slopes or slopes along the length of the fluid channel; and (e) a meniscus configured and arranged to stagnate and position liquid flowing along the length of the fluid channel.

[0021] In some embodiments provided herein, the capillary barrier extends substantially across the width of the fluid channel. In some embodiments provided herein, the capillary barrier is also configured and arranged to create a liquid-liquid interface. In some embodiments provided herein, the trapezoidal shape is an isosceles trapezoid. In some embodiments provided herein, the trapezoidal shape is a right-angled trapezoid comprising two substantially right angles. In some embodiments provided herein, the trapezoidal shape is a scalene trapezoid.

[0022] In some embodiments provided herein, the capillary barrier is a "plateau capillary barrier." In some embodiments provided herein, the capillary barrier is a "cliff capillary barrier." In some embodiments provided herein, the fluid device includes a cliff capillary barrier and a plateau capillary barrier in the same fluid loop. In some embodiments provided herein, the fluid device further includes a sample channel that includes a cliff capillary barrier. In some embodiments provided herein, the fluid device further includes a plateau capillary barrier located between buffer channels.

[0023] One aspect of this disclosure provides an isotachymeter electrophoresis (ITP) system comprising: (a) an interface configured for engaging a fluid device, wherein the fluid device includes one or more branch fluid loops, each branch fluid loop including a plurality of loading reservoirs, the plurality of loading reservoirs including a trailing electrolyte reservoir, a first leading electrolyte reservoir, and a first elution buffer reservoir, and wherein the interface includes: (i) a pneumatic manifold including a plurality of manifold pneumatic channels opening to one or more manifold ports and communicating with a positive or negative pneumatic pressure source, each manifold port being configured to engage one or more pneumatic ports of the fluid device when the fluid device is engaged with the interface; and (ii) a plurality of electrodes, each electrode communicating with a voltage or current source, including a first electrode, a second electrode, and a third electrode, wherein the plurality of electrodes are configured to be positioned, respectively, in the trailing electrolyte reservoir, the first leading electrolyte reservoir, and the first elution buffer reservoir when the fluid device is engaged with the interface; (b) a positive or negative pneumatic pressure source communicating with the pneumatic manifold; and (c) a voltage or current source communicating with the electrodes.

[0024] In some embodiments provided herein, the isotachophoresis system includes a motor to engage an interface with a fluid device. In some embodiments provided herein, the fluid device engages with the interface. In some embodiments provided herein, the pneumatic manifold further includes a valve for controlling pneumatic pressure on a pneumatic passage leading to at least one of the branch fluid loops. In some embodiments provided herein, the isotachophoresis system further includes: (d) a ridge having a long, narrow end, a heating element configured to heat the end, and an actuator configured to press the ridge end against the fluid device engaged with the interface to close multiple fluid channels in the microfluidic device. In some embodiments provided herein, the system is configured such that the multiple fluid channels can be closed using a heat-sealable material, PCR film, paraffin film, plastic wrap, adhesive layer, or a material not secured by a seal.

[0025] In some embodiments provided herein, the system is configured such that multiple fluid passages within the fluid device can be closed by a carrier block. In some embodiments provided herein, the system is configured such that multiple fluid passages within the fluid device can be closed by a mechanical actuator block having a rubber sealing member.

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

[0027] In some embodiments of the aspects provided herein, the display shows a measure of light detected by a light sensor. In some embodiments of the aspects provided herein, the display shows voltage or current on a fluid loop. 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 comprising one or more light sources configured to direct light to a fluid channel of the fluid loop and one or more light sensors to detect light emitted from the fluid channel of the fluid loop. In some embodiments of the aspects provided herein, the interface further includes one or more alignment marks to align the fluid device in a particular orientation. In some embodiments of the aspects provided herein, the system further includes software that regulates the electrodes in response to temperature, current, or voltage. In some embodiments of the aspects provided herein, the fluid device further includes multiple branch fluid loops, each branch fluid loop including independent circuitry. In some embodiments of the aspects provided herein, each of the branch fluid loops is coupled to the same voltage or current source or different voltage or current sources.

[0028] One aspect of this disclosure provides a method for creating a fluid loop, comprising: (a) providing a fluid device, wherein the fluid device includes at least one branched fluid loop, the branched fluid loop including a trailing electrolyte buffer reservoir, a first channel, a first leading electrolyte buffer reservoir, a sample loading reservoir, a second leading electrolyte buffer reservoir, and a first elution buffer reservoir, all of which are in fluid communication with each other, wherein: (i) the trailing electrolyte buffer reservoir includes a trailing electrolyte buffer; (ii) the first leading electrolyte buffer reservoir includes a first leading electrolyte buffer; (iii) the second leading electrolyte buffer reservoir includes a second electrolyte buffer different from the first electrolyte buffer; and (iv) the first elution buffer reservoir includes a first elution buffer. (a) flushing; (b) applying pneumatic pressure to the trailing electrolyte buffer reservoir and the leading electrolyte buffer reservoir such that the trailing electrolyte buffer and the leading electrolyte buffer each enter and remain in the first channel, with 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 channel; and (d) applying pneumatic 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 loop substantially simultaneously.

[0029] In some embodiments provided herein, the pneumatic pressure is a positive or negative pneumatic pressure. In some embodiments provided herein, the applied pneumatic pressure in operation (b) causes the trailing electrolyte buffer to stagnate at a first capillary barrier within the first channel, and the leading electrolyte buffer to stagnate at a second capillary barrier within the first channel. In some embodiments provided herein, the applied pneumatic pressure in operation (d) causes the second leading electrolyte buffer to stagnate at a third capillary barrier within the fluid loop, and the first elution buffer to stagnate at a fourth capillary barrier within the fluid channel.

[0030] In some embodiments provided herein, the first capillary barrier and the second capillary barrier are cliff capillary barriers or slope capillary barriers. In some embodiments provided herein, the third capillary barrier and the fourth capillary barrier are platform capillary barriers. In some embodiments provided herein, the third capillary barrier and the fourth capillary barrier each have a burst pressure lower than the burst pressure of the first capillary barrier or the second capillary barrier. In some embodiments provided herein, the sample includes a wetting agent.

[0031] One aspect of this disclosure provides a fluid apparatus comprising one or more branched fluid loops, each of said branched fluid loops comprising an isotachyphoresis (“ITP”) branch and an elution branch in communication with said ITP branch, wherein: (a) said ITP branch comprises a trailing electrolyte buffer reservoir, a sample channel, a leading electrolyte buffer channel, a first leading electrolyte buffer reservoir, and a second leading electrolyte buffer reservoir, all of which are in communication with each other, wherein: (i) said sample channel is separated from said trailing electrolyte reservoir by a first cliff capillary barrier and from said leading electrolyte buffer channel by a second cliff capillary barrier; (ii) said leading electrolyte reservoir is separated from said second leading electrolyte reservoir by a first platform capillary barrier; and (b) said elution branch comprises an elution channel, a first elution buffer reservoir, and a second elution buffer reservoir, all of which are in communication with each other, wherein: (i) said first elution buffer reservoir is separated from said second elution buffer reservoir by a second platform capillary barrier, and (ii) said leading electrolyte buffer channel is separated from at least a portion of said elution channel by a third platform capillary barrier.

[0032] One aspect of this disclosure provides a method for creating a fluid loop, comprising: (a) Providing a fluid apparatus according to one aspect of the present invention, 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 leading electrolyte buffer; (iv) the first elution buffer reservoir contains a first elution buffer; and (v) the second elution buffer reservoir contains a second elution buffer; (b) applying negative pneumatic pressure to the first cliff capillary barrier and the second cliff capillary barrier to initiate the trailing electrolyte buffer and the first leading electrolyte buffer at the cliff capillary barrier; (c) loading a sample into the sample channel, wherein the sample contains a wetting agent sufficient to create a fluid connection between the first cliff capillary barrier and the second cliff capillary barrier; and (d) applying negative pneumatic pressure to the first platform capillary barrier, the second platform capillary barrier, and the third platform capillary barrier to create a fluid connection between the first platform capillary barrier, the second platform capillary barrier, and the third platform capillary barrier.

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

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

[0035] In some embodiments of the aspects provided herein, the method further includes adding a topper liquid to the sample reservoir. In some embodiments of the aspects provided herein, the method further includes peaking the sample with a trailing electrolyte buffer. In some embodiments of the aspects provided herein, the method further includes 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 0V to about 1500V. In some embodiments of the aspects provided herein, the method further includes applying a negative pneumatic pressure of about 0 mpsi to about 200 mpsi. In some embodiments of the aspects provided herein, the auxiliary applied negative pneumatic pressure is between about 10 mpsi and about 80 mpsi.

[0036] One aspect of this disclosure provides a fluid device comprising a fluid channel 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, wherein the capillary barrier includes: (i) a sidewall disposed on or integrated into an inner surface of the second wall and extending substantially between the first wall and the third wall; (ii) a first lateral sidewall and a second lateral sidewall respectively connected to, integrated into, or adjacent to the first wall and the third wall, wherein the first lateral sidewall and the second lateral sidewall each have a cross-section having a trapezoidal shape; (iii) a platform surface substantially parallel to the second wall and located between the second wall and the fourth wall; and (iv) a ramp connecting the second wall to the platform surface, wherein the ramp slopes or descends along the length of the fluid channel.

[0037] In some embodiments provided herein, the trapezoidal shape is an isosceles trapezoid. In some embodiments provided herein, the trapezoidal shape is a right-angled trapezoid comprising two substantially right angles. In some embodiments provided herein, the trapezoidal shape is an scalene trapezoid. In some embodiments provided herein, the capillary barrier is a "plateau capillary barrier". In some embodiments provided herein, the capillary barrier is a "cliff capillary barrier". In some embodiments provided herein, the fluid device includes a cliff capillary barrier and a plateau capillary barrier in the same fluid loop. In some embodiments provided herein, the device further includes a sample channel that includes a cliff capillary barrier. In some embodiments provided herein, the device further includes a plateau capillary barrier located between buffer channels.

[0038] One aspect of this disclosure provides a fluid device comprising a fluid channel including a capillary barrier projecting into the fluid channel from a first wall thereof, wherein the capillary barrier includes (i) two lateral sides each having a trapezoidal cross-section; (ii) a plateau side substantially parallel to the first wall of the channel; and (iii) a ramp having one edge intersecting the plateau side to form an inner obtuse angle of the capillary barrier and an opposite edge intersecting the first wall of the channel to form an inner acute angle of the capillary barrier.

[0039] In some embodiments provided herein, the capillary barrier further includes a side connecting the platform side to the first wall. In some embodiments provided herein, the side connecting the platform side to the first wall is substantially perpendicular to the first wall. In some embodiments provided herein, the side connecting the platform side to the first wall intersects the first wall at an acute angle. In some embodiments provided herein, at least one of the lateral sides is substantially parallel to or integrated into the second wall of the fluid channel.

[0040] One aspect of this disclosure provides a fluid system comprising: (a) a first isotachophoresis circuit in a microfluidic chip, comprising: (i) a first sample reservoir; (ii) a trailing electrolyte buffer reservoir containing a trailing electrolyte buffer in fluid communication with the sample reservoir; and (iii) a leading electrolyte buffer channel containing a leading electrolyte buffer in fluid communication with the sample reservoir; (b) a sensor configured to detect temperature changes in the leading electrolyte buffer channel; and (c) means configured to monitor voltage or current in the first isotachophoresis circuit and supply a constant current within the first isotachophoresis circuit.

[0041] In some embodiments provided herein, the lead electrolyte channel includes an elution channel. In some embodiments provided herein, the sensor is configured and arranged to detect temperature changes in the elution channel. In some embodiments provided herein, the fluid system further includes an elution orifice. In some embodiments provided herein, the first isotachophoresis loop further includes an elution channel containing an elution buffer.

[0042] One aspect of this disclosure provides a fluid system comprising: (a) a first isotachymeter electrophoresis circuit in a microfluidic chip, including: (i) a first sample reservoir in fluid communication with a first fluid channel; (ii) a first buffer reservoir, a second buffer reservoir, and a third buffer reservoir in fluid communication with the first fluid channel, wherein the first buffer reservoir and the second buffer reservoir are separated by a first capillary barrier; and (iii) an elution reservoir in fluid communication with the first fluid channel; (b) a sensor configured to detect temperature changes in the first fluid channel within the first isotachymeter electrophoresis region; and (c) means configured to monitor voltage or current and supply a constant current within the first isotachymeter electrophoresis circuit.

[0043] In some embodiments provided herein, the first fluid channel includes a second capillary barrier adjacent to the first sample reservoir. In some embodiments provided herein, the first capillary barrier is a plateau capillary barrier, and the second capillary barrier is a cliff capillary barrier. In some embodiments provided herein, the first capillary barrier is a cliff capillary barrier, a plateau capillary barrier, or a slope capillary barrier. In some embodiments provided herein, the first fluid channel includes a cliff capillary barrier and a narrowing downstream of the cliff barrier. In some embodiments provided herein, the system further includes a temperature sensor disposed downstream of the narrowing.

[0044] One aspect of this disclosure provides a fluid system comprising: a fluid chip including a plurality of loops, each of said loops including an elution channel in fluid communication with an elution reservoir; and a mechanical member including a ridge, said mechanical member being configured to simultaneously apply mechanical pressure to the plurality of said elution channels via said ridge to at least partially close said elution channels by plastic deformation of at least one wall of said elution channel.

[0045] In some embodiments provided herein, the system further includes a base film bonded to a substrate layer, the base film forming the wall of each of the elution channels, wherein the base film and the substrate layer each comprise a material having the same melting point. In some embodiments provided herein, each elution channel includes a bend, and wherein the ridge at least partially closes each elution channel at two locations spanning the bend. In some embodiments provided herein, the ridge completely closes the channel.

[0046] One aspect of this disclosure provides a method for retrieving an analyte from a determination, comprising: introducing the analyte into one of the loops in the fluid system of the aspect provided herein; causing the analyte to migrate to the elution channel in the one of the loops; and engaging the mechanical member to apply mechanical pressure to the plurality of elution channels via the ridge so as to at least partially close the elution channel by plastic deformation of at least one wall of the elution channel.

[0047] One aspect of this disclosure provides a fluid device comprising: a first liquid channel; a gas channel in fluid communication with the first liquid channel; a pneumatic port in fluid communication with the gas channel; and a breathable hydrophobic membrane disposed across the pneumatic port, wherein the hydrophobic membrane is not liquid-permeable and is configured to inhibit liquid from leaving the pneumatic port when a negative pressure is applied to the gas channel via the pneumatic port.

[0048] In some embodiments provided herein, the device further includes a gasket disposed above the pneumatic port. In some embodiments provided herein, the device further includes a narrowing element between the liquid and gas passages to prevent liquid from leaving the gas passages. In some embodiments provided herein, the gasket is secured in place by a cover layer including a channel communicating with the gas passage through the port. In some embodiments provided herein, the cover layer includes an interference fit configured to maintain compressive force on the gasket.

[0049] One aspect of this disclosure provides a method comprising: (a) providing a fluid loop including: (i) an elution well adjacent to an elution channel containing an elution buffer, wherein the elution channel is connected to a lead electrolyte channel containing a lead electrolyte buffer; and (ii) a capillary barrier located at an interface between the elution buffer and the lead electrolyte buffer; (b) allowing the interface between the lead electrolyte buffer and the elution buffer to flow toward the elution well; and (c) stagnating the flow at the interface between the lead electrolyte buffer and the elution buffer such that the capillary barrier is completely submerged by the lead electrolyte buffer.

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

[0051] One aspect of this disclosure provides a method comprising: (a) providing a fluid apparatus including a fluid circuit having a trailing electrolyte buffer reservoir, a sample channel, a lead electrolyte buffer channel, and an elution reservoir, all of which are in communication with each other, wherein: (i) the lead electrolyte buffer channel is fluidly connected to the elution reservoir via an orifice in the lead electrolyte buffer channel located below the elution reservoir; (ii) The trailing electrolyte buffer reservoir contains a trailing electrolyte buffer; (ii) the sample channel contains an analyte; (iii) the leading electrolyte buffer channel contains a leading electrolyte buffer; (iv) the elution reservoir contains an elution buffer; and (b) an electric current is applied to the fluid loop to move the analyte to the elution reservoir, wherein the electric current is configured and arranged to generate a first temperature at the interface between the analyte and the trailing electrolyte buffer, and a second temperature at the interface between the sample and the leading electrolyte buffer, wherein a temperature difference exists between the first temperature and the second temperature; and wherein the analyte is facilitated into the elution reservoir by the temperature difference when it reaches the orifice in the leading electrolyte buffer channel located below the elution reservoir.

[0052] In some embodiments provided herein, the method further includes removing the analyte from the elution reservoir.

[0053] One aspect of this disclosure provides a method for quantifying a nucleic acid sample, the method comprising: providing a fluid apparatus including an isotachymeter electrophoresis (ITP) channel, the ITP channel containing a nucleic acid sample, wherein the nucleic acid sample includes nucleic acids complexed with an intercalation dye; performing ITP in the fluid channel to focus the nucleic acids complexed with the intercalation dye; and, after performing ITP, quantifying the nucleic acids complexed with the intercalation dye within the channel by measuring the intensity of the intercalation dye, wherein the intercalation dye includes SYTOTM 13. or One or more of them.

[0054] In some embodiments provided herein, the dye includes SYTOTM 13. In some embodiments provided herein, the dye includes... In some embodiments provided herein, the dye includes In some embodiments provided herein, the dye includes In some embodiments provided herein, the nucleic acid complexed with the insert dye is located in a region of the ITP channel containing a lead electrolyte buffer or elution buffer. In some embodiments provided herein, the nucleic acid complexed with the insert dye comprises RNA or DNA, or a combination thereof. In some embodiments provided herein, the nucleic acid sample also includes contaminants. In some embodiments provided herein, the contaminants include proteins, cell debris, lipids, plasma membranes, small molecules, or combinations thereof. In some embodiments provided herein, performing ITP in the fluid channel results in the separation of the nucleic acid complexed with the insert dye from the contaminants.

[0055] One aspect of this disclosure provides a fluid apparatus comprising one or more branched fluid loops, wherein each of the branched fluid loops includes an isotachyphoresis (“ITP”) branch and an elution branch communicating with the ITP branch, wherein: the ITP branch includes a trailing electrolyte buffer reservoir, a sample channel, a lead electrolyte buffer channel, a first lead electrolyte buffer reservoir, and a second lead electrolyte buffer reservoir, all of which are in communication with each other; and the elution branch includes an elution channel and an elution well, the elution well including a first through-hole and a second through-hole communicating with the elution channel.

[0056] In some embodiments provided herein, the first and second through-holes are circular. In some embodiments provided herein, the first through-hole has an elliptical shape. In some embodiments provided herein, the first through-hole has a maximum dimension of less than 1.5 mm, or less than 1 mm. In some embodiments provided herein, the second through-hole has a maximum dimension of less than 1.5 mm. In some embodiments provided herein, the second through-hole has a maximum dimension of less than 1 mm. In some embodiments provided herein, the first and second through-holes are on the same vertical plane within the elution well. In some embodiments provided herein, the first and second through-holes are on different vertical planes within the elution well. In some embodiments provided herein, the first and second through-holes are aligned with the longitudinal axis of the elution channel. In some embodiments provided herein, the first through-hole is configured to constrain a pipette tip at a predetermined coupling position. In some embodiments provided herein, the first through-hole comprises a circular cross-section. In some embodiments provided herein, the first through-hole comprises an elliptical cross-section. In some embodiments provided herein, the first through-hole comprises a D-shaped cross-section. In some embodiments provided herein, the first through-hole includes a guide wall disposed at an angle ranging from about 60 degrees to about 90 degrees relative to the channel. In some embodiments provided herein, the elution orifice includes one or more vertical gates that separate the first through-hole from the second through-hole. In some embodiments provided herein, the elution orifice includes a circular cross-section. In some embodiments provided herein, the elution orifice includes an elongated cross-section.

[0057] One aspect of this disclosure provides a fluid device comprising: a first channel terminating at one end in a first through-hole; a second channel terminating at one end in a second through-hole; and a fluid reservoir defined by a wall having a height of not more than 25 mm, not more than 15 mm, not more than 10 mm, or more than 10 mm; wherein the reservoir is in fluid communication with each of the two fluid channels through the first and second through-holes, and wherein the first through-hole enters the reservoir at a position lower than the second through-hole.

[0058] In some embodiments provided herein, the first channel is in communication with a first electrode; and the second channel is in communication with a second electrode, wherein the channels and the reservoir contain a conductive fluid, and wherein a voltage applied between the first and second electrodes generates a current traveling through the reservoir. In some embodiments provided herein, the wall has a height in the range of about 8 mm to about 10 mm. In some embodiments provided herein, the first and second through-holes have areas of about 0.2 mm² to 7 mm² and about 0.2 mm² to 7 mm², respectively. In some embodiments provided herein, the first and second through-holes have areas of about 0.8 mm² to 1.5 mm² and about 1 mm² to 2.75 mm², respectively. In some embodiments provided herein, the second through-hole enters the reservoir via a platform located in the reservoir about 1 mm to about 6 mm above the point where the first through-hole enters the reservoir. In some embodiments provided herein, the volume of the reservoir between the first and second through-holes is not greater than about 2.5 ml, 1 ml, or 0.5 ml. In some embodiments provided herein, the volume of the reservoir between the first through-hole and the second through-hole is 0.1 mL.

[0059] One aspect of this disclosure provides a method comprising: providing any fluid device described herein, wherein the channel and reservoir contain a conductive fluid, and the first channel further contains an ionic analyte; applying a voltage between a first electrode and a second electrode to generate a current traveling through the reservoir; and moving the analyte into the reservoir through a first through-hole.

[0060] In some embodiments provided herein, the analyte comprises nucleic acid, such as RNA or DNA. In some embodiments provided herein, the method further includes performing isovelocity electrophoresis in a first channel to move the analyte into a reservoir.

[0061] One aspect of this disclosure provides a fluid device comprising: a fluid channel having a length and a width; a reservoir positioned above the fluid channel and fluidly connected to the fluid channel through one or more through-holes; wherein at least a portion of each through-hole extends substantially co-exists with the fluid channel over the width of the fluid channel and has a shape, wherein when the fluid channel and the reservoir contain a conductive fluid and an electric current flows through the fluid channel, at least 5%, at least 6%, at least 7%, at least 10%, or at least 20% of the electric current flows through the reservoir.

[0062] In some embodiments provided herein, the reservoir includes a through-hole. In some embodiments provided herein, the reservoir includes two through-holes arranged along the longitudinal axis of the channel. In some embodiments provided herein, the one or both through-holes have a square, rectangular, elliptical shape, or an elongated dimension in the width direction. In some embodiments provided herein, the one or both through-holes include one or both sides spanning the width of the fluid channel or the width of the reservoir, wherein each side is at least 75% linear.

[0063] One aspect of this disclosure provides a method comprising: providing any of the devices described herein, wherein the channel and the reservoir contain a conductive fluid, and the reservoir contains an ionic analyte; and passing an electric current through the channel, wherein the electric current causes at least some of the analyte to move from the reservoir into the channel.

[0064] One aspect of this disclosure provides a fluid apparatus comprising one or more branched fluid loops, wherein each of the branched fluid loops includes an isotachymetry electrophoresis (“ITP”) branch, wherein: the ITP branch includes a trailing electrolyte buffer reservoir, a sample channel, a leading electrolyte buffer channel, a first leading electrolyte buffer reservoir and a second leading electrolyte buffer reservoir, all of which are in communication with each other, and the sample reservoir includes a first through-hole communicating with the sample channel.

[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 size ranging from about 0.5 mm to about 5 mm. In some embodiments of the aspects provided herein, the first through-hole has a maximum size of about 1.5 mm. In some embodiments of the aspects provided herein, the first through-hole has a maximum size 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 ranging from 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 includes a second through-hole communicating with the sample channel. In some embodiments of the aspects provided herein, the first through-hole and the second through-hole are separated by a filler block. In some embodiments of the aspects provided herein, the filler block has a height ranging from about 0.2 mm to about 2 mm within the channel. In some embodiments of the aspects provided herein, the filler block has a height of approximately 1.2 mm within the channel. 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 size ranging from approximately 0.5 mm to approximately 5 mm. In some embodiments of the aspects provided herein, the second through-hole has a maximum size of approximately 1.5 mm. In some embodiments of the aspects provided herein, the second through-hole has a maximum size of approximately 1 mm. In some embodiments of the aspects provided herein, the second through-hole has a volume of less than approximately 15 μL. In some embodiments of the aspects provided herein, the second through-hole has a volume of approximately 7 μL. In some embodiments of the aspects provided herein, the second through-hole has a width ranging from approximately 80% to approximately 120% of the width of the sample channel. In some embodiments of the aspects provided herein, the second through-hole has a width ranging from approximately 80% to approximately 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 over the length of the sample reservoir above the top surface of the sample channel. In some embodiments provided herein, the sample reservoir has a conical shape, with its narrower portion having a diameter ranging from about 0.1 mm to about 4 mm.In some embodiments provided herein, the sample reservoir has a conical shape, with its narrower portion having a diameter ranging from about 1 mm to about 4 mm. In some embodiments provided herein, the sample reservoir has an elliptical shape.

[0066] One aspect of this disclosure provides an apparatus for performing vertical isokinetic electrophoresis, the apparatus comprising: one or more cylindrical columns including an internal channel defined by an inner wall of the cylindrical column, each cylindrical column including: a first step including a first gel plug disposed at a first position within the internal channel and a first space disposed within the internal channel between the first gel plug and an upper end of the cylindrical column; a second step including a second gel plug disposed at a second position within the internal channel and a second space disposed within the internal channel between the first gel plug and the second gel plug, the second position being located below the first position and oriented in accordance with gravity; and a third step including a third gel plug disposed at a third position within the internal channel and a third space disposed within the internal channel between the second gel plug and the third gel plug, the third position being located below the second position and oriented in accordance with gravity.

[0067] In some embodiments of the various aspects provided herein, one or more cylindrical columns comprise a plurality of cylindrical columns arranged to conform to standard microtiter plate dimensions. In some embodiments of the various aspects provided herein, the one or more cylindrical columns have a cross-sectional column area of ​​approximately 9 mm × 9 mm. In some embodiments of the various aspects provided herein, the first space contains a trailing electrolyte buffer, the second space contains an analyte, and the third space contains a first leading electrolyte buffer. In some embodiments of the various aspects provided herein, the device further includes a fourth step comprising a fourth gel plug disposed at a fourth position within the internal channel and a fourth space within the internal channel disposed between the third gel plug and the fourth gel plug, the fourth position being below the third position and oriented in accordance with gravity. In some embodiments of the various aspects provided herein, the fourth space contains an elution buffer. In some embodiments of the various aspects provided herein, the device further includes a fifth step comprising a fifth gel plug disposed at a fifth position within the internal channel and a fifth space within the internal channel disposed between the fourth gel plug and the fifth gel plug, the fifth position being below the fourth position and oriented in accordance with gravity. In some embodiments provided herein, the fifth space contains a second leading electrolyte buffer.

[0068] One aspect of this disclosure provides a method for focusing an analyte, the method comprising: introducing the analyte into a second space of a second step above a second gel plug of any vertical or columnar ITP device described herein; and applying an electric current to the device to cause the analyte to move in a gravitational direction from the second space through a second gel plug and into the third space.

[0069] The method of claim 255 further comprises applying the current to the device to cause the analyte to move in the direction of gravity from the third space through the third gel plug and into the fourth space.

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

[0071] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a fluid device: (i) a tissue sample comprising lysed solid tissue, wherein the lysed solid tissue comprises nucleic acids and contaminants; (ii) a trailing electrolyte buffer containing a first trailing electrolyte ion having an effective migration rate less than the effective migration rate of the nucleic acid; and (iii) a leading electrolyte buffer containing a first leading electrolyte ion having a second effective migration rate greater than the effective migration rate of the nucleic acid; and (b) applying an electric field within the fluid device to perform isovelocity electrophoresis with the first trailing electrolyte ion, the nucleic acid, and the first leading electrolyte ion, 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 trailing 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 trailing electrolyte buffer, and the leading electrolyte buffer are loaded into a first region of the microfluidic chip. Some embodiments of one aspect of the present invention may further include performing at least one sample preparation procedure selected from the group consisting of the first region of the microfluidic chip on the tissue sample: (1) removing embedding material, (2) disrupting tissue, (3) lysing cells, (4) uncrosslinking the nucleic acids, (5) digesting proteins, and (6) digesting the nucleic acids. In some embodiments of the aspects provided herein, the isovelocity electrophoresis is performed in a second region of the microfluidic chip, wherein the second region is separated from and fluidly connected to the first region. 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 contains 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 tissue (FFPE). In some embodiments of the aspects provided herein, the lysed solid tissue comprises urea or thiourea. In some embodiments of the aspects provided herein, the lysed solid tissue further includes disrupting cell-cell 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 cross-linked nucleic acids, embedding materials, tissue fragments, fixation chemicals, proteins, inhibitors, and combinations thereof. In some embodiments of the aspects provided herein, the contaminant comprises cross-linked nucleic acids. In some embodiments of the aspects provided herein, the tissue sample is combined with the trailing electrolyte buffer prior to loading. In some embodiments of the aspects provided herein, the tissue sample is combined with the leading electrolyte buffer prior to 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 lysed in the leading electrolyte buffer prior to the loading of the tissue sample. In some embodiments provided herein, the solid tissue is lysed in the trailing electrolyte buffer prior to loading the tissue sample.In some embodiments of the aspects provided herein, the sample preparation procedure includes removing embedding material by incubating the tissue sample in the fluid device at a temperature of at least about 37°C for a duration 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 duration is from about 1 minute to about 120 minutes. In some embodiments of the aspects provided herein, the sample preparation procedure includes destroying tissue or lysing cells by applying mechanical stress to the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure includes destroying 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 includes destroying tissue or lysing cells by contacting the tissue sample with a solution having a pH of at least 10 or by digesting the tissue sample with protein hydrolysis. In some embodiments of the aspects provided herein, the protein hydrolysis is performed at a temperature greater than about 25°C. In some embodiments of the aspects provided herein, the sample preparation procedure includes disrupting tissue or lysing cells by applying at least one surfactant to the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure includes disrupting tissue or lysing cells by applying a solution containing urea to the tissue or cell sample. In some embodiments of the aspects provided herein, the solution further contains thiourea. In some embodiments of the aspects provided herein, the concentration of urea in the solution is in the range of about 4 M to about 9 M, and the concentration of 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 urea in the solution is about 6.5 M to about 7.5 M, and the concentration of thiourea in the solution is about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the sample preparation procedure includes uncrosslinking the nucleic acid by digesting the crosslinked protein with proteinase K. In some embodiments of the aspects provided herein, the sample preparation procedure includes digesting the nucleic acid with a DNase or RNase. Some embodiments of the aspects provided herein further include eluting an output solution containing the purified nucleic acid from an outlet reservoir of the fluid device. In some embodiments provided herein, the concentration of the purified nucleic acid in the output solution is at least about twice as high as the concentration of the nucleic acid in the tissue sample.In some embodiments of the aspects provided herein, the purified nucleic acid in the tissue sample and the output solution comprises cross-linked nucleic acid, and the concentration of the cross-linked nucleic acid in the output solution is at least about one-half lower than the concentration of the cross-linked 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 at least one-half lower than the concentration of the contaminant in the tissue sample. In some embodiments of the aspects provided herein, the first trailing electrolyte ion comprises hexanoic 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 trailing electrolyte buffer comprises a second trailing electrolyte ion having a different effective mobility than the first trailing electrolyte ion. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) or MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the first trailing electrolyte ion comprises MOPS. In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a second trailing electrolyte ion having a second effective mobility, wherein the magnitude of the second effective mobility is substantially the same as or lower than the 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 region of the microfluidic chip, and performing an enzymatic reaction on the tissue sample in the second region of the microfluidic chip. In some embodiments provided herein, the first sample processing procedure includes removing embedding material, disrupting tissue or cell lysis, and the enzymatic reaction includes decrosslinking the nucleic acid, digesting proteins, or digesting nucleic acids. In some embodiments provided herein, the first region and the second region are each heated to a temperature above 37°C.In some embodiments provided herein, the first region is heated to a temperature of about 60°C to 100°C during the first sample processing procedure, and the second region is heated to a temperature of 40°C to 60°C.

[0073] One aspect of this disclosure provides a method for simultaneously purifying nucleic acids from at least two different samples, the method comprising: (a) loading a first channel of a microfluidic chip with: (i) a first sample containing a first nucleic acid and a first contaminant; (ii) a first tailing electrolyte buffer containing a first tailing ion, wherein the effective mobility of the first tailing ion is less than the effective mobility of the first nucleic acid; and (iii) The microfluidic chip comprises: (a) a first leader electrolyte buffer containing a first leader ion, wherein the effective mobility of the first leader ion is greater than the effective mobility of the first nucleic acid; (b) loading the following substances into a second channel of the microfluidic chip: (i) a second sample containing a second nucleic acid and a second contaminant; (ii) a second tailing electrolyte buffer containing a second tailing ion, wherein the effective mobility of the second tailing ion is less than the effective mobility of the second nucleic acid; and (iii) a second leader electrolyte buffer containing a second leader ion, wherein the effective mobility of the second leader ion is greater than the effective mobility of the second nucleic acid; and (c) applying a first electric field within the microfluidic chip to perform isotachymetric electrophoresis in the first channel using the first tailing ion, the first nucleic acid, and the first leader ion, and applying a second electric field to perform isotachymetric electrophoresis in the second channel using the second tailing ion, the second nucleic acid, and the second leader ion, thereby simultaneously purifying the first nucleic acid from the first contaminant and purifying the second nucleic acid from the second contaminant.

[0074] In some embodiments provided herein, the first sample and the second sample are different sample types. In some embodiments provided herein, the first nucleic acid and the second nucleic acid are nucleic acids of different types or lengths. In some embodiments provided herein, the first trailing electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or tissue disruptor. In some embodiments provided herein, the lysing agent or the tissue disruptor comprises one or more reagents selected from solutions with a pH greater than about 12, proteases, urea, thiourea, and surfactants. In some embodiments provided herein, the first sample comprises lysed solid tissue. In some embodiments provided herein, the second sample comprises lysed cells. In some embodiments provided herein, the first sample does not contact the second sample during the isotachophoresis process. Some embodiments of the aspects provided herein further include loading the following substances into a third channel of the microfluidic chip: (i) a third sample containing a third nucleic acid and a third contaminant; (ii) a third tailing electrolyte buffer containing a third tailing ion, wherein the effective mobility of the third tailing ion is less than the effective mobility of the third nucleic acid; and (iii) a third leading electrolyte buffer containing a third leading ion, wherein the effective mobility of the third leading ion is greater than the effective mobility of the third nucleic acid, wherein an electric field is applied within the microfluidic chip to perform isovelocity electrophoresis in the third channel using the third tailing ion, the third nucleic acid, and the third leading ion, 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 by a single electrode pair. In some embodiments of the aspects provided herein, the first and second electric fields are generated by different electrode pairs. In some embodiments of the aspects provided herein, the first and second channels are coupled to independent sensors. In some embodiments provided herein, feedback from the independent sensors is used to independently control the first and second electric fields. In some embodiments provided herein, the independent sensors sense voltage, and the feedback is used to control the current (or resistance) within the first and second channels. In some embodiments provided herein, the nucleic acid comprises DNA. In some embodiments provided herein, the nucleic acid comprises RNA.

[0075] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances onto a fluid device: (i) a sample containing fixed cells, fixed tissue, or embedded tissue, wherein the sample contains nucleic acids; (ii) a tail electrolyte buffer containing a tail electrolyte having a lower effective migration rate than the nucleic acids; and (iii) a lead electrolyte buffer containing a lead electrolyte having a higher effective migration rate than the nucleic acids; and (b) applying an electric field to the fluid device to perform isovelocity electrophoresis using the tail electrolyte, the nucleic acids, and the lead electrolyte, thereby purifying the nucleic acids from contaminants in the sample.

[0076] In some embodiments of the aspects provided herein, the contaminant is selected from cross-linked nucleic acids, embedding materials, immobilization chemicals, 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 tissue embedded in paraffin. 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 tissue biopsy. In some embodiments of the aspects provided herein, the sample is an autopsied formalin-fixed paraffin-embedded (FFPE) sample. Some embodiments of the aspects provided herein further include comparing the characteristics of the nucleic acid with nucleic acids from other samples, wherein said characteristics are expression levels, nucleic acid sequence, molecular weight, nucleic acid integrity, nucleic acid strand type (e.g., double-stranded vs. single-stranded), 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 trailing electrolyte buffer has a pH greater than about 7. Some embodiments of the aspects provided herein further include incubating the tissue sample in the fluid device at a temperature of at least about 37°C for at least about 1 minute before 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 duration is from about 1 minute to about 120 minutes. In some embodiments of the aspects provided herein, the leading electrolyte buffer contains proteinase K. Some embodiments of the aspects provided herein further include removing protein cross-links from the nucleic acid using the proteinase K. Some embodiments of the aspects provided herein further include removing protein cross-links from the nucleic acid by heating after applying the electric field. Some embodiments of the aspects provided herein further include eluting an output solution containing the purified nucleic acid from an outlet reservoir of the fluid device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about twice the concentration of the nucleic acid in the tissue sample. In some embodiments provided herein, the concentration of the cross-linked nucleic acid in the output solution is at least about one-half lower than the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments provided herein, the output solution has a volume equal to or less than about 50 μL. In some embodiments 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 greater than 25 μL. In some embodiments of the aspects provided herein, the trailing electrolyte has a higher effective migration rate than the contaminant. In some embodiments of the aspects provided herein, the trailing electrolyte comprises (i) a first ion having a higher effective migration rate than the contaminant, and (ii) a second ion having an effective migration rate substantially the same as or lower than that of the contaminant. In some embodiments of the aspects provided herein, the isotachophoresis quenches the pH of the tissue sample to approximately 7.5. Some embodiments of the aspects provided herein further include dewaxing the sample prior to loading. Some embodiments of the aspects provided herein further include detecting the concentration of the nucleic acid. In some embodiments of the aspects provided herein, the concentration is less than or equal to about 1 picogram per microliter (pg / μL).

[0077] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a fluid device: (i) a tissue sample comprising lysed solid tissue and nucleic acids; (ii) a trailing electrolyte buffer containing a trailing electrolyte ion having a first effective mobility, wherein the magnitude of the first effective mobility is lower than the magnitude of the effective mobility of the nucleic acid; (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir containing a first leading electrolyte ion having a second effective mobility, wherein the magnitude of the second effective mobility is greater than the magnitude of the effective mobility of the nucleic acid; and (iv) a second leading electrolyte buffer in a second leading electrolyte reservoir containing a second leading electrolyte ion having a third effective mobility, wherein the magnitude of the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acid; wherein the first leading electrolyte buffer is different from the second leading electrolyte buffer. (b) Purifying the nucleic acid from the contaminants in the tissue sample by performing isotachophoresis for the first time using the trailing electrolyte ion, the nucleic acid, and the first leading electrolyte ion; and (c) Performing isotachophoresis for the second time using the trailing electrolyte ion, the nucleic acid, and the second leading electrolyte ion.

[0078] In some embodiments provided herein, the second isotachophoresis includes changing the applied current from the first channel to the second channel. In some embodiments 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 provided herein, the magnitude of the second effective mobility is greater than the magnitude of the third effective mobility. In some embodiments provided herein, the first leading electrolyte ion is different from the second leading electrolyte ion. In some embodiments 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 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, and 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 removing the nucleic acid from the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the first isotachyphoresis and the second isotachyphoresis are performed by applying a single electric field. In some embodiments of the aspects provided herein, the first isotachyphoresis and the second isotachyphoresis 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 contains 50 mM Tris HCl.

[0079] One aspect of this disclosure provides a microfluidic device comprising: (a) a first isotachymeter electrophoresis region in a microfluidic chip, the first isotachymeter electrophoresis region comprising: (i) a first sample reservoir in fluid communication with a first fluid channel, (ii) a first buffer reservoir in fluid communication with the first fluid channel, and (iii) a second buffer reservoir in fluid communication with the first channel; and (b) a second isotachymeter electrophoresis region in the microfluidic chip, the second isotachymeter electrophoresis region comprising: (i) a second sample reservoir in fluid communication with a second fluid channel, (ii) a third buffer reservoir in fluid communication with the second fluid channel, and (iii) a fourth buffer reservoir in fluid communication with the second channel, wherein the first isotachymeter electrophoresis region is not in fluid communication with the second isotachymeter electrophoresis region, and wherein the microfluidic device is configured to independently control a first circuit for applying current to the first isotachymeter electrophoresis region and a second circuit for applying current to the second isotachymeter electrophoresis region.

[0080] In some embodiments of the aspects provided herein, the leakage rate between the first and second isotachophoretic electrophoresis zones is less than 1 μl / hour. In some embodiments of the aspects provided herein, the current leakage between the first and second isotachophoretic electrophoresis zones is less than 1 μA. In some embodiments of the aspects provided herein, the impedance is greater than 1 megohm. In some embodiments of the aspects provided herein, the first fluid channel contains a liquid volume greater than 100 μl. In some embodiments of the aspects provided herein, the first fluid channel and the second fluid channel are separated by a distance at least 4 / 5 smaller than the width of the first channel. In some embodiments of the aspects provided herein, the microfluidic device is configured to simultaneously control the first circuit with the second circuit. Some embodiments of the aspects provided herein further include an elution reservoir in fluid communication with the first channel, wherein a temperature sensor is located within 5 mm of the elution reservoir.

[0081] One aspect of this disclosure provides a method comprising: (a) providing an electrofluidic apparatus including a sample input reservoir in fluid communication with a channel; (b) loading a sample volume into the sample input reservoir; (c) moving at least 50% of the sample volume from the sample input reservoir to the channel without adding additional volume to the sample input reservoir; and (d) applying an ion current through the channel.

[0082] In some embodiments of the aspects provided herein, the movement is carried out by means of gravity. In some embodiments of the aspects provided herein, the ion current substantially does not pass through the channel. In some embodiments of the aspects provided herein, the at least 50% of the sample volume includes at least 80% of the sample volume. In some embodiments of the aspects provided herein, the sample volume contains nucleic acids. In some embodiments of the aspects provided herein, the sample volume contains tissue samples or formalin-fixed paraffin-embedded (FFPE) samples. In some embodiments of the aspects provided herein, the application of the ion current includes isovelocity electrophoresis. In some embodiments of the aspects provided herein, the total sample volume loaded into the sample input reservoir is less than or equal to the internal volume of the input reservoir. In some embodiments of the aspects provided herein, the sample input reservoir includes a top region connected to a bottom region via a conical region, wherein the top region has a first diameter and the bottom region has a second diameter, wherein the first diameter is at least twice as long as the second diameter to facilitate the movement of at least 50% of the sample volume from the sample input reservoir to the channel. In some embodiments of the aspects provided herein, the sample volume is at least 25 μl. In some embodiments of the various aspects provided herein, the sample volume is at least 50 μl. In some embodiments of the various aspects provided herein, the sample volume is at least 100 μl.

[0083] One aspect of this disclosure provides a microfluidic chip comprising: a first sample input reservoir, wherein the first sample input reservoir includes a top region connected to a bottom region via a conical region, wherein the top region has a first internal hydraulic diameter and the bottom region has a second internal hydraulic diameter, wherein the first internal hydraulic diameter is at least twice as long as the second internal hydraulic diameter, and wherein the first sample input reservoir is in fluid communication with a first channel; a first buffer reservoir in fluid communication with the first channel, wherein the first sample reservoir is configured such that the free surface of the liquid in the first sample reservoir has a negligible buffer head height difference relative to the liquid in the first buffer reservoir; and a second buffer reservoir in fluid communication with the first channel.

[0084] In some embodiments provided herein, the first internal hydraulic diameter is in the range of about 1 mm to about 15 mm. In some embodiments provided herein, the second internal hydraulic diameter is in the range of about 0.5 mm to about 5 mm. In some embodiments provided herein, the first sample reservoir is configured to contain at least 100 μl of sample volume. In some embodiments provided herein, the microfluidic chip is configured to move at least 50% of the sample volume from the first sample reservoir to the first channel when a vacuum is applied thereto. In some embodiments provided herein, the microfluidic chip is configured to perform isovelocity electrophoresis on the sample entering the first channel.

[0085] One aspect of this disclosure provides a method for extracting nucleic acids, the method comprising: (a) exposing a biological sample containing cells or tissue to a solution containing urea or thiourea to lyse the cells or tissue within the biological sample and produce cell lysates; (b) introducing the cell lysates into an apparatus; and (c) performing isovelocity electrophoresis using the apparatus to separate nucleic acids from the cell lysates.

[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 comprises urea and thiourea. In some embodiments of the aspects provided herein, the solution comprises urea and thiourea in a ratio of about 2:1. In some embodiments of the aspects provided herein, the concentration of urea in the solution is about 4 M to about 9 M, and the concentration of 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 urea in the solution is about 6.5 M to about 7.5 M, and the concentration of thiourea in the solution is about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the solution comprises a trailing electrolyte ion or a leading electrolyte ion, or both a trailing electrolyte ion and a leading electrolyte ion.

[0087] One aspect of this disclosure provides a method for purifying high molecular weight nucleic acids from tissue samples, the method comprising: (a) loading the following substances into a fluid apparatus: (i) The method comprises: (i) a cell sample containing genomic DNA and contaminants, wherein the cell sample is contacted with a lysis buffer before or after loading the cell sample into the fluid device; (ii) a trailing electrolyte buffer containing a trailing electrolyte ion having a first effective migration rate, wherein the first effective migration rate is less than the effective migration rate of the high molecular weight nucleic acid and greater than the amount of the contaminant; and (iii) a first leading electrolyte buffer containing a first leading electrolyte ion having a second effective migration rate, wherein the second effective migration rate is greater than the effective migration rate of the high molecular weight nucleic acid; (b) isotachophoresis using the trailing electrolyte ion, the high molecular weight nucleic acid, and the first leading electrolyte ion to separate the high molecular weight nucleic acid from the contaminant and enrich the high molecular weight nucleic acid in the isotachophoresis region; and (c) eluting the genomic DNA into a solution in an output reservoir, wherein more than 50% by mass of the nucleic acid in the solution is greater than 30 kilobases.

[0088] In some embodiments provided herein, the lysis buffer does not include an alkaline buffer. In some embodiments provided herein, the lysis buffer contains octylphenol ethoxylate. In some embodiments provided herein, the solution contains more than 50% by mass of nucleic acid greater than 50 kilobases.

[0089] One aspect of this disclosure provides a method for performing isotachophoresis, the method comprising: (a) providing a fluid apparatus including a first channel in fluid communication with a sample input reservoir containing a tissue sample containing 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 fluid apparatus to perform isotachophoresis, wherein the isotachophoresis occurs without direct contact between the tissue sample and the first and second electrodes.

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

[0091] One aspect of this disclosure provides a microfluidic system comprising: (a) a microfluidic chip including a first channel and a first reservoir in fluid communication with the first channel, wherein the first channel and the first reservoir meet at a first connector; and (b) a mechanical member including a first tooth, wherein the mechanical member is configured to apply mechanical pressure to the first channel via the first tooth to at least partially close the first channel and increase the fluid resistance between the first channel and the first reservoir through plastic deformation of at least one wall of the first channel.

[0092] In some embodiments provided herein, the microfluidic chip further includes a second reservoir in fluid communication with the first reservoir and a second channel connecting the first reservoir and the second reservoir, and wherein the mechanical component further includes a second tooth configured to apply mechanical pressure to the second channel to plastically close the second channel and prevent fluid communication between the first reservoir and the second reservoir. In some embodiments provided herein, the first tooth is configured to deliver mechanical pressure to the first connector to close the first channel by plastic deformation of at least one wall of the first channel. In some embodiments provided herein, the first tooth is configured to heat the first channel. In some embodiments provided herein, the mechanical component comprises a material with a Young's modulus greater than that of the first channel. In some embodiments provided herein, the microfluidic system is configured to perform isovelocity electrophoresis. In some embodiments provided herein, the first tooth is thermally coupled to a heating element. In some embodiments provided herein, the first tooth is heated to a temperature above the glass transition temperature of the at least one wall of the first channel. Some embodiments of the aspects provided herein include a method for completing a process in a fluid system, the method comprising using the microfluidic system to at least partially close the first channel by plastic deformation, thereby increasing the resistance to fluid flow between the first channel and the first reservoir. In some embodiments of the aspects provided herein, the first tooth of the mechanical component applies a force of at least 0.25 lbs to the first channel. In some embodiments of the aspects provided herein, the process in the fluid system is isokinetic electrophoresis.

[0093] One aspect of this disclosure provides a method for isotachyphoresis of a sample containing nucleic acids, the method comprising: (a) loading the sample containing nucleic acids into a first reservoir of a microfluidic chip; (b) loading a trailing electrolyte buffer into a second reservoir of the microfluidic chip, wherein the trailing electrolyte buffer contains a first trailing electrolyte ion having an effective mobility less than the effective mobility of the nucleic acid; (c) loading a leading electrolyte buffer into a third reservoir of the microfluidic chip, wherein the third reservoir contains a first leading electrolyte ion having a second effective mobility greater than the effective mobility of the nucleic acid; (d) applying an electric field within the microfluidic chip to perform isotachyphoresis with the first trailing electrolyte ion, the nucleic acid, and the first leading electrolyte ion, thereby confining the nucleic acid or a portion thereof within an isotachyphoresis region; and (e) using a temperature sensor to sense temperature changes in or near the isotachyphoresis region, wherein feedback from the temperature sensor is used to control the electric field.

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

[0095] One aspect of this 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 channel; (ii) a first buffer reservoir, a second buffer reservoir, and a third buffer reservoir in fluid communication with the first fluid channel, wherein the first buffer reservoir and the second buffer reservoir are separated by a capillary barrier; and (iii) an elution reservoir in fluid communication with the first fluid channel; (b) a sensor configured to detect temperature changes in the first fluid channel within the first isotachophoresis region; and (c) means positioned to provide current within the first channel within the first isotachophoresis region.

[0096] Some embodiments of the aspects provided herein further include a controller configured to trigger a 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 within the range of about 0.2°C to 5°C. In some embodiments of the aspects provided herein, the microfluidic device is further configured to separate nucleic acid samples in the elution reservoir after the sensor detects a temperature change. In some embodiments of the aspects provided herein, the sensing of the nucleic acid is performed using a sensor located at most 8 mm within the elution reservoir. In some embodiments of the aspects provided herein, the first channel includes a single sensor.

[0097] One aspect of this disclosure provides 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 tail electrolyte buffer containing a tail electrolyte; and (c) a leading electrolyte buffer containing a leading electrolyte.

[0098] In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a mixture of at least two electrolytes having different effective migration rates. In some embodiments of the aspects provided herein, the mixture comprises (i) a first electrolyte having an effective migration rate lower than that of nucleic acids and a higher effective migration rate than that of contaminants, and (ii) a second electrolyte having an effective migration rate lower than that of said contaminants. In some embodiments of the aspects provided herein, the first electrolyte comprises hexanoic acid. In some embodiments of the aspects provided herein, the second electrolyte comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid). In some embodiments of the aspects provided herein, the kit further comprises a sample buffer, wherein the sample buffer comprises any combination of a lead electrolyte buffer, a trailing electrolyte buffer, or urea. In some embodiments of the aspects provided herein, the kit further comprises a sample buffer containing urea and thiourea.

[0099] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a fluid device: (i) a tissue sample containing nucleic acids and contaminants, wherein the tissue sample is not an unlysaturated whole blood sample; (ii) a trailing electrolyte buffer containing trailing electrolyte ions having an effective migration rate greater than the effective migration rate of the contaminants and less than the effective migration rate of the nucleic acids; and (iii) a leading electrolyte buffer containing leading electrolyte ions having a second effective migration rate, wherein the second effective migration rate is greater than the effective migration rate of the nucleic acids; and (b) applying an electric field within the fluid device to perform isovelocity electrophoresis with 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 provided herein, the tissue sample is not a whole blood sample. In some embodiments provided herein, the trailing electrolyte ion comprises hexanoic acid. In some embodiments provided herein, the leading electrolyte ion comprises chloride. In some embodiments provided herein, the trailing electrolyte buffer comprises a second trailing electrolyte ion having a different effective mobility than the first trailing electrolyte ion. In some embodiments provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid). In some embodiments provided herein, the second trailing electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid. In some embodiments provided herein, the second trailing electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES, and the first trailing electrolyte ion comprises MOPS. In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a second trailing electrolyte ion having a second effective mobility, wherein the magnitude of the second effective mobility is substantially the same as or less than the magnitude of the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the contaminant is selected from cross-linked nucleic acids, embedding materials, immobilization chemicals, proteins, inhibitors, and combinations thereof. In some embodiments of the aspects provided herein, the contaminant comprises cross-linked nucleic acids. In some embodiments of the aspects provided herein, the tissue sample is combined with the trailing electrolyte buffer prior to loading. In some embodiments of the aspects provided herein, the tissue sample is combined with the lead electrolyte buffer prior to loading. In some embodiments of the aspects provided herein, the loading of the lead electrolyte buffer is performed prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the method further comprises eluting an output solution containing the purified nucleic acid from an outlet reservoir of the fluid device. In some embodiments provided herein, the concentration of the purified nucleic acid in the output solution is at least about twice as high as the concentration of the nucleic acid in the tissue sample. In some embodiments provided herein, the concentration of the cross-linked nucleic acid in the output solution is at least about half as low as the concentration of the cross-linked nucleic acid in the tissue sample.In some embodiments of the aspects provided herein, the output solution does not contain 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 tissue (FFPE). In some embodiments of the aspects provided herein, the method further includes lysing or destroying the tissue sample prior to loading. In some embodiments of the aspects provided herein, the lysis or destruction is performed using urea or thiourea.

[0101] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a first channel of a fluid device: (i) a first tissue sample comprising a first nucleic acid and a first contaminant; (ii) a first tailing electrolyte buffer comprising a first tailing ion, wherein the effective mobility of the first tailing ion is less than the effective mobility of the first nucleic acid; and (iii) a first leading electrolyte buffer comprising a first leading ion, wherein the effective mobility of the first leading ion is greater than the effective mobility of the first nucleic acid; (b) loading the following substances into a second channel of the fluid device: (iv) a second tissue sample comprising a second nucleic acid and a second contaminant; and (v) a package containing... (vi) a second tailing electrolyte buffer containing a second tailing ion, wherein the amount of the second tailing ion is less than the amount of the effective mobility of the second nucleic acid; and (vi) a second leading electrolyte buffer containing a second leading ion, wherein the amount of the effective mobility of the second leading ion is greater than the amount of the effective mobility of the second nucleic acid; and (c) applying an electric field within the fluid device to perform isotachophoresis in the first channel using the first tailing ion, the first nucleic acid, and the first leading ion, and in the second channel using the second tailing ion, the second nucleic acid, and the second leading ion, thereby purifying the first nucleic acid from the first contaminant and purifying the second nucleic acid from the second contaminant.

[0102] In some embodiments provided herein, the first trailing electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or a tissue disrupting agent. In some embodiments provided herein, the second trailing electrolyte buffer or the second leading electrolyte buffer further comprises a lysing agent or a tissue disrupting agent. In some embodiments provided herein, the lysing agent or the tissue disrupting agent comprises one or more agents selected from solutions with a pH greater than about 12, proteases, urea, thiourea, and surfactants.

[0103] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a first region of a fluid device: (i) a tissue sample containing nucleic acids and contaminants; (ii) a tailing electrolyte buffer containing tailing ions, wherein the effective mobility of the tailing ions is less than the effective mobility of the nucleic acids; and (iii) a leading electrolyte buffer containing leading ions, wherein the effective mobility of the leading ions is greater than the effective mobility of the nucleic acids; and (b) applying an electric field to the fluid device to perform isovelocity electrophoresis in a second region of the fluid device using the tailing ions, the nucleic acids, and the leading ions, thereby purifying the nucleic acids from the contaminants, wherein during the application of the electric field, the first region is maintained at a first temperature, and the second region is maintained at a second temperature different from the first temperature.

[0104] In some embodiments provided herein, the trailing electrolyte buffer or the leading electrolyte buffer further comprises a lysing agent or a tissue disrupting agent. In some embodiments provided herein, the lysing agent or the tissue disrupting agent comprises one or more agents selected from solutions with a pH greater than about 12, proteases, urea, thiourea, and surfactants. In some embodiments provided herein, the first temperature is from about 4°C to about 40°C. In some embodiments provided herein, the first temperature is from about 40°C to about 80°C.

[0105] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a first zone of a fluid device: (i) a tissue sample containing nucleic acids; (ii) a tailing electrolyte buffer containing tailing ions, wherein the effective mobility of the tailing ions is less than the effective mobility of the nucleic acids; and (iii) a leading electrolyte buffer containing leading ions, wherein the effective mobility of the leading ions is greater than the effective mobility of the nucleic acids; (b) in the first zone, performing at least one sample preparation selected from the group consisting of: (1) removing embedding material; (2) disrupting tissue; (3) lysing cells; (4) decrosslinking nucleic acids; (5) digesting proteins; and (6) digesting nucleic acids; and (c) applying an electric field within the fluid device to perform isovelocity electrophoresis in a second zone of the fluid device using the tailing ions, the nucleic acids, and the leading ions, thereby purifying the nucleic acids from contaminants in the tissue sample.

[0106] In some embodiments of the aspects provided herein, the removal of the embedding material or the lysed cells includes incubating the tissue sample in the fluid apparatus at a temperature of at least about 37°C for a duration 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 duration is from about 1 minute to about 60 minutes. In some embodiments of the aspects provided herein, the destruction of tissue or the lysed cells includes applying mechanical stress to the sample. In some embodiments of the aspects provided herein, the destruction of tissue or the lysed cells includes applying heat to the 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 65°C. In some embodiments of the aspects provided herein, the destruction of tissue or the lysed cells comprises a solution with a pH of at least 12. In some embodiments of the aspects provided herein, the destruction of tissue or the lysed cells comprises proteolytic digestion. 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 temperature is from about 30°C to about 65°C. In some embodiments provided herein, the destruction of tissue or the lysis of cells comprises applying at least one surfactant to the tissue or cells. In some embodiments provided herein, the destruction of tissue or the lysis of cells comprises applying a solution containing urea to the tissue or cells. In some embodiments provided herein, the solution further comprises thiourea. In some embodiments provided herein, the concentration of urea in the solution is from about 4 M to about 9 M, and the concentration of thiourea in the solution is from about 0.5 M to about 3.5 M. In some embodiments provided herein, the concentration of urea in the solution is from about 6.5 M to about 7.5 M, and the concentration of thiourea in the solution is from about 1.5 M to about 2.5 M. In some embodiments provided herein, the decrosslinking of nucleic acids comprises digesting crosslinked proteins with proteinase K. In some embodiments provided herein, the digestion of nucleic acids is performed using a DNase or an RNase.

[0107] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances onto a fluid device: (i) a tissue sample containing nucleic acids, wherein the tissue sample is embedded or immobilized; (ii) a tailing electrolyte buffer containing a tailing electrolyte having a lower effective migration rate than the nucleic acids; and (iii) a leading electrolyte buffer containing a leading electrolyte having a higher effective migration rate than the nucleic acids; and (b) applying an electric field to the fluid device to perform isovelocity electrophoresis using the tailing electrolyte, the nucleic acids, and the leading electrolyte, thereby purifying the nucleic acids from contaminants in the tissue sample.

[0108] In some embodiments provided herein, the contaminant is selected from cross-linked nucleic acids, embedding materials, immobilization chemicals, enzymes, and inhibitors. In some embodiments provided herein, the embedding material comprises paraffin. In some embodiments provided herein, the tissue sample is formalin-fixed. In some embodiments provided herein, the tissue sample is embedded and fixed. In some embodiments provided herein, the tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In some embodiments provided herein, the tissue sample is an anatomically dissected tissue sample. In some embodiments provided herein, the anatomically dissected tissue sample is an anatomically dissected FFPE sample. In some embodiments provided herein, the method further includes the step of comparing the characteristics of the nucleic acid with nucleic acids from other samples. In some embodiments provided herein, the characteristic is expression level. In some embodiments provided herein, the characteristic is nucleic acid sequence. In some embodiments provided herein, the characteristic is molecular weight. In some embodiments provided herein, the characteristic is nucleic acid integrity. In some embodiments provided herein, the characteristic is nucleic acid purity. In some embodiments provided herein, the method further includes the step of administering a drug based on the characteristics 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 trailing electrolyte buffer has a pH of about 7. In some embodiments of the aspects provided herein, the trailing electrolyte buffer has a pH greater than about 7. In some embodiments of the aspects provided herein, the method further includes incubating the tissue sample in the fluid device at a temperature of at least about 37 °C for a duration of at least about 1 minute before 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 duration is from about 1 minute to about 60 minutes. In some embodiments of the aspects provided herein, the leading electrolyte buffer contains proteinase K. In some embodiments of the aspects provided herein, the method further includes removing protein cross-links from the nucleic acid using the proteinase K. In some embodiments of the aspects provided herein, the method further includes removing protein cross-links from the nucleic acid by heating after applying the electric field. In some embodiments of the aspects provided herein, the method further includes eluting an output solution containing the purified nucleic acid from an outlet reservoir of the fluid 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 times 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 one-half 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 does not contain the contaminant. In some embodiments of the aspects provided herein, the output solution has a volume equal to or less than about 50 μL. 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 trailing electrolyte has a higher effective migration rate than the contaminant. In some embodiments of the aspects provided herein, the trailing electrolyte comprises (i) a first ion having a higher effective migration rate than the contaminant, and (ii) a second ion having a migration rate substantially the same as or lower than that of the contaminant. In some embodiments of the aspects provided herein, the isovelocity electrophoresis quenches the pH of the tissue sample to about 7. In some embodiments of the aspects provided herein, the method further includes dewaxing the tissue sample prior to loading. 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 characteristics of the nucleic acid with the characteristics of different nucleic acids from different tissue samples. In some embodiments of the aspects provided herein, the method further includes the step of detecting the concentration of the nucleic acid. In some embodiments of the aspects provided herein, the concentration is less than or equal to about 1 picogram per microliter (pg / μL). In some embodiments of the aspects provided herein, the concentration is less than or equal to about 0.5 pg / μL. In some embodiments of the aspects provided herein, the concentration is at least about 1 picogram per microliter (pg / μL).

[0109] One aspect of this disclosure provides a fluid apparatus comprising: a sample purification region including: (a) a first region; (b) a sample inlet located in the first region; (c) a trailing electrolyte reservoir in fluid communication with the first region; (d) a second region in fluid communication with the first region; (e) a leading electrolyte reservoir in fluid communication with the second region; (f) a sample outlet in fluid communication with the second region; (g) a first heater in thermal communication with the first region; and (h) a second heater configured to transfer heat to the second region, wherein the first region and the second region are substantially thermally isolated.

[0110] One aspect of this disclosure provides a fluid apparatus comprising: a sample purification region including: (a) a first region; (b) a sample inlet located in the first region; (c) a trailing electrolyte reservoir in fluid communication with the first region; (d) a second region in fluid communication with the first region; (e) a leading electrolyte reservoir in fluid communication with the second region; (f) a sample outlet in fluid communication with the second region; and (g) a heater in thermal communication with the first region and the second region.

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

[0112] One aspect of this disclosure provides a kit comprising the device provided herein, a tailing electrolyte buffer containing a tailing electrolyte, and a leading electrolyte buffer containing a leading electrolyte.

[0113] In some embodiments provided herein, the trailing electrolyte buffer comprises a mixture of at least two electrolytes having different effective migration rates. In some embodiments provided herein, the mixture comprises (i) a first electrolyte having a lower effective migration rate than nucleic acids and a higher effective migration rate than contaminants, and (ii) a second electrolyte having a lower effective migration rate than said contaminants. In some embodiments provided herein, the contaminant comprises cross-linked nucleic acids. In some embodiments provided herein, the first electrolyte comprises hexanoic acid. In some embodiments provided herein, the second electrolyte comprises HEPES.

[0114] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a fluid device: (i) a tissue sample containing nucleic acids; (ii) a trailing electrolyte buffer containing trailing electrolyte ions having a first effective mobility, wherein the magnitude of the first effective mobility is lower than the magnitude of the effective mobility of the nucleic acids; (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir containing first leading electrolyte ions having a second effective mobility, wherein the magnitude of the second effective mobility is greater than the magnitude of the effective mobility of the nucleic acids; and (iv) a second leading electrolyte buffer in a second leading electrolyte reservoir containing second leading electrolyte ions having a third effective mobility, wherein the magnitude of the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acids, wherein the first leading electrolyte buffer is different from the second leading electrolyte buffer; (b) performing isovelocity electrophoresis for the first time with the trailing electrolyte ions, the nucleic acids, and the first leading electrolyte ions to purify the nucleic acids from contaminants in the tissue sample; and (c) The tailing electrolyte ions, the nucleic acid, and the second leading electrolyte ions are used to perform a second isotachophoresis.

[0115] In some embodiments provided herein, the second isotachophoresis includes changing the applied current from a first channel to a second channel. In some embodiments 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 provided herein, 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 times. In some embodiments provided herein, the first leading electrolyte ion is different from the second leading electrolyte ion. In some embodiments 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 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, and 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. 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 trailing electrolyte buffer is loaded into a trailing electrolyte reservoir, which is separated from the first and second leading electrolyte reservoirs. In some embodiments of the aspects provided herein, the first and second isotachyphoresis are performed by applying an electric field. In some embodiments of the aspects provided herein, the first and second isotachyphoresis are performed by applying more than one electric field.

[0116] One aspect of this disclosure provides a fluid apparatus comprising: a sample purification region including: (a) a channel comprising a first region and a second region in fluid communication with the first region; (b) a sample inlet, a tail electrolyte reservoir comprising a tail electrolyte buffer and a first lead electrolyte reservoir comprising a first lead electrolyte buffer, each in fluid communication with the first region; and (c) a second lead electrolyte reservoir comprising a second lead electrolyte buffer in fluid communication with the second region, wherein the second lead electrolyte buffer is different from the first lead electrolyte buffer.

[0117] In some embodiments provided herein, the sample inlet is capable of receiving a sample containing at least some non-liquid biological material. In some embodiments provided herein, the second pre-lead electrolyte buffer contains a pre-lead electrolyte co-ion different from the first pre-lead electrolyte buffer. In some embodiments provided herein, the first pre-lead electrolyte buffer contains a first pre-lead electrolyte ion, and the second pre-lead electrolyte buffer contains a second pre-lead electrolyte ion identical to the first pre-lead electrolyte ion, wherein the concentration of the first pre-lead electrolyte ion in the first pre-lead electrolyte buffer differs from the concentration of the second pre-lead electrolyte ion in the second pre-lead electrolyte buffer. In some embodiments provided herein, the first pre-lead electrolyte buffer contains a first pre-lead electrolyte ion, and the second pre-lead electrolyte buffer contains a second pre-lead electrolyte ion, wherein the concentration of the first pre-lead electrolyte ion in the first pre-lead electrolyte buffer differs from the concentration of the second pre-lead electrolyte ion in the second pre-lead electrolyte buffer by at least 1.5 times. In some embodiments provided herein, the first pre-lead electrolyte buffer contains a first pre-lead electrolyte ion, and the second pre-lead electrolyte buffer contains a second pre-lead electrolyte ion different from the first pre-lead electrolyte ion. In some embodiments provided herein, the first lead electrolyte buffer contains a first lead electrolyte ion, and the second lead electrolyte buffer contains a second lead electrolyte ion that is the same as the first lead electrolyte ion, wherein the concentration of the first lead electrolyte ion in the first lead electrolyte buffer is the same as the concentration of the second lead electrolyte ion in the second lead electrolyte buffer, and wherein the first lead electrolyte buffer contains a third lead electrolyte ion.

[0118] One aspect of this disclosure provides a method comprising: (a) providing an electrofluidic apparatus including a reservoir in fluid communication with a channel; (b) loading a sample volume into the reservoir; (c) moving at least 50% of the sample volume from the reservoir to the channel; and (d) applying an ion current through the channel.

[0119] In some embodiments provided herein, the movement is carried out by means of gravity. In some embodiments provided herein, the ion current substantially does not pass through the reservoir. In some embodiments provided herein, at least 50% of the sample volume comprises at least 80% of the sample volume. In some embodiments provided herein, the sample volume contains nucleic acids. In some embodiments provided herein, the sample volume contains tissue samples. In some embodiments provided herein, the sample volume contains formalin-fixed paraffin-embedded (FFPE) samples. In some embodiments provided herein, the application of the ion current comprises isotachyplasty (ITP). Incorporation

[0120] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by reference. Attached Figure Description

[0121] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of the invention, in which:

[0122] Figure 1A An exemplary protocol for sample processing and nucleic acid extraction or purification is shown.

[0123] Figure 1B An exemplary scheme for automating sample processing and nucleic acid extraction or purification is shown.

[0124] Figure 2A An exemplary schematic diagram shows the separation and purification of DNA and RNA from contaminants by medium-speed electrophoresis.

[0125] Figure 2B An exemplary schematic diagram illustrates the isochronous electrophoresis separation and purification of nucleic acids from paraffin and other possible sample contaminants, simultaneously with protease-mediated tissue disruption and decrosslinking of nucleic acids.

[0126] Figure 3 Exemplary results of DNA extraction and purification by automating isotachophoresis in a fluid apparatus are shown, compared to exemplary results from a typical solid-phase column extraction kit.

[0127] Figure 4AExemplary results of unbiased (e.g., relative to sequence) extraction of GC-rich and AT-rich synthetic DNA oligonucleotides mixed in proportion to sample concentrations using isotachophoresis are shown.

[0128] Figure 4B Exemplary results of DNA molecular weight gradient purification before and after purification using isovelocity electrophoresis with no bias (e.g., relative to size or molecular weight) are shown; a comparison with two solid-phase column-based nucleic acid purification methods is also shown.

[0129] Figure 5A An exemplary schematic diagram of a channel with a sample preparation area and an isotachophoresis purification area is shown.

[0130] Figure 5B An exemplary fluid device cassette is shown, comprising eight parallel fluid channels and a reservoir for simultaneously processing up to eight samples (e.g., ...). Figure 5A (As shown).

[0131] Figure 5C It shows how to use such Figure 5B An exemplary top view of a channel of the fluid device box and its connected reservoir is shown, further illustrating the channel through which external pressure or vacuum is applied to the fluid device box using a gas port.

[0132] Figure 5D As shown Figure 5B An exemplary side view of the fluid device box shown.

[0133] Figure 5E As shown Figure 5B An exemplary end view of the fluid device box shown.

[0134] Figure 6A An exemplary top view of a fluid device box is shown.

[0135] Figure 6B An exemplary side view of a fluid device box is shown.

[0136] Figure 6C An exemplary bottom view of the fluid device box is shown.

[0137] Figure 6D An exemplary three-dimensional top-view schematic diagram of a fluid device box is shown.

[0138] Figure 7A An exemplary top view of a fluid device box is shown.

[0139] Figure 7B An exemplary side view of a fluid device box is shown.

[0140] Figure 7CAn exemplary bottom view of the fluid device box is shown.

[0141] Figure 7D An exemplary three-dimensional bottom-view full-view schematic diagram of a fluid device box is shown.

[0142] Figure 8A An exemplary top view of a fluid device box is shown.

[0143] Figure 8B An exemplary side view of a fluid device box is shown.

[0144] Figure 8C An exemplary bottom view of the fluid device box is shown.

[0145] Figure 8D An exemplary three-dimensional bottom-view full-view schematic diagram of a fluid device box is shown.

[0146] Figure 9A As shown Figure 5B The diagram shown is an exemplary schematic of a fluid device housing containing eight parallel channels.

[0147] Figure 9B An exemplary schematic diagram of two thermal controllers is shown, each thermal controller being coupled with, as shown in the diagram. Figure 9A The regions of the eight parallel channels shown are aligned.

[0148] Figure 10A An exemplary gas channel is shown, which may include a capillary barrier.

[0149] Figure 10B yes Figure 10A An enlarged schematic diagram of the gas channel.

[0150] Figure 11 An exemplary low-loss sample reservoir is shown.

[0151] Figure 12A An exemplary mechanical component is shown, which can be used to apply pressure to close the passage of a fluid device.

[0152] Figure 12B An exemplary comb-shaped mechanical component is shown.

[0153] Figure 12C The alignment of channels in comb-shaped mechanical components and fluid devices is shown.

[0154] Figure 13A An exemplary benchtop device for automating sample preparation and isotachophoresis on a fluid device box is shown.

[0155] Figure 13B An exemplary computer control system is shown that is programmed or otherwise configured to implement the methods provided herein.

[0156] Figure 14 Exemplary results are shown of fluorescence-based measurement and quantification of a titration series of nucleic acids using isotachophoresis.

[0157] Figure 15 A schematic diagram of an exemplary design is shown, comprising a connected reservoir, a non-contact electrode (which can be used as a conductivity sensor), and a fluid channel for automatically loading fluid into the channel / device and for automating isokinetic electrophoresis.

[0158] Figure 16 A graph showing the voltage measurements in the ITP channel over time during operation is shown.

[0159] Figure 17 Two from Figure 16 A graph showing the derivative analysis of the voltage measurements.

[0160] Figure 18 An example of conductivity measurements over time in the ITP channel near the elution reservoir is shown.

[0161] Figure 19 An exemplary schematic diagram of a C4D sensor implementation is shown.

[0162] Figure 20A An exemplary temperature map of an ITP channel, captured using a thermal imaging camera, is shown.

[0163] Figure 20B It shows in Figure 20A A graph showing the temperature at the cursor position 1 over time.

[0164] Figure 21 The graph shows the temperature measurements and temperature derivatives over time during ITP operation.

[0165] Figure 22A An exemplary schematic diagram of a vertical (or columnar) ITP setup is shown.

[0166] Figure 22B An exemplary image of a vertical ITP setup with DNA ITP bands is shown.

[0167] Figure 23 Exemplary images and corresponding fluorescence intensity traces are shown of the extraction and separation of amplifiable (e.g., uncrosslinked) DNA from crosslinked DNA in FFPE samples using isotachophoresis.

[0168] Figure 24A An exemplary image is shown showing DNA extraction and purification from an FFPE sample using isodynamic electrophoresis.

[0169] Figure 24BExemplary DNA yields, measured by quantitative PCR of DNA extracted and purified from FFPE samples using isospeed electrophoresis, are shown compared to exemplary results from a typical solid-phase column extraction kit.

[0170] Figure 25A An image of a single-channel ITP chip loaded with nucleic acids (RNA extracted and digested from human cells) stained with useful dyes for visualization is shown.

[0171] Figure 25B An image of a single-channel ITP chip loaded with nucleic acids (RNA extracted and digested from human cells) stained with useful dyes for visualization is shown.

[0172] Figure 26A Images of RNA ITP bands in the chip channels during purification are shown.

[0173] Figure 26B Images of total nucleic acid ITP bands in the chip channels during the purification process are shown.

[0174] Figure 26C It shows Figure 26A The image shows an electrophoresis diagram of the RNA mass of the sample.

[0175] Figure 26D It shows Figure 26B The image shows an electrophoresis diagram of the RNA mass of the sample.

[0176] Figure 27A Results for DNA yield (ng) of ITP (square) as a function of the percentage of whole blood volume in the starting sample are shown compared to column (rhomboid, Qiagen QiaAmp) extraction from mouse whole blood.

[0177] Figure 27B An image of total nucleic acids in the ITP bands during ITP purification of lysed mouse whole blood on a chip is shown.

[0178] Figure 27C and Figure 27D Images of the ITP chip channels under white light and fluorescence overlays are shown, illustrating the physical separation of heme in the sample / lead electrolyte channels from the elution channels and reservoirs before and after ITP purification of 50% volume of whole blood lysate. Nucleic acids were stained with green dye for visualization in the elution wells. Figure 27C The chip prior to ITP is shown (blood lysate and ITP buffer loaded in the chip; buffer only in the elution wells). Figure 27D The image shows the chip after ITP (blood lysate and ITP buffer loaded on the chip; purified DNA in the elution wells).

[0179] Figure 27E The chip is shown after ITP purification (50% volume of blood).

[0180] Figure 27F The chip is shown after ITP purification (25% volume of blood).

[0181] Figure 28 The results of high molecular weight DNA purification by ITP are shown compared to solid-phase extraction.

[0182] Figure 29A A fluid device containing eight closed channels is shown.

[0183] Figure 29B An enlarged microscopic view of the second channel closure position adjacent to the elution reservoir of each channel is shown.

[0184] Figure 29C The calculated percentage of closure is shown as a function of the force applied to the fluid device.

[0185] Figure 29D The conductivity measurements with the channel closed are shown.

[0186] Figure 30 The graph shows the voltage measurements and voltage derivatives over time during ITP operation.

[0187] Figure 31A The image shows a micrograph of an ITP band with focused DNA in each of eight samples in the sample channel area of ​​the device.

[0188] Figure 31B The independent voltage signal data of each of the eight channels over time under a fixed current are shown.

[0189] Figure 31C Photomicrographs of the same eight ITP bands of focused DNA from a sample eluted in an elution reservoir are shown.

[0190] Figure 31D It is used for Figure 31B The image shows an amplified portion of the triggered voltage tracking (monitoring).

[0191] Figure 32A An exemplary method for preparing samples from cultured mammalian cells and for ITPs used for DNA purification is shown.

[0192] Figure 32B An exemplary method for preparing samples from tissue samples and for ITP for DNA purification is shown.

[0193] Figure 33 An exemplary processing workflow for automating ITP is shown.

[0194] Figure 34 A circuit configured to detect and prevent current leakage during ITP is shown.

[0195] Figures 35A-35B An exemplary fluid device comprising two interlocking parts is shown.

[0196] Figures 36A-36B An example of a fluid device comprising multiple parts is shown.

[0197] Figures 37A-37B An example of a fluid device comprising three parts is shown.

[0198] Figure 38 An exemplary channel diagram of eight parallel channels on the bottom side of a chip for a three-part fluid device is shown.

[0199] Figure 39 An exemplary multi-part fluid device is shown.

[0200] Figure 40 An exemplary fluid circuit including voltage and temperature sensing is shown.

[0201] Figures 41A-41B An exemplary “cliff capillary barrier” is shown.

[0202] Figures 42A-42B An exemplary “platform capillary barrier” is shown.

[0203] Figure 43 An exemplary channel or fluid loop is shown, highlighting the initial fluid interface location after loading.

[0204] Figure 44 An exemplary channel or fluid loop is shown, highlighting the location of the final fluid interface after loading.

[0205] Figure 45A An example of a fluid layer on a chip is shown.

[0206] Figure 45B A cross-sectional view of the pneumatic port is shown.

[0207] Figure 45C The implementation of this design is shown.

[0208] Figure 46A An exemplary microfluidic channel is shown, in which an empty sample channel can be detected.

[0209] Figure 46B The sequence of events used to detect whether the channel is empty or filled is shown.

[0210] Figure 46CThe pressure trace and control signal trace from the implementation of this technology are shown.

[0211] Figures 47A-47B An exemplary pneumatic control scheme for staged liquid loading is shown.

[0212] Figures 48A-48H An exemplary sample inlet reservoir designed for direct injection is shown.

[0213] Figure 49A and Figure 49B An exemplary low-dispersion elution channel is shown.

[0214] Figure 50A1 A fluid surface of a substrate is shown having a channel terminating in a through-hole that communicates with a reservoir on an opposite reservoir surface of the substrate.

[0215] Figures 50A-50B A technical diagram of an exemplary elution reservoir on a chip device is shown.

[0216] Figures 50C-50E yes Figure 50A Background subtraction fluorescence images of the fluid apparatus in different elution steps of the design.

[0217] Figure 50F It is a flowchart representing the steps in the elution process.

[0218] Figure 51A An exemplary fluid reservoir that can be used for nucleic acid elution is shown.

[0219] Figure 51B A second embodiment of the reservoir is shown, which has further variations for injection molding compatibility.

[0220] Figure 51C and Figure 51D Two views are shown of a third embodiment of the reservoir design.

[0221] Figure 51E A comparison of nucleic acid dwell time between the disclosed design and the reference design is shown.

[0222] Figure 51F The cross-section of the reference design is shown. It is a drawn vertical cylinder without any internal structure.

[0223] Figures 52A-52D A fluid reservoir consisting of elliptical through-holes is shown, its dimensions designed to allow easy positioning of the pipette tip for reliable fluid recovery from the device.

[0224] Figures 53A-53C Another exemplary fluid reservoir is shown.

[0225] Figure 53D A top view of an exemplary elution reservoir is shown. Figure 53E It shows Figure 53D A cross-sectional view of the washing reservoir.

[0226] Figure 54A An exemplary mechanical component is shown, which can be used to apply pressure to close the passage of a fluid device.

[0227] Figure 54B An exemplary ridge-shaped mechanical component is shown.

[0228] Figure 54C The alignment of the ridge-shaped mechanical component with the fluid device channel is shown.

[0229] Figure 54D An exploded assembly diagram of a mechanical actuator coupled to a ridge structure to close a channel is shown.

[0230] Figure 55 The pneumatic control block diagrams for the β prototype and production instruments are depicted.

[0231] Figures 56A-56B , Figure 57 and Figures 58A-58C An alternative mechanism for closing a channel is shown without utilizing the plastic deformation of the fluid device caused by heat or pressure.

[0232] Figures 59A-59C An exemplary benchtop device is shown for performing automated isotachophoresis and / or sample preparation on a fluid device cartridge.

[0233] Figure 60 An exemplary image is shown that can be displayed to the user to instruct and guide the user through the storage loading process.

[0234] Figure 61 A pneumatic manifold is shown that can facilitate the integration of microfluidic chips with instruments.

[0235] Figure 62 The cross-section of the chip in the instrument is shown.

[0236] Figure 63 A vertical manifold motion mechanism is described, which includes a mechanical assembly design for alignment and automatic retraction motion.

[0237] Figure 64 The design of a horizontal manifold motion mechanism is shown, which includes a mechanical assembly design for horizontal motion using a rack and pinion.

[0238] Figure 65 A heat pipe with a thermoelectric cooler design is depicted for maintaining a specified temperature in areas located away from the thermoelectric cooler.

[0239] Figure 66 Another exemplary benchtop device is shown for automated isotachophoresis and / or sample preparation on a fluid device cartridge.

[0240] Figure 67 The peak area response diagrams of various nucleic acid binding dyes to nucleic acid quality are shown.

[0241] Figure 68 The peak width response diagrams of various nucleic acid binding dyes to nucleic acid quality are shown.

[0242] Figures 69A-69C Depicting Incompatibility with PCR.

[0243] Figure 70 The compatibility of PicoGreen with Qubit dsDNA assays was described.

[0244] Figure 71 The compatibility of Syto13 with Qubit dsDNA assay was described.

[0245] Figures 72A-72B The compatibility of PicoGreen with PCR was described.

[0246] Figures 73A-73C The compatibility of Syto13 with PCR was demonstrated. gDNA was purified using isospeed electrophoresis in both the presence and absence of Syto13.

[0247] Figure 74 PicoGreen's compatibility with amplicon-based sequencing library preparation was described.

[0248] Figure 75 The compatibility of Syto13 with amplicon-based sequencing library preparation was demonstrated.

[0249] Figures 76A-76B The incompatibility between PicoGreen and whole-genome sequencing library preparation is described.

[0250] Figures 77A-77B The compatibility of Syto13 with next-generation sequencing library preparation was described.

[0251] Figure 78 This demonstrates the incompatibility between PicoGreen and whole-genome sequencing library preparation.

[0252] Figure 79 The compatibility of Syto13 with next-generation sequencing library preparation was described.

[0253] Figures 80A-80DThe compatibility of Syto13 with next-generation whole-exome sequencing library preparation is described.

[0254] Figure 81 A block diagram describing an optical signal processing algorithm is shown.

[0255] Figure 82 A diagram shows the design of a mechatronic assembly for illuminating and detecting the fluorescence of a sample bound to a dye.

[0256] Figure 83 The optical path for achieving the excitation of sample-bound dye fluorescence and the capture of emitted light from the sample-bound dye fluorescence is shown.

[0257] Figures 84A-84F A control scheme for verifying how a circuit created by electrodes in a channel can be verified is shown.

[0258] Figure 85 A channel closure device is shown—a toothed component with a mechanical actuator.

[0259] Figure 86 A contrast image of the stained analyte material in the channel is shown.

[0260] Figure 87A The conductivity data obtained by measuring the conductivity of the eluted material using a conductivity meter are shown.

[0261] Figure 87B A contrast image of the stained analyte material in the channel is shown.

[0262] Figure 88A A typical trace of the temperature signal captured by the IR sensor during operation is shown.

[0263] Figure 88B It shows Figure 88A The first derivative of the temperature data in the image.

[0264] Figure 88C A comparison of the residuals after subtracting the fit (0016) from the data and after subtracting the null hypothesis (0015) from the data is shown to produce the likelihood ratio.

[0265] Figure 88D A block diagram of the triggering process is shown.

[0266] Figure 88E This demonstrates the successful triggering of the nucleic acid extraction process.

[0267] Figure 88F The failed trigger run is shown.

[0268] Figure 89AThe capillary barrier between the sample (in this case, the sample prepared in the lead electrolyte) and the lead electrolyte buffer is shown.

[0269] Figure 89B The passages of nucleic acids with added slow ions (3-(N-morpholino)propanesulfonic acid) and controls are shown.

[0270] Figure 89C It shows the morphology of the nucleic acid at a later point in the extraction process.

[0271] Figure 89D The capillary barrier between the lead electrolyte buffer and the elution buffer is shown.

[0272] Figure 89E An example of adjusting the buffer interface is shown.

[0273] Figure 89F This demonstrates how adjusting the interface position can facilitate nucleic acid passage.

[0274] Figures 90A-90C The lysis efficiency of three different cell lines is shown.

[0275] Figures 91A-91B An exemplary temperature measurement result is shown, demonstrating the use of an infrared thermal sensor to trigger a reduction or elimination of current in one of the channels.

[0276] Figure 92A A block diagram of the sample channel to LE channel triggering process is shown.

[0277] Figure 92B An exemplary trace for the voltage, voltage derivative, and measurement error used for sample channel to LE channel triggering is shown.

[0278] Figure 92C A block diagram of the LE channel triggering process is shown.

[0279] Figure 92D An exemplary trace is shown for the voltage, voltage derivative, and measurement error used to trigger at the narrowing point behind the capillary barrier within the LE channel.

[0280] Figure 92E A block diagram of the elution triggering process used is shown.

[0281] Figure 92F An exemplary trace for the voltage, voltage derivative, and measurement error used for elution triggering is shown. Detailed Implementation

[0282] Overview

[0283] Sample preparation is the first step in almost all genomics and transcriptomics analyses and can be a major source of variability. Sample preparation can also be manual-intensive, especially when the samples are formalin-fixed paraffin-embedded (FFPE) samples containing cross-linked proteins.

[0284] This disclosure provides methods and apparatus for improving the efficiency of nucleic acid extraction and purification from tissue and cell samples, including samples that have been treated in some way, such as paraffin-embedded samples or chemically fixed samples (e.g., FFPE samples, samples containing solid tissue). The methods provided herein include preparing such treated samples on or off a chip prior to isotachyphoresis using methods incorporating leading and trailing electrolyte ions. In some cases, the method includes treating (e.g., by removing embedding material, lysis, enzymatic destruction) the fixed solid tissue in a trailing or leading electrolyte buffer prior to isotachyphoresis of the sample. The method may also include using a second leading electrolyte buffer of lower ionic strength to produce a sample compatible with downstream processes such as amplification or other enzymatic assays. The apparatus and systems provided herein include devices suitable for isotachyphoresis of tissue-derived samples, including microfluidic devices with parallel processing features and an automated feedback control mechanism that may include a thermal sensor detecting temperature changes within the sample processing channel.

[0285] The methods and apparatus of this disclosure provide improved nucleic acid recovery from samples, particularly from low-abundance samples (e.g., less than 100 ng of nucleic acids), samples with relatively large volumes (e.g., total volume greater than 25 μl, 50 μl, 100 μl, or more), or liquid samples containing solid particles. The methods and apparatus provided herein also offer high reproducibility and reduced bias from short nucleic acids. The apparatus provided herein integrates sample preparation (e.g., removal of crosslinking or embedding materials) and nucleic acid extraction operations into a single device. The apparatus and methods of this disclosure also provide compatibility with process automation, integration with downstream processes, integration with online quantification (e.g., at single-pico resolution), and / or integration with nucleic acid length and sequence distribution analysis.

[0286] The methods described herein are generally isotachophoresis methods performed under conditions suitable for extracting nucleic acids from certain samples, particularly FFPE samples. In some cases, the disclosed methods include isotachophoresis using a trailing electrolyte buffer containing at least two ions with different effective mobility values. The method may also include isotachophoresis using two different leading electrolyte buffers (one of which can be used as a sample elution buffer). The method may include process automation and parallel processing of multiple samples.

[0287] This disclosure also includes protocols for using buffer and spacer chemicals. These buffer and spacer chemicals may include the use of a variety of electrolytes for ITP. For example, a trailing electrolyte may comprise a mixture of electrolyte types capable of separating non-crosslinked nucleic acids from crosslinked nucleic acids, while simultaneously separating non-crosslinked nucleic acids or both crosslinked and non-crosslinked nucleic acids from contaminants within the sample.

[0288] The apparatus described herein includes injection fluidization apparatus having parallel sample processing channels capable of multiplexing ITP and ITP apparatus having two or more regions connected to a thermal device. The techniques of this disclosure can employ ITP to simultaneously collect, purify, and focus extracted RNA and DNA to quantify total nucleic acids extracted on a chip (e.g., by online ITP-assisted concentration to very small volumes or labeling with inserted fluorescent dyes) and deliver nucleic acids downstream to parallel output reservoirs compatible with robotic pipetting.

[0289] The techniques disclosed herein enable the purification of sample materials (e.g., nucleic acids) without binding the sample material to a solid support. The techniques disclosed herein also enable the purification of sample materials (e.g., nucleic acids) without using liquid-phase extraction. This allows purification to be performed without relying on differences in solubility.

[0290] The operation of the devices disclosed herein can be automated, largely automated, or partially automated. In some cases, the methods of this disclosure involve only a single out-of-chip mixing step of dispensing a sample (e.g., the FFPE portion) into a solution (e.g., an alkaline solution, a lysis solution, or a buffer solution containing urea and / or thiourea), followed by loading the sample into a reservoir of a fluid device for further on-device sample preparation (e.g., dewaxing, tissue disruption and cell lysis, protease digestion, protein hydrolysis, or other treatments, including protein denaturation or nuclease digestion) and nucleic acid extraction, purification, enrichment, online quantification, and sorting or grading by size (e.g., size selection). In some cases, the methods of this disclosure include dispensing samples (e.g., FFPE fractions or other tissue samples) into reservoirs or channels of a fluid device (e.g., a cartridge) pre-filled with solutions (e.g., alkaline solutions, lysis solutions, or buffer solutions containing urea and / or thiourea) for on-board sample preparation (e.g., dewaxing, tissue disruption and cell lysis, protease digestion or other treatments, including protein denaturation or nuclease digestion) and nucleic acid extraction, purification, enrichment, online quantification, and size sorting or grading (e.g., size selection). In some cases, the methods of this disclosure include disrupting tissue and / or lysing cells of samples outside the chip, followed by loading samples (which may be homogeneous or may be a heterogeneous mixture of lysed solid tissue and nucleic acids) into reservoirs of a fluid device for further on-board sample preparation (e.g., dewaxing, protease digestion or other treatments, including protein denaturation or nuclease digestion) and nucleic acid extraction, purification, enrichment, online quantification, and size sorting or grading (e.g., size selection). Nuclease digestion may include removing DNA for DNA-free RNA extraction or removing RNA for RNA-free DNA extraction. The fluid apparatus described herein can be used with benchtop systems to automate electric field-based methods for extracting DNA and RNA from samples.

[0291] The devices disclosed herein include systems that automate and integrate on-chip heating (e.g., to temperatures from 37°C to 80°C), sample preparation (e.g., dewaxing, tissue disruption, and cell lysis), buffer replacement, nucleic acid extraction and purification, enrichment of non-crosslinked or amplifiable nucleic acids (e.g., by separating them 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, this disclosure includes a standard, robotically automated, microtiter-compatible eight-channel box and an integrated benchtop controller prototype that provides automated control of the loading of buffers and other fluids, application of temperature and electric fields to the device, and automated start and end of parallel sample processing. The system can be readily modified in the future to provide higher throughput for larger diagnostic or clinical laboratories (e.g., 96-well sample formats), as needed.

[0292] For example, Figure 1A A schematic diagram of an exemplary process for sample processing and nucleic acid extraction using the techniques of this disclosure is shown. A sample (101) may be provided and subjected to any pretreatment step 102, such as mixing with a buffer, lysis, or removal of embedding material (if present). The sample (and, for example, buffer) may then be loaded onto a fluid device (103). Sample preparation steps (104) may then be performed on the fluid device, such as removal of embedding material (if present and if not previously removed during pretreatment), tissue disruption, cell lysis, protein or proteolytic digestion, and, for example, nuclease digestion. Isotachymetry (105) may then be performed to separate and purify nucleic acids from contaminants within the sample, such as cell debris, plasma membranes, small molecules, embedding material, cross-linked nucleic acids, fixatives such as formalin, inhibitors, enzymes such as digestive enzymes, or restriction enzymes. Other steps may occur concurrently with isotachymetry, such as decrosslinking of cross-linked nucleic acids (e.g., using heating or protease digestion). Nucleic acids may be detected and quantified during or after isotachymetry (106). Once extracted or purified, the nucleic acid can be eluted and recovered from the device (107).

[0293] Figure 32A This paper illustrates a non-limiting exemplary method for preparing samples from cultured mammalian cells and for ITP (intracytoplasmic protein transfer) for DNA purification using the methods and apparatus provided herein. Typically, the steps may include separation (step 151) and cell washing (step 152), cell lysis (steps 153-155) and protein degradation (steps 156-157), and homogenizing the DNA released from the lysate (steps 158-159) while maintaining appropriate ion content for downstream ITP.

[0294] In step 151, cultured mammalian cells can be precipitated from live, healthy (e.g., >90% viability) logarithmic phase cell cultures by centrifugation (e.g., 250 g x 5 min). In at least some cases, such as when using adherent or semi-adherent cells, trypsin digestion can be performed prior to precipitation. The used culture medium can be discarded, and the cells can be washed in fresh culture medium, precipitated, resuspended in fresh culture medium, and counted. A cell suspension of appropriate density (e.g., 100,000 live cells per ITP extraction lane) can be deposited in microcentrifuge tubes (e.g., 2 ml Eppendorf Lo-Bind microcentrifuge tubes) for downstream processing (e.g., steps 152-163). In some cases, as will be apparent to those skilled in the art, resuspended fluorescence-activated cell sorting (FACS) can be used to separate and count cells of interest into receiving tubes, which can then be prepared as described herein for ITP.

[0295] In step 152, the cells can be washed with a buffer such as phosphate-buffered saline (PBS). The cells can be precipitated by centrifugation (e.g., 250 g x 5 min). The cell culture medium can then be discarded, and the precipitate can be resuspended in PBS (e.g., 190 μL 1X PBS (Ca2+ or Mg2+-free)). The resuspended cells can be centrifuged to form a precipitate, and the PBS supernatant can be removed from the precipitated cells.

[0296] In step 153, the cell pellet can be lysed by resuspending it in a lysis buffer, such as a proprietary alkaline CCD lysis buffer (“L1”), by pipetting (e.g., pipetting 5 times using a P1000 pipette). Lysis can be performed alternatively or in combination using other lysis techniques known to those skilled in the art, such as sonication, manual grinding, bead milling, homogenization, freezing, enzymatic digestion, and / or chemical destruction. The lysed cells can then be vortexed (e.g., for 3 seconds). Typically, the lysis buffer in step 153 is highly alkaline. In some cases, the alkaline solution may comprise 30–120 mM NaOH at a pH of about 10–13 (40–80 mM NaOH in some cases). An exemplary alkaline solution may comprise 80 mM NaOH, 11 mM DTT, and 0.5% v / v Igepal CA-630.

[0297] In step 154, the cells can be incubated in lysis buffer at room temperature (e.g., 2 min) to allow the lysis process to lyse the cells.

[0298] In step 155, lysis can be stopped and the sample pH can be neutralized. For example, a proprietary acidic quenching buffer (“quenching”) can be applied to the lysed cell sample. In some cases, the quenching buffer may contain only LE, contain LE and TE, or contain only TE. In some cases, including LE and TE or only TE in the quenching buffer can reduce nucleic acid retention at the capillary barrier, particularly at 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, quenching may be unnecessary when the lysis solution is at a neutral or non-alkaline pH. In such cases, the lysis solution may include LE, LE and TE, or TE. Quenching is often useful when an alkaline lysis buffer is used during the lysis step (e.g., for cultured cell applications or primary human cells).

[0299] The sample can be mixed by pipetting (e.g., by pipetting up and down five times) and then vortexing (e.g., for 3 seconds).

[0300] In step 156, the lysed cell suspension may be treated to denature the proteins in the cell lysate. Optionally, RNA (or DNA, if the sample molecule of interest to ITP is RNA) may be degraded. For example, proteinase K (e.g., 20 mg / mL) and optional RNase A (e.g., 10 mg / mL) may be added. The sample may be vortexed (e.g., 3 seconds) to mix and then pulsed (e.g., 2 min), followed by incubation at room temperature.

[0301] In step 157, the sample can be incubated at 56°C for 10 min.

[0302] In step 158, the sample may be homogenized by vortexing (e.g., 3 seconds) and pulsed rotation.

[0303] In step 159, the sample may then be cooled to room temperature (e.g., 5 min). In some cases, where the sample is prepared in an LE, TE may be added after cooling to reduce or minimize the retention of nucleic acids at the capillary barrier, particularly at the capillary barrier between the sample and the LE.

[0304] In step 160, a sample surfactant (e.g., MOPS) may optionally be added to the sample.

[0305] In step 161, the sample may optionally be frozen for later use.

[0306] In step 162, the sample can be heated to 20°C.

[0307] In step 163, nucleic acid dyes or staining agents may optionally be added to the sample lysate for visualization and detection of nucleic acids (e.g., for quantification of nucleic acid quality). For example, when performing online quantification of nucleic acid quality during the ITP process, nucleic acid staining agents, such as nucleic acid binding or insertion dyes, may be used.

[0308] In step 164, the sample may be loaded into one or more sample reservoirs. Prior to loading, the sample may be pulsed vortexed (e.g., twice) and pulsed rotated to agitate and mix the sample. The ITP channel may be pre-primed with the leading electrolyte buffer, trailing electrolyte buffer, and / or other buffers described herein. Alternatively, the sample may be loaded simultaneously with the remaining liquid in the channel.

[0309] In step 165, ITP can be performed. During the ITP process, the DNA in the sample can be purified (i.e. concentrated) as it moves through the channels until it reaches the elution reservoir as described herein. The sample DNA can then be quantified as described herein.

[0310] In step 166, the eluted sample can be recovered from the elution reservoir by elution (e.g., by pipetting).

[0311] Figure 32B Non-limiting exemplary methods are shown for preparing samples from tissue samples (e.g., fresh or FFPE tissue samples) and for ITP for DNA purification using the methods and apparatus provided herein. Typically, these steps may include trimming excess paraffin from the tissue (e.g., when using FFPE tissue sections mounted on a glass slide) (step 171), tissue lysis (steps 172-175), and protein degradation (steps 176-177), as well as reverse cross-linking of the DNA released from the lysate (steps 178-179), while maintaining appropriate ion content for downstream ITP.

[0312] In step 171, excess paraffin may optionally be trimmed or removed from the FFPE tissue sample (e.g., prior to further sample preparation steps). The FFPE tissue sample may be obtained directly from a paraffin block or a section mounted on a glass slide.

[0313] In 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 glass slide or otherwise placing a fresh or FFPE tissue sample into the tube.

[0314] In step 173, tissue can be lysed by pipetting resuspension in a lysis buffer, such as a proprietary alkaline CCD lysis buffer (“L1”) (e.g., pipetting 5 times using a P1000 pipette). Lysis can be performed alternatively or in combination using other lysis techniques known to those skilled in the art, such as sonication, manual grinding, bead milling, homogenization, freezing, enzymatic digestion, and / or chemical destruction. The lysed tissue can then be vortexed (e.g., for 5 seconds). Typically, the lysis buffer in step 173 is highly alkaline. In some cases, the alkaline solution may comprise 30–120 mM NaOH at a pH of about 10–13 (40–80 mM NaOH in some cases). An exemplary alkaline solution may comprise 80 mM NaOH, 11 mM DTT, and 0.5% v / v Igepal CA-630.

[0315] In step 174, the tissue may be incubated in lysis buffer at 80°C (e.g., 3 min) to allow the lysis process to lyse the tissue. The sample can then be mixed by pipetting (e.g., by five up-and-down pipettings) and then vortexed (e.g., 3 seconds).

[0316] In step 175, the lysed tissue can be incubated at room temperature (e.g., 3 min) to cool it down.

[0317] In step 176, the lysed tissue suspension may be treated to denature the proteins in the tissue lysate. Optionally, RNA (or DNA, if the sample molecule of interest to ITP is DNA) may be degraded. For example, proteinase K (e.g., 20 mg / mL) may be added.

[0318] In step 177, the sample can be incubated at 56°C for 1 hour. After incubation, the sample can be vortexed and pulsed.

[0319] In step 178, the sample can be incubated at 90°C for 1 hour.

[0320] In step 179, the sample may then be cooled to room temperature (e.g., 5 min).

[0321] In step 180, RNase A (e.g., 10 mg / mL) may optionally be added.

[0322] In step 181, the sample can be incubated at 25°C for 5 minutes. After incubation, the sample can be vortexed and pulsed.

[0323] In step 182, a sample lubricant (e.g., MOPS) may optionally be added to the sample.

[0324] In step 183, the sample may optionally be frozen for later use.

[0325] In step 184, the sample can be heated to 20°C.

[0326] In step 185, nucleic acid dyes or staining agents may optionally be added to the sample lysate for visualization and detection of nucleic acids (e.g., for quantification of nucleic acid quality). For example, when performing online quantification of nucleic acid quality during the ITP process, nucleic acid staining agents, such as nucleic acid binding or insertion dyes, may be used.

[0327] In step 186, the sample may be loaded into one or more sample reservoirs. Prior to loading, the sample may be pulsed vortexed (e.g., twice) and pulsed rotated to agitate and mix the sample. The ITP channel may be pre-started with the leading electrolyte buffer, trailing electrolyte buffer, and / or other buffers described herein. Alternatively, the sample may be loaded simultaneously with the remaining liquid in the channel.

[0328] In step 187, ITP can be performed. During the ITP process, the DNA in the sample can be purified (i.e. concentrated) as it moves through the channels until it reaches the elution reservoir as described herein. The sample DNA can then be quantified as described herein.

[0329] Similarly, in step 187, the eluted sample can be recovered from the elution reservoir by elution (e.g., by pipetting).

[0330] Figure 1BAn exemplary processing workflow for automating ITP is illustrated. In step 110, a scheme can be selected, such as by using a graphical user interface on a benchtop device. This user interface software can enable ease of use or manual operation. For example, the user can select from a menu (e.g., a drop-down menu). Alternatively, the device can scan a barcode (e.g., an optical barcode, RFID chip) associated with the sample or fluid device chip, which can indicate the scheme to be performed. In step 111, the instrument cover can be opened (e.g., manually or automatically by a motor). The motorized cover opening is compatible with robotic laboratory automation. In step 112, the user can load the chip (e.g., a fluid device) onto the benchtop instrument. The chip may contain a monolithic, multichannel SLAS standard microtiter plate (MTP) footprint for automated ITP. In step 113, ITP liquid can be loaded into the chip wells. Reservoirs for the ITP fluid and user samples can be designed for easy loading, such as via a multichannel pipette (e.g., in the form of a 9mm pitch SLAS standard microtiter plate). The geometry of the channel connecting the reservoir to the ITP channel (e.g., a capillary barrier) can resist gravity-driven flow or wetting of liquid into the channel prior to operation. These structures prevent fluid from defining locations within the ITP channel (including establishing the leading electrolyte / tailing electrolyte interface) and enable bubble-free loading. In some cases, pneumatic actuation can be applied to prime the channel prior to operation. Chip materials can be selected to prevent or resist fluid wetting or wicking into the channel (e.g., plastics with hydrophobic properties or high contact angles). Users can load ITP reagents and buffers onto the chip (e.g., five different fluids); alternatively, pre-loaded reagents can be provided to the chip. In step 114, the user or device can close the device cap. Sample loading can be actuated via a gas or air port on the chip. Wetting and / or gravity-driven flow can be used to fill the channel with liquid, for example, without active pressure application.

[0331] In step 115, the instrument can apply pressure to load fluid into the chip, thereby activating the channel. In step 116, the device can check whether the channel has been properly activated. For example, optical (e.g., reflective), electrical, pressure, and / or flow rate sensors can be used to check whether the fluid has been loaded into the correct position within the chip. Sensors and device software enable real-time monitoring and control of liquid loading. ITP reagent and buffer loading can be performed before loading the sample onto the chip to avoid wasting sample material in case of incorrect loading. If the channel is not properly activated, the device can issue an error report (130). In step 117, the device cap can be opened. In step 118, the sample can be loaded onto the device. Sample loading can be performed manually by the user or can be performed automatically, such as by a laboratory automation robot. Other sample preparation steps can also be performed. For example, paraffin-embedded samples (e.g., FFPE) can be loaded, and the device can then control the temperature within the sample reservoir to dewax the sample. In step 119, the device cap can be closed. In step 120, the device can perform a self-test. For example, electrical feedback from device electrodes coupled to on-chip storage can be used for self-testing of successful liquid initiation (e.g., bubble detection). Optical sensors can be used to provide feedback on the liquid initiation 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 self-test determines that the device has not been properly initiated, the device can issue an error report (131).

[0332] In step 121, ITP-based purification can be performed. Feedback control and process timing using sensors (e.g., triggers) as described herein can be used to control ITP purification and / or automate it. The device can determine whether purification has been successfully performed, and if not, it can issue an error report (132). In step 122, sensors on the device (e.g., optical sensors) can be used to quantify the sample, for example by fluorescence, UV, or other optical detection. Sample size classification can also be performed. If the device determines that the sample has not been properly quantified or detects other problems, it can issue an error report (133). In step 123, changes in conductivity can be detected, which can be used to indicate the timing for ending the ITP run (e.g., when nucleic acids reach a designated elution location or reservoir). Other detection methods described herein (such as temperature or drive voltage) can also be used to determine the run end timing or other triggers. For example, temperature or voltage sensors can be used to control the electric field applied to channels within the device to automate the ITP process. As an example, an electric field can be applied to a channel to initiate ITP purification. Sensed voltage changes can be used to trigger the initiation of temperature or other sensing at a fixed location within the channel (such as at or near the elution reservoir). The voltage can change as the ITP region containing confined nucleic acids moves. Changes in the ITP region, indicating a decrease in the cross-sectional area of ​​the channel, can be sensed by a voltage sensor, and feedback can be used to alter the electric field, for example, by reducing the applied current. Temperature changes can be detected when the ITP region passes through a temperature sensor at or near the elution reservoir, and feedback from the sensor can be used to control the electric field, for example, by removing it 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 terminates, nucleic acids can be located or isolated within the elution reservoir or region. In step 125, the device can close the channel, which can fix the elution volume to maintain a constant elution volume (e.g., by resisting or preventing flow into the elution reservoir or outlet reservoir during removal of the elution volume). Fixing the elution volume can improve ease of use and can help in reporting the concentration of eluted sample material. In step 126, the device cover may be opened (e.g., by the user or automatically).

[0333] In step 127, the purified sample can be extracted from the device. As discussed herein, the chip and / or device can be designed for a given elution volume. Removal of 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 engaging the ITP chip with another fluidic chip or system (e.g., without an elution reservoir). The purified sample material can then be subjected to further operations using other fluidic systems, 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 volume and / or sample concentration. The device may contain algorithms or other software for converting measurements (e.g., fluorescence signals) into sample quantification and can report this data to the user. In step 129, the process ends.

[0334] Figure 33 A detailed, non-limiting exemplary processing workflow for automating ITP is illustrated using the methods, devices, and systems described herein.

[0335] In step 250, the user can turn on the instrument.

[0336] In step 251, instrument initialization may optionally be performed. Initialization may include one or more of the following checks / steps, such as: (a) checking pressure control tolerances at 0 psi and -0.1 psi; (b) confirming negligible flow rate in the channel with the negative pressure valve closed; (c) observing the proportional threshold; (d) checking the HVS temperature; (e) checking the HVS voltage rail; (f) confirming no voltage when the HVS is disabled; (g) checking the optics temperature; (h) checking the optics voltage rail; (i) confirming an increase in the pickoff value when each LED is turned on; (j) confirming an increase in the detector value when each LED is turned on; and / or (k) confirming the infrared sensor at room temperature or near room temperature.

[0337] In step 252, the instrument may optionally display a "Start" or "Home" menu to the user.

[0338] In step 253, the user can choose to click "Start New Run" on the instrument display.

[0339] In step 254, the instrument door can be opened.

[0340] In step 255, the user can place the new microfluidic chip on the instrument stage.

[0341] In step 256, a “chip in place” sensor may optionally be used to detect whether the chip has been placed on the instrument platform and / or whether the chip has been correctly placed or oriented on the platform.

[0342] In step 257, the instrument may optionally read the barcode identifier on the chip. For example, a user may use a handheld scanner.

[0343] In step 258, the instrument may optionally select the program to run based on the scanned barcode identifier.

[0344] In step 259, the instrument may optionally check the temperature of the instrument (e.g., the heater). For example, the temperature of the heating / cooling element may be read by the instrument. The instrument may read the temperature detected by an optional thermal sensor. The temperature range of the instrument can be confirmed, for example, by checking that the difference between the instrument temperature and the temperature detected by the thermal sensor is no greater than about 5°C.

[0345] In 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.) onto the chip. For example, the instrument may display visual and / or color-coded instructions to the user (e.g., such as...). Figure 60 (As shown).

[0346] In step 261, the user can pipette the buffer solution onto the chip.

[0347] In step 262, the user can close the instrument door, for example by pressing a button on the display.

[0348] In step 263, the instrument may load one or more buffer solutions into the chip. The display may optionally provide the user with an indication that loading is in progress, for example by displaying a "Startup in Progress" message.

[0349] In step 264, the instrument may optionally be checked to ensure that the buffer reagent has been loaded and that the channel has been properly started with the buffer. Loading can be confirmed, for example, by testing the conductivity between the two high-voltage electrodes, as described herein. For example, 10 μA can be drawn from one electrode and 10 μA can be poured in from the other. The voltage difference between the two electrodes can be measured, as described herein. Channel start-up can be confirmed before, during, or after confirming buffer loading. Channel start-up can be confirmed, for example, by ensuring the conductivity between the source electrode and the ground electrode.

[0350] In step 265, after confirming that one or more buffer solutions have been correctly loaded, the instrument can open the door / cap to allow the user to access the chip.

[0351] In step 266, the instrument may optionally display instructions to the user on how to load the sample onto the chip.

[0352] In step 267, the user can pipette the sample onto the chip. The user can optionally remove the seal from the sample well of the chip to achieve sample loading. As described herein, after sample loading, the user can optionally add an additional volume of "top layer" to the sample reservoir.

[0353] In step 268, the user can close the instrument door, for example by pressing a button on the display.

[0354] In step 269, the instrument may optionally verify that the sample has been correctly loaded. As described herein, sample loading can be verified by checking the conductivity of each electrode to ground. After sample loading, while one or more buffer solutions remain unloaded, the instrument may load the remaining buffer solutions onto the chip.

[0355] In step 270, the instrument can begin ITP operation. After loading the chip, the instrument may optionally wait for a predetermined amount of time to allow the fluid within the chip to reach equilibrium. Initiating ITP operation may require activating the high-voltage (“HV”) power supply, adjusting the chip temperature to the operating temperature (“T_run”), and / or turning on the optical detection system (e.g., a light-emitting diode). For example, the operating temperature of a typical ITP procedure can be in the range of approximately 15°C to approximately 23°C.

[0356] In step 271, the instrument may optionally record and process the detected voltage signal, as described herein.

[0357] In step 272, the instrument may optionally detect a change in voltage as a trigger to initiate the ITP, as described herein. The change in voltage may optionally act as a trigger to change the polarity of the drive electrode and / or the drive voltage, as described herein.

[0358] In step 273, the instrument may optionally perform optical detection.

[0359] In step 274, the instrument may optionally process the detected optical signal.

[0360] 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. The temperature change may optionally serve as a trigger to terminate the ITP, as described herein.

[0361] In step 276, the instrument may optionally detect a change in voltage as a trigger to terminate the ITP, as described herein.

[0362] In step 277, the instrument may optionally be triggered to shut down the high-voltage power supply, thereby ending ITP operation. The instrument may also shut down the optical system.

[0363] In step 278, the instrument may optionally use a channel closure device to seal the channel, as described herein.

[0364] In step 279, the instrument may optionally display an indicator or message to the user to remind them that the ITP has been completed.

[0365] In step 280, the instrument may optionally display to the user any quantitative data collected during ITP operation.

[0366] In step 281, the user can return to the machine or be at the machine.

[0367] In step 282, the instrument may optionally maintain the chip at a fixed temperature until the user returns to the instrument, as shown in step 281. For example, this fixed temperature may be in the range of about 4°C to about 20°C.

[0368] In step 283, the user may optionally open the instrument door / cover, for example by pressing a button on the display.

[0369] In step 284, the user may optionally recover the sample from the elution reservoir. Alternatively or in combination, the instrument may optionally automatically recover the sample from the elution reservoir. The sample may then be optionally used for further downstream analysis as desired by the user.

[0370] In step 285, the user can remove the used chip from the instrument.

[0371] In step 286, the chip placement sensor may optionally detect the user's removal of the chip. The barcode information stored on the instrument may be cleared.

[0372] In step 287, if necessary, the user may optionally prepare their reagents and samples for an additional ITP run.

[0373] In step 288, the user can close the door.

[0374] In step 289, the user may optionally repeat steps 253 through 288 using new chips, buffer solutions, samples, etc., as many times as needed. During idle runs between operations, the instrument temperature may optionally be adjusted, for example, to a temperature range of approximately 20°C to approximately 25°C.

[0375] In step 290, the user may optionally transfer the data collected during ITP operation, for example, via a USB port on the instrument or via a wireless connection.

[0376] In step 291, the user may optionally shut down the instrument. In some cases, the instrument can be programmed to shut down after a predetermined idle time when the chip-in-place sensor confirms that there is no chip in the instrument.

[0377] Table 1 shows the usage Figure 33 The typical operating time of each step of the ITP process of the exemplary method described herein, whether these steps are manual (performed by the user) or automatic (performed by the instrument).

[0378] Table 1

[0379] These issues may be particularly important for resolving precious, hard-to-collect, or low-abundance samples (e.g., samples containing less than 100 ng of nucleic acids or containing low abundance of undamaged or uncrosslinked nucleic acids). For such samples, current protocols may lack reproducibility, introduce sample material loss, introduce bias towards short or long nucleic acid targets, introduce bias towards nucleic acid target sequences, and / or lack reproducibility. Such protocols may also lack compatibility with process automation or downstream analysis. Current protocols for nucleic acid preparation may include liquid phase extraction (LPE) such as phenol-chloroform extraction or Trizol extraction, and solid phase extraction (SPE). SPE-type methods may use structures including filled beads, monolithic porous structures, and / or magnetic beads. In some cases, LPE and SPE-type methods can lead to mechanical shearing during processing, which may cause fragmentation and / or reduce the yield of long or high molecular weight nucleic acids.

[0380] The isotachophoresis method and apparatus described herein are particularly well-suited for extracting nucleic acids from lysates of solid or semi-solid tissues. Solid-phase extraction (SPE) techniques typically process lysates by pumping the entire lysate sample volume through a column to selectively adsorb nucleic acids onto the column surface. This pumping of complex lysates (which may contain liquid-particle mixtures) through a porous column can lead to column clogging or fouling, thereby reducing the efficiency of nucleic acid extraction. In contrast, the isotachophoresis method and apparatus described herein typically do not involve pumping or “filtering” the entire lysate sample volume through a column. Instead, an electric field can be applied to the lysate to cause charged, solvated nucleic acids dispersed throughout the composite sample lysate to migrate through and out of the continuous liquid phase of the sample. Nucleic acids can contain relatively high electrophoretic mobility values ​​relative to other solutes, debris, or contaminants in the sample lysate. Solutes in the sample may have relatively low electrophoretic mobility and be too low to focus on the isotachophoretic region located at the interface between the leading and trailing electrolytes. Applying an electric field can induce nucleic acid migration while leaving behind particles and / or other tissue debris (including, for example, cell debris, unlysed cells, or tissues that can connect cells to other cells). Therefore, the isotachophoresis method and apparatus presented herein are well-suited for extracting charged solvated nucleic acids from complex lysed solid tissue samples without having to process the entire mixture through a column as in SPE.

[0381] As used herein, “particle” can refer to a component of a sample mixture or a mixture of sample lysates that is a phase distinct from the continuous liquid phase of the sample (e.g., an aqueous solution). Particles can be non-liquid components of a sample mixture. Particles can be, for example, suspended solid particles or colloids suspended in the sample. Such particles can have a variety of characteristic length scales ranging from about 1 nanometer (nm) to about 1 millimeter (mm). In some cases, particles may not be single-celled organisms or cells.

[0382] Compared to typical SPE methods, the isotachophoresis method and apparatus presented in this paper can provide a reduced strain rate as the sample moves through the channel. In some cases, the strain rate of the method and apparatus presented in this paper is less than about 250 s⁻¹. -1 500s -1 750s -1 1000s -1 2000 s -1 3000s -1 4000s -1 5000s -1 6000s -1 7000s -1 8000s -1 9000s -1 Or 10,000s -1In some cases, the strain rate of the methods and apparatus presented in this paper is greater than approximately 250 s. -1 500s -1 750s -1 1000s -1 2000s -1 3000s -1 4000s -1 5000 s -1 6000s -1 7000s -1 8000s -1 9000s -1 Or 10,000s -1 In some cases, the method presented in this paper can be performed without centrifugation.

[0383] Isotachyphoresis chemical process and operation

[0384] Figure 2A An exemplary schematic diagram of an isotachophoresis (ITP) process for purifying nucleic acids is shown. A sample 201 (e.g., a lysed solid tissue sample) containing nucleic acids (DNA and RNA) 202 and contaminants 203 can be loaded with trailing electrolyte (TE) 204 into an isotachophoresis channel 200 containing a leading electrolyte (LE) 205. Under the influence of an electric field 220 applied to the isotachophoresis channel 210, nucleic acids 212 can migrate away from contaminants 213. The electric field can also cause trailing electrolyte 214 to migrate through a channel that is typically located behind the nucleic acids, and typically cause leading electrolyte 215 to migrate through a channel that is typically in front of the nucleic acids. The effective migration rate of the leading electrolyte is greater than that of the nucleic acids, and in turn, the effective migration rate of the nucleic acids is greater than that of the trailing electrolyte, while the effective migration rate of the trailing electrolyte is greater than that of the contaminants.

[0385] Figure 2BAn exemplary schematic diagram illustrates a process of simultaneously decrosslinking nucleic acids and crosslinked nucleic acids with contaminants (e.g., paraffin) using isotachyphoresis (ITP) on a fluid apparatus. In some cases, the contaminants may contain crosslinked nucleic acids. Paraffin-embedded samples can be loaded onto the fluid apparatus in an alkaline buffer and incubated for 10–30 minutes at approximately pH 10 and approximately 50°C to approximately 80°C for tissue lysis and initial dewaxing. Incubation can be performed before or simultaneously with isotachyphoresis under an applied electric field. Alternatively, the sample can be loaded into a lead electrolyte buffer. After incubation, at a first time point 240, the sample containing crosslinked nucleic acids 236 and paraffin 237 can be located in an ITP channel with a trailing electrolyte 232. In front of the ITP channel, in the lead electrolyte (LE) region 238, are the lead 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 contacts and decrosslinks the crosslinked nucleic acids, producing uncrosslinked nucleic acids 235, which focus in the ITP region 239 between the trailing and leading electrolytes. The decrease in pH (e.g., in the range of about 10-12 to about 7 (or about 6.5 to about 8.5)) provides an environment suitable for enzyme activity and improved nucleic acid chemical stability. At a third time point 260, proteinase K decrosslinks more nucleic acids, producing free proteins 234, and the decrosslinked nucleic acids further migrate upstream from paraffin, free proteins, and other contaminants. This process can be operated by a fluidic apparatus or automated by a benchtop system.

[0386] In some cases, samples may be loaded in a sample buffer containing a concentration of lead electrolytes 205 and 231, different from the concentration of lead electrolytes 205 and 231 used for isotachophoresis. In other cases, samples may be loaded in a sample buffer containing a second lead electrolyte, different from lead electrolyte 215. The second lead electrolyte may have an effective migration rate greater than the effective migration rate of nucleic acids. The second lead electrolyte may have an effective migration rate less than the effective migration rate of lead electrolyte 215.

[0387] In some cases, the pH of a sample can be quenched by performing isotachophoresis. In some cases, the pH of the sample can be quenched to a range of approximately 6.5 to approximately 8.5 (e.g., approximately 7 or 7.5).

[0388] Various lead and follow-up electrolytes can be used for ITP. Lead electrolytes can be selected to have a higher effective migration rate than the extraction target (e.g., nucleic acid), and follow-up electrolytes can be selected to have a lower effective migration rate than the extraction target. Lead and / or follow-up electrolytes can be present at concentrations from about 10 mM to about 200 mM. Lead and / or follow-up electrolytes can be present at concentrations 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 electrolyte and / or trailing electrolyte may be present at concentrations 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 electrolyte and / or trailing electrolyte may be present at concentrations of up to 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 a specific ITP condition may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different ion species. The trailing electrolyte used in a specific ITP condition may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different ion species. Different types of ions in the leading electrolyte and / or trailing electrolyte may be present at different concentrations. Different concentrations of ions (e.g., in tailing electrolytes or leading electrolytes) can be selected to manipulate the size of the spacer region. The spacer region can be used to further separate one type of target from another, such as separating uncrosslinked nucleic acids from protein-crosslinked nucleic acids.

[0389] The trailing electrolyte can contain a mixture of ions with different effective migration values. Using a first trailing electrolyte ion with a first effective migration value and a second trailing electrolyte ion with a second effective migration value lower than that of the first ion can be used to separate non-crosslinked nucleic acids from protein-crosslinked nucleic acids, while simultaneously separating both nucleic acids (or at least uncrosslinked nucleic acids) from contaminants. In this case, non-crosslinked nucleic acids may have a larger effective migration value than the first trailing electrolyte ion, the first trailing electrolyte ion may have a larger effective migration value than the crosslinked nucleic acid, and conversely, crosslinked nucleic acids may have a larger effective migration value than the second trailing electrolyte ion, and conversely, the second trailing electrolyte ion may have a larger effective migration value than the contaminant. For example, crosslinked and non-crosslinked nucleic acids can be enriched separately by isovelocity electrophoresis using a leader electrolyte and two trailing electrolytes (such as hexanoic acid as the first ion and HEPES as the second ion).

[0390] Electrolyte ions can also be selected based on acidity (e.g., pKa). Ions with specific pKas can be selected, for example, to induce pH changes along ITP channels. Ions can also be selected for non-electrophoretic reasons, such as compatibility with downstream processes (e.g., enzymatic processes like PCR or next-generation sequencing library preparation). For example, hexanoic acid, MOPS, and HEPES can be selected to achieve good downstream enzymatic compatibility.

[0391] Exemplary leading electrolyte ions include, but are not limited to, hydrochloric acid, acetic acid, 2-chloroisocrotonic acid, salicylic acid, clotrimazole, nicotinic acid, gallic acid, trichlorolactic acid, butyric acid, p-aminobenzenesulfonic acid, benzoic acid, crotonic acid, trichloroacrylate, propionic acid, levulinic acid, sorbic acid, orotic acid, valeric acid, picric acid, 2-naphthalenesulfonic acid, saccharin, dinitrophenol, p-toluenesulfonic acid, aspartic acid, trimethacrylic acid, isohexanoic acid, hexanoic acid, octylsulfonic acid, nitrophenol, GABA, dimethylarsonic acid, trimethylpyruvic acid, ethylmaleic acid, ethylfumaric acid, benzoic acid, heptanoic acid, mandelic acid, cinnamic acid, cresol, glutamic acid, MES, their isomers, and combinations thereof.

[0392] Exemplary trailing electrolyte ions include, but are not limited to, caprylic acid, gluconic acid, vanillic acid, decyl sulfonic acid, aspirin, glucuronic acid, nonanoic acid, benzyl aspartic acid, ascorbic acid, dodecyl sulfonic acid, MOPS (3-(N-morpholino)propanesulfonic acid), dichlorophenol, hexanoic acid, decanoic acid, tyrosine, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), their isomers, and combinations thereof.

[0393] Using mixtures of different trailing electrolyte ions can be used to achieve in-bracket separations (e.g., separation of non-crosslinked nucleic acids from crosslinked nucleic acids and contaminants), improved mobility, compatibility with downstream assays, favorable surface energy or contact angle between fluid and fluid device materials, buffering capacity, and total ion solubility.

[0394] A lead electrolyte can be loaded into a lead electrolyte buffer. The lead electrolyte buffer may contain one or more lead electrolytes to compress the target nucleic acid in the channel after the lead electrolyte is applied during ITP. The lead electrolyte can also separate the target nucleic acid from any contaminants or inhibitors that have a greater effective migration rate than the target nucleic acid. The lead electrolyte buffer may, for example, contain chloride. The lead electrolyte buffer may contain a sufficient concentration of chloride such that the separating power of the lead electrolyte buffer is greater than the ionic strength of the sample nucleic acid. The lead electrolyte buffer may contain a pH compatible with nucleic acid stability. The lead electrolyte buffer may contain surfactants (e.g., Tween) to reduce or minimize electroosmotic flow. The lead electrolyte buffer may contain surfactants (e.g., Brij-35) to reduce or minimize surface adsorption. The lead electrolyte buffer may contain one or more surfactants as described herein. The concentration of one or more surfactants can be adjusted to ensure that the LE does not uncontrollably permeate the capillary barrier (e.g., platform capillary barrier) described herein.

[0395] In some implementations, a leading electrolyte can be loaded into a high-concentration leading electrolyte buffer, which can act as a buffer against a lower-concentration leading electrolyte buffer. The high-concentration leading electrolyte buffer can have sufficient buffering capacity to prevent pH changes during electrolysis that occurs throughout the ITP run. The high-concentration leading electrolyte buffer can contain one or more leading electrolytes, which can be the same as the leading electrolyte in the leading electrolyte buffer but at a higher concentration. The high-concentration leading electrolyte buffer can contain Tris and chloride. The high-concentration leading electrolyte buffer can contain a Tris to chloride ratio configured to maximize buffering capacity, such as a high Tris:chloride ratio, to provide a Tris source during ITP run. The high-concentration leading electrolyte buffer can contain 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 is loaded onto the capillary barrier when negative pressure is applied. The concentration of one or more surfactants can be adjusted to ensure that the LE does not uncontrollably permeate the capillary barrier (e.g., a ramp barrier) described herein.

[0396] In some implementations, a lead electrolyte can be loaded into the elution buffer. The elution buffer may contain one or more lead electrolytes. One or more lead electrolytes in the elution buffer may have a lower ionic strength than the lead electrolyte in the lead electrolyte buffer. One or more lead electrolytes can enable switching of the ITP band from a lead electrolyte buffer with a higher ionic strength to an elution buffer with a lower ionic strength. The elution buffer can 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, such as 10 mM Tris-HCl. The elution buffer may contain a pH compatible with nucleic acid stability. The lead electrolyte buffer may contain a surfactant (e.g., Tween) to reduce or minimize electroosmotic flow. The lead electrolyte buffer may contain a surfactant to reduce or minimize bubble growth in the fluid channels (e.g., during temperature measurements described herein). The lead electrolyte buffer may contain a surfactant to reduce or minimize surface adsorption. The elution buffer may contain one or more surfactants as described herein. The concentration of one or more surfactants can be adjusted to ensure that the elution buffer does not uncontrollably permeate the capillary barrier (e.g., ramp barrier) described herein.

[0397] In some implementations, a lead electrolyte may be loaded into a high-concentration elution buffer that can buffer a lower-concentration elution buffer (e.g., a lower concentration suitable for extraction and use in the downstream assays described herein). The high-concentration elution buffer may have sufficient buffering capacity to prevent pH changes during electrolysis that occurs throughout the ITP run. The high-concentration elution buffer may have a minimal (e.g., less than 1 μl) amount of residue between the high-concentration elution buffer and the elution buffer. The high-concentration elution buffer may contain sufficiently low ion concentrations so that such residues do not affect downstream compatibility. The high-concentration elution buffer may contain Tris and chloride. The high-concentration elution buffer may contain Tris and chloride at a ratio configured to maximize buffering capacity, such as a high Tris:chloride ratio, to provide a Tris source during ITP runs. The high-concentration elution buffer may contain sufficiently high ion concentrations of Tris and chloride to achieve robust buffering while minimizing the effects of residues. High-concentration elution buffers may contain one or more surfactants to ensure that the high-concentration elution buffer wets the walls of the high-concentration elution buffer reservoir and is loaded onto the capillary barrier when negative pressure is applied. The concentration of one or more surfactants may be adjusted to ensure that the high-concentration elution buffer does not uncontrollably permeate the capillary barrier (e.g., a ramp barrier) described herein.

[0398] Tail electrolytes can be contained in a tail electrolyte buffer. The tail electrolyte buffer may contain one or more tail electrolytes that compress the target nucleic acid in the channel before the tail electrolyte is applied during ITP. The tail electrolyte can also separate the target nucleic acid from any contaminants or inhibitors that have a lower effective migration rate than the target nucleic acid. The tail electrolyte buffer may have sufficient buffering capacity to prevent pH changes during electrolysis that occurs throughout the ITP run. The tail electrolyte buffer may contain, for example, hexanoic acid. The tail electrolyte buffer may contain MOPS. The tail electrolyte buffer may contain both hexanoic acid and MOPS. The tail electrolyte buffer may contain high concentrations of hexanoic acid. High concentrations of hexanoic acid can lead to overwetting of the fluid. MOPS can be added to the hexanoic acid in the tail electrolyte buffer to provide the necessary buffering capacity in the tail electrolyte reservoir without increasing the wetting of excessively high concentrations of hexanoic acid. The tail electrolyte buffer may contain a pH compatible with nucleic acid stability.

[0399] The lead electrolyte buffer may contain one or more surfactants described herein (e.g., Tween). The concentration of one or more surfactants may be adjusted to ensure that the TE does not uncontrollably permeate the capillary barrier described herein (e.g., plateau capillary barrier). The concentration of one or more surfactants may be adjusted to prevent or reduce the generation of bubbles during electrolysis, avoiding the generation of large bubbles that could cause fluid fluctuations and thus interfere with the ITP band, voltage trace, and / or temperature trace, or the generation of very large bubbles that could move and negatively affect triggering.

[0400] Isotachymeter electrophoresis can quench the pH of a sample to neutral or near-neutral. Ions that affect local pH (e.g., sodium ions (Na+)) can migrate from the sample region during isotachymeter electrophoresis, thereby shifting the pH of the sample region to neutral.

[0401] Isokinetic electrophoresis can be performed within a certain range of voltage, current, and electric field strength. For example, it can be performed at voltages from approximately 100V to approximately 1500V. Isokinetic electrophoresis can be performed at voltages of approximately 100V, 200V, 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1100V, 1200V, 1300V, 1400V, or 15000V. Isokinetic electrophoresis can be performed at voltages of at least approximately 100V, 200V, 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1100V, 1200V, 1300V, 1400V, or 15000V. Isotachyphoresis can be performed at voltages of up to approximately 100V, 200V, 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1100V, 1200V, 1300V, 1400V, or 15000V. Isotachyphoresis can be performed at currents of approximately 10nA to approximately 10mA. Isokinetic electrophoresis can be performed at currents of approximately 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. Isotachyphoresis can be performed at currents of at least approximately 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. Isotachyphoresis can be performed at currents of up to approximately 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. Isotachyphoresis can be performed at electric field strengths ranging from approximately 10 V / cm to approximately 100 V / cm.Isotachymetry can be performed at field strengths of approximately 10 V / cm, 15 V / cm, 20 V / cm, 25 V / cm, 30 V / cm, 35 V / cm, 40 V / cm, 45 V / cm, 50 V / cm, 55 V / cm, 60 V / cm, 65 V / cm, 70 V / cm, 75 V / cm, 80 V / cm, 85 V / cm, 90 V / cm, 95 V / cm, or 100 V / cm. Isotachymetry can be performed at field strengths of at least approximately 10 V / cm, 15 V / cm, 20 V / cm, 25 V / cm, 30 V / cm, 35 V / cm, 40 V / cm, 45 V / cm, 50 V / cm, 55 V / cm, 60 V / cm, 65 V / cm, 70 V / cm, 75 V / cm, 80 V / cm, 85 V / cm, 90 V / cm, 95 V / cm, or 100 V / cm. Isotachymetry can be performed at electric field strengths of up to approximately 10 V / cm, 15 V / cm, 20 V / cm, 25 V / cm, 30 V / cm, 35 V / cm, 40 V / cm, 45 V / cm, 50 V / cm, 55 V / cm, 60 V / cm, 65 V / cm, 70 V / cm, 75 V / cm, 80 V / cm, 85 V / cm, 90 V / cm, 95 V / cm, or 100 V / cm.

[0402] Isotachyphoresis can be used to concentrate nucleic acids in a sample. The concentration of nucleic acids in the sample can be increased by at least approximately 2, 5, 10, 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, or 1,000,000,000. The operation time for concentrating nucleic acids using isotachyphoresis can be less than or equal to approximately 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. In some cases, isotachophoresis can be used to increase the concentration of nucleic acids in a sample by 1,000,000-fold in less than or equal to about 2 minutes. In some cases (e.g., samples from 25 μL of blood lysate), isotachophoresis can be used to increase the concentration of nucleic acids in a sample by 100,000-fold in less than or equal to about 5 minutes.

[0403] The technology disclosed herein can be used to reduce the concentration of cross-linked nucleic acids in a sample. The concentration of cross-linked nucleic acids in the sample can be reduced by at least approximately 2, 5, 10, 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, or 1,000,000,000. Isotachyphoresis can also be used to reduce the concentration of contaminants in a sample. The concentration of contaminants in the sample can be reduced by at least approximately 2, 5, 10, 100, 1,000, 10,000, 100,000, 100,000, 10,000,000, 100,000,000, or 1,000,000,000.

[0404] Nucleic acid samples 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. Nucleic acid samples may contain 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, 9 ng, 10 ng, 20 ng, 30 ng. 40ng, 50ng, 60ng, 70ng, 80ng, 90ng, 100ng, 200ng, 300ng, 400ng, 500ng, 600ng, 700ng, 800ng, 900ng, 1μg, 2μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12μg, 13μg, 14μg, 15μg, 16μg, 17μg, 18μg, 19μg, 20μg, 21μg, 22μg, 23μg, 24μg or 25μg.

[0405] Nucleic acid samples may contain 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. Nucleic acid samples may contain lengths 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 longer. The techniques of this disclosure can be used to extract different sample types in different channels of a fluidic apparatus. For example, different channels can be used to extract nucleic acids of different lengths and / or different types.

[0406] In some cases, the characteristics of a nucleic acid sample can be compared with one or more nucleic acids from another sample. These characteristics can be, for example, expression levels, nucleic acid sequence, molecular weight, nucleic acid integrity, nucleic acid chain type, or nucleic acid purity.

[0407] Nucleic acid samples may have specific quality before and / or after extraction or other processing. Nucleic acid quality can be assessed by a variety of metrics, including but not limited to RNA integrity number (RIN), DNA integrity number (DIN), size distribution (e.g., using electrophoresis), and amplification capacity (e.g., by PCR) or by other enzymatic treatments (e.g., fragmentation, ligation, α-tailing, or hybridization for next-generation sequencing library preparation). The technology disclosed herein can be used to extract or process nucleic acids and provides RINs 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, Nucleic acids extracted or processed at levels 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, 8.0, 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 technology disclosed herein can be used to extract or process nucleic acids, and provides RINs of up to approximately 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, Nucleic acids extracted or processed at levels 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, 8.0, 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 technology disclosed herein can be used to extract or process nucleic acids, and provides DIN values ​​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. Nucleic acids extracted or processed at 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 technology disclosed herein can be used to extract or process nucleic acids, and provides DIN values ​​of up to 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, Nucleic acids extracted or processed at levels 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 technology disclosed herein can be used to extract or process nucleic acids, and provides extracted or processed nucleic acids such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% by mass of nucleic acids in a sample have 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 higher. In some cases, about 90% to about 100% by mass of the processed nucleic acid is about 10 bp to about 1000 bp, about 200 bp to about 2000 bp, or about 200-5000 bp.

[0408] Isotropic electrophoresis can be used to extract nucleic acids at a certain extraction efficiency or yield (described as the percentage of nucleic acids produced from a given starting amount of nucleic acid). The techniques of this disclosure can provide extracted nucleic acids at yields 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%. Even for low input amounts of nucleic acids (including less than or equal to about 10... 4 10 ng 3 ng, 10 2 ng, 10 1 ng、 10 0 ng, 10 -1 ng or 10 -2 The technology disclosed herein can also provide high yields. For example, Figure 3 Exemplary nucleic acid yields are shown for a range of different input amounts and nucleic acid sources. High nucleic acid yields and / or low losses can be important for next-generation sequencing library preparation. Nucleic acid recovery rates can be 100% or close to 100%.

[0409] The techniques disclosed herein can extract nucleic acids with low or no sequence bias. That is, the sequence composition of the extracted and purified nucleic acids (e.g., the ratio of GC-rich nucleic acids to AT-rich nucleic acids) can be similar to or identical to the sequence composition of the input nucleic acids (see, for example, Figure 4A The difference between the sequence composition of the extracted nucleic acid and the sequence composition of the input nucleic acid can be less than or equal to approximately 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0410] The technology disclosed herein can extract nucleic acids with low or no length bias. That is, the length distribution of the extracted nucleic acids (e.g., the proportion of nucleic acids of different sizes) can be similar to or the same as the length distribution of the input nucleic acids (see, for example, ...). Figure 4B The length distribution of the extracted nucleic acid may differ from that of the input nucleic acid by less than or equal to about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, short nucleic acids (e.g., about 10 kB to about 300 kB), long 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 longer), or both short and long nucleic acids can be extracted with reduced loss or bias. In some cases, solid-phase columns may lose up to 100% of the short and / or long nucleic acid material. The techniques of this disclosure can recover nucleotides ranging in size from a single base to hundreds of kilobases. The technology disclosed herein can recover at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of short and / or long nucleic acids present in a sample.

[0411] The techniques disclosed herein can lead to the removal of contaminants from samples. Contaminants may include, but are not limited to, embedding materials, cell debris, extracellular matrix components, tissue debris, embedding fragments, lipids, carbohydrates, enzymes, ligation byproducts, primers, unbound probes or ligants, divalent metals, detergents, preservatives, fixatives, anticoagulants, collagen fibers, and PCR inhibitors. Contaminants may originate from tissues or cells in the sample, from preservatives or embedding materials used on the sample, or from previous preparations, reactions, or assays performed on the sample. For example, enzymes such as restriction nucleases can be used to prepare DNA for fingerprint analysis assays, and the DNA can be separated from the enzyme after digestion (e.g., DNase digestion).

[0412] sample

[0413] The techniques disclosed herein can be used to process various sample types, including but not limited to 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, adhesion junctions), samples containing lysed solid tissues and nucleic acids, multiphase samples, heterogeneous liquids or solutions (e.g., tissues, whole blood, or unlysed cell suspensions), biological samples containing genomic DNA, lysed and unlysed whole blood, plasma, and serum, oral swabs, dried blood spots, and other forensic samples, fresh or fresh frozen (FF) tissues, cells cultured or harvested from blood or tissues (lysed and unlysed), fixed cells, feces and bodily fluids (e.g., saliva, urine), or any combination thereof. Non-limiting examples of solid organs include the liver, pancreas, brain, heart, gallbladder, colon, lungs, and reproductive organs. For both eukaryotes and prokaryotes, samples may include cellular nucleic acids and cell-free nucleic acids. Fixed samples may be chemically or physically fixed (e.g., heated or frozen). For example, samples may be chemically fixed using fixatives such as formalin, neutral buffered formalin (NBF), formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, mercuric chloride, zinc salts, Bouin's fluid, ethanol-formaldehyde-acetic acid (AFA or FAA), citrate-acetone-formaldehyde (CAF), acetone, methanol, ethanol, Clarke's solution, Carnoy's fluid, or Puchtler's methacarn. Embedded samples may be embedded in materials including, but not limited to, waxes (e.g., paraffin), agar, gelatin, or plastic resins. Formalin-fixed paraffin-embedded (FFPE) samples can be processed using the techniques described in this disclosure. Samples may include oral swabs, bloodstains, and other forensic samples. Samples may include clinical samples, fine-needle aspirates, biopsies, whole blood, lysed blood, serum, plasma, urine, cell culture lysates or freshly harvested cell lysates (e.g., blood cells, isolated fresh tissue, stem cells), blood cells, circulating cells (e.g., circulating tumor cells (CTCs)), nucleic acids from blood or other body fluids, and other sample categories. Cell-free nucleic acids (e.g., cfDNA or cfRNA) can be recovered using the techniques described in this disclosure, such as from unlysaturated whole blood; typically, cell-free nucleic acids are circulating cell-free nucleic acids. Samples may come from a variety of sources, including but not limited to normal tissues, benign tumors, malignant tumors, stem cells, human tissues, animal tissues, plant tissues, bacteria, viruses, and environmental sources (e.g., water).Human or animal tissues may include, but are not limited to, epithelial tissue, connective tissue (e.g., blood, bone), muscle tissue (e.g., smooth muscle, skeletal muscle, cardiac muscle), and nervous tissue (e.g., brain, spinal cord).

[0414] The sample may contain one or more suspended particles. These particles can range in size from colloidal to visible. The particles may have a size of 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, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, etc. 275μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm or 1 millimeter (mm) size. The one or more particles may have a maximum size of approximately 1 nanometer (nm), 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 600nm, 700nm, 800nm, 900nm, 950nm, 1 micrometer (μm), 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 150μm, 175μm, 200μm, 225μm, 250μm. The particle sizes are 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 can be the same size or different sizes. A sample can, for example, contain multiple particles ranging in size from 1 nm to 500 μm.

[0415] Samples of various volumes can be processed on fluid devices (e.g., for the extraction and purification of 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. Sample volumes (with or without buffer) can be up to approximately 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 approximately 1 nL to approximately 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.

[0416] Samples with varying cell numbers can be processed on fluidic devices (e.g., for nucleic acid extraction and purification). For example, samples may contain approximately 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, 100, 90, 80, 70, 60, 50, 40, 90, 80, 70, 60, 50, or 40 cells. 30 cells, 20 cells, 10 cells, 5 cells, 2 cells, or 1 cell. In some cases, the sample contains at least approximately 10,000,000 cells, 5,000,000 cells, 1,000,000 cells, 500,000 cells, 100,000 cells, 50,000 cells, 20,000 cells, 15,000 cells, 10,000 cells, 9,000 cells, 8,000 cells, 7,000 cells, 6,000 cells, 5,000 cells, 4,500 cells, 4,000 cells, 3,500 cells, 3,000 cells, 2,500 cells, 2,000 cells, 1,500 cells, 1,000 cells, 900 cells, 800 cells, 700 cells, etc. 1 cell, 600 cells, 500 cells, 400 cells, 300 cells, 200 cells or 100 cells.

[0417] Samples of varying qualities can be processed on fluidic devices (e.g., for the extraction and purification of nucleic acids). For example, a sample may contain approximately 0.001 mg to approximately 10 mg of tissue. Samples may contain up to approximately 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 may 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 may contain approximately 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.

[0418] Samples containing varying amounts of nucleic acids can be processed on fluidic instruments (e.g., for the extraction and purification of nucleic acids). For example, samples may contain less than or equal to about 1 microgram (1 μg), 100 nanograms (ng), 10 ng, 1 ng, 100 picograms (pg), 10 pg, or 1 pg of nucleic acid. In some cases, samples may contain greater than or equal to about 1 microgram (1 μg), 100 nanograms (ng), 10 ng, 1 ng, 100 picograms (pg), 10 pg, or 1 pg of nucleic acid.

[0419] Samples can be loaded into a sample buffer. The sample buffer may contain a lysis agent or surfactant to lyse the input sample during off-chip processing, thereby providing access to the target nucleic acid. The sample buffer may contain one or more lead electrolytes. The sample buffer may be sufficiently wettable to be automatically loaded into the sample channels due to 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 fluid channel walls. The sample buffer may contain an ionic content optimized to have sufficient salt for lysis and / or nucleic acid preservation while still allowing for nucleic acid separation. Those skilled in the art will understand that the higher the ionic content of the sample buffer (or any buffer described herein), the greater the current required to transfer the charge of the sample buffer.

[0420] Samples can be loaded into a buffer containing a trailing electrolyte or a leading electrolyte. Samples can be loaded into a buffer containing a second leading electrolyte, which is different from the leading electrolyte used for ITP. Samples can be loaded into a buffer such as an alkaline aqueous buffer or a neutral aqueous buffer. Exemplary alkaline solutions or buffers (e.g., for DNA extraction) may contain 30-120 mM NaOH (in some cases, 40-80 mM NaOH) at a pH of about 10-13 (in some cases, with at least one other component). In some cases, when samples are lysed by treatment with an alkaline solution or buffer before being loaded onto the chip, the lysed sample can subsequently be quenched by adding an acidic solution or buffer to bring the pH of the lysed sample to the range of about 7.5 to about 8.5 before isotachophoresis. Exemplary aqueous buffers (e.g., for DNA or RNA extraction) may contain 2-150 mM Tris-HCl (pH about 7 to about 8) or BisTris-HCl (pH about 5.8 to about 7.3), having at least one other component. Other components used in the buffer may include nonionic surfactants or detergents, ionic or zwitterionic surfactants or detergents, dissociation agents, disulfide bond reducing agents, proteases, nucleases, and other additives or components that are digested, denatured, destroyed, or degraded for the purpose of extracting, purifying, enriching, or otherwise isolating nucleic acids.

[0421] Samples can be loaded into a buffer containing added trailing or leading electrolytes to reduce or minimize nucleic acid retention at capillary barriers, particularly at the capillary barrier between the sample and the elution buffer (LE). For example, a small amount of trailing electrolyte (e.g., MOPS and / or hexanoic acid) can be added to the lysate sample to intentionally slow down the compression of the DNA ITP band. It is not desirable to be bound by any particular theory, but it is thought that this may help maintain DNA in a more dispersed state as it passes through the contraction space of the capillary barrier (e.g., the cliff capillary barrier at the junction between the sample and the LE), which could further impair the passage of a more compact ITP band. Because the incorporated TE has a slower migration rate than DNA and LE, the TE may lag behind once the ITP band enters the LE buffer, allowing the ITP band to be sufficiently compressed before reaching the elution reservoir.

[0422] Nonionic surfactants or detergents may include, but are not limited to, surfactants from the following categories: octylphenol ethoxylates, polysorbates, poloxamers, or polyoxyethylene. Octylphenol ethoxylate surfactants may include, but are not limited to, branched octylphenoxy polyethylene glycol (IGEPAL CA-630) and tert-octylphenoxy polyethylene glycol (Triton). TM X-100) or other polyethylene oxide chains having aromatic lipophilic or hydrophobic groups. Polysorbate surfactants may include, but are not limited to, polyethylene glycol dehydrated sorbitol monolaurate. Polyethylene glycol dehydrated sorbitan monooleate Or dehydrated sorbitan monooleate Poloxamer surfactants (i.e., block copolymers based on ethylene oxide and propylene oxide) may include, but are not limited to, polyoxyethylene-polyoxypropylene block copolymers. Or polyethylene glycol-polypropylene glycol block copolymer Polyoxyethylene surfactants may include, but are not limited to, nonylphenoxy polyethylene glycol (NP-40).

[0423] Nonionic surfactants or detergents may include, but are not limited to, (For example, CA-630), Triton TM X-100 NP-40, including Other block copolymers of (e.g., F-68 or F-127), Polyethylene glycol-modified polymers or copolymers. Nonionic surfactants or detergents can be used to reduce or prevent biomolecule adsorption onto channel walls, or to control the wetting and / or surface tension properties of fluids to control sample loading into fluid devices. Nonionic surfactants or detergents can be present at concentrations 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 may include, but are not limited to, sodium dodecyl sulfate (e.g., at 0.01-2% w / v), sodium dodecylbenzenesulfonate (e.g., at 0.01-2% w / v), sodium cholesterol sulfate (e.g., at 0.01%-2% w / v), and sodium deoxycholate (e.g., at about 10-1000 mM). The dissociation agent may include, but is not limited to, urea (e.g., about 0.5-9.5M, or in some cases, 5-9.5M), thiourea, butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, lithium chloride, magnesium chloride, phenol, and propanol. For example, 7.0M urea and 2.0M thiourea can be used in a 5-50mM Tris-HCl (in some cases, 10-20mM Tris-HCl) buffer solution for RNA or DNA extraction, or for total nucleic acid extraction. The ratio of urea to 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 may include, but are not limited to, DTT (e.g., at about 0.1-40 mM, or in some cases about 10 mM) and β-mercaptoethanol (e.g., at about 0.5-2%, or in some cases about 1%). Proteases may include, but are not limited to, proteinase K, proteases, endopeptides (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Nucleases may include, but are not limited to, nonspecific nucleases such as DNases including 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 may also include specific nucleases (e.g., restriction enzymes) that can cleave specific nucleic acid sequences and produce predictable fragment sizes and fragment size distributions. In some cases, one or more methods or processes provided herein are performed without the use of nucleases, DNases, or RNases. For example, methods provided herein include RNA extraction without the use of DNases.

[0424] Restriction enzymes may include, but are not limited to, type I to 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 approximately 20°C to approximately 37°C. Other nucleic acid modifying enzymes, such as transposases, ligases, polymerases, and phosphatases, may be used. Other protein or polynucleotide digestive agents or degrading agents, such as lysozyme, may be used.

[0425] Samples may undergo varying degrees of pretreatment before being loaded onto a fluidic device. In some cases, samples may simply be loaded into a buffer solution before being loaded onto the fluidic device, and any other necessary or required sample preparation steps may be performed on the device. In other cases, samples may be added to a sample reservoir pre-filled with a processing fluid, such as a solution or buffer solution. In still other cases, samples may undergo embedding material removal, tissue disruption, cell lysis, or digestion before being loaded onto the fluidic device. In one example, the sample is dewaxed before being loaded onto the fluidic device, and nucleic acid decrosslinking is performed on the fluidic device. In another example, the sample is dewaxed, disrupted, and lysed before being loaded onto the fluidic device, and optionally, nucleic acid decrosslinking is performed on the fluidic device. In yet another example, the sample is dewaxed before being loaded onto the fluidic device, and tissue disruption and cell lysis are performed on the fluidic device. In yet another example, the sample is loaded onto the fluidic device, and dewaxing, tissue disruption, cell lysis, and nucleic acid decrosslinking are all performed on the fluidic device. Sample preparation steps are further discussed in this disclosure.

[0426] Sample preparation

[0427] Sample preparation can be performed prior to isotachophoresis. Sample preparation may involve steps including, but not limited to, removal of embedding materials, tissue disruption, cell lysis, protein digestion, removal of nucleic acid crosslinks, isothermal enzymatic processes, enzymatic amplification, enzymatic digestion, disruption of cell-cell junctions, disruption of the extracellular matrix, disruption of connective tissue, and combinations thereof. Sample preparation may involve techniques such as polymerase chain reaction (PCR) or other nucleic acid amplification, isolation or purification of materials of interest (e.g., cells, nucleic acids), probe hybridization, and antibody hybridization (e.g., antibody-nucleosome hybridization). In some cases, samples can be prepared for further analysis by isolating a portion of cellular material from the sample. For example, circulating tumor cells can be isolated from heterogeneous cell populations using cell sorting equipment such as flow cytometry or magnetized columns. In another instance, peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs) can be isolated from blood samples. Sample preparation can be performed on-device or off-device. In some cases, some sample preparation steps are performed off-device, and the sample is subsequently loaded onto a fluidic device where additional sample preparation steps are performed.

[0428] Biological material (e.g., cells, tissues, nucleic acids) in embedded samples can be removed from the embedding material. For example, paraffin-embedded samples can be dewaxed. Removal of the embedding material can be performed using techniques including, but not limited to, heat treatment, chemical treatment (e.g., acid or alkali), enzymatic treatment, and combinations thereof. Dewaxing can be performed by chemically treating the sample, by heat treatment, by enzymatic treatment, or by other methods. For example, dewaxing can be performed at elevated temperatures (e.g., from about 50°C to about 80°C) in the presence of a neutral buffer or a slightly acidic buffer (e.g., as low as about pH 5.5), a slightly alkaline buffer (up to about pH 9), or an alkaline solution (e.g., pH from about 12 to about 13). Removal of the embedding material can be performed outside or on the device. In one example, the embedded sample can be incubated in a container at an elevated temperature and then loaded onto a fluid device. In another example, the embedded sample can be loaded onto a fluid device and incubated on the device (e.g., in a channel or reservoir) at an elevated temperature.

[0429] The embedded material can be removed by heat treatment. The incubation for removing the embedded material can be carried out at temperatures 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. The incubation for removing the embedded material can also be carried out at temperatures 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°C to about 99.5°C. Incubation for removing embedded material can be performed for a duration 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 for removing embedded material can also be performed for a duration 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 for removing embedded material can be performed, for example, at a temperature of at least about 37°C for a duration of at least about 1 minute. Incubation for removing embedded material can be performed in the presence of an alkaline buffer or a neutral buffer (e.g., lysis buffer). Alkaline buffers (e.g., lysis buffers) may 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. Neutral buffers may have a pH of about 7.0 (e.g., about 7 to about 8).

[0430] Tissues or cells can be disrupted or lysed to release nucleic acids for isolation, purification, or extraction. Tissue disruption or cell lysis can be achieved using techniques including, but not limited to, mechanical stress, acoustic treatment, electroporation, osmotic pressure, chemical treatment (e.g., acid or alkali), enzymatic treatment, thermal treatment, and combinations thereof. For example, pressure can be used to drive tissue through a structure (e.g., a channel, a resin such as a glass frit or porous resin or glass material) to mechanically disrupt the tissue or lyse cells. In some cases, the trailing electrolyte buffer may contain one or more tissue disruptors and / or cell lysants. In some cases, the leading electrolyte buffer may contain one or more tissue disruptors and / or cell lysants. In some cases, the removal of the embedding material can be achieved through 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 the removal of the embedding material, tissue disruption, and cell lysis. Tissue disruption or cell lysis can be performed off-device or on-device. In one instance, the tissue sample is disrupted in a container and subsequently loaded onto a fluid device. In another instance, a tissue sample previously loaded onto a fluid device was destroyed on the device.

[0431] Samples containing tissues or cells can be lysed using a lysis solution or buffer compatible with isotachyphoresis before or after loading onto a fluidic apparatus. Isotachyphoresis-compatible lysis buffers may include nonionic surfactants or detergents, ionic or zwitterionic surfactants or detergents, dissociative agents, disulfide bond reducing agents, proteases, nucleases, and other additives or components digested, denatured, destroyed, or degraded for the purpose of extracting, purifying, enriching (concentrating), or otherwise isolating nucleic acids. In some cases, the lysis buffer may contain an alkaline buffer. In some cases, the lysis buffer may not contain an alkaline buffer. Exemplary lysis buffers may include 0.5M to 9.5M, 4M to 9M, or 6.5M to 7M urea as described herein. Exemplary lysis buffers may include 0.5M to 3.5M or 1.5M to 2.5M thiourea as described herein. Exemplary lysis buffers may include 0.5-9.5M urea and thiourea, such as 7M urea and 2M thiourea having nonionic surfactants as described herein. Urea, used alone or in combination with thiourea, can be used to lyse cells for nucleic acid purification. In combination, urea and thiourea work synergistically to lyse cells and provide an uncharged, isotropic electrophoresis-compatible buffer for nucleic acid purification.

[0432] Exemplary lysis buffers may include nonionic surfactants, such as 0.05-0.5% v / v IGEPAL CA-630 as described herein. In some cases, the lysis buffer may contain one or more trailing electrolytes. In some cases, the lysis buffer may contain a trailing electrolyte buffer with additives for tissue destruction or cell lysis as described herein. In some cases, the lysis buffer may contain one or more leading electrolytes. In some cases, the lysis buffer may contain a leading electrolyte buffer with additives for tissue destruction or cell lysis as described herein. In some cases, the lysis buffer may contain one or more leading electrolytes and one or more trailing electrolytes. In some cases, the lysis buffer may contain one or more leading electrolytes and one or more trailing electrolytes with additives for tissue destruction or cell lysis as described herein.

[0433] In some cases, the methods or procedures described herein may involve lysing cell or tissue samples using a lysis buffer that minimizes mechanical damage to DNA and / or RNA during the lysis reaction. For example, cells or tissues may be lysed in a buffer solution containing HCl (e.g., 1 mM, 5 mM, 10 mM HCl), Tris (e.g., 5 mM, 10 mM Tris), and a nonionic surfactant. A nonionic detergent (e.g., IGEPAL CA-630) may be present in the lysis buffer at about 1%, about 2%, about 3%, about 4%, or higher, or less than about 1%. Cells or tissues may be lysed in the lysis buffer by gentle mixing (e.g., 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 may be introduced into an isotachophoresis apparatus to purify desired analytes such as high molecular weight DNA fragments.

[0434] Proteins in a sample can be digested, for example, by enzymatic digestion with proteases. Proteases may include, but are not limited to, proteinase K, proteases, endopeptides (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Other protein or polynucleotide digestive agents or degrading agents, such as lysozyme, may be used. Protein digestion can remove cross-linked proteins from cross-linked nucleic acids, thereby converting them into non-cross-linked nucleic acids. Protein digestion can occur at room temperature or at elevated temperatures (e.g., greater than about 25 °C) as described herein.

[0435] The sample can be processed on a device (e.g., an electric device or system having at least one reservoir connected to at least one channel) such that the sample volume passes through the reservoir into the channel, wherein less than 20% of the sample volume remains in the reservoir, and an ion current can subsequently be applied through the sample volume in the channel. The ion current may substantially not pass through the channel. 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.

[0436] The sample can be processed on an apparatus (e.g., an electric device or system having at least one reservoir connected to at least one channel) such that the sample volume entering the channel through the reservoir is at least 50% of the sample volume loaded into the reservoir, and an ion current can subsequently be applied through the sample volume in the channel. In some cases, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of the sample volume moves from the reservoir to the channel. In some cases, the total volume loaded into the reservoir is less than or equal to the internal volume of the reservoir. The ion current may substantially not pass through the channel. In some cases, applying the ion current includes performing isovelocity electrophoresis.

[0437] In some implementations, after the sample volume has been loaded into the reservoir and subsequently into the channel, a top buffer can be added to the reservoir to facilitate the movement of the sample volume into the channel. A volume of top buffer can be added to the reservoir to "push" additional sample volume into the channel. For example, a volume of top buffer greater than or equal to the remaining sample volume in the reservoir can be added to the reservoir to allow at least a portion of the remaining sample volume to move into the channel. The top buffer may, for example, contain the same buffer as the sample buffer, but without the analyte.

[0438] Isokinetic electrophoresis equipment

[0439] Isotachyphoresis and / or sample preparation (e.g., dewaxing, digestion, lysis) can be performed in fluidic devices (e.g., microfluidic chips). Figure 5AA schematic diagram of a channel is shown, comprising a sample preparation (e.g., dewaxing) zone 500 with a sample inlet 501 and a trailing electrolyte reservoir 502, 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 before voltage is applied. 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, dewaxing may be performed outside the chip initially, or since the starting material may be unnecessary. In this case, the channel may include a lysis and digestion zone (e.g., pH 7, 56°C) and a crosslink removal and purification zone (e.g., isotachophoresis) zone (e.g., pH 7, 80°C). In another example, dewaxing may be performed outside the chip initially, or since the starting material may be unnecessary. In this case, the channel may include a lysis and digestion zone (e.g., pH 7, temperature T1) and a crosslink removal and / or purification zone (e.g., isotachophoresis) zone (e.g., pH 7, temperature T2). In another example, dewaxing may be performed outside the chip initially, or since the starting material may be unnecessary. In this case, the channel may include a destruction and / or lysis zone (e.g., pH 7, temperature T1) and a digestion and / or purification zone (e.g., isotachophoresis) zone (e.g., pH 7, temperature T2). In another example, dewaxing may be performed outside the chip initially, or since the starting material may be unnecessary, in which case the channel may 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 some cases, the channel may include three zones, such as a disruption and / or lysis zone (e.g., pH 7, temperature T1), an isothermal enzymatic amplification zone (e.g., pH 7, temperature T2), and a purification (e.g., isotachophoresis) zone (e.g., pH 7, temperature T3). Figure 5B An exemplary fluid device housing with eight parallel channels is shown, each channel as follows: Figure 5A As shown. Figure 5C It shows Figure 5B The diagram shows a top view of the fluid device, while Figure 5D and Figure 5ESide and end views are shown respectively. The device may include a sample inlet or reservoir 530, an ITP electrolyte buffer reservoir 531, and a sample elution outlet or reservoir 532. The channels and / or reservoirs may be coupled to one or more pneumatic ports. Each of the eight parallel channels of the fluid device can be operated independently from each of the other channels. In some cases, each channel has a dedicated set of electrodes and circuitry to drive the ITP. The electrodes may be located, for example, in the trailing electrolyte reservoir 502 and the leading electrolyte reservoir 511, such that the electrodes do not directly contact the sample material.

[0440] In some cases, there may be little or no fluid or ion flow between parallel channels. In some cases, parallel channels may not be fluidly connected to each other. The fluid leakage rate between parallel channels may be less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 μL / hour.

[0441] In some cases, there may be little or no electrical communication between parallel channels, making them electrically isolated from each other. Each parallel channel can be electrically controlled independently to apply an independent electric field to each channel. In some cases, the current leakage between channels is less than about 0.1 μA, 0.2 μA, 0.3 μA, 0.4 μA, 0.5 μA, 0.6 μA, 0.7 μA, 0.8 μA, 0.9 μA, or 1 μA. In some cases, the impedance between channels can be greater than 0.1 MΩ, 0.2 MΩ, 0.3 MΩ, 0.4 MΩ, 0.5 MΩ, 0.6 MΩ, 0.7 MΩ, 0.8 MΩ, 0.9 MΩ, 1 MΩ, 5 MΩ, 10 MΩ, 20 MΩ, 30 MΩ, 40 MΩ, or 50 MΩ.

[0442] In some cases, each parallel channel can be coupled to the same current or voltage source and is electrically controlled independently. In other cases, each parallel channel can be coupled to different current or voltage sources and is electrically controlled independently.

[0443] Figure 34 A loop configured to detect and prevent current leakage in a parallel channel during ITP is illustrated. The loop can be configured to monitor the current entering the ITP system from a single HV electrode and the current leaking through the high-voltage board. By measuring both currents at regular intervals or continuously, the current entering the chip can be precisely controlled even when the leakage current varies. This allows the electrodes to operate in a net-pull or net-sink configuration. The loop may include a voltage source V. SThe system includes a sink current controller 3406, a source current controller 3408, and two current measurement loops 3405 and 3409, which measure the current flowing through the controlled current sources 3406 and 3408. The controlled current sources can be controlled by a microprocessor using digital-to-analog (DAC) converters 3402 and 3403. DAC converter 3402 controls the current entering the electrodes and leakage path. DAC converter 3403 controls the current entering the leakage path. The microprocessor can read the measured current using analog-to-digital (ADC) converters 3401 and 3404. ADC 3401 measures the power supply V. S The ADC 3404 can measure the current at the leakage path, where the current is directed to ground. Current control sources 3406 and 3408 can be connected to the load 3407, such that current source 3408 can draw current from the load 3407 and current source 3406 can draw current from the load 3407. The current flowing into the load 3407 can be expressed as I. RL The current flowing through the controlled current source 3408 can be expressed as I. m1 The current flowing through the controlled current source 3406 can be expressed as I. m2 Therefore, the current flowing into or out of the 3407 can be determined by equation I. RL =I m1 -I m2 Description. Current I m1 The current commanded by the controlled source 3408 (will be controlled by I) c1 (represented by I) and leakage current (by I) L1 (Indicated by) the components, this leakage current is due to parasitic conductive paths present in any physically feasible loop. Current I m1 Equation I can be used m1 =I c1 +I L1 Describe, and similarly, the current I m2 Available I m2 =I c2 +I L2 Description. Therefore, by extension, the current flowing into or out of the 3407 can be expressed by equation I. RL =I c1 +I L1 -I c2 -I L2 Description. In some applications, it may be desirable to command a circuit to be in an off state so that I... RL =0. Therefore, the controlled current source is typically commanded to make I = 0. c1 =0 and I c2 =0, however, due to leakage current I L1 and I L2During the off state, the current flowing into or out of load 3407 can be determined by I. RL =I L1 -I L2 Describe some non-zero currents. To represent the off-state current I... RL Reduce to a value significantly less than the leakage current I L1 Or I L2 The value allows the circuit to manipulate additional current through the controlled current source 3406 or 3408 until it flows into I. RL The current balance is zero. For example, the loop can be set to I. c1 =I c2 +I L2 -I L1 Or I c2 =I c1 +I L1 -I L2 In either case, the resulting current I RL Will decrease to I RL =0. In other words, the circuit can regulate the current from current sources 3406 and 3408 to balance and offset the leakage current, thereby driving the net current between the parallel channels to be 0.

[0444] In many applications, such as isovelocity electrophoresis, where a controlled current source is applied to a load for a period of time and then removed from the load for a period of time, it may be desirable to minimize leakage of the current source loop to the load when the controlled current source is removed. When the control loop is intended to be shut down, many or all of the loop components used to construct the current source can allow some parasitic leakage current to flow through the loop. Minimizing this leakage typically requires the use of more complex and higher-quality components exhibiting lower parasitic leakage properties; however, this is costly and often requires more physical volume to implement the loop. The disclosed content allows for the reduction of leakage current applied to the load by manipulating the leakage current away from the load. Therefore, the disclosed content can allow for the use of simpler loop components in the construction of the current source, achieving a current source with loop components optimized for other purposes such as low cost or physical size, resulting in lower leakage current. Current leakage can be caused by liquid leakage between fluid channels in which material layers close the fluid channels in the substrate surface. Ensuring a strong bond of the layers across the entire substrate surface can reduce such leakage. Current leakage can also be caused by the movement of liquid between ports of different fluid loops, particularly between ports that act as sources of negative pressure leading to the fluid passage. Providing a hydrophobic barrier at such ports can reduce this type of leakage.

[0445] In some cases, each zone on the isotachophoresis apparatus can be heated. In some cases, these zones are heated to the same temperature. In some cases, the zones are heated to different temperatures. In some cases, the first zone can be heated to a temperature above 37°C, for example, in the range of about 60°C to about 100°C. In some cases, the second zone can be heated to a temperature above 37°C, for example, in the range of about 40°C to about 60°C.

[0446] Isotachyphoresis fluid apparatus may include one or more reservoirs, including but not limited to buffer loading reservoirs, sample loading reservoirs (including reservoirs that accept solid, multiphase, or other heterogeneous liquids or solutions such as tissue, whole blood, or unlysed cell suspensions), lead electrolyte reservoirs, tail electrolyte reservoirs, reagent reservoirs, elution reservoirs (e.g., for unloading processed samples), and gas or air reservoirs. In some cases, a single physical reservoir may be used for multiple purposes, such as buffer loading and sample loading. Liquid or air reservoirs may be used to apply external pressure for liquid loading (e.g., applying positive pressure to a liquid orifice or applying a vacuum only to a gas reservoir). Those skilled in the art will understand that any reservoir described herein may be used to load or retrieve any of the buffers and / or samples described herein.

[0447] The reservoir can be thermally connected to a heating or cooling source, thereby allowing temperature control of the reservoir and any materials within it (e.g., reagents, samples, products). For example, when within a fluid device, the elution reservoir can be thermally controlled to control the temperature of the eluted products (e.g., to maintain structural integrity).

[0448] Reagent reservoirs can be used to load one or more reagents to process samples before, during, or after isotachyphoresis. Reagents may include digestion reagents, amplification reagents, reverse transcription reagents, linear polymer solutions for size-based separation, probes for hybridization reactions, ligation reagents, dyes (e.g., insert dyes as described herein), tracers, markers, and other reagents. Reagent reservoirs may be connected to a reaction channel or a reaction section of another channel where reactions can occur. Heating or cooling (e.g., using a thermal controller as discussed herein) may be applied to catalyze reactions (e.g., enzymatic reactions with nucleic acids or proteins), to hybridize or unwind nucleic acids, or to remove insert dyes from nucleic acids (e.g., prior to elution). Heating and cooling may also be used to control a fixed operating temperature for ITP (e.g., cooling may be applied to reduce the effects of Joule heating) or to maintain a reservoir (e.g., an elution reservoir) at a fixed temperature (e.g., cooler than room temperature), such as for the stable storage of purified nucleic acids. Light (e.g., using a light source as discussed herein) may be applied for purposes including optical interrogation, fluorescence excitation, and reaction energy or catalysis.

[0449] A gas or air reservoir or gas or air outlet may be connected via a gas passage to a liquid passage within the fluid device to allow purging of air or other gases from the fluid device (e.g., during liquid filling of the fluid device). A gas or air reservoir or pneumatic pressure port may be connected via a gas passage to a liquid passage to allow pumping of fluid onto or into the fluid device (e.g., for pumping fluid from the reservoir into the passage).

[0450] The device may contain multiple purification (e.g., isotachophoresis) zones connected to each other. For example, a second isotachophoresis zone may be separated from and run in parallel with a first isotachophoresis zone, thereby allowing sample bands to be separated in a specific ratio (e.g., based on the current ratio between the two zones) for parallel processing.

[0451] Fluid apparatus may contain multiple parallel purification zones (see, for example, Figure 5CFor example, a fluidic apparatus may contain more than one set of purification zones, each set having associated reservoirs, inlets, outlets, channels, and any other components described herein (e.g., sample preparation zones, electrodes, heaters, detectors), these components being parallel, separate from each other, and each capable of independently processing samples. A fluidic apparatus may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48, 96 or more parallel purification zones. A fluidic apparatus may contain multiple parallel channels. A fluidic apparatus may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48, 96 or more parallel channels. Parallel components, such as purification zones or channels, can be side-by-side, located in different equipment layers (e.g., horizontal or vertical layers), or arranged differently. Parallel components can be identical or can be designed differently but functionally equivalent or nearly equivalent. For example, parallel channels can have different geometries to allow for a smaller overall fluidic apparatus footprint while still having similar functionality. Alternatively, parallel components can be designed to function differently, such as processing different types of samples in parallel or subjecting samples to different operations. In some cases, parallel components can be designed to subject different sample types to different operations in parallel. In some cases, parallel components can be designed to subject the same sample type to different operations in parallel. In some cases, parallel components can be designed to subject different sample types to the same operations in parallel. In some cases, parallel components can be designed to subject the same sample type to the same operations in parallel. In some cases, parallel components can be designed to subject two or more samples to one or more operations in parallel simultaneously and / or independently. In some cases, the leakage rate between two or more channels (or between two or more purification zones) can be less than 0.5 μl / hour, less than 1 μl / hour, less than 5 μl / hour, or less than 10 μl / hour. In some embodiments, the current leakage rate between two or more channels (or between two or more purification zones) can be less than 0.5 μA, less than 1 μA, less than 5 μA, or less than 10 μA. In some embodiments, the impedance between channels or regions can be greater than 0.5 megohms, greater than 1 megohm, greater than 5 megohms, or greater than 10 megohms.

[0452] As discussed in this article, fluid devices can be designed to handle different sample volumes. For example, Figure 6A , Figure 6B , Figure 6C and Figure 6DTop, side, bottom, and three-quarter top views of a rapid purification ITP fluid apparatus 600 for sample volumes greater than or equal to about 200 μL are shown. The apparatus includes channels 600 connected via through-holes or orifices as described herein to a sample inlet port 601, an ITP buffer port 602, and a sample outlet (elution) port 603. The ITP buffer port 602 may include an elution buffer reservoir 605, a lead electrolyte reservoir 606, a lead electrolyte buffer reservoir 607, and a trailing electrolyte reservoir 608. An elution reservoir 603 may be connected to the elution buffer reservoir 605 via an elution buffer channel 609. Capillary barriers (e.g., platform capillary barriers, ramp capillary barriers, or cliff capillary barriers as described herein) may be provided in the elution buffer channel 609 to reduce or prevent mixing or pressure-driven flow between the contents of the elution buffer reservoir 605 and the elution reservoir 603. A lead electrolyte reservoir 606 can be connected to a lead electrolyte buffer reservoir 607 via a lead electrolyte buffer channel 610. A capillary barrier (e.g., a platform capillary barrier, ramp capillary barrier, or cliff capillary barrier) can be provided in the lead electrolyte buffer channel 610 to reduce or prevent mixing or pressure-driven flow between the contents of the lead electrolyte buffer reservoir 607 and the lead electrolyte reservoir 606. Buffer reservoir 605 may contain an elution buffer electrolyte with an ionic strength higher than that of the electrolyte in elution reservoir 603, while buffer reservoir 607 may contain a lead electrolyte with an ionic strength higher than that of the electrolyte in lead electrolyte reservoir 606. The device may further include a pneumatic port 604 along its edge, configured to couple to a pneumatic device, such as a vacuum source on a benchtop instrument. Pneumatic port 604 can be coupled to channel 600 and the reservoir via a gas channel as described herein. Aspiration applied at pneumatic port 604 loads the sample, lead electrolyte, and elution buffer into channel 600. In some cases, the trailing electrolyte buffer fluid is retained in trailing electrolyte reservoir 608. Aspiration can be applied simultaneously or sequentially to pneumatic port 604 to load channel 600 simultaneously or in stages, respectively. The sample can be loaded into a first region or subchannel of channel 600, which extends from trailing electrolyte reservoir 608 to capillary barrier 611 in channel 600 with a 180° low-dispersion turn. Capillary barrier 611 provides an interface between the sample and lead electrolyte buffer during loading to limit, reduce, or prevent mixing or pressure-driven flow. Capillary barrier 611 may include cliff capillary barriers as described herein. A capillary barrier (e.g., cliff capillary barrier, ramp capillary barrier, or platform capillary barrier) may be provided between the trailing electrolyte reservoir 608 and the first zone or subchannel to limit, reduce, or prevent mixing or pressure-driven flow between the contents of the trailing electrolyte reservoir 608 and the sample.A leading electrolyte can be loaded into a second region or subchannel of channel 600, extending from capillary barrier 611 to capillary barrier 612. Capillary barrier 612 (e.g., a platform capillary barrier, ramp capillary barrier, or cliff capillary barrier) can provide an interface between the leading electrolyte buffer and the elution buffer. Elution buffer can be loaded into a third region or subchannel of channel 600, extending from capillary barrier 612 to elution reservoir 603. In some embodiments, ITP buffer orifice 602 may further include a trailing electrolyte buffer reservoir (not shown) containing a trailing electrolyte with an ionic strength higher than that of the electrolyte in trailing electrolyte reservoir 608. The trailing electrolyte buffer reservoir can be connected to trailing electrolyte reservoir 608 via a trailing electrolyte buffer channel (not shown). The trailing electrolyte buffer channel may include capillary barriers (e.g., ramp capillary barriers, platform capillary barriers, or cliff capillary barriers) to limit, reduce, or prevent mixing or pressure-driven flow between the trailing electrolyte buffer reservoir and the contents of the trailing electrolyte reservoir 608.

[0453] Electrodes may be located, for example, in a trailing electrolyte reservoir 608, a trailing electrolyte buffer reservoir (not shown), a leading electrolyte reservoir 606, and / or a leading electrolyte buffer reservoir 607, such that the electrodes do not directly contact the sample material. In response to feedback from a sensor (e.g., a voltage, current, conductivity, or temperature sensor as described herein), the electrodes may be triggered to alter or control the applied electric field. For example, a pathway of nucleic acids within the ITP region from the second region of channel 600 to the third region of channel 600 may be detected, and feedback from the detector may trigger a change in the applied current. For example, the current may be increased, decreased, or stopped according to the instrument's protocol. For example, the current may be paused (e.g., temporarily reduced to zero) to enable on-chip quantification of nucleic acids. Alternatively or in combination, the current may be reduced to slow isovelocity electrophoresis within the third region, thereby further concentrating nucleic acids that may have been dispersed during the transition from the leading electrolyte buffer to the elution buffer (or the second leading electrolyte buffer) before reaching the elution well 603.

[0454] The methods and procedures provided herein include those using any of the devices provided herein. The devices provided herein, having multiple channels for parallel processing of multiple samples, can be used in a variety of environments. In some cases, the method may include using the device to process multiple samples that share a common characteristic (e.g., solid tissue lysate, cell lysate, solid tissue, fixed tissue) (e.g., by performing isotachyphoresis on these samples). In some cases, the multiple samples may be different samples. For example, the method may involve performing isotachyphoresis on a tissue sample in one region of the device while simultaneously, but independently, performing isotachyphoresis on different samples (e.g., cell samples or samples containing cross-linked nucleic acids).

[0455] In some cases, the methods or multiplexing procedures described herein may involve isotachophoresis of a sample in a channel, parallel to isotachophoresis of a second sample in a second channel using the same or similar leading electrolyte and / or trailing electrolyte buffer. In some cases, a sample in one channel is treated with a first leading electrolyte buffer, and samples in different channels are treated with a second leading electrolyte buffer different from the first. For example, the first leading electrolyte buffer may contain one or more leading electrolyte ions that are different from the leading electrolyte ions contained in the second leading electrolyte buffer. In another example, the first leading electrolyte buffer may contain one or more leading electrolyte ions that are the same as the leading electrolyte ions contained in the second leading electrolyte buffer, but the concentration of such leading electrolyte ions in the first leading electrolyte buffer differs from the concentration of such ions in the second leading electrolyte buffer. In some cases, the methods or procedures described herein may involve isotachophoresis of a sample in a channel, parallel to isotachophoresis of a second sample in a second channel using the same or similar trailing electrolyte or trailing electrolyte buffer. In some cases, a first trailing electrolyte buffer is used to treat a sample in one channel, and a second trailing electrolyte buffer, different from the first, is used to treat samples in different channels. For example, the first trailing electrolyte buffer may contain one or more trailing electrolyte ions that are different from those contained in the second trailing electrolyte buffer. In another example, the first trailing electrolyte buffer may contain one or more trailing electrolyte ions that are the same as those contained in the second trailing electrolyte buffer, but the concentration of these trailing electrolyte ions in the first trailing electrolyte buffer differs from the concentration in the second trailing electrolyte buffer.

[0456] In some embodiments, one or more reservoirs may be connected to two channels or sub-channels. For example, elution reservoir 603 may be connected to both channel 600 and elution buffer channel 609. Alternatively or in combination, lead electrolyte reservoir 606 may be connected to both channel 600 and lead electrolyte buffer channel 610. Alternatively or in combination, trailing electrolyte reservoir 608 may be connected to both channel 600 and trailing electrolyte buffer channel. Alternatively or in combination, sample inlet port 601 may be connected to the midpoint of channel 600, such that channel 600 extends to the left (as a first sub-channel) and right (as a second sub-channel) of inlet port 601. Two channels or sub-channels can be connected to one or more reservoirs, wherein the angle between the two channels (scanned in the main plane of the fluid device) is at least about 5°, 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 135°, 140°, 150°, 160°, 170° or 180°. Two channels or sub-channels can be connected to one or more reservoirs, wherein the angle between the two channels (scanned in the main plane of the fluid device) is up to about 5°, 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 135°, 140°, 150°, 160°, 170° or 180°.

[0457] For example, the device may include eight channels as shown in the figure. Each channel can hold a sample volume of approximately 50 μL to approximately 275 μL and a total volume of approximately 500 μL. The 180° low-dispersion steering in each channel facilitates such a large sample volume in an 8-channel multichannel plate with a standard SLAS footprint.

[0458] Figure 7A , Figure 7B , Figure 7C and Figure 7D Top, side, bottom, and three-quarter bottom views of a rapid purification ITP fluid device 700 for sample volumes less than or equal to about 100 μL are shown. The device includes a sample inlet port 701, an ITP buffer port 702, and a sample outlet (elution) port 703. Device 700 may be substantially similar to device 600, but has a different channel geometry (and corresponding reservoir geometry) excluding 180° turns within the channels.

[0459] For example, the device may include eight channels as shown in the figure. Each channel can hold a sample volume of approximately 10 μL to approximately 100 μL. Devices with smaller sample volumes can be used for PCR cleaning or other reaction cleaning applications or for smaller sample sizes (e.g., samples with a small number of cells or tissues).

[0460] Figure 8A , Figure 8B , Figure 8C and Figure 8D Top, side, bottom, and three-quarter bottom views of another rapid purification ITP fluid device 800 for sample volumes less than or equal to approximately 100 μL are shown. This device includes a sample inlet well 801, an ITP buffer well 802, and a sample outlet (elution) well 803. Device 800 may be substantially similar to devices 600 and 700, but contains multiple different channel geometries on a single chip.

[0461] Any fluid device described herein may include one or more electrodes that apply an electric field to the fluid device or a portion thereof. The applied electric field can be used to perform isovelocity electrophoresis. A fluid device may include one or more electrodes that apply a single electric field to all channels of the fluid device. A fluid device may include one or more electrodes that apply more than one electric field to the fluid device, such as one electric field per channel of the device. In some cases, the first and second electric fields are generated by a single electrode pair. In some cases, the first and second electric fields are generated by different electrode pairs. The electric fields may be applied simultaneously, sequentially, and / or independently, or applied to each other. Electrodes may be external, such as wires falling into a reservoir. Electrodes may be internal, such as microfabricated, printed, or other embedded elements included in the fabrication of the fluid device. Electrode materials may include, but are not limited to, metals (e.g., platinum, titanium) and carbon.

[0462] One or more electrodes of a fluid apparatus may be part of one or more circuits that apply an electric field to the fluid apparatus or a portion thereof. A fluid apparatus may contain one or more circuits that apply a single electric field to all channels or isokinetic electrophoresis zones of a region of the fluid apparatus. A fluid apparatus may contain one or more circuits that apply more than one electric field to the fluid apparatus, such as one electric field per channel on the apparatus. In some cases, the first and second electric fields may be generated by a single circuit. In some cases, the first and second electric fields may be generated by different circuits. Electric fields may be applied simultaneously, sequentially, and / or independently, or to each other, by one or more circuits. In some cases, the apparatus (or benchtop instrument) may be configured to control a first circuit simultaneously with and independently of a second circuit.

[0463] Electrodes can be located in reservoirs such as trailing and leading electrolyte reservoirs, which can be separated from the sample reservoir via buffer channels. In some cases, electrodes are located in buffer channels or buffer reservoirs. The location of electrodes in electrolyte reservoirs or electrolyte buffer reservoirs can isolate the electrodes from analytes such as nucleic acids to reduce or eliminate contamination of the electrodes by sample materials. This method can allow for the reuse of electrodes without cross-contamination between samples. In one example, a trailing electrolyte reservoir or trailing electrolyte channel is connected via a buffer channel to a buffer reservoir containing trailing electrolyte ions and an electrode, and the trailing electrolyte reservoir is also connected to a sample reservoir or sample channel, which in turn is connected to a leading electrolyte reservoir via a leading electrolyte channel; the leading electrolyte reservoir is also connected via a buffer channel to a buffer reservoir containing a leading electrolyte and an electrode. In another example, or as a continuation of the previous example, an elution reservoir containing elution buffer is connected via an elution channel to a leading electrolyte reservoir, and is also connected to a buffer reservoir containing elution buffer electrolyte and an electrode. Buffer channels between a buffer reservoir and its corresponding reservoir may include capillary barriers and / or low cross-sectional areas to limit, reduce, or prevent mixing and pressure-driven flow as described herein. Buffer reservoirs may contain electrolytes with the same or higher ionic strength as their corresponding reservoirs. For example, an elution reservoir may be connected to a buffer reservoir containing an elution buffer electrolyte with the same or higher ionic strength or concentration as the elution reservoir. A trailing electrolyte reservoir may be connected to a buffer reservoir containing a trailing electrolyte with the same or higher ionic strength or concentration as the trailing electrolyte reservoir. A leading electrolyte reservoir may be connected to a buffer reservoir containing a leading electrolyte with the same or higher ionic strength or concentration as the leading electrolyte reservoir. Providing dedicated buffer reservoirs for connection to elution reservoirs, trailing electrolyte reservoirs, and / or leading electrolyte reservoirs with higher ionic strengths can provide additional ion pools to maintain pH and conductivity in the channels as the sample moves through them.

[0464] Fluid equipment can be used with one or more thermal controllers. For example, Figure 9A A schematic diagram of an eight-fold sample preparation and isovelocity electrophoresis apparatus is shown, which includes Figure 5A The design shown has eight parallel channels 900. Figure 9BSchematic diagrams of first and second thermal controllers 901, 902 are shown. The first thermal controller 901 at temperature T1 (e.g., 80°C) is aligned with the sample preparation area of ​​the channel, and the second thermal controller 902 at temperature T2 (e.g., 50°C) is aligned with the isovelocity electrophoresis area of ​​the channel. In some cases, additional thermal controllers may be aligned with additional areas of the channel (not shown), for example, a third thermal controller at temperature T3 may be aligned with a third area at temperature T3. In some cases, each area of ​​each channel may have its own separate thermal controller, rather than sharing a common thermal controller with corresponding areas of other channels. In other cases, all areas or channels may share a single thermal controller. In still other cases, multiple, but fewer than all areas or channels may share a single thermal controller. The thermal controller may include components including, but not limited to, resistance heaters, fluid-based heating or cooling systems, and Peltier devices. The thermal controller may be made of materials including, but not limited to, metals (e.g., platinum, titanium, copper, gold), carbon, and indium tin oxide (ITO). A thermal controller may include a temperature sensor that can be used to monitor the controlled temperature and provide temperature feedback for thermal control. As further discussed in this disclosure, the thermal controller may be used in conjunction with a computer control system. In some cases, the thermal controller operates without temperature feedback. The thermal controller may be integrated into the fluid device or located externally, such as within a benchtop system.

[0465] Fluid apparatuses can be used with one or more light sources. The light source can be integrated into the fluid apparatus or located externally, such as within a benchtop system or in a separate device. The light source can provide illumination for optical interrogation, fluorescence excitation, temperature sensing, reaction energy or catalysis, and other purposes.

[0466] Fluid devices can be designed such that their outermost frame or dimensions conform to microtiter plate standards (e.g., SLAS microtiter plate standards). Fluid devices can be designed to use defined ports of a microtiter plate (e.g., SLAS standard microtiter plate) as liquid reservoirs, with pneumatically actuated ports located on unused surfaces outside the liquid reservoir. The pneumatic ports can be positioned at the edges of the fluid device with a layout compatible with microtiter plates, thereby avoiding cross-contamination via pneumatic actuation of the liquid reservoir and facilitating access to the ports with pneumatic hardware. A subset of the defined ports may also be used for pneumatic actuation, among other functions. In some cases, the fluid device can be designed and manufactured with two interlocking components: first, an insert comprising a channel unit (e.g., a layer with a flat surface conducive to film adhesion), holes, and pneumatic ports; and second, an outer ring or cover conforming to microtiter plate standards (e.g., SLAS microtiter plate size standards), including alignment features for aligning the fluid device with a benchtop system and mating features for interlocking with the first component. These holes can be connected to form bosses, which can be more compatible with injection molding. In some cases, the fluid device can be designed and manufactured as three connected parts; first, a chip or substrate, which includes holes and pneumatic ports on its top surface and etched or molded channels on its bottom surface; second, a material layer (e.g., a thin film) that seals to the bottom surface of the chip to form a closed channel (together sufficient to form a fluid chip); and third, a cover ring or cover that conforms to microtiter plate standards (e.g., SLAS microtiter plate size standards), including alignment features for aligning the fluid device with a benchtop system and mating features for interlocking with the first part. Such a device can also be referred to as a cartridge.

[0467] Figures 35A-35B An example of a fluid device 3500 comprising two interlocking sections is shown. Figure 35A A cover 3501 is shown, which is mounted onto the microfluidic chip insertion portion 3502, as shown. Figure 35B As shown. Figure 35B An exploded view of the chip 3502 and the cap 3501 is shown. The chip or substrate 3502 may have a first side and a second side. The first side may include a plurality of reservoirs 3508 configured to contain liquid. The second side may include a plurality of channels. The reservoirs 3508 may communicate with the channels via through-holes in the substrate 3502. A hydrophobic membrane 3503 may be sandwiched between the chip 3502 and the cap 3501. The cap 3501 may be configured to trap and compress two hydrophobic membrane filters 3503, which may function as valves that allow air but not fluid to pass through. When the cap 3501 is assembled onto the microfluidic chip 3502, a compressible washer 3504 (e.g., ...) on the underside of the cap 3501... Figure 35A(As shown) A constant downward compressive force can be provided on chip 3502 to create a seal to prevent or reduce leakage between channels and / or instruments. This downward force can be initially created during assembly when two sets of features engage between chip 3502 and cap 3501. First, a set of snaps 3505 around the cap engages with mating features 3506 on chip 3502, ensuring alignment between cap 3501 and chip 3502 and providing initial compression from gasket features to membrane 3503. As additional force is applied during assembly, interference fit features 3507 on the cap can engage with the outer wall of a limited set of fluid reservoirs 3508 on chip 3502. Interference fit 3507 can be designed to maintain a fixed displacement, thus maintaining a fixed compressive force on gasket 3504 when the assembly force is removed. A fixed height support 3509 adjacent to interference fit feature 3507 can limit the distance that cap 3502 can be pressed down onto reservoir 3508. Cover 3501 may include a mating surface 3510 for use with pneumatic equipment, such as the pneumatic manifold interface described herein.

[0468] For example, chip 3502 may include any number of interference fit features 3507 as desired by those skilled in the art. For example, chip 3502 may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 interference fit features 3507. As those skilled in the art will understand, the retention capability of the interference fit features 3507 may depend on the number of interference fit features 3507 on chip 3502. For example, four interference fit features 3507 may have a retention capability of 4 lb-f / feature 3507, while for the same decoupling force, eight interference fit features 3507 may have a retention capability of 2 lb-f / feature 3507. The interference fit features 3507 may have radial interference in the range of about 10 μm to about 120 μm, for example, in the range of about 30 μm to about 60 μm, such as about 45 μm.

[0469] Figures 36A-36B An example of a fluid dev...

Claims

1. A fluid apparatus comprising an isotachymeter electrophoresis (ITP) circuit, the ITP circuit comprising: The first channel includes a first capillary barrier and a second capillary barrier spaced apart. A first loading reservoir is in fluid communication with the first channel via a first orifice in the first channel, wherein the first orifice is positioned between a first capillary barrier and a second capillary barrier to allow a first liquid entering the first channel via the first orifice to flow along the first channel in one direction and stagnate at the first capillary barrier, and to flow along the first channel in another direction and stagnate at the second capillary barrier, wherein one or both of the first capillary barrier and the second capillary barrier are cliff capillary barriers or platform capillary barriers, wherein the first capillary barrier and the second capillary barrier substantially extend the width of the first channel.

2. The fluid apparatus of claim 1, wherein the first liquid entering the first channel via the first orifice flows along a path to the first capillary barrier or the second capillary barrier, the first capillary barrier or the second capillary barrier being longer than the width of the first channel.

3. The fluid apparatus of claim 1, wherein the first liquid flowing into the first channel via the first orifice causes the meniscus of the first liquid to stagnate at the first capillary barrier or the second capillary barrier, or at both the first capillary barrier and the second capillary barrier.

4. The fluid device of claim 1, wherein the first capillary barrier is configured and arranged to be destroyed by the first liquid when a first rupture pressure is applied to the ITP circuit, and the second capillary barrier is configured and arranged to be destroyed by the first liquid when a second rupture pressure is applied to the ITP circuit, wherein the first rupture pressure and the second rupture pressure are equal.

5. The fluid device of claim 1, wherein the first capillary barrier is configured and arranged to be destroyed by the first liquid when a first rupture pressure is applied to the ITP circuit, and the second capillary barrier is configured and arranged to be destroyed by the first liquid when a second rupture pressure is applied to the ITP circuit, wherein the first rupture pressure is greater than the second rupture pressure.

6. The fluid device of claim 1, wherein the first capillary barrier is a cliff capillary barrier.

7. The fluid device of claim 1, wherein the second capillary barrier is a cliff capillary barrier.

8. The fluid apparatus of claim 1, wherein the platform capillary barrier is configured and arranged such that an air gap is formed between the first liquid and the second liquid after the first liquid stagnates at the platform capillary barrier and after the second liquid flows toward the platform capillary barrier in another direction and stagnates at the platform capillary barrier relative to the first liquid.

9. The fluid apparatus of claim 1, wherein the ITP circuit includes a second channel in fluid communication with the first channel, and the first capillary barrier is configured and arranged to impede the flow of the second liquid as the second liquid flows along the second channel, such that a liquid-liquid interface is formed between the first liquid and the second liquid at the first capillary barrier.

10. The fluid apparatus of claim 1, wherein the fluid apparatus further comprises a substrate having a first surface and a second surface, wherein the first surface includes a plurality of reservoirs including the first loading reservoir, and the second surface includes a plurality of channels including the first channel, wherein the plurality of reservoirs are in communication with the plurality of channels via through-holes in the substrate.

11. The fluid apparatus of claim 1, wherein the ITP circuit further comprises a second loading reservoir and a second channel, wherein the second loading reservoir is in fluid communication with the second channel via a second orifice, and the second channel comprises a third capillary barrier, wherein the third capillary barrier is configured and arranged to utilize capillary forces to stagnate the flow of liquid along the second channel at a meniscus of the third capillary barrier.

12. The fluid device of claim 11, wherein the ITP circuit further comprises a third loading reservoir fluidly connected to a third channel via a third orifice, wherein the third channel is fluidly connected to the second loading reservoir, wherein the third channel includes a fourth capillary barrier positioned between the second orifice and the third orifice.

13. The fluid apparatus of claim 12, wherein the first channel or the first loading reservoir comprises a sample buffer; the second channel or the second loading reservoir comprises a first lead electrolyte buffer; or the third channel or the third loading reservoir comprises a second lead electrolyte buffer.

14. The fluid device of claim 12, further comprising a fourth channel or a fourth loading reservoir in fluid communication with the first channel and adjacent to the first capillary barrier, wherein the fourth channel or the fourth loading reservoir comprises a trailing electrolyte buffer.

15. The fluid apparatus of claim 1, wherein the ITP circuit includes an elution channel connected to the elution connection and the first elution reservoir.

16. The fluid device of claim 1, wherein the ITP circuit includes a first lead electrolyte buffer reservoir connected to a second lead electrolyte buffer reservoir via a buffer channel, wherein the buffer channel contains a first and a second lead electrolyte buffer that meet at an interface, at which a platform capillary barrier is located.

17. The fluid device of claim 1, wherein the first capillary barrier or the second capillary barrier or both are adjacent to an air passage, the air passage comprising a narrowing element.

18. The fluid apparatus of claim 1, further comprising at least two additional ITP loops, each additional ITP loop including an isotachycardia electrophoresis (ITP) branch, the ITP branch including a first loading reservoir and a first channel, wherein the first loading reservoir is in fluid communication with the first channel via a first connector, and the first channel including a first capillary barrier and a second capillary barrier positioned on either side of the first connector, wherein the first capillary barrier is configured and arranged to impede flow of a liquid meniscus along the first channel by capillary force at the first capillary barrier, and the second capillary barrier is configured and arranged to impede flow of another liquid meniscus at the second capillary barrier by capillary force.

19. The fluid apparatus of claim 1, further comprising at least five additional fluid loops, each additional loop including an isotonic electrophoresis (ITP) branch, the ITP branch including a first loading reservoir and a first channel, wherein the first loading reservoir is in fluid communication with the first channel via a first connector, and the first channel including a first capillary barrier and a second capillary barrier positioned on either side of the first connector, wherein the first capillary barrier is configured and arranged to impede flow of a liquid meniscus along the first channel by capillary force at the first capillary barrier, and the second capillary barrier is configured and arranged to impede flow of another liquid meniscus at the second capillary barrier by capillary force.

20. The fluid apparatus of claim 1, wherein the first loading reservoir is a sample reservoir closed by a removable material.

21. The fluid device of claim 1, further comprising one or more pneumatic channels that open at one or more pneumatic ports and communicate with each of the capillary barriers.

22. The fluid device of claim 21, wherein the head height of the one or more pneumatic ports relative to the substrate is shorter than that of the first loading reservoir.

23. The fluid apparatus of claim 1, wherein the first channel is a sample channel with a depth of less than 2 mm or a volume of 10 μL to 1 ml.

24. The fluid device of claim 1, wherein the first loading reservoir comprises (a) a conical cross-section in the region of the first loading reservoir adjacent to the substrate, and (b) a cylindrical through-hole or aperture penetrating the substrate.

25. The fluid device of claim 1, wherein the first loading reservoir includes (a) an inlet channel for ambient air at one end, and (b) a first orifice penetrating the substrate at the other end of the first loading reservoir, wherein the first loading reservoir has a truncated conical shape, wherein a wider region of the truncated conical shape is located at the inlet channel for ambient air, and a narrower region is located at the first orifice penetrating the substrate.

26. The fluid device of claim 21, wherein the pneumatic port on the substrate is inserted into the surface of a first side of the substrate at a depth of 1 μm to 1 mm, or protrudes from the surface of the first side of the substrate at a height of 0 μm to 2 mm.

27. A method of loading a fluid device according to claim 1, wherein the fluid device further comprises a second loading reservoir or channel, a third loading reservoir or channel, a fourth loading reservoir or channel, a fifth loading reservoir or channel, or a sixth loading reservoir or channel, and the method comprises loading different buffer solutions into the first channel or the first loading reservoir, the second channel or the second loading reservoir, the third channel or the third loading reservoir, the fourth channel or the fourth loading reservoir, the fifth channel or the fifth loading reservoir, or the sixth channel or the sixth loading reservoir.

28. The method of claim 27, wherein the fluid device includes the first channel, the first channel including the platform capillary barrier adjacent to a second channel, and the loading of the buffer solution includes: Load the first buffer solution into the first channel or the first loading reservoir and load the second buffer solution into the second channel or the second loading reservoir; A first positive pneumatic pressure or a first negative pneumatic pressure is applied to the fluid device, causing the first buffer solution and the second buffer solution to stagnate at the base of the slope within the capillary barrier of the platform, wherein the application of the first positive pneumatic pressure or the first negative pneumatic pressure includes increasing or decreasing the first positive pneumatic pressure or the first negative pneumatic pressure in a fixed increment.

29. The method of claim 28, further comprising applying a second positive pneumatic pressure or a second negative pneumatic pressure to the fluid device such that the first buffer solution and the second buffer solution flow along a ramp on either side of the platform capillary barrier, wherein the application of the second positive pneumatic pressure or the second negative pneumatic pressure comprises increasing or decreasing the second positive pneumatic pressure or the second negative pneumatic pressure in a fixed increment.

30. The method of claim 29, wherein the first buffer and the second buffer are stagnant at the platform of the platform capillary barrier and have an air gap therebetween, the air gap being located above or below the platform of the platform capillary barrier, and the method further comprising applying a third positive pneumatic pressure or a third negative pneumatic pressure to the fluid device such that the first buffer and the second buffer enter the air gap, thereby forming a liquid-liquid interface between the first buffer and the second buffer above or below the platform of the platform capillary barrier.

31. The fluid device of claim 1, wherein the first capillary barrier is a platform capillary barrier.

32. The fluid device of claim 1, wherein the second capillary barrier is a platform capillary barrier.

33. The fluid device of claim 1, wherein the cliff capillary barrier comprises: A ramp protruding from the surface of the first channel at a first angle; Platform area; as well as A cliff region extending from the platform area to the surface of the first channel, wherein the cliff region intersects the surface at a second angle that is substantially steeper than the first angle.

34. The fluid device of claim 1, wherein the platform capillary barrier comprises: A first ramp protruding from the surface of the first channel at a first angle; Platform area; as well as A second slope extends from the platform region to the surface of the first channel, wherein the fourth angle at which the second slope of the platform capillary barrier intersects the surface of the first channel and the first angle are inclined to each other.

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