Isotachophoresis for nucleic acid purification

Abstract

To provide isotachophoresis for purification of nucleic acids.SOLUTION: 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 for processing samples and extracting nucleic acids, e.g., by isotachophoresis. In particular, the systems and related methods can allow extraction of nucleic acids, including non-crosslinked nucleic acids, from samples such as tissue or cells. The systems and devices can also be used for multiplex parallel sample processing.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 288,930, filed January 29, 2016, and entitled "Isotachophoresis for the Purification of Nucleic Acids" (Attorney Subject Docket No: 43647-712.101), the entire contents of which are incorporated herein by reference.

[0002] Statement of Federally Funded Research This invention was made with United States government support under grant number 1R43HG007620-01 awarded by the National Institutes of Health. The United States government has certain rights in this invention. [Background technology]

[0003] Formalin-fixed, paraffin-embedded (FFPE) samples have been collected, prepared, stored, and archived in large tissue banks for over a century. As of 2008, over 400 million FFPE samples were stored in biobanks worldwide, and this number continues to grow. These samples often contain clinical information, such as primary diagnosis, treatment regimens, and follow-up data, making them an important source of information for therapeutic drug development and genomic and transcriptomic biomarker discovery. Summary of the Invention [Means for solving the problem]

[0004] Sample preparation methods for extracting and purifying nucleic acids from FFPE samples remain manual and labor-intensive. FFPE extraction and purification techniques vary widely but often involve steps that are difficult to automate and accelerate: dewaxing, centrifugation, buffer exchange, temperature control, cross-link reduction, and enzymatic treatment. FFPE generally refers to the use of formalin to cross-link proteins in a sample and embedding the sample in paraffin (wax). FFPE processing of a sample often enables the sample to be preserved for extended periods of time and can be particularly useful for long-term storage. Cross-linking proteins bind DNA and RNA in the sample, generally rendering it unavailable for downstream applications such as amplification, library preparation, or sequencing.

[0005] Removal of paraffin and protein crosslinks in FFPE samples can be a difficult process. Deparaffinization is traditionally performed using highly flammable xylene. Alternately or sequentially, samples may be treated with other solvents, mineral oils, and alkaline chemicals and / or high temperatures. After deparaffinization, proteins in the sample may be treated with different agents or subjected to conditions that may require additional time and effort.

[0006] At the end of digestion and denaturation, a mixture of cross-linked and non-cross-linked nucleic acids may remain.Removal of non-cross-linked material may be important for high quality, resulting from assays such as amplification or sequencing.In some cases, if the proportion of non-cross-linked material is too small, downstream assays may not be able to be performed, resulting in the loss of not only the sample itself, but also labor, time and information resources.

[0007] Isotachophoresis (ITP) is a method for electrophoresis in which a leading electrolyte (LE) with a higher magnitude of effective mobility and a trailing electrolyte (TRAIL) with a lower magnitude of effective mobility (e.g., relative to the LE). ITP is an electrophoretic technique that uses a discontinuous buffer containing an electrolyte (TE) to collect sample species with an effective mobility magnitude higher than the leading electrolyte but lower than the leading electrolyte. ITP can selectively collect nucleic acids from a sample by over 10,000-fold in less than five minutes. The present disclosure provides methods and devices for using and automating ITP for sample preparation, including extraction, purification, enrichment, and highly sensitive quantification, and is particularly useful for preparing and purifying nucleic acids from FFPE samples and other biological samples.

[0008] While sample preparation is important for genomic analysis, it remains a primary source of analytical variability and can require significant manual labor. The present disclosure includes techniques and devices to address this challenge, such as by using on-chip isotachophoresis (ITP) for nucleic acid extraction and purification. These techniques are designed to enable higher yields and higher quality nucleic acid sample preparation, and to reduce the number of samples from FFPE and other archived or fresh samples to more usable samples (e.g., quality-check rejects). The present invention includes a method for enriching (concentrating) non-crosslinked nucleic acids to produce a reduced rejection (reduced crosslink rejection).

[0009] The present disclosure includes techniques and devices for automating nucleic acid sample preparation from samples including solid tissue samples, lysed solid tissue samples, archived or fixed tissue samples (e.g., FFPE), whole blood, plasma and serum, buccal swabs, dried bloodstains and other forensic samples, fresh or fresh frozen (FF) tissue, biopsy tissue, organ tissue, solid organ tissue, samples containing intercellular junctions (e.g., gap junctions, tight junctions, adherens junctions), cultured or harvested cells from blood or tissue, stool, and bodily fluids (e.g., saliva, urine), or any combination thereof. Samples can include cellular and cell-free nucleic acids for both eukaryotic and prokaryotic organisms, or any combination thereof. The techniques of the present disclosure can be faster, less manual, and more suitable for both small and large starting amounts of tissue than existing methods, achieving higher yields from samples and higher quality analysis of the samples.

[0010] An aspect of the present disclosure provides a method for sample purification, comprising: (a) loading into a fluidic device: (i) a tissue sample comprising lysed solid tissue, the lysed solid tissue comprising nucleic acids and contaminants; (ii) a final electrolyte buffer comprising first final electrolyte ions having an effective mobility smaller than the effective mobility of the nucleic acid; and (iii) a leading electrolyte buffer comprising first leading electrolyte ions having a second effective mobility, the second effective mobility being larger than the effective mobility of the nucleic acid; and (b) applying an electric field within the fluidic device to perform isotachophoresis using the first final electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby purifying the nucleic acid from the contaminants in the tissue sample.

[0011] In some embodiments of the aspects provided herein, the effective mobility of the first final 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 final electrolyte buffer, and the leading electrolyte buffer are loaded into a first zone of the microfluidic chip. Some embodiments of the aspects provided herein can further include subjecting the tissue sample to at least one sample preparation procedure selected from the group consisting of (1) removal of embedding material, (2) tissue disruption, (3) cell lysis, (4) decrosslinking of the nucleic acid, (5) protein digestion, and (6) digestion of the nucleic acid in the first zone of the microfluidic chip. In some embodiments of the aspects provided herein, the isotachophoresis is performed in a second zone of the microfluidic chip, the second zone being separated from the first zone and fluidly connected to the first zone. In some embodiments of the aspects provided herein, the solid tissue is derived from a solid organ. In some embodiments of the aspects provided herein, the lysed solid tissue comprises a chemical fixative. In some embodiments of the aspects provided herein, the chemical fixative is formalin. In some embodiments of the aspects provided herein, the solid tissue is formalin-fixed, paraffin-embedded tissue (FFPE). In some embodiments of the aspects provided herein, the lysed solid tissue comprises urea or thiourea. Some embodiments of the aspects provided herein further comprise disrupting intercellular junctions, extracellular matrix, or connective tissue to obtain the lysed solid tissue. In some embodiments of the aspects provided herein, the lysed solid tissue comprises solid particles. In some embodiments of the aspects provided herein, the nucleic acid comprises dispersed or solvated nucleic acid. In some embodiments of the aspects provided herein, the contaminant is selected from the group consisting of crosslinked nucleic acids, embedding materials, tissue debris, chemical fixatives, proteins, inhibitors, and combinations thereof.In some embodiments of the aspects provided herein, the contaminant comprises a cross-linked nucleic acid. In some embodiments of the aspects provided herein, the tissue sample is combined with the final electrolyte buffer prior to the loading. In some embodiments of the aspects provided herein, the tissue sample is combined with the leading electrolyte buffer prior to the loading. In some embodiments of the aspects provided herein, the loading of the leading electrolyte buffer occurs prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the solid tissue is dissolved in the leading electrolyte buffer prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the solid tissue is dissolved in the final electrolyte buffer prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure comprises removing an embedding material by incubating the tissue sample in the fluidic device at a temperature of at least about 37° C. for a period of at least about 1 minute prior to the application of the electric field. In some embodiments of the aspects provided herein, the temperature is about 40°C to about 80°C. In some embodiments of the aspects provided herein, the time period is about 1 minute to about 120 minutes. In some embodiments of the aspects provided herein, the sample preparation procedure comprises disrupting tissue or lysing cells by applying mechanical stress to the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure comprises disrupting tissue or lysing cells by applying heat to the tissue sample. In some embodiments of the aspects provided herein, the application of heat brings the temperature of the tissue sample to about 30°C to about 80°C. In some embodiments of the aspects provided herein, the sample preparation procedure comprises disrupting tissue or lysing cells by contacting the tissue sample with a solution having a pH of at least 10 or by proteolytic digestion of the tissue sample. In some embodiments of the aspects provided herein, the proteolytic digestion is performed at a temperature greater than about 25°C.In some embodiments of the aspects provided herein, the sample preparation procedure includes disrupting tissue or lysing cells by applying at least one detergent 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 includes thiourea. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is in the range of about 4 M to about 9 M, and the concentration of the thiourea in the solution is in the range of about 0.5 M to about 3.5 M. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is in the range of about 6.5 M to about 7.5 M, and the concentration of the thiourea in the solution is in the range of about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the sample preparation procedure includes decrosslinking the nucleic acid by digesting crosslinked proteins with proteinase K. In some embodiments of the aspects provided herein, the sample preparation procedure includes digesting the nucleic acid using DNase or RNase. Some embodiments of the aspects provided herein further include eluting an output solution containing the purified nucleic acid from an output reservoir of the fluidic device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the tissue sample and the purified nucleic acid in the output solution include a cross-linked nucleic acid, and the concentration of the cross-linked nucleic acid in the output solution is at most about half 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 that is at most half the concentration of the contaminant in the tissue sample. In some embodiments of the aspects provided herein, the first final electrolyte ion includes caproic acid.In some embodiments of the aspects provided herein, the first leading electrolyte ion comprises chloride ion. In some embodiments of the aspects provided herein, the final electrolyte buffer comprises a second final electrolyte ion having a different effective mobility than the first final electrolyte ion. In some embodiments of the aspects provided herein, the second final electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments of the aspects provided herein, the second final electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and the first final electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the second final electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid) and the first final electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the second final electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and the first final electrolyte ion comprises MOPS. In some embodiments of the aspects provided herein, the final electrolyte buffer comprises a second final electrolyte ion having a second effective mobility, the second effective mobility being approximately the same magnitude as or smaller than the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the tissue sample loaded into the fluidic device has a volume of at least 50 μl. Some embodiments of the aspects provided herein further include performing a first sample processing procedure on the tissue sample in the first zone of the microfluidic chip and performing an enzymatic reaction on the tissue sample in a second zone of the microfluidic chip. In some embodiments of the aspects provided herein, the first sample processing procedure comprises removal of embedding material, tissue disruption, or cell lysis, and the enzymatic reaction comprises decrosslinking the nucleic acid, digesting a protein, or digesting a nucleic acid.In some embodiments of the aspects provided herein, the first zone and the second zone are each heated to a temperature greater than 37° C. In some embodiments of the aspects provided herein, the first zone is heated to a temperature of about 60° C. to 100° C. and the second zone is heated to a temperature of about 40° C. to 60° C. during the first sample processing procedure.

[0012] An aspect of the present disclosure includes a method for detecting nucleic acids, the method comprising: (a) loading into a first channel of a microfluidic chip: (i) a first sample comprising a first nucleic acid and a first contaminant; (ii) a first final electrolyte buffer comprising a first final ion, wherein the magnitude of the effective mobility of the first final ion is smaller than the magnitude of the effective mobility of the first nucleic acid; and (iii) a first leading electrolyte buffer comprising a first leading ion, wherein the magnitude of the effective mobility of the first leading ion is larger than the magnitude of the effective mobility of the first nucleic acid; (b) loading into a second channel of the microfluidic chip: (i) a second sample comprising a second nucleic acid and a second contaminant; and (ii) a second final electrolyte buffer comprising a second final ion, wherein the magnitude of the effective mobility of the second final ion is smaller than the magnitude of the effective mobility of the second nucleic acid. and (iii) a second leading electrolyte buffer containing a second leading ion, wherein the magnitude of the effective mobility of the second leading ion is greater than the magnitude of the effective mobility of the second nucleic acid; and (c) applying a first electric field within the microfluidic chip to perform isotachophoresis in the first channel using the first leading ion, the first nucleic acid, and the first leading ion, and applying a second electric field to perform isotachophoresis in the second channel using the second leading ion, the second nucleic acid, and the second leading ion, thereby simultaneously purifying the first nucleic acid from the first contaminant and the second nucleic acid from the second contaminant.

[0013] In some embodiments of the aspects provided herein, the first sample and the second sample are different sample types. In some embodiments of the aspects provided herein, the first nucleic acid and the second nucleic acid are nucleic acids of different types or lengths. In some embodiments of the aspects provided herein, the first final electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or tissue disruption agent. In some embodiments of the aspects provided herein, the lysing agent or tissue disruption agent comprises one or more agents selected from the group consisting of a solution having a pH greater than about 12, a proteinase, urea, thiourea, and a surfactant. In some embodiments of the aspects provided herein, the first sample comprises lysed solid tissue. In some embodiments of the aspects provided herein, the second sample comprises lysed cells. In some embodiments of the aspects provided herein, the first sample does not contact the second sample during the isotachophoresis. Some embodiments of the aspects provided herein further include loading into a third channel of the microfluidic chip: (i) a third sample comprising a third nucleic acid and a third contaminant; (ii) a third final electrolyte buffer comprising a third final ion, wherein the magnitude of the effective mobility of the third final ion is smaller than the magnitude of the effective mobility of the third nucleic acid; and (iii) a third leading electrolyte buffer comprising a third leading ion, wherein the magnitude of the effective mobility of the third leading ion is larger than the magnitude of the effective mobility of the third nucleic acid; and applying the electric field within the microfluidic chip to perform the isotachophoresis in the third channel using the third final ion, the third nucleic acid, and the third leading ion, thereby simultaneously purifying the first nucleic acid from the first contaminant, the second nucleic acid from the second contaminant, and the third nucleic acid from the third contaminant. In some embodiments of the aspects provided herein, the first and second electric fields arise from a single electrode pair.In some embodiments of the aspects provided herein, the first and second electric fields are generated from different electrode pairs. In some embodiments of the aspects provided herein, the first and second channels are coupled to independent sensors. In some embodiments of the aspects provided herein, feedback from the independent sensors is used to independently control the first and second electric fields. In some embodiments of the aspects provided herein, the independent sensors sense voltage, and the feedback is used to control current (or resistance) in the first and second channels. In some embodiments of the aspects provided herein, the nucleic acid comprises DNA. In some embodiments of the aspects provided herein, the nucleic acid comprises RNA.

[0014] An aspect of the present disclosure provides a method for sample purification, comprising: (a) loading into a fluidic device: (i) a sample comprising fixed cells, fixed tissue, or embedded tissue, the sample comprising nucleic acids; (ii) a final electrolyte buffer comprising a final electrolyte, the final electrolyte having a lower effective mobility than the nucleic acids; and (iii) a leading electrolyte buffer comprising a leading electrolyte, the leading electrolyte having a higher effective mobility than the nucleic acids; and (b) applying an electric field to the fluidic device to perform isotachophoresis using the final electrolyte ions, the nucleic acids, and the leading electrolyte, thereby purifying the nucleic acids from contaminants in the sample.

[0015] In some embodiments of the aspects provided herein, the contaminant is selected from the group consisting of cross-linked nucleic acids, embedding materials, chemical fixatives, enzymes, and inhibitors. In some embodiments of the aspects provided herein, the sample comprises the fixed cells, the fixed tissue, or both the fixed cells and the fixed tissue. In some embodiments of the aspects provided herein, the sample is formalin-fixed. In some embodiments of the aspects provided herein, the sample comprises the embedded tissue. In some embodiments of the aspects provided herein, the sample comprises the 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 a tissue biopsy. In some embodiments of the aspects provided herein, the sample is a resected formalin-fixed, paraffin-embedded (FFPE) sample. Some embodiments of the aspects provided herein further include comparing the nucleic acid characteristic with a nucleic acid characteristic from another sample, the characteristic being expression level, nucleic acid sequence, molecular weight, nucleic acid integrity, nucleic acid strandedness (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 final electrolyte buffer has a pH greater than about 7. Some embodiments of the aspects provided herein further include incubating the tissue sample in the fluidic device at a temperature of at least about 37°C for a period of at least about 1 minute prior to the application of the electric field. In some embodiments of the aspects provided herein, the temperature is between about 40°C and about 80°C. In some embodiments of the aspects provided herein, the period is between about 1 minute and about 120 minutes. In some embodiments of the aspects provided herein, the leading electrolyte buffer comprises proteinase K. Some embodiments of the aspects provided herein further comprise using proteinase K to remove protein crosslinks from the nucleic acid.Some embodiments of the aspects provided herein further include removing protein crosslinks from the nucleic acid using heat after the application of the electric field. Some embodiments of the aspects provided herein further include eluting an output solution containing the purified nucleic acid from an output reservoir of the fluidic device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the concentration of the crosslinked nucleic acid in the output solution is at most about half the concentration of the crosslinked nucleic acid in the tissue sample. 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 greater than 25 μL. In some embodiments of the aspects provided herein, the final electrolyte has a higher effective mobility than the contaminant. In some embodiments of the aspects provided herein, the final electrolyte includes (i) a first ion having an effective mobility magnitude greater than that of the contaminant, and (ii) a second ion having an effective mobility magnitude approximately the same as or smaller than that of the contaminant. In some embodiments of the aspects provided herein, performing the isotachophoresis includes quenching the pH of the tissue sample to about 7.5. Some embodiments of the aspects provided herein further include deparaffinizing the sample before 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 / microliter (pg / μL).

[0016] An embodiment of the present disclosure provides a method for detecting nucleic acids in a fluidic device, comprising: (i) a tissue sample comprising lysed solid tissue and nucleic acids; (ii) a final electrolyte buffer comprising final electrolyte ions having a first effective mobility, the first effective mobility being an order of magnitude smaller than the effective mobility of the nucleic acid; (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir, the first leading electrolyte buffer comprising first leading electrolyte ions having a second effective mobility, the second effective mobility being an order of magnitude larger than the effective mobility of the nucleic acid; and (iv) a second leading electrolyte buffer in a second leading electrolyte reservoir, the first leading electrolyte buffer comprising first leading electrolyte ions having a third effective mobility. The method includes the steps of: (a) introducing a second leading electrolyte buffer containing a second leading electrolyte ion, wherein the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acid, and the first leading electrolyte buffer is different from the second leading electrolyte buffer; (b) performing a first isotachophoresis run using the final electrolyte ion, the nucleic acid, and the first leading electrolyte ion, thereby purifying the nucleic acid from the contaminants in the tissue sample; and (c) performing a second isotachophoresis run using the final electrolyte ion, the nucleic acid, and the second leading electrolyte ion.

[0017] In some embodiments of the aspects provided herein, performing the second isotachophoresis step includes varying the applied current from the first channel to the second channel. In some embodiments of the aspects provided herein, the first leading electrolyte ions are the same as the second leading electrolyte ions, and the concentration of the first leading electrolyte ions in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ions in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the second effective mobility has a magnitude greater than the magnitude of the third effective mobility. In some embodiments of the aspects provided herein, the first leading electrolyte ions are different from the second leading electrolyte ions. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer includes a third leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer includes a third leading electrolyte ion. 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 isotachophoresis step and the second isotachophoresis step are performed by applying a single electric field. In some embodiments of the aspects provided herein, the first isotachophoresis step and the second isotachophoresis step are performed by applying more than one electric field.In some embodiments of the aspects provided herein, the concentration of the second leading electrolyte ion in the second leading electrolyte buffer is less than 50 mM. In some embodiments of the aspects provided herein, the second leading electrolyte buffer comprises 50 mM Tris HCl.

[0018] An embodiment of the present disclosure includes: (a) a first isotachophoresis region in a microfluidic chip, the first isotachophoresis region including: (i) a first sample reservoir in fluid communication with a first fluid 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) (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 microfluidic device comprising a second isotachophoresis region in the microfluidic chip, the second isotachophoresis region comprising a fourth buffer reservoir in fluid communication with the second channel, wherein the first isotachophoresis region is not in fluid communication with the second isotachophoresis region, and the microfluidic device is configured to independently control a first electrical circuit that applies current to the first isotachophoresis region and a second electrical circuit that applies current to the second isotachophoresis region.

[0019] In some embodiments of the aspects provided herein, the leak rate between the first and second isotachophoresis regions is less than 1 μl per hour. In some embodiments of the aspects provided herein, the current leakage between the first and second isotachophoresis regions is less than 1 μA. In some embodiments of the aspects provided herein, the impedance is greater than 1 megaohm. In some embodiments of the aspects provided herein, the first fluidic channel holds a liquid volume greater than 100 μl. In some embodiments of the aspects provided herein, the first fluidic channel is separated from the second fluidic channel by a distance that is at most one-fifth the width of the first channel. In some embodiments of the aspects provided herein, the microfluidic device is configured to control the first electrical circuit simultaneously with the second electrical circuit. Some embodiments of the aspects provided herein further include an elution reservoir in fluid communication with the first channel, and the temperature sensor is located within 5 mm of the elution reservoir.

[0020] An embodiment of the present disclosure provides a method comprising the steps of: (a) providing an electrokinetic fluid device including a sample input reservoir in fluid communication with a channel; (b) loading a sample volume into the sample input reservoir; (c) transferring at least 50% of the sample volume from the sample input reservoir to the channel without adding additional volume to the sample input reservoir; and (d) applying an ionic current to the channel.

[0021] In some embodiments of the aspects provided herein, the moving step is performed using gravity. In some embodiments of the aspects provided herein, the ionic current does not substantially flow through the channel. In some embodiments of the aspects provided herein, the at least 50% of the sample volume accounts for at least 80% of the sample volume. In some embodiments of the aspects provided herein, the sample volume comprises nucleic acid. In some embodiments of the aspects provided herein, the sample volume comprises a tissue sample or a formalin-fixed, paraffin-embedded (FFPE) sample. In some embodiments of the aspects provided herein, the applying an ionic current comprises performing isotachophoresis. 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 an upper region connected to a bottom region via a tapered region, the upper region having a first diameter and the bottom region having a second diameter, the first diameter being at least two times longer than the second diameter to facilitate the transfer 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 aspects provided herein, the sample volume is at least 50 μl. In some embodiments of the aspects provided herein, the sample volume is at least 100 μl.

[0022] Aspects of the present disclosure provide a microfluidic chip including: a first sample input reservoir, the first sample input reservoir including a top region connected to a bottom region via a tapered region, the top region having a first inner hydraulic diameter and the bottom region having a second inner hydraulic diameter, the first inner hydraulic diameter being at least two times longer than the second inner hydraulic diameter, the first sample input reservoir being in fluid communication with a first channel; a first buffer reservoir in fluid communication with the first channel, the first sample reservoir being configured such that a free surface of liquid in the first sample reservoir has a negligible buffer head height relative to liquid in the first buffer reservoir; and a second buffer reservoir in fluid communication with the first channel.

[0023] In some embodiments of the aspects provided herein, the first hydraulic inner diameter ranges from about 1 mm to about 15 mm. In some embodiments of the aspects provided herein, the second hydraulic inner diameter ranges from about 0.5 mm to about 5 mm. In some embodiments of the aspects provided herein, the first sample reservoir is configured to hold a sample volume of at least 100 μl. In some embodiments of the aspects provided herein, the microfluidic chip is configured such that at least 50% of the sample volume is transferred from the first sample reservoir to the first channel when a vacuum is applied to the microfluidic chip. In some embodiments of the aspects provided herein, the microfluidic chip is configured to perform isotachophoresis on a sample entering the first channel.

[0024] Aspects of the present disclosure provide a method for extracting nucleic acids, comprising: (a) exposing a biological sample containing cells or tissue to a solution containing urea or thiourea, thereby lysing the cells or tissue in the biological sample to produce a cell lysate; (b) introducing the cell lysate into a device; and (c) performing isotachophoresis using the device to isolate nucleic acids from the cell lysate.

[0025] Some embodiments of the aspects provided herein further include digesting the sample with proteinase K. In some embodiments of the aspects provided herein, the solution includes urea and thiourea. In some embodiments of the aspects provided herein, the solution includes a ratio of urea to thiourea of ​​about 2 to 1. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is about 4 M to about 9 M, and the concentration of the thiourea in the solution is about 0.5 M to about 3.5 M. In some embodiments of the aspects provided herein, the concentration of the urea in the solution is about 6.5 M to about 7.5 M, and the concentration of the thiourea in the solution is about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the solution includes a terminal electrolyte ion or a leading electrolyte ion, or both a terminal electrolyte ion and a leading electrolyte ion.

[0026] An aspect of the present disclosure provides a method for preparing a fluidic device comprising: (a) a fluidic device containing: (i) a cell sample comprising genomic DNA and a contaminant, wherein the cell sample is contacted with a lysis buffer before or after loading the cell sample into the fluidic device; (ii) a final electrolyte buffer comprising a final electrolyte ion having a first effective mobility, the first effective mobility being smaller in magnitude than the effective mobility of a high molecular weight nucleic acid and larger in magnitude than the contaminant; and (iii) a first leading electrolyte buffer comprising a first leading electrolyte ion having a second effective mobility, the second effective mobility being (b) introducing the first leading electrolyte buffer solution having a magnitude greater than the magnitude of the effective mobility of the high molecular weight nucleic acid; (b) performing isotachophoresis using the final electrolyte ion, the high molecular weight nucleic acid, and the first leading electrolyte ion, thereby separating the high molecular weight nucleic acid from the contaminants and enriching the high molecular weight nucleic acid in the isotachophoresis zone; and (c) eluting the genomic DNA into a solution in an output reservoir, wherein more than 50% of the mass of nucleic acids in the solution is greater than 30 kilobases.

[0027] In some embodiments of the aspects provided herein, the lysis buffer does not comprise an alkaline buffer. In some embodiments of the aspects provided herein, the lysis buffer comprises octylphenol ethoxylate. In some embodiments of the aspects provided herein, more than 50% of the mass of nucleic acids in the solution is greater than 50 kilobases.

[0028] An aspect of the present disclosure provides a method for performing isotachophoresis, comprising the steps of: (a) providing a fluidic device including a first channel in fluid communication with a sample input reservoir containing a tissue sample comprising lysed solid tissue, a first buffer reservoir containing a first leading electrolyte buffer, and a second buffer reservoir containing a final 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 final electrolyte buffer in the second buffer reservoir; and (d) applying an electric field within the fluidic device to perform isotachophoresis, wherein the isotachophoresis is performed without direct contact between the tissue sample and the first and second electrodes.

[0029] In some embodiments of the aspects provided herein, the fluidic 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 of the aspects provided herein, the third buffer reservoir and the first buffer reservoir are connected by a second channel including 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 of the aspects provided herein, the fluidic device further includes an elution reservoir. In some embodiments of the aspects provided herein, the elution reservoir is in fluid communication with a fourth buffer reservoir.

[0030] An embodiment of the present 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 junction; and (b) a mechanical member including first teeth, the mechanical member configured to apply mechanical pressure to the first channel via the first teeth to at least partially close the first channel by plastic deformation of at least one wall of the first channel and to increase fluid resistance between the first channel and the first reservoir.

[0031] In some embodiments of the aspects provided herein, the microfluidic chip further includes a second reservoir in fluid communication with the first reservoir and a second channel connecting the first reservoir and the second reservoir, and the mechanical member further includes second teeth 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 of the aspects provided herein, the first teeth are configured to apply mechanical pressure to the first junction to close the first channel by plastic deformation of at least one wall of the first channel. In some embodiments of the aspects provided herein, the first teeth are configured to heat the first channel. In some embodiments of the aspects provided herein, the mechanical member includes a material having a Young's modulus greater than the Young's modulus of the first channel. In some embodiments of the aspects provided herein, the microfluidic system is configured to perform isotachophoresis. In some embodiments of the aspects provided herein, the first tine is thermally coupled to a heating element. In some embodiments of the aspects provided herein, the first tine 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 of using the microfluidic system to complete a process in a fluid system, the method including at least partially closing the first channel by plastic deformation, thereby increasing resistance to fluid flow between the first channel and the first reservoir. In some embodiments of the aspects provided herein, the first tine of the mechanical member applies a force of at least 0.25 lb to the first channel. In some embodiments of the aspects provided herein, the process in the fluid system is isotachophoresis.

[0032] An aspect of the present disclosure is a method for detecting nucleic acids comprising the steps of: (a) loading the sample containing nucleic acids into a first reservoir of a microfluidic chip; (b) loading a final electrolyte buffer into a second reservoir of the microfluidic chip, the final electrolyte buffer comprising first final electrolyte ions having an effective mobility that is less than the magnitude of the effective mobility of the nucleic acids; and (c) loading a leading electrolyte buffer into a third reservoir of the microfluidic chip, the third reservoir comprising first leading electrolyte ions having a second effective mobility that is less than the magnitude of the effective mobility of the nucleic acids. (d) applying an electric field within the microfluidic chip to perform isotachophoresis using the first final electrolyte ion, the nucleic acid, and the first leading electrolyte ion, thereby confining the nucleic acid or a portion thereof in an isotachophoresis zone; and (e) using a temperature sensor to sense temperature changes in or near the isotachophoresis zone, wherein feedback from the temperature sensor is used to control the electric field.

[0033] In some embodiments of the aspects provided herein, the controlling of the electric field causes the nucleic acid, or a portion thereof, to be located in an elution reservoir or in a region of the microfluidic chip. In some embodiments of the aspects provided herein, the temperature sensor is positioned within at most 8 mm of the elution reservoir. In some embodiments of the aspects provided herein, the temperature change is in the range of about 0.2°C to 5°C. In some embodiments of the aspects provided herein, the applying of an electric field causes the leading electrolyte and the final electrolyte to meet at an isotachophoresis interface, and the temperature sensor senses the isotachophoresis interface.

[0034] An embodiment of the present disclosure provides a microfluidic device including: (a) a first isotachophoresis region in a microfluidic chip, the first isotachophoresis region including: (i) a first sample reservoir in fluid communication with a first fluid channel; (ii) first, second, and third buffer reservoirs in fluid communication with the first fluid channel, the first and second buffer reservoirs being separated by a capillary barrier; and (iii) an elution reservoir in fluid communication with the first fluid channel; (b) a sensor configured to detect a temperature change in the first fluid channel within the first isotachophoresis region; and (c) an apparatus positioned to provide an electric current within the first channel within the first isotachophoresis region.

[0035] Some embodiments of the aspects provided herein further include a controller configured to activate a reduction or termination of the current when the sensor receives a thermal signal. In some embodiments of the aspects provided herein, the temperature change is an increase in temperature in 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 isolate a sample of nucleic acid 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 by a sensor positioned within at most 8 mm of the elution reservoir. In some embodiments of the aspects provided herein, the first channel includes a single sensor.

[0036] An embodiment of the present 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 final electrolyte buffer comprising a final electrolyte; and (c) a leading electrolyte buffer comprising a leading electrolyte.

[0037] In some embodiments of the aspects provided herein, the final electrolyte buffer comprises a mixture of at least two electrolytes having different effective mobilities. In some embodiments of the aspects provided herein, the mixture comprises (i) a first electrolyte having an effective mobility magnitude lower than that of the nucleic acid and higher than that of the contaminant, and (ii) a second electrolyte having an effective mobility magnitude lower than that of the contaminant. In some embodiments of the aspects provided herein, the first electrolyte comprises caproic acid. In some embodiments of the aspects provided herein, the second electrolyte comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments of the aspects provided herein, the kit further comprises a sample buffer, wherein the sample buffer comprises any combination of a leading electrolyte buffer, a final electrolyte buffer, or urea. In some embodiments of the aspects provided herein, the kit further comprises a sample buffer comprising urea and thiourea.

[0038] An aspect of the present disclosure provides a method for sample purification, comprising: (a) loading into a fluidic device: (i) a tissue sample containing nucleic acids and contaminants, the tissue sample not including an unlysed whole blood sample; (ii) a final electrolyte buffer containing final electrolyte ions having an effective mobility greater than the effective mobility of the contaminants and less than the effective mobility of the nucleic acids; and (iii) a leading electrolyte buffer containing leading electrolyte ions having a second effective mobility, wherein the second effective mobility is greater than the effective mobility of the nucleic acids; and (b) applying an electric field within the fluidic device to perform isotachophoresis using the final electrolyte ions, the nucleic acids, and the leading electrolyte ions, thereby purifying the nucleic acids from the contaminants in the tissue sample.

[0039] In some embodiments of the aspects provided herein, the tissue sample is not a whole blood sample. In some embodiments of the aspects provided herein, the final electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the leading electrolyte ion comprises chloride ion. In some embodiments of the aspects provided herein, the final electrolyte buffer comprises a second final electrolyte ion having a different effective mobility than the first final electrolyte ion. In some embodiments of the aspects provided herein, the second final electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments of the aspects provided herein, the second final electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments of the aspects provided herein, the second final electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and the first final electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the second final electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid) and the first final electrolyte ion comprises caproic acid. In some embodiments of the aspects provided herein, the second final electrolyte ion comprises HEPES and the first final electrolyte ion comprises MOPS. In some embodiments of the aspects provided herein, the final electrolyte buffer comprises a second final electrolyte ion having a second effective mobility, the second effective mobility being about the same magnitude as or smaller than the magnitude of the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the contaminant is selected from the group consisting of cross-linked nucleic acids, embedding materials, chemical fixatives, 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 mixed with the final electrolyte buffer prior to loading.In some embodiments of the aspects provided herein, the tissue sample is mixed with the lead electrolyte buffer prior to the loading. In some embodiments of the aspects provided herein, the loading of the lead electrolyte buffer occurs prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the method further includes eluting an output solution containing the purified nucleic acid from an output reservoir of the fluidic device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the concentration of the cross-linked nucleic acid in the output solution is at most about half the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the output solution is free of the contaminants. In some embodiments of the aspects provided herein, the tissue sample is fresh tissue. In some embodiments of the aspects provided herein, the tissue sample is fresh frozen (FF) tissue. In some embodiments of the aspects provided herein, the tissue sample is formalin-fixed, paraffin-embedded (FFPE) tissue. In some embodiments of the aspects provided herein, the method further comprises lysing or disrupting the tissue sample prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the lysing or disrupting is performed using urea or thiourea.

[0040] An aspect of the present disclosure is a method for detecting nucleic acids comprising the steps of: (a) loading into a first channel of a fluidic device: (i) a first tissue sample comprising a first nucleic acid and a first contaminant; (ii) a first final electrolyte buffer comprising a first final ion, wherein the magnitude of the effective mobility of the first final ion is less than the magnitude of the effective mobility of the first nucleic acid; and (iii) a first leading electrolyte buffer comprising a first leading ion, wherein the magnitude of the effective mobility of the first leading ion is greater than the magnitude of the effective mobility of the first nucleic acid; (b) loading into a second channel of the fluidic device: (iv) a second tissue sample comprising a second nucleic acid and a second contaminant; and (v) a second final electrolyte buffer comprising a second final ion, wherein the magnitude of the second final ion is greater than the magnitude of the effective mobility of the first nucleic acid. and (vi) a second leading electrolyte buffer containing a second leading ion, the second leading ion having an effective mobility magnitude greater than the effective mobility of the second nucleic acid; and (c) applying an electric field within the fluidic device to perform isotachophoresis using the first leading ion, the first nucleic acid, and the first leading ion in the first channel, and to perform isotachophoresis using the second leading ion, the second nucleic acid, and the second leading ion in the second channel, thereby purifying the first nucleic acid from the first contaminant and purifying the second nucleic acid from the second contaminant.

[0041] In some embodiments of the aspects provided herein, the first final electrolyte buffer or the first leading electrolyte buffer further comprises a lysis agent or tissue disruption agent. In some embodiments of the aspects provided herein, the second final electrolyte buffer or the second leading electrolyte buffer further comprises a lysis agent or tissue disruption agent. In some embodiments of the aspects provided herein, the lysis agent or tissue disruption agent comprises one or more agents selected from the group consisting of a solution having a pH greater than about 12, a proteinase, urea, thiourea, and a surfactant.

[0042] An aspect of the present disclosure provides a sample purification method, comprising: (a) loading into a first zone of a fluidic device: (i) a tissue sample containing nucleic acids and contaminants; (ii) a final electrolyte buffer containing final ions, wherein the magnitude of the effective mobility of the final ions is smaller than the magnitude of the effective mobility of the nucleic acid; and (iii) a leading electrolyte buffer containing leading ions, wherein the magnitude of the effective mobility of the leading ions is larger than the magnitude of the effective mobility of the nucleic acid; and (b) applying an electric field to the fluidic device to perform isotachophoresis using the final ions, the nucleic acid, and the leading ions in a second zone of the fluidic device, thereby purifying the nucleic acid from the contaminants; wherein during the applying step, the first zone is maintained at a first temperature and the second zone is maintained at a second temperature different from the first temperature.

[0043] In some embodiments of the aspects provided herein, the final electrolyte buffer or the leading electrolyte buffer further comprises a lysis or tissue disruption agent. In some embodiments of the aspects provided herein, the lysis or tissue disruption agent comprises one or more agents selected from the group consisting of a solution having a pH greater than about 12, a proteinase, urea, thiourea, and a surfactant. In some embodiments of the aspects provided herein, the first temperature is between about 4°C and about 40°C. In some embodiments of the aspects provided herein, the first temperature is between about 40°C and about 80°C.

[0044] An aspect of the present disclosure relates to a method for detecting nucleic acid fragments, the method comprising: (a) loading into a first zone of a fluidic device: (i) a tissue sample containing nucleic acids; (ii) a final electrolyte buffer containing final ions, the final ions having an effective mobility magnitude less than the effective mobility magnitude of the nucleic acids; and (iii) a leading electrolyte buffer containing leading ions, the leading ions having an effective mobility magnitude greater than the effective mobility magnitude of the nucleic acids; (b) disposing in the first zone: (1) an embedding material; (1) subjecting the tissue sample to at least one sample preparation step selected from the group consisting of (1) removal of ions from the tissue sample, (2) tissue disruption, (3) cell lysis, (4) decrosslinking of nucleic acids, (5) protein digestion, and (6) nucleic acid digestion; and (c) applying an electric field within the fluidic device to perform isotachophoresis using the terminal ions, the nucleic acid, and the leading ions in a second zone of the fluidic device, thereby purifying the nucleic acid from contaminants in the tissue sample.

[0045] In some embodiments of the aspects provided herein, the removal of embedding material or the lysis of cells comprises incubating the tissue sample in the fluidic device at a temperature of at least about 37°C for a period of at least about 1 minute prior to the application of the electric field. In some embodiments of the aspects provided herein, the temperature is from about 40°C to about 80°C. In some embodiments of the aspects provided herein, the period is from about 1 minute to about 60 minutes. In some embodiments of the aspects provided herein, the disruption of tissue or the lysis of cells comprises applying mechanical stress to the sample. In some embodiments of the aspects provided herein, the disruption of tissue or the lysis of cells comprises applying heat to the sample. In some embodiments of the aspects provided herein, the application of heat brings the temperature of the tissue sample to about 30°C to about 65°C. In some embodiments of the aspects provided herein, the disruption of tissue or the lysis of cells comprises a solution of at least pH 12. In some embodiments of the aspects provided herein, the disruption of tissue or the lysis of cells comprises proteolytic digestion. In some embodiments of the aspects provided herein, the proteolytic digestion is carried out at a temperature greater than about 25°C. In some embodiments of the aspects provided herein, the temperature is between about 30°C and about 65°C. In some embodiments of the aspects provided herein, the tissue disruption or cell lysis comprises applying at least one detergent to the tissue or cells. In some embodiments of the aspects provided herein, the tissue disruption or cell lysis comprises applying a solution comprising urea to the tissue or cells. In some embodiments of the aspects provided herein, the solution further comprises thiourea. In some embodiments of the aspects provided herein, the concentration of urea in the solution is between about 4M and about 9M, and the concentration of thiourea in the solution is between about 0.5M and about 3.5M.In some embodiments of the aspects provided herein, the concentration of the urea in the solution is about 6.5 M to about 7.5 M, and the concentration of the thiourea in the solution is about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the step of decrosslinking the nucleic acid comprises digesting crosslinked proteins using proteinase K. In some embodiments of the aspects provided herein, the step of digesting the nucleic acid is performed using DNase or RNase.

[0046] An aspect of the present disclosure provides a method for sample purification, comprising: (a) loading into a fluidic device: (i) a tissue sample containing nucleic acids, the tissue sample being embedded or fixed; (ii) a final electrolyte buffer solution containing a final electrolyte, the final electrolyte having a lower effective mobility than the nucleic acid; and (iii) a leading electrolyte buffer solution containing a leading electrolyte, the leading electrolyte having a higher effective mobility than the nucleic acid; and (b) applying an electric field to the fluidic device to perform isotachophoresis using the final electrolyte, the nucleic acid, and the leading electrolyte, thereby purifying the nucleic acid from contaminants in the tissue sample.

[0047] In some embodiments of the aspects provided herein, the contaminant is selected from the group consisting of cross-linked nucleic acids, embedding materials, chemical fixatives, enzymes, and inhibitors. In some embodiments of the aspects provided herein, the embedding material comprises paraffin. In some embodiments of the aspects provided herein, the tissue sample is formalin-fixed. In some embodiments of the aspects provided herein, the tissue sample is embedded and fixed. In some embodiments of the aspects provided herein, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. In some embodiments of the aspects provided herein, the tissue sample is a resected tissue sample. In some embodiments of the aspects provided herein, the resected tissue sample is a resected FFPE sample. In some embodiments of the aspects provided herein, the method further comprises comparing the nucleic acid feature with a nucleic acid feature from another sample. In some embodiments of the aspects provided herein, the feature is an expression level. In some embodiments of the aspects provided herein, the feature is a nucleic acid sequence. In some embodiments of the aspects provided herein, the characteristic is molecular weight. In some embodiments of the aspects provided herein, the characteristic is nucleic acid integrity. In some embodiments of the aspects provided herein, the characteristic is nucleic acid purity. In some embodiments of the aspects provided herein, the method further comprises administering a drug based on the characteristic of the nucleic acid. In some embodiments of the aspects provided herein, the tissue sample is a tumor sample. In some embodiments of the aspects provided herein, the final electrolyte buffer has a pH of about 7. In some embodiments of the aspects provided herein, the final electrolyte buffer has a pH greater than about 7. In some embodiments of the aspects provided herein, the method further comprises incubating the tissue sample in the fluidic device at a temperature of at least about 37° C. for a period of at least about 1 minute prior to the application of the electric field.In some embodiments of the aspects provided herein, the temperature is about 40°C to about 80°C. In some embodiments of the aspects provided herein, the time period is about 1 minute to about 60 minutes. In some embodiments of the aspects provided herein, the lead electrolyte buffer comprises proteinase K. In some embodiments of the aspects provided herein, the method further comprises using proteinase K to remove protein crosslinks from the nucleic acid. In some embodiments of the aspects provided herein, the method further comprises using heat to remove protein crosslinks from the nucleic acid after the application of the electric field. In some embodiments of the aspects provided herein, the method further comprises eluting an output solution containing the purified nucleic acid from an output reservoir of the fluidic device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the concentration of the cross-linked nucleic acid in the effluent solution is at most about half the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the effluent solution does not contain the contaminant. In some embodiments of the aspects provided herein, the effluent 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 less than about 500 μL. In some embodiments of the aspects provided herein, the final electrolyte has a higher effective mobility than the contaminant. In some embodiments of the aspects provided herein, the final electrolyte includes (i) a first ion having an effective mobility magnitude greater than the contaminant, and (ii) a second ion having an effective mobility magnitude approximately the same as or smaller than the contaminant.In some embodiments of the aspects provided herein, the isotachophoresis is performed by quenching the pH of the tissue sample to about 7. In some embodiments of the aspects provided herein, the method further includes deparaffinizing the tissue sample before the loading. In some embodiments of the aspects provided herein, the tissue sample is a conventional formalin-fixed, paraffin-embedded (FFPE) sample, further comprising comparing the nucleic acid signature with different nucleic acid signatures from different tissue samples. In some embodiments of the aspects provided herein, the method further includes 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).

[0048] An embodiment of the present disclosure provides a fluidic device including: (a) a first zone; (b) a sample inlet disposed in the first zone; (c) a reservoir for a final electrolyte in fluid communication with the first zone; (d) a second zone in fluid communication with the first zone; (e) a reservoir for a leading electrolyte in fluid communication with the second zone; (f) a sample outlet in fluid communication with the second zone; (g) a first heater in thermal communication with the first zone; and (h) a second heater configured to transfer heat to the second zone, wherein the first zone is substantially thermally isolated from the second zone.

[0049] An embodiment of the present disclosure provides a fluidic device including a sample purification region including: (a) a first zone; (b) a sample inlet disposed in the first zone; (c) a reservoir for a final electrolyte in fluid communication with the first zone; (d) a second zone in fluid communication with the first zone; (e) a reservoir for a leading electrolyte in fluid communication with the second zone; (f) a sample outlet in fluid communication with the second zone; and (g) a heater in thermal communication with the first zone and the second zone.

[0050] In some embodiments of the aspects provided herein, the device further includes a second sample purification region. In some embodiments of the aspects provided herein, the first zone is a deparaffinization zone. In some embodiments of the aspects provided herein, the first zone is a disruption zone. In some embodiments of the aspects provided herein, the second zone is an isotachophoresis zone. In some embodiments of the aspects provided herein, the first zone or the second zone has a width of less than about 1 mm. In some embodiments of the aspects provided herein, the first zone or the second zone has a width of less than about 0.5 mm.

[0051] Aspects of the present disclosure provide kits that include a device provided herein, a final electrolyte buffer solution that includes a final electrolyte, and a leading electrolyte buffer solution that includes a leading electrolyte.

[0052] In some embodiments of the aspects provided herein, the final electrolyte buffer comprises a mixture of at least two electrolytes having different effective mobilities. In some embodiments of the aspects provided herein, the mixture comprises (i) a first electrolyte having an effective mobility magnitude smaller than that of the nucleic acid and larger than that of the contaminant, and (ii) a second electrolyte having an effective mobility magnitude smaller than that of the contaminant. In some embodiments of the aspects provided herein, the contaminant comprises a cross-linked nucleic acid. In some embodiments of the aspects provided herein, the first electrolyte comprises caproic acid. In some embodiments of the aspects provided herein, the second electrolyte comprises HEPES.

[0053] An embodiment of the present disclosure provides a fluidic device comprising: (a) a fluidic device having: (i) a tissue sample containing nucleic acids; (ii) a final electrolyte buffer containing final electrolyte ions having a first effective mobility, the first effective mobility being an order of magnitude smaller than the effective mobility of the nucleic acid; (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir, the first leading electrolyte buffer containing first leading electrolyte ions having a second effective mobility, the second effective mobility being an order of magnitude larger than the effective mobility of the nucleic acid; and (iv) a second leading electrolyte buffer in a second leading electrolyte reservoir, the first leading electrolyte buffer containing first leading electrolyte ions having a third effective mobility. The method includes the steps of: (a) introducing a second leading electrolyte buffer containing leading electrolyte ions, wherein the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acid, and the first leading electrolyte buffer is different from the second leading electrolyte buffer; (b) performing a first isotachophoresis run using the final electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby purifying the nucleic acid from the contaminants in the tissue sample; and (c) performing a second isotachophoresis run using the final electrolyte ions, the nucleic acid, and the second leading electrolyte ions.

[0054] In some embodiments of the aspects provided herein, performing the second isotachophoresis step includes varying the applied current from the first channel to the second channel. In some embodiments of the aspects provided herein, the first leading electrolyte ions are the same as the second leading electrolyte ions, and the concentration of the first leading electrolyte ions in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ions in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the concentration of the first leading electrolyte ions in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ions in the second leading electrolyte buffer by at least 1.5x. In some embodiments of the aspects provided herein, the first leading electrolyte ions are different from the second leading electrolyte ions. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer includes a third leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer includes a third leading electrolyte ion. In some embodiments of the aspects provided herein, the method further includes collecting the nucleic acid in the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the method further includes removing the nucleic acid from the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the final electrolyte buffer is loaded into a final electrolyte reservoir that is separate from the first leading electrolyte reservoir and the second leading electrolyte reservoir.In some embodiments of the aspects provided herein, the first isotachophoresis step and the second isotachophoresis step are performed by applying one electric field. In some embodiments of the aspects provided herein, the first isotachophoresis step and the second isotachophoresis step are performed by applying more than one electric field.

[0055] An embodiment of the present disclosure provides a fluidic device including a sample purification region including: (a) a channel including a first zone and a second zone in fluid communication with the first zone; (b) a sample inlet, a final electrolyte reservoir containing a final electrolyte buffer, and a first leading electrolyte reservoir containing a first leading electrolyte buffer, each in fluid communication with the first zone; and (c) a second leading electrolyte reservoir containing a second leading electrolyte buffer, wherein the second leading electrolyte buffer is in fluid communication with the second zone and the second leading electrolyte buffer is different from the first leading electrolyte buffer.

[0056] In some embodiments of the aspects provided herein, the sample inlet is capable of receiving a sample containing at least some non-liquid biological material. In some embodiments of the aspects provided herein, the second leading electrolyte buffer contains a different leading electrolyte co-ion from the first leading electrolyte buffer. In some embodiments of the aspects provided herein, the first leading electrolyte buffer contains a first leading electrolyte ion, the second leading electrolyte buffer contains a second leading electrolyte ion that is the same as the first leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the first leading electrolyte buffer comprises a first leading electrolyte ion and the second leading electrolyte buffer comprises a second leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer differs from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer by a factor of at least 1.5x. In some embodiments of the aspects provided herein, the first leading electrolyte buffer comprises a first leading electrolyte ion and the second leading electrolyte buffer comprises a second leading electrolyte ion that is different from the first leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte buffer contains a first leading electrolyte ion, the second leading electrolyte buffer contains a second leading electrolyte ion that is the same as the first leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer contains a third leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte buffer contains a first leading electrolyte ion, the second leading electrolyte buffer contains a second leading electrolyte ion that is the same as the first leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer contains a third leading electrolyte ion.

[0057] An embodiment of the present disclosure provides a method comprising the steps of: (a) providing an electrokinetic fluid device including a reservoir in fluid communication with a channel; (b) loading a sample volume into the reservoir; (c) transferring at least 50% of the sample volume from the reservoir to the channel; and (d) applying an ionic current to the channel.

[0058] In some embodiments of the aspects provided herein, the moving step is performed using gravity. In some embodiments of the aspects provided herein, the ionic current does not substantially flow in the reservoir. In some embodiments of the aspects provided herein, the at least 50% of the sample volume accounts for at least 80% of the sample volume. In some embodiments of the aspects provided herein, the sample volume comprises nucleic acid. In some embodiments of the aspects provided herein, the sample volume comprises a tissue sample. In some embodiments of the aspects provided herein, the sample volume comprises a formalin-fixed, paraffin-embedded (FFPE) sample. In some embodiments of the aspects provided herein, the applying an ionic current comprises performing isotachophoresis (ITP).

[0059]

[0013] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure have been shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. Incorporation by Reference

[0060] All publications, patents, and patent applications specified in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0061] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. The present invention provides, for example, the following items. (Item 1) (a) Fluidic device (i) a tissue sample comprising lysed solid tissue, the lysed solid tissue comprising nucleic acids and contaminants; (ii) a final electrolyte buffer comprising a first final electrolyte ion having an effective mobility that is less than the magnitude of the effective mobility of the nucleic acid; and (iii) introducing a leading electrolyte buffer containing a first leading electrolyte ion having a second effective mobility, the second effective mobility having an order of magnitude greater than the order of magnitude of the effective mobility of the nucleic acid; and (b) applying an electric field within the fluidic device to perform isotachophoresis using the first final electrolyte ion, the nucleic acid, and the first leading electrolyte ion, thereby purifying the nucleic acid from the contaminants in the tissue sample. A sample purification method comprising: (Item 2) 2. The method of claim 1, wherein the effective mobility of the first final electrolyte ion has a magnitude greater than the magnitude of the effective mobility of the contaminant. (Item 3) 2. The method of claim 1, wherein the fluidic device is a microfluidic chip, and the tissue sample, the final electrolyte buffer, and the leading electrolyte buffer are loaded into a first zone of the microfluidic chip. (Item 4) 4. The method of any one of items 1 to 3, further comprising subjecting the tissue sample in the first zone of the microfluidic chip to at least one sample preparation procedure selected from the group consisting of: (1) removal of embedding material, (2) tissue disruption, (3) cell lysis, (4) decrosslinking of the nucleic acid, (5) digestion of protein, and (6) digestion of the nucleic acid. (Item 5) 5. The method of any one of items 1 to 4, wherein the isotachophoresis is performed in a second zone of the microfluidic chip, the second zone being separated from and fluidly connected to the first zone. (Item 6) 6. The method of any one of items 1 to 5, wherein the solid tissue is derived from a solid organ. (Item 7) 7. The method of any one of items 1 to 6, wherein the lysed solid tissue comprises a chemical fixative. (Item 8) 8. The method according to item 7, wherein the chemical fixative is formalin. (Item 9) 9. The method of claim 8, wherein the solid tissue is a formalin-fixed, paraffin-embedded tissue (FFPE). (Item 10) 10. The method of any one of items 1 to 9, wherein the lysed solid tissue comprises urea or thiourea. (Item 11) 11. The method according to any one of items 1 to 10, further comprising the step of disrupting intercellular junctions, extracellular matrix or connective tissue to obtain said dissolved solid tissue. (Item 12) 11. The method of any one of items 1 to 10, wherein the lysed solid tissue comprises solid particles. (Item 13) 13. The method of any one of items 1 to 12, wherein the nucleic acid comprises dispersed or solvated nucleic acid. (Item 14) 14. The method of any one of items 1 to 13, wherein the contaminant is selected from the group consisting of cross-linked nucleic acids, embedding materials, tissue strips, chemical fixatives, proteins, inhibitors and combinations thereof. (Item 15) 15. The method of any one of items 1 to 14, wherein the contaminant comprises cross-linked nucleic acid. (Item 16) 16. The method of any one of items 1 to 15, wherein the tissue sample is combined with the final electrolyte buffer prior to the loading step. (Item 17) 17. The method of any one of items 1 to 16, wherein the tissue sample is combined with the lead electrolyte buffer prior to the loading step. (Item 18) 18. The method of any one of items 1 to 17, wherein the step of loading the lead electrolyte buffer is performed before the step of loading the tissue sample. (Item 19) 19. The method of any one of items 1 to 18, wherein the solid tissue is dissolved in the lead electrolyte buffer prior to the step of loading the tissue sample. (Item 20) 20. The method of any one of items 1 to 19, wherein the solid tissue is dissolved in the final electrolyte buffer prior to the step of loading the tissue sample. (Item 21) 21. The method of any one of items 4 to 20, wherein the sample preparation procedure comprises removing embedding material by incubating the tissue sample in the fluidic device at a temperature of at least about 37°C for a period of at least about 1 minute prior to the applying the electric field. (Item 22) 22. The method according to item 21, wherein the temperature is about 40°C to about 80°C. (Item 23) 22. The method according to item 21, wherein the period is from about 1 minute to about 120 minutes. (Item 24) 24. The method of any one of items 4 to 23, wherein the sample preparation procedure comprises a step of disrupting tissue or lysing cells by applying mechanical stress to the tissue sample. (Item 25) 26. The method of any one of claims 4 to 24, wherein the sample preparation procedure includes applying heat to the tissue sample to disrupt tissue or lyse cells. 26. The method according to any one of items 4 to 25, wherein the application of heat results in a temperature of the tissue sample within a range of about 30°C to about 80°C. (Item 27) 27. The method of any one of items 4 to 26, wherein the sample preparation procedure comprises disrupting tissue or lysing cells by contacting the tissue sample with a solution having a pH of at least 10 or proteolytically digesting the tissue sample. (Item 28) 28. The method of claim 27, wherein the proteolytic digestion is carried out at a temperature greater than about 25°C. (Item 29) 29. The method of any one of items 4 to 28, wherein the sample preparation procedure comprises a step of disrupting tissue or lysing cells by applying at least one detergent to the tissue sample. (Item 30) 30. The method of any one of items 4 to 29, wherein the sample preparation procedure comprises disrupting tissue or lysing cells by applying a solution comprising urea to the tissue or cell sample. (Item 31) 31. The method of claim 30, wherein the solution further comprises thiourea. (Item 32) Item 32. The method according to Item 31, wherein the concentration of the urea in the solution is within a range of about 4 M to about 9 M, and the concentration of the thiourea in the solution is within a range of about 0.5 M to about 3.5 M. (Item 33) Item 32. The method according to Item 31, wherein the concentration of the urea in the solution is about 6.5 M to about 7.5 M, and the concentration of the thiourea in the solution is about 1.5 M to about 2.5 M. (Item 34) 34. The method of any one of items 4 to 33, wherein the sample preparation procedure comprises decrosslinking the nucleic acids by digesting crosslinked proteins with proteinase K. (Item 35) 35. The method of any one of items 4 to 34, wherein the sample preparation procedure comprises digesting the nucleic acids with DNase or RNase. (Item 36) 36. The method of any one of items 1 to 35, further comprising eluting an output solution containing the purified nucleic acid from an output reservoir of the fluidic device. (Item 37) 37. The method of claim 36, wherein the concentration of the purified nucleic acid in the effluent solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. (Item 38) 37. The method of claim 36, wherein the tissue sample and the purified nucleic acid in the effluent solution comprise cross-linked nucleic acids, and the concentration of the cross-linked nucleic acids in the effluent solution is at most about half the concentration of the cross-linked nucleic acids in the tissue sample. (Item 39) 37. The method of claim 36, wherein the contaminant is present in the effluent solution at a concentration that is at most two-fold lower than the concentration of the contaminant in the tissue sample. (Item 40) 40. The method of any one of the preceding claims, wherein the first final electrolyte ion comprises caproic acid. (Item 41) 41. The method of any one of the preceding claims, wherein the first leading electrolyte ions comprise chloride ions. (Item 42) 42. The method of any one of the preceding claims, wherein the final electrolyte buffer comprises a second final electrolyte ion having a different effective mobility than the first final electrolyte ion. (Item 43) 43. The method of claim 42, wherein the second final electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or MOPS (3-(N-morpholino)propanesulfonic acid). (Item 44) 43. The method of claim 42, wherein the second final electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and the first final electrolyte ion comprises caproic acid. (Item 45) Item 46. The method of item 42, wherein the second final electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid) and the first final electrolyte ion comprises caproic acid. 43. The method of claim 42, wherein the second final electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and the first final electrolyte ion comprises MOPS. (Item 47) 48. The method of claim 1, wherein the final electrolyte buffer comprises a second final electrolyte ion having a second effective mobility, the second effective mobility being approximately the same magnitude as or smaller than the effective mobility of the contaminant. 48. The method of any one of items 1 to 47, wherein the tissue sample loaded into the fluidic device has a volume of at least 50 μl. (Item 49) 49. The method of any one of items 1 to 48, further comprising the steps of subjecting the tissue sample to a first sample processing procedure in the first zone of the microfluidic chip and subjecting the tissue sample to an enzymatic reaction in a second zone of the microfluidic chip. (Item 50) 50. The method of claim 49, wherein the first sample processing procedure comprises removal of embedding material, tissue disruption or cell lysis, and the enzymatic reaction comprises decrosslinking of the nucleic acid, digestion of a protein or digestion of a nucleic acid. (Item 51) 50. The method of claim 49, wherein the first zone and the second zone are each heated to a temperature greater than 37°C. (Item 52) 50. The method of claim 49, wherein the first zone is heated to a temperature of about 60°C to 100°C during the first sample processing procedure and the second zone is heated to a temperature of 40°C to 60°C. (Item 53) (a) loading into a first channel of a microfluidic chip: (i) a first sample comprising a first nucleic acid and a first contaminant; (ii) a first final electrolyte buffer comprising a first final ion, wherein the magnitude of the effective mobility of the first final ion is smaller than the magnitude of the effective mobility of the first nucleic acid; and (iii) a first leading electrolyte buffer comprising a first leading ion, wherein the magnitude of the effective mobility of the first leading ion is larger than the magnitude of the effective mobility of the first nucleic acid; (b) loading into a second channel of the microfluidic chip: (i) a second sample comprising a second nucleic acid and a second contaminant; (ii) a second final electrolyte buffer comprising a second final ion, the magnitude of the second final ion being smaller than the magnitude of the effective mobility of the second nucleic acid; and (iii) a second leading electrolyte buffer comprising a second leading ion, the magnitude of the effective mobility of the second leading ion being larger than the magnitude of the effective mobility of the second nucleic acid; and (c) applying a first electric field within the microfluidic chip to perform isotachophoresis in the first channel using the first terminal ions, the first nucleic acid, and the first leading ions, and applying a second electric field to perform isotachophoresis in the second channel using the second terminal ions, the second nucleic acid, and the second leading ions, thereby simultaneously purifying the first nucleic acid from the first contaminant and the second nucleic acid from the second contaminant. A method for simultaneously purifying nucleic acids from at least two different samples, comprising: (Item 54) 54. The method of claim 53, wherein the first sample and the second sample are different sample types. (Item 55) 54. The method of claim 53, wherein the first nucleic acid and the second nucleic acid are nucleic acids of different types or lengths. (Item 56) 54. The method of any one of items 1 to 53, wherein the first final electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or tissue disrupting agent. (Item 57) 57. The method of claim 56, wherein the lysing agent or tissue disruption agent comprises one or more agents selected from the group consisting of a solution having a pH greater than about 12, proteinase, urea, thiourea, and a surfactant. (Item 58) 58. The method of any one of items 53 to 57, wherein the first sample comprises lysed solid tissue. (Item 59) 59. The method of claim 58, wherein the second sample comprises lysed cells. (Item 60) 60. The method of any one of items 53 to 59, wherein the first sample does not contact the second sample during the step of performing isotachophoresis. (Item 61) 61. The method of any one of items 53 to 60, further comprising the step of: loading a third channel of the microfluidic chip with (i) a third sample comprising a third nucleic acid and a third contaminant; (ii) a third final electrolyte buffer comprising a third final ion, wherein the magnitude of the effective mobility of the third final ion is smaller than the magnitude of the effective mobility of the third nucleic acid; and (iii) a third leading electrolyte buffer comprising a third leading ion, wherein the magnitude of the effective mobility of the third leading ion is larger than the magnitude of the effective mobility of the third nucleic acid; and applying the electric field within the microfluidic chip to perform the isotachophoresis in the third channel using the third final ion, the third nucleic acid, and the third leading ion, thereby simultaneously purifying the first nucleic acid from the first contaminant, the second nucleic acid from the second contaminant, and the third nucleic acid from the third contaminant. (Item 62) 62. The method of any one of items 53 to 61, wherein the first and second electric fields are generated from a single electrode pair. (Item 63) 63. The method of any one of items 53 to 62, wherein the first and second electric fields are generated from different pairs of electrodes. (Item 64) Item 64. The method of item 63, wherein the first and second channels are coupled to independent sensors. (Item 65) Item 65. The method of item 64, wherein feedback from the independent sensors is used to independently control the first and second electric fields. (Item 66) Item 65. The method of item 64, wherein the independent sensors detect voltage and the feedback is used to control current in the first and second channels. (Item 67) 67. The method of any one of items 1 to 66, wherein the nucleic acid comprises DNA. (Item 68) 67. The method of any one of items 1 to 66, wherein the nucleic acid comprises RNA. (Item 69) (a) Fluidic device (i) a sample comprising fixed cells, fixed tissues or embedded tissues, which sample comprises nucleic acids; (ii) a final electrolyte buffer comprising a final electrolyte, the final electrolyte having a lower effective mobility than the nucleic acid; and (iii) charging a leading electrolyte buffer containing a leading electrolyte, the leading electrolyte having a higher effective mobility than the nucleic acid; and (b) applying an electric field to the fluidic device to perform isotachophoresis using the final electrolyte, the nucleic acid, and the leading electrolyte, thereby purifying the nucleic acid from contaminants in the sample; A sample purification method comprising: (Item 70) 70. The method of claim 69, wherein the contaminant is selected from the group consisting of cross-linked nucleic acids, embedding materials, chemical fixatives, enzymes, and inhibitors. (Item 71) 70. The method of claim 69, wherein the sample comprises the fixed cells, the fixed tissue, or both the fixed cells and the fixed tissue. (Item 72) 70. The method of claim 69, wherein the sample is formalin-fixed. (Item 73) 70. The method of claim 69, wherein the sample comprises the embedded tissue. (Item 74) 70. The method of claim 69, wherein the sample comprises the tissue embedded in paraffin. (Item 75) 70. The method of claim 69, wherein the sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. (Item 76) 70. The method of claim 69, wherein the sample comprises a tissue biopsy. (Item 77) 76. The method of claim 75, wherein the sample is a resected formalin-fixed, paraffin-embedded (FFPE) sample. (Item 78) 70. The method of claim 69, further comprising comparing the nucleic acid feature with a nucleic acid feature from another sample, wherein the feature is expression level, nucleic acid sequence, molecular weight, nucleic acid integrity, nucleic acid purity, or nucleic acid strandedness. (Item 79) 70. The method of item 69, wherein the sample is a tumor sample. (Item 80) 70. The method of claim 69, wherein the final electrolyte buffer has a pH greater than about 7. (Item 81) 70. The method of claim 69, further comprising incubating the tissue sample in the fluidic device at a temperature of at least about 37°C for a period of at least about 1 minute prior to the applying the electric field. (Item 82) Item 82. The method according to item 81, wherein the temperature is about 40°C to about 80°C. (Item 83) Item 82. The method according to item 81, wherein the period is from about 1 minute to about 120 minutes. (Item 84) 61. The method of claim 60, wherein the lead electrolyte buffer comprises proteinase K. (Item 85) 85. The method of claim 84, further comprising the step of removing protein crosslinks from the nucleic acid using proteinase K. (Item 86) 61. The method of claim 60, further comprising the step of removing protein crosslinks from the nucleic acid using heat after the step of applying the electric field. (Item 87) 61. The method of claim 60, further comprising eluting an output solution containing the purified nucleic acid from an output reservoir of the fluidic device. (Item 88) 88. The method of claim 87, wherein the concentration of the purified nucleic acid in the effluent solution is at least about two-fold higher than the concentration of the nucleic acid in the tissue sample. (Item 89) 88. The method of claim 87, wherein the concentration of the cross-linked nucleic acid in the effluent solution is at most about half the concentration of the cross-linked nucleic acid in the tissue sample. (Item 90) 88. The method of claim 87, wherein the discharge solution has a volume equal to or less than about 50 μL. (Item 91) 61. The method of claim 60, wherein the tissue sample has a mass of at least about 1 ng. (Item 92) 61. The method of claim 60, wherein the tissue sample has a volume of more than 25 μL. (Item 93) Item 61. The method of item 60, wherein the final electrolyte has a higher effective mobility than the contaminant. (Item 94) Item 61. The method of item 60, wherein the final electrolyte comprises (i) a first ion having an effective mobility magnitude greater than that of the contaminant, and (ii) a second ion having an effective mobility magnitude about the same as or less than that of the contaminant. (Item 95) Item 61. The method of item 60, wherein the step of performing isotachophoresis quenches the pH of the tissue sample to about 7.5. (Item 96) Item 61. The method of item 60, further comprising the step of deparaffinizing the sample before the loading step. (Item 97) Item 61. The method of item 60, further comprising detecting the concentration of the nucleic acid. (Item 98) 89. The method of claim 88, wherein the concentration is less than or equal to about 1 picogram per microliter (pg / μL). (Item 99) (a) A fluidic device, (i) tissue samples containing lysed solid tissue and nucleic acids; (ii) a final electrolyte buffer comprising a final electrolyte ion having a first effective mobility, the first effective mobility having an order of magnitude less than the order of magnitude of the effective mobility of the nucleic acid; (iii) a first leading electrolyte buffer solution in a first leading electrolyte reservoir, the first leading electrolyte buffer solution comprising a first leading electrolyte ion having a second effective mobility, the second effective mobility being greater in magnitude than the effective mobility of the nucleic acid; and (iv) charging in a second leading electrolyte reservoir a second leading electrolyte buffer containing a second leading electrolyte ion having a third effective mobility, the third effective mobility being greater in magnitude than the effective mobility of the nucleic acid, wherein the first leading electrolyte buffer is different from the second leading electrolyte buffer. (b) performing a first isotachophoresis using the final electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby purifying the nucleic acid from contaminants in the tissue sample; and (c) performing a second isotachophoresis using the final electrolyte ions, the nucleic acid, and the second leading electrolyte ions; A sample purification method comprising: (Item 100) Item 100. The method of item 99, wherein performing the second isotachophoresis comprises varying the applied current from the first channel to the second channel. (Item 101) 101. The method of claim 99 or 100, wherein 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. (Item 102) 102. The method of claim 99, 100, or 101, wherein the second effective mobility has a magnitude greater than the magnitude of the third effective mobility. (Item 103) 103. The method of any one of items 99 to 102, wherein the first leading electrolyte ion is different from the second leading electrolyte ion. (Item 104) 104. The method of any one of items 99 to 103, wherein the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer contains a third leading electrolyte ion. (Item 105) 105. The method of any one of items 99 to 104, wherein the first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer contains a third leading electrolyte ion. (Item 106) 106. The method of any one of items 99 to 105, further comprising collecting the nucleic acid in the second leading electrolyte reservoir and removing the nucleic acid from the second leading electrolyte reservoir. (Item 107) 107. The method of any one of items 99 to 106, wherein the first isotachophoresis step and the second isotachophoresis step are performed by applying a single electric field. (Item 108) 108. The method of any one of items 99 to 107, wherein the steps of performing the first isotachophoresis and performing the second isotachophoresis are performed by applying more than one electric field. (Item 109) 109. The method of any one of items 99 to 108, wherein the concentration of the second leading electrolyte ion in the second leading electrolyte buffer is less than 50 mM. (Item 110) 109. The method of any one of items 99 to 108, wherein the second leading electrolyte buffer comprises 50 mM Tris HCl. (Item 111) (a) i. a first sample reservoir in fluid communication with the first fluid channel; ii. a first buffer reservoir in fluid communication with the first fluid channel; and iii. a first isotachophoresis region in the microfluidic chip comprising a second buffer reservoir in fluid communication with the first channel; and (b) i. a second sample reservoir in fluid communication with the second fluid channel; ii. a third buffer reservoir in fluid communication with the second fluid channel; and iii. a second isotachophoresis region in the microfluidic chip comprising a fourth buffer reservoir in fluid communication with the second channel. A microfluidic device comprising: a microfluidic device, wherein the first isotachophoresis region is not in fluid communication with the second isotachophoresis region, and the microfluidic device is configured to independently control a first electrical circuit that applies current to the first isotachophoresis region and a second electrical circuit that applies current to the second isotachophoresis region. (Item 112) 112. The microfluidic device according to item 111, wherein the leak rate between the first isotachophoresis zone and the second isotachophoresis zone is less than 1 μl / hr. (Item 113) Item 113. The microfluidic device of item 111 or 112, wherein the leakage current between the first region and the second region is less than 1 μA. (Item 114) 114. The microfluidic device of items 111, 112 or 113, wherein the impedance is greater than 1 megaohm. (Item 115) 10. The microfluidic device of any one of the preceding items, wherein the first fluid channel holds a liquid volume of more than 100 μl. (Item 116) 10. The microfluidic device of any one of the preceding items, wherein the first fluid channel is separated from the second fluid channel by a distance that is at most one-fifth the width of the first channel. (Item 117) 10. The microfluidic device of any one of the preceding items, wherein the microfluidic device is configured to control the first electrical circuit simultaneously with and independently of the second electrical circuit. (Item 118) 10. The microfluidic device of any one of the preceding items, further comprising an elution reservoir in fluid communication with the first channel, wherein the temperature sensor is positioned within 5 mm of the elution reservoir. (Item 119) (a) providing an electrokinetic fluid device comprising a sample input reservoir in fluid communication with a channel; (b) loading a sample volume into the sample input reservoir; (c) transferring at least 50% of the sample volume from the sample input reservoir to the channel without adding additional volume to the sample input reservoir; and (d) applying an ionic current through the channel. A method comprising: (Item 120) Item 120. The method of item 119, wherein the moving step is performed using gravity. (Item 121) 121. The method of claim 119 or 120, wherein the ionic current does not substantially pass through the channel. (Item 122) 122. The method of claim 119, 120, or 121, wherein said at least 50% of the sample volume comprises at least 80% of the sample volume. (Item 123) 10. The method of any one of the preceding items, wherein the sample volume comprises nucleic acid. (Item 124) 10. The method of any one of the preceding items, wherein the sample volume comprises a tissue sample or a formalin-fixed, paraffin-embedded (FFPE) sample. (Item 125) 10. The method of any one of the preceding items, wherein the step of applying an ionic current comprises performing isotachophoresis. (Item 126) 127. The method of claim 126, wherein the total sample volume loaded into the sample input reservoir is less than or equal to the internal volume of the input reservoir. 10. The method of claim 9, wherein the sample input reservoir includes a top region connected to a bottom region via a tapered region, the top region having a first diameter and the bottom region having a second diameter, the first diameter being at least two times longer than the second diameter, thereby facilitating the step of transferring at least 50% of the sample volume from the sample input reservoir to the channel. (Item 128) 10. The method of any one of the preceding items, wherein the sample volume deposited in the sample input reservoir is at least 25 μl. (Item 129) a first sample input reservoir comprising a top region connected to a bottom region via a tapered region, the top region having a first hydraulic inner diameter and the bottom region having a second hydraulic inner diameter, the first hydraulic inner diameter being at least two times larger than the second hydraulic inner diameter, the first sample input reservoir being in fluid communication with a first channel; a first buffer reservoir in fluid communication with the first channel, the first sample reservoir configured such that a free surface of 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; A microfluidic chip comprising: (Item 130) Item 130. The microfluidic chip according to item 129, wherein the first hydraulic inner diameter is in the range of about 1 mm to about 15 mm. (Item 131) Item 131. The microfluidic chip according to item 129 or item 130, wherein the second hydraulic inner diameter is in the range of about 0.5 mm to about 5 mm. (Item 132) 10. The microfluidic chip of any one of the preceding items, wherein the first sample reservoir is configured to hold a sample volume of at least 100 μl. (Item 133) 10. The microfluidic chip of any one of the preceding items, configured to transfer at least 50% of the sample volume from the first sample reservoir to the first channel when a vacuum is applied to the microfluidic chip. (Item 134) 10. The microfluidic chip of any one of the preceding items, configured to perform isotachophoresis on a sample entering the first channel. (Item 135) (a) exposing a biological sample containing cells or tissues to a solution containing urea or thiourea, thereby lysing the cells or tissues in the biological sample to produce a cell lysate; (b) introducing the cell lysate into a device; and (c) performing isotachophoresis using the device to isolate nucleic acids from the cell lysate. A method for extracting nucleic acid, comprising: (Item 136) 10. The method of any one of the preceding items, further comprising digesting the sample with proteinase K. (Item 137) 138. The method of claim 137, wherein the solution comprises urea and thiourea. 10. The method of any one of the preceding items, wherein the solution comprises a ratio of urea to thiourea of ​​about 2 to 1. (Item 139) 10. The method of claim 1, wherein the concentration of the urea in the solution is about 4 M to about 9 M, and the concentration of the thiourea in the solution is about 0.5 M to about 3.5 M. (Item 140) 10. The method of claim 1, wherein the concentration of the urea in the solution is about 6.5 M to about 7.5 M, and the concentration of the thiourea in the solution is about 1.5 M to about 2.5 M. (Item 141) 10. The method of any one of the preceding items, wherein the solution comprises terminal electrolyte ions or leading electrolyte ions, or both terminal and leading electrolyte ions. (Item 142) 1. A method for purifying high molecular weight nucleic acids from a tissue sample, comprising: (a) A fluidic device, (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 fluidic device; (ii) a final electrolyte buffer comprising a final electrolyte ion having a first effective mobility, the first effective mobility having an order of magnitude less than the order of magnitude of the effective mobility of the high molecular weight nucleic acid and greater than the order of magnitude of the contaminant; and (iii) introducing a first leading electrolyte buffer containing a first leading electrolyte ion having a second effective mobility, the second effective mobility being greater in magnitude than the effective mobility of the high molecular weight nucleic acid; (b) performing isotachophoresis using the final electrolyte ions, the high molecular weight nucleic acids, and the first leading electrolyte ions, thereby separating the high molecular weight nucleic acids from the contaminants and enriching the high molecular weight nucleic acids in the isotachophoresis zone; and (c) eluting the genomic DNA into a solution in an output reservoir, wherein greater than 50% of the mass of nucleic acids in the solution is greater than 30 kilobases. (Item 143) 143. The method of claim 142, wherein the lysis buffer does not contain an alkaline buffer. (Item 144) 10. The method of any one of the preceding items, wherein the lysis buffer comprises octylphenol ethoxylate. (Item 145) 10. The method of any one of the preceding items, wherein more than 50% of the mass of nucleic acids in the solution is greater than 50 kilobases. (Item 146) (a) providing a fluidic device comprising a first channel in fluid communication with a sample input reservoir containing a tissue sample comprising lysed solid tissue, a first buffer reservoir containing a first leading electrolyte buffer, and a second buffer reservoir containing a final electrolyte buffer; (b) contacting a first electrode with the first lead electrolyte buffer in the first buffer reservoir; (c) contacting a second electrode with the final electrolyte buffer in the second buffer reservoir; and (d) applying an electric field within the fluidic device to perform isotachophoresis, wherein the isotachophoresis is performed without direct contact between the tissue sample and the first and second electrodes. A method for performing isotachophoresis, comprising: (Item 147) Item 147. The method of item 146, wherein the fluidic device further comprises 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 leading electrolyte buffer than the first buffer reservoir. (Item 148) 10. The method of claim 9, wherein the third buffer reservoir and the first buffer reservoir are connected by 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. (Item 149) 10. The method of any one of the preceding items, wherein the fluidic device further comprises an elution reservoir. (Item 150) 10. The method of any one of the preceding items, wherein the elution reservoir is in fluid communication with a fourth buffer reservoir. (Item 151) (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 junction; and (b) a mechanical member including first teeth configured to apply mechanical pressure to the first channel via the first teeth to at least partially close the first channel by plastic deformation of at least one wall of the first channel and to increase fluid resistance between the first channel and the first reservoir; A microfluidic system comprising: (Item 152) Item 152. The microfluidic system of item 151, wherein the microfluidic chip further comprises a second reservoir in fluid communication with the first reservoir and a second channel connecting the first reservoir and the second reservoir, and the mechanical member further comprises second teeth 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. (Item 153) 10. The microfluidic system of claim 1, wherein the first tooth is configured to provide mechanical pressure at the first junction to close the first channel by plastic deformation of at least one wall of the first channel. (Item 154) 10. The microfluidic system of claim 1, wherein the first tooth is configured to heat the first channel. (Item 155) 10. The microfluidic system of claim 1, wherein the mechanical member comprises a material having a Young's modulus greater than the Young's modulus of the first channel. (Item 156) 10. The microfluidic system of any one of the preceding items, configured to perform isotachophoresis. (Item 157) 10. The microfluidic system of any one of the preceding items, wherein the first tooth is thermally coupled to a heating element. (Item 158) 10. The microfluidic system of claim 1, wherein the first tooth is heated to a temperature above the glass transition temperature of the at least one wall of the first channel. (Item 159) Item 152. A method of completing a process in a fluidic system using the microfluidic system of item 151, comprising the step of at least partially closing the first channel by plastic deformation, thereby increasing resistance to fluid flow between the first channel and the first reservoir. (Item 160) Item 159. The method of item 159, wherein the first tooth of the mechanical member applies a force of at least 0.25 lb to the first channel. (Item 161) Item 159. The method of item 159, wherein the process in the fluid system is isotachophoresis. (Item 162) (a) loading a sample containing nucleic acids into a first reservoir of a microfluidic chip; (b) loading a final electrolyte buffer into a second reservoir of the microfluidic chip, the final electrolyte buffer comprising a first final electrolyte ion having an effective mobility that is smaller than the effective mobility of the nucleic acid; (c) loading a third reservoir of the microfluidic chip with a leading electrolyte buffer, the third reservoir containing a first leading electrolyte ion having a second effective mobility, the second effective mobility being greater than the magnitude of the effective mobility of the nucleic acid; (d) applying an electric field within the microfluidic chip to perform isotachophoresis using the first final electrolyte ion, the nucleic acid, and the first leading electrolyte ion, thereby confining the nucleic acid or a portion thereof in an isotachophoresis zone; and (e) using a temperature sensor to sense temperature changes in or near the isotachophoresis zone and controlling the electric field using feedback from the temperature sensor. A method for performing isotachophoresis on a sample containing nucleic acid, comprising: (Item 163) 163. The method of claim 162, wherein the controlling of the electric field results in the location of the nucleic acid or a portion thereof in an elution reservoir or in a region of the microfluidic chip. (Item 164) 164. The method of claim 162 or 163, wherein the temperature sensor is positioned within at most 8 mm of the elution reservoir. (Item 165) 165. The method of claim 162, 163 or 164, wherein the temperature change is in the range of about 0.2°C to 5°C. (Item 166) 10. The method of any one of the preceding items, wherein the applied electric field causes the leading electrolyte and the final electrolyte to meet at an isotachophoretic interface, and the temperature sensor senses the isotachophoretic interface. (Item 167) (a) i. a first sample reservoir in fluid communication with the first fluid channel; ii. first, second, and third buffer reservoirs in fluid communication with the first fluid channel, the first and second buffer reservoirs being separated by a capillary barrier; and iii. an elution reservoir in fluid communication with the first fluid channel; a first isotachophoresis region in a microfluidic chip, comprising: (b) a sensor configured to detect a temperature change in the first fluid channel within the first isotachophoresis region; and (c) a device positioned to provide a current within the first channel within the first isotachophoresis region. A microfluidic device comprising: (Item 168) Item 168. The microfluidic device of Item 167, further comprising a controller configured to activate a reduction or removal of the current when the sensor receives a thermal signal. (Item 169) Item 169. The microfluidic device according to item 167 or 168, wherein the temperature change is a temperature increase in the range of about 0.2°C to 5°C. (Item 170) 10. The microfluidic device of any one of the preceding items, further configured to isolate a sample of nucleic acids in the elution reservoir after the sensor detects a change in temperature. (Item 171) 10. The microfluidic device of any one of the preceding items, wherein the sensing of the nucleic acid is performed by a sensor positioned within at most 8 mm of the elution reservoir. (Item 172) 10. The microfluidic device of any one of the preceding items, wherein the first channel comprises a single sensor. (Item 173) (a) the microfluidic device according to item 111, the microfluidic device according to item 167, or the microfluidic chip according to item 129; (b) a final electrolyte buffer solution containing a final electrolyte; and (c) Lead electrolyte buffer solution containing the lead electrolyte Kit including: (Item 174) Item 174. The kit of item 173, wherein the final electrolyte buffer comprises a mixture of at least two electrolytes having different effective mobilities. (Item 175) Item 175. The kit of item 174, wherein the mixture comprises (i) a first electrolyte having an effective mobility magnitude smaller than the nucleic acid and larger than the contaminant, and (ii) a second electrolyte having an effective mobility magnitude smaller than the contaminant. (Item 176) Item 176. The kit of item 175, wherein the first electrolyte comprises caproic acid. (Item 177) 177. The kit of claim 175 or 176, wherein the second electrolyte comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). (Item 178) 178. The kit of any one of items 175 to 177, further comprising a sample buffer, wherein the sample buffer comprises a leading electrolyte buffer, a final electrolyte buffer, or urea in any combination. (Item 179) 179. The kit of item 178, further comprising a sample buffer containing urea and thiourea. [Brief explanation of the drawings]

[0062] [Figure 1A] FIG. 1A shows an exemplary protocol for sample processing and nucleic acid extraction or purification.

[0063] [Figure 1B] FIG. 1B shows an exemplary protocol for automated sample processing and nucleic acid extraction or purification.

[0064] [Figure 2A] FIG. 2A shows an exemplary schematic diagram of isotachophoretic separation and purification of DNA and RNA from contaminants.

[0065] [Figure 2B] FIG. 2B shows an exemplary schematic diagram of isotachophoretic separation and purification of nucleic acids from paraffin and other possible sample contaminants associated with proteinase-mediated tissue disruption and nucleic acid decrosslinking.

[0066] [Figure 3] FIG. 3 shows exemplary results of DNA extraction and purification by automated isotachophoresis in a fluidic device compared to exemplary results from a typical solid-phase column extraction kit.

[0067] [Figure 4A] FIG. 4A shows exemplary results of unbiased (eg, sequence-wise) extraction of GC-rich and AT-rich synthetic DNA oligonucleotides mixed at a sample concentration ratio using isotachophoresis.

[0068] [Figure 4B] Figure 4B shows exemplary results before and after purification of an unbiased (e.g., size or molecular weight) DNA molecular weight ladder using isotachophoresis. A comparison with two solid-phase column-based nucleic acid purification methods is provided.

[0069] [Figure 5A] FIG. 5A shows an exemplary schematic of a channel having a sample preparation zone and an isotachophoretic purification zone.

[0070] [Figure 5B] FIG. 5B shows an exemplary fluidic device cartridge shown in FIG. 5A, with eight parallel fluidic channels and reservoirs for simultaneous processing of up to eight samples.

[0071] [Figure 5C] Figure 5C shows an exemplary top-down schematic view of one channel and its connected reservoir for the fluidic device cartridge shown in Figure 5B, further illustrating the use of gas ports to apply external pressure or vacuum to the channels within the fluidic device cartridge.

[0072] [Figure 5D]FIG. 5D shows an exemplary schematic side view of the fluidic device cartridge shown in FIG. 5B.

[0073] [Figure 5E] FIG. 5E shows an exemplary schematic end view of the fluidic device cartridge shown in FIG. 5B.

[0074] [Figure 6A] FIG. 6A shows an exemplary top schematic view of a fluidic device cartridge.

[0075] [Figure 6B] FIG. 6B shows an exemplary schematic side view of a fluidic device cartridge.

[0076] [Figure 6C] FIG. 6C shows an exemplary bottom schematic view of a fluidic device cartridge.

[0077] [Figure 6D] FIG. 6D shows a three-dimensional exemplary top-down schematic view of a fluidic device cartridge.

[0078] [Figure 7A] FIG. 7A shows an exemplary top schematic view of a fluidic device cartridge.

[0079] [Figure 7B] FIG. 7B shows an exemplary schematic side view of a fluidic device cartridge.

[0080] [Figure 7C] FIG. 7C shows an exemplary bottom schematic view of a fluidic device cartridge.

[0081] [Figure 7D] FIG. 7D shows a three-dimensional exemplary bottom-up schematic view of a fluidic device cartridge.

[0082] [Figure 8A] FIG. 8A shows an exemplary top schematic view of a fluidic device cartridge.

[0083] [Figure 8B] FIG. 8B shows an exemplary schematic side view of a fluidic device cartridge.

[0084] [Figure 8C] FIG. 8C shows an exemplary bottom schematic view of a fluidic device cartridge.

[0085] [Figure 8D] FIG. 8D shows a three-dimensional exemplary bottom-up schematic view of a fluidic device cartridge.

[0086] [Figure 9A] FIG. 9A shows an exemplary schematic diagram of a fluidic device cartridge containing the eight parallel channels shown in FIG. 5B.

[0087] [Figure 9B] FIG. 9B shows an exemplary schematic of two thermal control devices, each aligned with the zones of eight parallel channels shown in FIG. 9A.

[0088] [Figure 10A] FIG. 10A shows an exemplary gas channel that can include a capillary barrier.

[0089] [Figure 10B] FIG. 10B is an enlarged schematic view of the gas channels of FIG. 10A.

[0090] [Figure 11] FIG. 11 shows an exemplary low-loss sample reservoir.

[0091] [Figure 12A]FIG. 12A shows an exemplary mechanical member that can be used to apply pressure to close off channels in a fluidic device.

[0092] [Figure 12B] FIG. 12B shows an exemplary comb-like mechanical element.

[0093] [Figure 12C] FIG. 12C shows the alignment of the comb-like mechanical members and the channels of the fluidic device.

[0094] [Figure 13A] FIG. 13A shows an exemplary benchtop device for performing automated sample preparation and isotachophoresis on a fluidic device cartridge.

[0095] [Figure 13B] FIG. 13B illustrates an exemplary computer control system that is programmed or otherwise configured to implement the methods provided herein.

[0096] [Figure 14] FIG. 14 shows exemplary results of fluorescence-based measurement and quantification of a titration series of nucleic acids using isotachophoresis.

[0097] [Figure 15] Figure 15 shows a schematic diagram of an exemplary design of a fluidic channel with connected reservoirs, non-contact electrodes (which can be used as conductivity sensors) and gas ports for automated loading of fluids into the channel / device and automated isotachophoresis.

[0098] [Figure 16] FIG. 16 shows a graph of voltage measurements over time in a running ITP channel.

[0099] [Figure 17] FIG. 17 shows two graphs of the differential analysis of the voltage measurements from FIG.

[0100] [Figure 18] FIG. 18 shows an example of conductivity measurements over time in an ITP channel near the elution reservoir.

[0101] [Figure 19] FIG. 19 shows an exemplary schematic diagram of a C4D sensor implementation.

[0102] [Figure 20A] FIG. 20A shows an exemplary temperature map of an ITP channel taken using a thermal imaging camera.

[0103] [Figure 20B] FIG. 20B shows a plot of temperature over time at the location of cursor 1 in FIG. 20A.

[0104] [Figure 21] FIG. 21 shows a graph of temperature measurements and the derivative of temperature over time during an ITP run.

[0105] [Figure 22A] FIG. 22A shows an exemplary schematic diagram of a vertical (or column) ITP setup.

[0106] [Figure 22B] FIG. 22B shows an exemplary image of a vertical ITP established by a DNA ITP band.

[0107] [Figure 23] FIG. 23 shows exemplary images and corresponding fluorescence intensity traces of the extraction and separation of amplifiable (e.g., de-crosslinked) DNA from crosslinked DNA derived from an FFPE sample using isotachophoresis.

[0108] [Figure 24A] FIG. 24A shows an exemplary image of DNA extraction and purification from FFPE samples using isotachophoresis.

[0109] [Figure 24B] Figure 24B shows exemplary DNA yields, as measured by quantitative PCR, for extraction and purification of DNA from FFPE samples using isotachophoresis, compared to exemplary results from a typical solid-phase column extraction kit.

[0110] [Figure 25] Figure 25A shows an image of a single-channel ITP chip loaded with nucleic acids (RNA extraction and digestion from human cells) stained with a dye for visualization. Figure 25B shows an image of a single-channel ITP chip loaded with nucleic acids (RNA extraction and digestion from human cells) stained with a dye for visualization.

[0111] [Figure 26A] FIG. 26A shows an image of the RNA ITP band in the chip channel during purification.

[0112] [Figure 26B] FIG. 26B shows an image of the ITP bands of total nucleic acids in the chip channel during purification.

[0113] [Figure 26C] FIG. 26C shows a graph of the RNA quality electropherogram for the sample shown in FIG. 26A.

[0114] [Figure 26D] FIG. 26D shows a graph of the RNA quality electropherogram for the sample shown in FIG. 26B.

[0115] [Figure 27A]FIG. 27A shows the results of DNA yield (ng) for ITP (squares) compared to column (diamonds, Qiagen QiaAmp) extraction of whole mouse blood as a function of the volume percent of whole blood in the starting sample.

[0116] [Figure 27B] FIG. 27B shows an image of total nucleic acid in the ITP band during ITP purification of lysed whole mouse blood on the chip.

[0117] [Figure 27C] Figures 27C and 27D show white-light and fluorescent overlay images of the ITP chip channels showing the physical separation of heme in the sample / lead electrolyte channel from the elution channel and reservoir before and after ITP purification of 50% (volume) whole blood lysate. Nucleic acids are stained with green dye for visualization in the elution well. Figure 27C shows the chip before ITP (blood lysate and ITP buffer loaded into the chip; buffer only in the elution well). Figure 27D shows the chip after ITP (blood lysate and ITP buffer loaded into the chip; purified DNA in the elution well). [Figure 27D] Figures 27C and 27D show white-light and fluorescent overlay images of the ITP chip channels showing the physical separation of heme in the sample / lead electrolyte channel from the elution channel and reservoir before and after ITP purification of 50% (volume) whole blood lysate. Nucleic acids are stained with green dye for visualization in the elution well. Figure 27C shows the chip before ITP (blood lysate and ITP buffer loaded into the chip; buffer only in the elution well). Figure 27D shows the chip after ITP (blood lysate and ITP buffer loaded into the chip; purified DNA in the elution well).

[0118] [Figure 27E] FIG. 27E shows the chip after ITP purification (50% blood by volume).

[0119] [Figure 27F] Figure 27F shows the chip after ITP purification (25% blood by volume).

[0120] [Figure 28] FIG. 28 shows the results of high molecular weight DNA purification with ITP compared to solid phase extraction.

[0121] [Figure 29A] FIG. 29A shows a fluidic device containing eight closed channels.

[0122] [Figure 29B] FIG. 29B shows a magnified microscopic view of the second channel closure position adjacent to the elution reservoir of each channel.

[0123] [Figure 29C] FIG. 29C shows the percent closure calculated as a function of the force applied to the fluidic device.

[0124] [Figure 29D] FIG. 29D shows the results of conductance measurements of channel closure.

[0125] [Figure 30] FIG. 30 shows a graph of voltage measurements and the derivative of the voltage over time during an ITP run.

[0126] [Figure 31A] FIG. 31A shows a micrograph of the ITP bands with focused DNA for each of the eight samples in the sample channel region of the device.

[0127] [Figure 31B] FIG. 31B shows independent voltage signal data at fixed current for each of the eight channels over time.

[0128] [Figure 31C]FIG. 31C shows a micrograph of the same eight ITP bands containing focused DNA from the eluted sample in the elution reservoir.

[0129] [Figure 31D] FIG. 31D is an enlarged section of the voltage monitoring used in the operation shown in FIG. 31B. DETAILED DESCRIPTION OF THE INVENTION

[0130] Overview

[0131] Sample preparation is the first step for almost all genomic and transcriptomic analyses and can still be a major source of analytical variability. Sample preparation can also be labor-intensive, especially when the samples are formalin-fixed, paraffin-embedded (FFPE) samples that contain cross-linked proteins.

[0132] The present disclosure provides methods and devices for improving the efficiency of nucleic acid extraction and purification from tissue and cell samples, including samples that have been processed in some manner, such as paraffin-embedded or chemically fixed samples (e.g., FFPE samples, samples containing solid tissue). The methods provided herein include methods for on-chip or off-chip preparation of such processed samples prior to isotachophoresis using methods that incorporate leading and final electrolyte ions. In some examples, the methods involve processing (e.g., removing embedding material, lysing, enzymatic disruption) fixed solid tissue in a final or leading electrolyte buffer prior to isotachophoresis of the sample. The methods can also include the use of a second leading electrolyte buffer with lower ionic strength to generate samples compatible with downstream processing, such as amplification or other enzymatic assays. The devices and systems provided herein include devices suitable for performing isotachophoresis on tissue-derived samples, including microfluidic devices with parallel processing features and automated feedback control mechanisms, which may include thermal sensors that detect temperature changes within the sample processing channel.

[0133] The disclosed methods and devices can achieve improved nucleic acid recovery from samples, particularly from low-abundance samples (e.g., less than 100 ng of nucleic acid), relatively large volumes (e.g., total volumes greater than 25 μl, total volumes greater than 50 μl, total volumes greater than 100 μl, or more), or liquid samples containing solid particles. The methods and devices provided herein can also achieve high reproducibility and reduced bias toward short-chain nucleic acids. The devices provided herein can integrate sample preparation (e.g., removal of crosslinking or embedding materials) and nucleic acid extraction operations within a single device. The disclosed devices and methods can also be compatible with process automation, integrated with downstream processes, integrated with in-line quantification (e.g., at single picogram resolution), and / or integrated with nucleic acid length and sequence distribution analysis.

[0134] The method provided herein is often the method of carrying out isotachophoresis under the conditions suitable for extracting the nucleic acid from certain samples, especially FFPE samples.In some examples, the disclosed method comprises carrying out isotachophoresis using a final electrolyte buffer solution that contains at least two kinds of ions with different effective mobility.This method can also comprise carrying out isotachophoresis using two different leading electrolyte buffer solutions, one of which can serve as the elution buffer solution for sample.This method can comprise the process automation and parallel processing of multiple samples.

[0135] The present disclosure also includes protocols using buffer and spacer chemistries. These buffer and spacer chemistries can include the use of multiple species of electrolytes to perform ITP. For example, the final electrolyte can include a mixture of electrolyte species that can separate either the non-crosslinked nucleic acids or both the crosslinked and non-crosslinked nucleic acids from contaminants in the sample while simultaneously separating the non-crosslinked nucleic acids from the crosslinked nucleic acids.

[0136] The devices provided herein include injection-molded fluidic devices with parallel sample processing channels capable of performing ITP in multiple modes, and ITP devices with two or more regions connected to a thermal device. The techniques disclosed herein can use ITP to simultaneously recover, purify, and collect extracted RNA and DNA, quantify the total amount of extracted nucleic acid on-chip (e.g., concentration by in-line ITP to very small volumes or labeling with intercalating fluorescent dyes), and deliver nucleic acids downstream to parallel outlet reservoirs compatible with robotic pipetting.

[0137] The techniques of the present disclosure can enable the purification of sample materials (e.g., nucleic acids) without binding the sample materials to a solid support. The techniques of the present disclosure can enable the purification of sample materials (e.g., nucleic acids) without the use of liquid phase extraction. This can enable purification without relying on solubility differences.

[0138] The operation of the disclosed devices can be automated, largely automated, or partially automated. In some cases, the disclosed methods involve only a single off-chip mixing step of dispensing a sample (e.g., FFPE sections) into a solution (e.g., an alkaline, lysis, or buffer solution containing urea and / or thourea), followed by loading the sample into a reservoir of a fluidic device for further on-device sample preparation (e.g., deparaffinization, tissue disruption and cell lysis, protease digestion, protein digestion, or other treatments including protein denaturation, or nuclease digestion), and nucleic acid extraction, purification, enrichment, in-line quantification, and sizing or fractionation (e.g., size selection). In some cases, the disclosed methods include dispensing a sample (e.g., an FFPE section or other tissue sample) into a reservoir or channel (e.g., a cartridge) of a fluidic device pre-filled with a solution (e.g., an alkaline solution, lysis solution, or buffer solution containing urea and / or thiourea) for on-device sample preparation (e.g., deparaffinization, tissue disruption and cell lysis, protease digestion, other treatments including protein denaturation, or nuclease digestion), and nucleic acid extraction, purification, enrichment, in-line quantification, and sizing or fractionation (e.g., size selection). In some cases, the disclosed methods include off-chip disruption of the sample's tissue and / or lysis of its cells, followed by further on-device sample preparation (e.g., deparaffinization, protease digestion, or other treatments including protein denaturation, or nuclease digestion), and loading the sample, which may be a homogenous or heterogeneous mixture of lysed solid tissue and nucleic acids, into a reservoir of a fluidic device for nucleic acid extraction, purification, enrichment, in-line quantification, and sizing or fractionation (e.g., size selection). Nuclease digestion can include DNA removal to perform DNA-free RNA extraction, or RNA removal to perform RNA-free DNA extraction. The fluidic devices provided herein can be used with benchtop systems to automate electric field-based methods for extracting DNA and RNA from samples.

[0139] The disclosed devices include systems capable of automating and integrating on-chip heating (e.g., temperatures between 37°C and 80°C), sample preparation (e.g., deparaffinization, tissue disruption, and cell lysis), buffer exchange, nucleic acid extraction and purification, enrichment of uncrosslinked or amplifiable nucleic acids (e.g., by separating and delivering them separately from crosslinked nucleic acids), and delivery of purified nucleic acids to an output reservoir, such as an array, compatible with manual or robotic pipetting. For example, the disclosure includes an eight-channel cartridge in a microtiter plate format compatible with standard robotic automation, as well as an integrated benchtop controller prototype capable of providing automated control of loading buffers and other fluids, application of temperature and electric fields to the device, and automated start and stop run processing of parallel samples. This system can be easily modified in the future, if needed, to achieve high throughput for use in larger diagnostic or clinical laboratories (e.g., 96-well sample formats).

[0140] For example, FIG. 1A shows a schematic of an exemplary method for sample processing and nucleic acid extraction using the techniques of the present disclosure. A sample is prepared at 101 and may undergo optional pretreatment steps 102, such as mixing with a buffer, lysis, or removal of embedding material (if present). The sample (and, e.g., buffer) may then be loaded into a fluidic device 103. Sample preparation steps 104, such as removal of embedding material (if present and not already removed during pretreatment), tissue disruption, cell lysis, protein or protein digestion, and (e.g.) nuclease digestion, may then be performed in the fluidic device. Isotachophoresis 105 is then performed to separate and purify the nucleic acids from contaminants within the sample (e.g., cellular debris, embedding material, cross-linked nucleic acids, fixatives such as formalin, inhibitors, enzymes such as digestion or restriction enzymes). Other steps, such as de-cross-linking of cross-linked nucleic acids (e.g., by heat or protease digestion), may be performed simultaneously with the isotachophoresis. During or after isotachophoresis, the nucleic acids can be detected and quantified at 106. Once the nucleic acids have been extracted or purified, they can then be eluted and recovered from the device 107 .

[0141] FIG. 1B shows an exemplary process workflow for automated ITP. In step 110, a protocol can be selected, such as using a graphical user interface on a benchtop device. The user interface software can enable ease of use or hands-free 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., optical barcode, RFID chip) associated with the sample or fluidic device chip, which can display the protocol to be performed. In step 111, the instrument lid can be opened (e.g., manually or automatically with a motor). Motorized lid opening is compatible with robotic laboratory automation. In step 112, the user can load a chip (e.g., a fluidic device) into the benchtop instrument. The chip can include a monolithic multichannel SLAS standard microtiter plate (MTP) footprint for automated ITP. In step 113, ITP liquid can be loaded into the chip wells. Reservoirs for ITP fluids and user samples can be designed for ease of loading, such as with a multichannel pipette (e.g., SLAS standard microtiter plate format with a 9 mm pitch). The geometric design of the channels connecting the reservoirs to the ITP channels (e.g., capillary barriers) can prevent gravity flow or liquid wetting into the channel prior to operation. These structures can stop fluids at defined locations within the ITP channel, including establishing the leading / final electrolyte interface, and enable bubble-free loading. In some cases, a pneumatic actuation can be applied to inject fluids into the channel prior to operation. Chip materials can be selected to prevent or resist wetting or wicking of fluids into the channel (e.g., plastics with hydrophobic properties or high contact angles). Users can load ITP reagents and buffers (e.g., five different fluids) onto the chip.Alternatively, the chip can be supplied with pre-loaded reagents. In step 114, the user or device can close the lid of the device. Sample loading can be actuated from a gas or pneumatic port on the chip. Wetting and / or gravity flow can be used, for example, to fill the channels with liquid without the application of active pressure.

[0142] In step 115, the instrument can apply pressure to load fluid into the chip and inject it into the channels. In step 116, the device can verify that the channels were properly prepared. For example, optical (e.g., reflectance), electrical, pressure, and / or flow sensors can be used to verify that the fluid was loaded to the correct location within the chip. The sensors and device software can enable real-time monitoring and control of fluid loading. Loading of ITP reagents and buffers can occur before loading the sample into the chip, so that if loading fails, sample material is not wasted. If the channel is not properly loaded, the device can report an error 130. In step 117, the device lid can be opened. In step 118, the sample can be loaded into the device. Sample loading can be performed manually by a user or automated, such as by a laboratory automation robot. Other sample preparation steps can also be performed. For example, a paraffin-embedded sample (e.g., FFPE) can be loaded, and the device can then control the temperature in the sample reservoir to deparaffinize the sample. In step 119, the lid of the device can be closed. In step 120, the device can perform a self-test. For example, electrical feedback from device electrodes interfacing with the on-chip reservoir can be used to self-test for successful liquid injection (e.g., air bubble detection). Optical sensors can be used to provide feedback on the status of the liquid injection (e.g., whether the liquid 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 is not properly prepared, the device can report an error 131.

[0143] In step 121, ITP-based purification can be performed. Feedback control and process timing (e.g., actuation) using sensors described herein can be used to control and / or automate ITP purification. The device can determine whether the purification was successful; if not, the device can generate an error report 132. In step 122, the sample can be quantified using an on-device sensor (e.g., an optical sensor), e.g., by fluorescence, UV, or other optical detection. Sample sizing can also be performed. If the device determines that the sample was not properly quantified or discovers other problems, the device can generate an error report 133. In step 123, a change in conductivity can be detected and used to indicate the timing of the end of the ITP run (e.g., when the nucleic acid reaches a designated elution location or reservoir). Other detection methods described herein, such as temperature or drive voltage, can also be used to determine the end of run timing or other actuation. To automate the ITP process, for example, a temperature or voltage sensor can be used to control the electric field applied to a channel in the device. As an example, an electric field can be applied to a channel to initiate ITP purification. The sensed voltage change may 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. As the ITP zone containing the trapped nucleic acid moves, the voltage may change. A change indicative of the ITP zone passing a channel feature, such as a region of reduced cross-sectional area, can be sensed by a voltage sensor, and feedback can be used, for example, to alter the electric field by decreasing the applied current. As the ITP zone passes a temperature sensor at or near the elution reservoir, a temperature change can be detected, and feedback from the sensor can be used to control the electric field, for example, by removing the electric field to terminate the ITP run. In step 124, the device can terminate the run, for example, based on the actuation signal.Once the ITP run is complete, the nucleic acids can be located or isolated in an elution reservoir or region. In step 125, the device can close the channel and fix the elution volume to maintain a constant volume for elution (e.g., by preventing or preventing flow into the elution or waste reservoir during pipetting out of the elution volume). Fixing the elution volume can aid in ease of use and can aid in reporting the concentration of the eluted sample material. In step 126, the lid of the device can be opened (e.g., by the user or automatically).

[0144] In step 127, the purified sample can be extracted from the device. The chip and / or device can be designed to achieve a predetermined elution volume, as discussed herein. Recovering the purified material from the device can be accomplished by pipetting or otherwise removing the material from the chip. Alternatively, sample extraction can be accomplished by interfacing the ITP chip with another fluidic chip or system (e.g., in the absence of an elution reservoir). Other operations can then be performed on the purified sample material using other fluidic systems, such as next-generation sequencing (NGS) library preparation, sample analysis such as 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 can contain algorithms or other software for converting measurements (e.g., fluorescent signals) into sample quantification values ​​and can report that data to the user. The process ends in step 129.

[0145] These issues can be particularly important to address for samples that are precious, difficult to recover, or low in abundance (e.g., less than 100 ng of nucleic acid, or samples with low levels of intact or non-crosslinked nucleic acid). For such samples, current protocols can lack reproducibility, result in sample material loss, bias toward short or long nucleic acid targets, bias toward the sequence of nucleic acid targets, and / or lack reproducibility. Such protocols can also lack compatibility with process automation or downstream analysis. Current protocols for nucleic acid preparation can include liquid-phase extraction (LPE), such as phenol-chloroform or Trizol extraction, and solid-phase extraction (SPE). SPE-type techniques can use structures including packed beads, monolithic porous structures, and / or magnetic beads. In some cases, LPE and SPE-type techniques can result in mechanical shearing during processing, which can induce fragmentation of long or high-molecular-weight nucleic acids and / or reduce their yield.

[0146] The isotachophoresis methods and devices provided herein are particularly well suited for extracting nucleic acids from solid or semi-solid tissue lysates. Solid-phase extraction (SPE) techniques typically process lysates by pumping the entire volume of the lysate sample through a column to selectively adsorb nucleic acids onto the column's surface. Such pumping of complex lysates, which may contain a liquid-particle mixture, can result in clogging or fouling of the column, potentially reducing the efficiency of nucleic acid extraction due to the porous column. In contrast, the isotachophoresis methods and devices described herein often do not involve pumping or "filtering" the entire volume of the lysate sample through a column. Rather, an electric field can be applied to the lysate to migrate charged, solvated nucleic acids dispersed throughout the complex sample lysate into or out of the sample's continuous liquid phase. Nucleic acids may have a relatively larger electrophoretic mobility magnitude than other solutes, debris, or contaminants in the sample lysate. Solutes in a sample may have relatively low electrophoretic mobilities, too low to be concentrated in the isotachophoresis zone located at the interface between the leading and final electrolytes. Application of an electric field can migrate nucleic acids while leaving behind particles and / or other tissue debris (e.g., cell debris, unlysed cells, or tissue that can bind cells to other cells). Thus, the isotachophoresis methods and devices provided herein may be well suited for extracting charged, solvated nucleic acids from complex lysed solid tissue samples without the need to process the entire mixture through a column, as in SPE.

[0147] As used herein, "particle" can refer to a component of a sample mixture or sample dissolution mixture that is a phase different from the continuous liquid phase (e.g., aqueous solution) of the sample. A particle can also be a non-liquid component of a sample mixture. A particle can be, for example, a suspended solid particle or a colloidal body suspended within a sample. Such particles can have various characteristic length scales ranging from about 1 nanometer (nm) to about 1 millimeter (mm). In some instances, a particle may not be a single-cell organism or a cell.

[0148] The isotachophoresis methods and devices provided herein can achieve a reduced strain rate as the sample moves through the channel compared to typical SPE methods. In some cases, the methods and devices provided herein can achieve a strain rate of about 250 s -1 , 500s -1 , 750s -1 , 1000s -1 , 2000s -1 , 3000s -1 , 4000s -1 , 5000s -1 , 6000s -1 , 7000s -1 , 8000s -1 , 9000s -1 or 10,000s -1 In some cases, the methods and devices provided herein have a strain rate of less than about 250 s -1 , 500s -1 , 750s -1 , 1000s -1 , 2000s -1 , 3000s -1 , 4000s -1 , 5000s -1 , 6000s -1 , 7000s -1 , 8000s -1 , 9000s -1 or 10,000s -1 In some cases, the methods provided herein can be performed without centrifugation.

[0149] Isotachophoresis Chemistry and Operation

[0150] FIG. 2A shows an exemplary schematic diagram of a nucleic acid purification process by isotachophoresis (ITP). A sample 201, e.g., a lysed solid tissue sample containing nucleic acids (DNA and RNA) 202 and contaminants 203, is loaded into a final electrolyte (TE) 204 and enters an isotachophoresis channel 200 containing a leading electrolyte (LE) 205. Under the influence of an electric field 220 applied to the isotachophoresis channel 210, the nucleic acids 212 migrate away from the contaminants 213. The electric field also migrates the final electrolyte 214 through the channel to a position generally behind the nucleic acids, and the leading electrolyte 215 through the channel generally ahead of the nucleic acids. The effective mobility of the leading electrolyte is greater than that of the nucleic acids, which in turn is greater than that of the final electrolyte, which is greater than that of the contaminants.

[0151] Figure 2B shows an exemplary schematic diagram of a process using isotachophoresis (ITP) on a fluidic device to simultaneously decrosslink nucleic acids and separate the decrosslinked nucleic acids from crosslinked nucleic acids and contaminants (e.g., paraffin). In some cases, the contaminants may include crosslinked nucleic acids. To perform tissue lysis and initial deparaffinization, a paraffin-embedded sample is loaded into the fluidic device in an alkaline buffer and incubated at a pH of about 10 and a temperature of about 50°C to about 80°C for 10 to 30 minutes. Incubation can occur before or during application of an electric field to perform isotachophoresis. Alternatively, the sample can be loaded into a leading electrolyte buffer. After incubation, at a first time point 240, the sample containing crosslinked nucleic acids 236 and paraffin 237 is placed in an ITP channel with a leading electrolyte 232. In the leading electrolyte (LE) zone 238, leading electrolyte 231 and proteinase K enzyme 233 are located ahead of the ITP channel. At a second time point 250, at 50°C, an ITP-driven pH quench lowers the pH, and the proteinase K enzyme contacts and decrosslinks the crosslinked nucleic acids, generating uncrosslinked nucleic acids 235, which collect in the ITP zone 239 between the destination and leading electrolytes. Lowering the pH (e.g., from about 10-12 to about 7 (or about 6.5-8.5)) may provide a more favorable environment for enzyme activity and improve the chemical stability of the nucleic acids. At a third time point 260, proteinase K decrosslinks even more nucleic acids, resulting in free proteins 234, and the decrosslinked nucleic acids move further upstream away from paraffin, free proteins, and other contaminants. Operation of this process can be automated using a fluidics device or benchtop system.

[0152] In some cases, the sample may be loaded into a sample buffer containing a concentration of leading electrolyte 205, 231 that is different from the concentration of the leading electrolyte 205, 231 used to perform the isotachophoresis. In some cases, the sample may be loaded into a sample buffer containing a second leading electrolyte that is different from the leading electrolyte 215. The second leading electrolyte can have an effective mobility magnitude that is greater than the effective mobility magnitude of the nucleic acid. The second leading electrolyte can have an effective mobility magnitude that is less than the effective mobility magnitude of the leading electrolyte 215.

[0153] In some cases, the pH of the sample may be quenched by performing isotachophoresis. In some examples, the pH of the sample may be quenched to a range of about 6.5 to about 8.5, for example, about 7 or 7.5.

[0154] ITP can be performed using a variety of leading and final electrolytes. The leading electrolyte can be selected to have a larger effective mobility magnitude than the extraction target (e.g., nucleic acid), and the final electrolyte can be selected to have a smaller effective mobility magnitude than the extraction target. The leading and / or final electrolyte can be present at a concentration of about 10 mM to about 200 mM. The leading and / or final electrolyte can be present at a concentration of about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The leading and / or final electrolytes can be present at a concentration of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM. The leading and / or final electrolytes can be present at a concentration of at most about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The leading electrolytes used in specific examples of ITPs can include one, two, three, four, five, six, seven, eight, nine, ten, or more different ionic species. The final electrolytes used in specific examples of ITPs can include one, two, three, four, five, six, seven, eight, nine, ten, or more different ionic species. Different ionic species in the leading and / or final electrolytes can be present at different concentrations. Different concentrations of ions, such as in the final or leading electrolytes, can be selected to manipulate the size of spacing zones. Spacing zones can be used to further separate one type of target from another, such as separating de-crosslinked nucleic acids from protein-crosslinked nucleic acids.

[0155] The final electrolyte can include a mixture of ions with different effective mobility magnitudes. Using a first final electrolyte ion with a first effective mobility magnitude and a second final electrolyte ion with a second effective mobility magnitude smaller than the first ion's effective mobility magnitude, it is possible to separate uncrosslinked nucleic acids from protein-crosslinked nucleic acids while simultaneously separating both (or at least the uncrosslinked nucleic acids) from contaminants. In such an example, the uncrosslinked nucleic acid can have a greater effective mobility magnitude than the first final electrolyte ion, which can have a greater effective mobility magnitude than the crosslinked nucleic acid; the crosslinked nucleic acid can then have a greater effective mobility magnitude than the second final electrolyte ion, which can then have a greater effective mobility magnitude than the contaminants. For example, crosslinked and uncrosslinked nucleic acids can be individually enriched by isotachophoresis using a leading electrolyte and two final electrolytes, such as caproic acid as the first ion and HEPES as the second ion.

[0156] Electrolyte ions can also be selected based on acidity (e.g., pKa). Ions with specific pKa can be selected to effect pH changes, for example, along the ITP channel. Ions can also be selected for non-electrophoretic reasons, such as compatibility with downstream processes (e.g., enzymatic processes such as PCR or next-generation sequencing library preparation). For example, caproic acid, MOPS, and HEPES can be selected for their good downstream enzyme compatibility.

[0157] Exemplary leading electrolyte ions include, but are not limited to, hydrochloric acid, acetic acid, 2-chloroisocrotonic acid, salicylic acid, chlorocrotonic acid, nicotinic acid, gallic acid, trichlorolactic acid, butyric acid, sulfanilic acid, benzoic acid, crotonic acid, trichloroacrylic acid, propionic acid, levulinic acid, sorbic acid, orotic acid, valeric acid, picric acid, 2-naphtalenesulfonic acid, saccharin, dinitrophenol, p-toluenesulfonic acid, aspartic acid, trimethylacrylic acid, isocaproic acid, caproic acid, octylsulfonic acid, nitrophenol, GABA, cacodylic acid, trimethylpyruvic acid, ethyl maleate, ethyl fumarate, toluic acid, enanthic acid, mandelic acid, cinnamic acid, cresol, glutamic acid, MES, isomers thereof, and combinations thereof.

[0158] Exemplary final electrolyte ions include, but are not limited to, caprylic acid, gluconic acid, vanillic acid, decyl sulfonic acid, aspirin, glucuronic acid, pelargonic acid, benzylaspartic acid, ascorbic acid, dodecyl sulfonic acid, MOPS (3-(N-morpholino)propanesulfonic acid), dichlorophenol, caproic acid, capric acid, tyrosine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), isomers thereof, and combinations thereof.

[0159] Mixtures of different ultimate electrolyte ions can be used to achieve bracketed separation by mobility (e.g., crosslinked nucleic acids from contaminants, crosslinked from non-crosslinked nucleic acids), compatibility with downstream assays, favorable surface energy or contact angle between fluid and fluidic device materials, buffering capacity, and total ion solubility.

[0160] Isotachophoresis can quench the pH of the sample to neutral or near neutral. Ions that affect the local pH (e.g., sodium ions (Na+)) are displaced from the sample zone during isotachophoresis, thereby shifting the pH in the sample zone toward neutral.

[0161] Isotachophoresis can be performed over a range of voltages, currents, and field strengths. For example, isotachophoresis can be performed at voltages of about 100V to about 1500V. Isotachophoresis can be performed at voltages of about 100V, 200V, 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1100V, 1200V, 1300V, 1400V, or 15000V. Isotachophoresis can be performed at voltages of at least about 100V, 200V, 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1100V, 1200V, 1300V, 1400V, or 15000V. Isotachophoresis can be performed at a voltage of at most about 100 V, 200 V, 300 V, 400 V, 500 V, 600 V, 700 V, 800 V, 900 V, 1000 V, 1100 V, 1200 V, 1300 V, 1400 V, or 15000 V. Isotachophoresis can be performed at a current of about 10 nA to about 10 mA. Isotachophoresis can be performed at a current of about 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA or 10 mA. Isotachophoresis can be performed at a current of at least about 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA or 10 mA. Isotachophoresis can be performed at currents of at most about 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA, or 10 mA. Isotachophoresis can be performed at field strengths of about 10 V / cm to about 100 V / cm.Isotachophoresis can be performed at a field strength of about 10V / cm, 15V / cm, 20V / cm, 25V / cm, 30V / cm, 35V / cm, 40V / cm, 45V / cm, 50V / cm, 55V / cm, 60V / cm, 65V / cm, 70V / cm, 75V / cm, 80V / cm, 85V / cm, 90V / cm, 95V / cm or 100V / cm. Isotachophoresis can be carried out at a field strength of at least about 10V / cm, 15V / cm, 20V / cm, 25V / cm, 30V / cm, 35V / cm, 40V / cm, 45V / cm, 50V / cm, 55V / cm, 60V / cm, 65V / cm, 70V / cm, 75V / cm, 80V / cm, 85V / cm, 90V / cm, 95V / cm, or 100V / cm. Isotachophoresis can be performed at a field strength of at most about 10V / cm, 15V / cm, 20V / cm, 25V / cm, 30V / cm, 35V / cm, 40V / cm, 45V / cm, 50V / cm, 55V / cm, 60V / cm, 65V / cm, 70V / cm, 75V / cm, 80V / cm, 85V / cm, 90V / cm, 95V / cm or 100V / cm.

[0162] Isotachophoresis can be used to concentrate nucleic acids in a sample. The concentration of nucleic acids in a sample can be increased by at least about 2-fold, 5-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, 100,000,000-fold, or 1,000,000,000-fold after isotachophoresis. The run time for concentrating nucleic acids by isotachophoresis can be less than or equal to about 5 hours, 4.5 hours, 4 hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 10 seconds, or 1 second. In some cases, isotachophoresis can be used to increase the concentration of nucleic acids in a sample by 1,000,000 times in less than or about 2 minutes. In some cases (e.g., from a 25 μL blood lysate sample), isotachophoresis can be used to increase the concentration of nucleic acids in a sample by 100,000 times in less than or about 5 minutes.

[0163] The technique of the present disclosure can be used to reduce the concentration of cross-linked nucleic acid in sample.After isotachophoresis, the concentration of cross-linked nucleic acid in sample can be reduced by at most about 2 times, 5 times, 10 times, 100 times, 1,000 times, 10,000 times, 100,000 times, 1,000,000 times, 10,000,000 times, 100,000,000 times or 1,000,000,000 times.Isotachophoresis can be used to reduce the concentration of contaminants in sample. The concentration of a contaminant in a sample can be reduced by at most about 2-fold, 5-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, 100,000,000-fold or 1,000,000,000-fold after isotachophoresis.

[0164] The nucleic acid sample can contain about 0.1 picograms (pg) to about 25 micrograms (μg). For example, the nucleic acid sample can contain about 5 pg to about 5 μg. The nucleic acid samples were prepared in the following concentrations: approximately 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 nanogram (ng), 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng The amount of sucrose in the solution may be 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, or 25 μg.

[0165] The nucleic acid sample can include deoxyribonucleic acid (DNA), single-stranded DNA, double-stranded DNA, genomic DNA, complementary DNA, ribonucleic acid (RNA), ribosomal RNA, transfer RNA, messenger RNA, microRNA, etc., or any combination thereof. The nucleic acid sample can include a length of at least about 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kB or more. Different sample types can be extracted in various channels of a fluidic device using the techniques disclosed herein. For example, different lengths and / or different types of nucleic acids can be extracted using various channels.

[0166] In some cases, a feature of a nucleic acid sample can be compared to one or more nucleic acids from another sample, and the feature can be, for example, expression level, nucleic acid sequence, molecular weight, nucleic acid integrity, nucleic acid strandedness, or nucleic acid purity.

[0167] Nucleic acid samples can be of a certain quality before and / or after extraction or other processing. Nucleic acid quality can be assessed by various indicators, including, but not limited to, RNA integrity number (RIN), DNA integrity number (DIN), size distribution (e.g., using electrophoresis) and amplification ability (e.g., by PCR), or can be enzymatically processed (e.g., fragmentation, ligation, a-tailing, or hybridization for next-generation sequencing library preparation). Using the techniques of the disclosure, nucleic acids are extracted and processed to produce a nucleic acid sequence of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, The extracted or processed nucleic acid may have a RIN of 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.Using the techniques of the disclosure, nucleic acids are extracted and processed to provide a nucleic acid sequence that is at most about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, The extracted or processed nucleic acid may have a RIN of 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. Using the techniques of the disclosure, nucleic acids are extracted and processed to produce a nucleic acid sequence of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, The extracted or processed nucleic acid may have a DIN of 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.Using the techniques of the disclosure, nucleic acids are extracted and processed to provide a nucleic acid sequence that is at most about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, The extracted or processed nucleic acid may have a DIN of 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 techniques of the present disclosure can be used to extract and process nucleic acids to result in 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% of the 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 greater. In some cases, about 90% to about 100% of the mass of the processed nucleic acids is between about 10 and about 1000 bp, between about 200 and about 2000 bp, or between about 200 and 5000 bp.

[0168] Isotachophoresis can be used to extract nucleic acids from a given starting amount of nucleic acid with an extraction efficiency or yield, characterized as a percentage yield of nucleic acid. The disclosed techniques can provide extracted nucleic acids with a yield of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%. The disclosed techniques can provide extracted nucleic acids with a yield of at least about 10%. 4nanogram (ng), 10 3 ng, 10 2 ng, 10 1 ng, 10 0 ng, 10 -1 ng or 10 -2 Even in the case of low input amounts of nucleic acid, including less than ng, high yields can be achieved. For example, Figure 3 shows exemplary nucleic acid yields from a wide range of different input amounts and sources of nucleic acid. High yields and / or low loss of nucleic acid can be important for next-generation sequencing library preparation. Nucleic acid recovery can be 100% or close to it.

[0169] The disclosed techniques can extract nucleic acids with little or no sequence bias. That is, the sequence composition (e.g., the ratio of GC-rich nucleic acids to AT-rich nucleic acids) of the extracted and purified nucleic acids can be similar to or the same as the sequence composition of the input nucleic acid (see, e.g., Figure 4A). The difference in the sequence composition of the extracted nucleic acid from the sequence composition of the input nucleic acid can be less than or equal to about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0170] The disclosed techniques can extract nucleic acids with little or no length bias. That is, the chain length distribution (e.g., proportion of nucleic acids of different sizes) of the extracted nucleic acids can be similar to or the same as the chain length distribution of the input nucleic acids (see, e.g., Figure 4B). The difference in the chain length distribution of the extracted nucleic acids from the chain length distribution of the input nucleic acids can be less than or equal to about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, short-chain nucleic acids (e.g., about 10 to about 300 bp), long-chain nucleic acids (e.g., about 10 kB, 20 kB, 30 kB, 40 kB, 50 kB, 60 kB, 70 kB, 80 kB, 90 kB, 100 kB, or more), or both short-chain and long-chain nucleic acids can be extracted with reduced loss or bias. Solid-phase columns can, in some cases, result in the loss of up to 100% of short-chain and / or long-chain nucleic acid material. The disclosed techniques can recover nucleotides ranging in size from a single base to hundreds of kilobases. The disclosed techniques 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 the short-chain and / or long-chain nucleic acids present in a sample.

[0171] The disclosed techniques can remove contaminants from samples. Contaminants can include, but are not limited to, embedding materials, cell debris, extracellular matrix components, tissue debris, embedding debris, lipids, carbohydrates, enzymes, ligation by-products, primers, unbound probes or ligations, divalent metals, detergents, preservatives, fixatives, anticoagulants, collagen fibers, and PCR inhibitors. Contaminants can come from the tissues or cells of the sample, preservatives or embedding materials used in the sample, or previous preparations, reactions, or assays performed on the sample. For example, enzymes such as restriction nucleases can be used to prepare DNA for fingerprinting assays and subsequent digestion (e.g., DNase digestion), and the DNA can be separated from the enzymes.

[0172] sample

[0173] The disclosed techniques can process a variety of sample types, including, but not limited to, biological samples, solid tissues, biospecimens, tissue biospecimens, liquid biospecimens, organs, tumors, fresh tissues, solid organs, archived tissues (e.g., FFPE), excised FFPE, freshly frozen tissues, fixed samples, embedded samples, lysed samples, unlysed samples, samples containing cell-cell junctions (e.g., gap junctions, tight junctions, adherens junctions), lysed solid tissues and samples containing nucleic acids, multiphasic samples, heterogeneous liquids or solutions (such as tissue, whole blood, or unlysed cell suspensions), biological samples containing genomic DNA, lysed and unlysed whole blood, plasma and serum, buccal swabs, dried bloodstains, and other forensic samples, fresh tissues or fresh frozen (FF) tissues, cells (lysed and unlysed) cultured or harvested from blood or tissues, fixed cells, stool, 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, lung, and reproductive organs. Samples can contain cellular and cell-free nucleic acids for both eukaryotes and prokaryotes. Fixed samples can be chemically or physically fixed (e.g., by heating or freezing). For example, samples can be chemically fixed with chemical fixatives such as formalin, neutral buffered formalin (NBF), formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, mercuric chloride, zinc salts, Bouin's fluid, alcohol-formalin-acetic acid (AFA or FAA), citrate-acetone-formalin (CAF), acetone, methanol, ethanol, Clarke's fluid, Carnoy's fluid, or Puchtler's methacarn. Embedded samples can be embedded in substances including, but not limited to, wax (e.g., paraffin), agar, gelatin, or plastic resins. Formalin-fixed paraffin-embedded (FFPE) samples can be processed using the techniques of the present disclosure. Samples can include buccal swabs, blood stains, and other forensic samples.Samples can include clinical samples, fine needle aspirates, biopsies, whole blood, lysed blood, serum, plasma, urine, cell culture lysates or freshly harvested cell (e.g., blood cells, dissociated fresh tissue, stem cells) lysates, blood cells, circulating cells (e.g., circulating tumor cells (CTCs)), nucleic acids derived from blood or other bodily fluids, and other sample categories. Cell-free nucleic acids (e.g., cfDNA or cfRNA) can be recovered from unlysed whole blood and the like using the techniques of the present disclosure. In many cases, the cell-free nucleic acids are circulating cell-free nucleic acids. Samples can be derived from a variety of sources, including, but not limited to, normal tissue, benign neoplasms, malignant neoplasms, stem cells, human tissue, animal tissue, plant tissue, bacteria, viruses, and environmental sources (e.g., water). Human or animal tissues can include, but are not limited to, epithelial tissue, connective tissue (e.g., blood, bone), muscle tissue (e.g., smooth muscle, musculoskeletal, cardiac muscle), and nervous tissue (e.g., brain, spinal cord).

[0174] The sample can include one or more particles in suspension. The one or more particles can range in size from colloidal to visible. The one or more particles can be at least about 1 nanometer (nm), 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 950 nm, 1 micrometer (μm), 10 μm, 20 μm, 30 μm ... The size may be 1 mm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm or 1 millimeter (mm). The one or more particles may be at most about 1 nanometer (nm), 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 950 nm, 1 micrometer (μm), 10 μm, 20 μm, 30 μm The particles may have a size of 1 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, or 1 millimeter (mm). One or more particles may be the same size or different sizes. A sample may contain multiple particles, for example, ranging in size from 1 nm to 500 μm.

[0175] Samples of various volumes can be processed on fluidic devices (eg, to extract and purify nucleic acids). For example, the sample volume (with or without buffer) can be at least about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 175 μL, 200 μL, 225 μL, 250 μL, 275 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. The sample volume (with or without buffer) can be at most about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. In some cases, the sample volume can be from about 1 nL to about 10 nL. 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, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. In some cases, the sample volume can be from about 1 nL to about 10 nL.

[0176] Samples containing various numbers of cells can be processed on the fluidic device (e.g., to extract and purify nucleic acids). For example, samples can be about 20,000 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, 90 The cell population may contain less than or equal to 0 cells, 800 cells, 700 cells, 600 cells, 500 cells, 400 cells, 300 cells, 200 cells, 100 cells, 90 cells, 80 cells, 70 cells, 60 cells, 50 cells, 40 cells, 30 cells, 20 cells, 10 cells, 5 cells, 2 cells, or 1 cell. In some cases, the sample contains at least about 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 Contains 4,000 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, 600 cells, 500 cells, 400 cells, 300 cells, 200 cells or 100 cells.

[0177] Samples of various masses can be processed on the fluidic device (e.g., to extract and purify nucleic acids). For example, a sample can contain from about 0.001 milligrams (mg) to about 10 mg of tissue. A sample can contain at most about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue. The sample can contain at least about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue. The sample can contain about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg of tissue.

[0178] Samples containing various amounts of nucleic acids can be processed on a fluidic device (e.g., to extract and purify nucleic acids). For example, a sample can contain 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, a sample can contain more 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.

[0179] The sample can be loaded into a buffer containing a terminal or leading electrolyte. The sample can be loaded into a buffer containing a second leading electrolyte different from the leading electrolyte used to perform ITP. The sample can be loaded into a buffer, such as an alkaline or neutral aqueous buffer. An exemplary alkaline solution or buffer (e.g., for DNA extraction) can include 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 additional component). In some cases, if the sample is lysed by treatment with an alkaline solution or buffer before loading onto the chip, the lysed sample can be subsequently quenched by adding an acidic solution or buffer to bring the pH of the lysed sample to a range of about 7.5 to about 8.5 before performing isotachophoresis. Exemplary aqueous buffers (e.g., for DNA or RNA extraction) can include 2-150 mM Tris-HCl (pH of about 7 to about 8) or BisTris-HCl (pH of about 5.8 to about 7.3), along with at least one additional component. Additional components used in the buffer can include non-ionic surfactants or detergents, ionic or zwitterionic surfactants or detergents, chaotropic agents, disulfide bond reducing agents, proteases, nucleases, and other additives or components that digest, denature, disrupt, or degrade nucleic acids to extract, purify, enrich, or otherwise isolate nucleic acids.

[0180] Nonionic surfactants or detergents can include, but are not limited to, surfactants from the following classes: octylphenol ethoxylate, polysorbate, poloxamer, or polyoxyethylene. Octylphenol ethoxylate surfactants can include, but are not limited to, branched octylphenoxypolyethoxyethanol (IGEPAL CA-630), t-octylphenoxypolyethoxyethanol (Triton™ X-100), or other polyethylene oxide chains with aromatic hydrocarbon lipophilic or hydrophobic groups. Polysorbate surfactants can include, but are not limited to, polyethylene glycol sorbitan monolaurate (Tween® 20), polyethylene glycol sorbitan monooleate (Tween® 80), or sorbitan monooleate (Span® 80). Poloxamer surfactants (i.e., block copolymers based on ethylene oxide and propylene oxide) can include, but are not limited to, polyoxyethylene-polyoxypropylene block copolymer (Pluronic® F-68) or polyethylene-polypropylene glycol block copolymer (Pluronic® F-127). Polyoxyethylene surfactants can include, but are not limited to, nonylphenoxypolyethoxylethanol (NP-40).

[0181] Non-ionic surfactants or detergents can include, but are not limited to, IGEPAL® (e.g., IGEPAL® CA-630), Triton™ X-100, Tween® 20, Tween® 80, NP-40, other block copolymers (including Pluronic® (e.g., F-68 or F-127)), Span® 80, and PEGylated polymers or copolymers. Non-ionic surfactants or detergents can be used to reduce or prevent adsorption of biological molecules to channel walls or to control the wetting and / or surface tension properties of fluids to control sample loading into fluidic devices. Non-ionic surfactants or detergents can be present at a concentration of approximately 0.0005-5% (v / v or w / v). For example, IGEPAL CA-630 can be used at approximately 0.05-0.5% v / v. Ionic surfactants or detergents include, but are not limited to, sodium dodecyl sulfate (e.g., 0.01-2% w / v), sodium dodecylbenzenesulfonate (e.g., 0.01-2% w / v), sodium cholesteryl sulfate (e.g., 0.01%-2% w / v), and sodium deoxycholate (e.g., about 10-1000 mM). Chaotropic agents include, but are not limited to, urea (e.g., about 0.5-9.5 M, or in some cases, 5-9.5 M), thiourea, butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, lithium chloride, magnesium chloride, phenol, and propanol. For example, for either RNA or DNA extraction, or for total nucleic acid extraction, 7.0 M urea and 2.0 M thiourea can be used in a 5-50 mM Tris-HCl (in some cases, 10-20 mM Tris-HCl) buffer solution. The ratio of urea 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 can include, but are not limited to, DTT (e.g., about 0.1-40 mM, or in some cases, about 10 mM) and beta-mercaptoethanol (e.g., about 0.5-2%, or in some cases, about 1%). Proteases can include, but are not limited to, proteinase K, proteases, endoproteinases (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Nucleases can include, but are not limited to, nonspecific nucleic acid digestion enzymes, such as DNases, including DNase I (e.g., for preparing DNA-free RNA extracts), and RNases, such as RNase A, RNase T, or combinations thereof (e.g., for preparing RNA-free DNA extracts). Nucleases can also include specific nucleic acid digestion enzymes (e.g., restriction enzymes) that can cleave at specific nucleic acid sequences and generate predictable fragment sizes and fragment size distributions. In some cases, one or more methods or processes provided herein are carried out without using nucleases, without using DNases, or without using RNases.For example, the methods provided herein include RNA extraction without using DNases.

[0182] Restriction enzymes can include, but are not limited to, type 1 to type 5 restriction enzymes, BamHI, EcoP15I, EcoRI, EcoRII, EcoRV, HaeIII, HgaI, HindIII, HinFI, KpnI, NotI, PstI, PvuII, SacI, SalI, SmaI, SpeI, SphI, XbaI, and StuI. Nucleases can be used at concentrations ranging from 50 to 400 μg / mL. Nuclease digestion can be performed at temperatures ranging from about 20°C to about 37°C. Other nucleic acid-modifying enzymes, such as transposases, ligases, polymerases, and phosphatases, can also be used. Other protein or polynucleotide digesting or degrading agents, such as lysozyme, can also be used.

[0183] Prior to loading into the fluidic device, samples can be subjected to varying degrees of pre-processing. In some cases, samples can simply be loaded into a buffer solution prior to loading into the fluidic device, and any other necessary or desired sample preparation steps can be performed on the device. In other cases, samples can be added to a sample reservoir pre-filled with a processing fluid, such as a solution or buffer. In other cases, samples can be subjected to embedding material removal, tissue disruption, cell lysis, or digestion prior to loading into the fluidic device. In one example, samples are deparaffinized and nucleic acid de-crosslinking is performed on the fluidic device prior to loading into the fluidic device. In another example, samples are deparaffinized, disrupted, and lysed prior to loading into the fluidic device, and optionally nucleic acid de-crosslinking is performed on the fluidic device. In another example, samples are deparaffinized and tissue disruption and cell lysis are performed on the fluidic device prior to loading into the fluidic device. In another example, samples are loaded into the fluidic device, and deparaffinization, tissue disruption, cell lysis, and nucleic acid de-crosslinking are all performed on the fluidic device. Sample preparation steps are discussed further in this disclosure.

[0184] Sample preparation

[0185] Samples can be prepared prior to isotachophoresis. Sample preparation can include steps including, but not limited to, removal of embedding material, tissue disruption, cell lysis, protein digestion, nucleic acid cross-linking, isothermal enzymatic processes, enzyme amplification, enzymatic digestion, disruption of intercellular junctions, disruption of extracellular matrix, disruption of connective tissue, and combinations thereof. Sample preparation can include techniques such as polymerase chain reaction (PCR) or other nucleic acid amplification, isolation or purification of the material of interest (e.g., cells, nucleic acids), probe hybridization, and antibody hybridization (e.g., hybridization of antibodies to nucleosomes). In some cases, samples can be prepared for further analysis by isolating a portion of material from cells derived from the sample. For example, circulating tumor cells can be isolated from a heterogeneous population of cells using a cell sorting device such as a flow cytometer or a magnetic column. In another example, peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs) can be isolated from a blood sample. Sample preparation can be performed on-device or off-device. In some cases, some sample preparation steps are performed off-device, and the sample is then loaded into a fluidic device where further sample preparation steps are performed.

[0186] Biological materials (e.g., cells, tissues, nucleic acids) in an embedded sample can be removed from the embedding material. For example, a paraffin-embedded sample can be deparaffinized. Removal of the embedding material can be achieved using techniques including, but not limited to, heat treatment, chemical treatment (e.g., acid or base), enzymatic treatment, and combinations thereof. Deparaffinization can be achieved by chemical treatment of the sample, heat treatment of the sample, enzymatic treatment of the sample, or other methods. For example, deparaffinization can be achieved at elevated temperatures (e.g., about 50°C to about 80°C) in the presence of a neutral buffer or a slightly acidic buffer (e.g., pH reduced to about 5.5), or a slightly basic (up to about pH 9) or alkaline solution (e.g., pH about 12 to about 13). Removal of the embedding material can be off-device or on-device. In one example, the embedded sample can be incubated at elevated temperatures in a container and then loaded into a fluidic device. In another example, the embedded sample can be loaded into a fluidic device and incubated on the device, for example in a channel or reservoir, at an elevated temperature.

[0187] The embedding material can be removed by heat treatment. Incubation for removing the embedding material can be performed at a temperature of at least about 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 96°C, 97°C, 98°C, 99°C, 99.5°C, or 100°C. Incubation for removing the embedding material can be performed at a temperature of about 40°C to about 80°C, about 50°C to about 80°C, about 50°C to about 99.9°C, or about 95 to about 99.5°C. Incubation to remove the embedding material can be carried out for at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, or 120 minutes. Incubation to remove the embedding material can be carried out for about 1 minute to about 20 minutes, about 1 minute to about 30 minutes, about 1 minute to about 60 minutes, about 1 minute to about 120 minutes, or about 5 minutes to about 20 minutes. Incubation to remove the embedding material can be carried out, for example, for at least about 1 minute at a temperature of at least about 37°C. Incubation to remove the embedding material can be performed in the presence of an alkaline or neutral buffer (e.g., a lysis buffer). The alkaline buffer (e.g., a lysis buffer) can have a pH of at least about 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, or 13.5. The neutral buffer can have a pH of about 7.0 (e.g., about 7 to about 8).

[0188] Tissues or cells can be disrupted or lysed to release nucleic acids for separation, purification, or extraction. Tissue disruption or cell lysis can be achieved using techniques including, but not limited to, mechanical stress, sonication, electroporation, osmotic pressure, chemical treatment (e.g., acid or base), enzymatic treatment, heat treatment, and combinations thereof. For example, pressure can be used to mechanically disrupt tissue or drive tissue through structures (e.g., channels, resins such as frits or porous resins, or glass materials) to lyse cells. In some cases, the final electrolyte buffer can contain one or more tissue disrupting agents and / or cell lysing agents. In some cases, the leading electrolyte buffer can contain one or more tissue disrupting agents and / or cell lysing agents. In some cases, removal of the embedding material can be achieved by the same treatment as tissue disruption or cell lysis. For example, removal of the embedding material, tissue disruption, and cell lysis can be achieved by incubation at high 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). Tissue disruption or cell lysis can be performed off-device or on-device. In one example, the tissue sample is disrupted in a container and subsequently loaded into the fluidic device. In another example, a tissue sample previously loaded into the fluidic device is disrupted in the device.

[0189] Samples containing tissues or cells can be lysed before or after loading into a fluidic device using a lysis solution or buffer compatible with isotachophoresis. Lysis buffers compatible with isotachophoresis can include non-ionic surfactants or detergents, ionic or zwitterionic surfactants or detergents, chaotropic agents, disulfide bond reducing agents, proteases, nucleases, and other additives or components that digest, denature, disrupt, or degrade nucleic acids for extraction, purification, enrichment, or other isolation. In some cases, the lysis buffer may include an alkaline buffer. In some cases, the lysis buffer may be free of an alkaline buffer. Exemplary lysis buffers can include 0.5M-9.5M, 4M-9M, or 6.5M-7M urea, as described herein. Exemplary lysis buffers can include 0.5M-3.5M, or 1.5M-2.5M thiourea, as described herein. An exemplary lysis buffer can include 0.5-9.5 M urea and thiourea, e.g., 7 M urea and 2 M thiourea, along with a nonionic detergent as described herein. Urea can be used alone or in combination with thiourea to lyse cells for nucleic acid purification. When combined, urea and thiourea can act synergistically to lyse cells and provide an uncharged, isotachophoresis-compatible buffer for nucleic acid purification.

[0190] An exemplary lysis buffer can include a non-ionic surfactant, such as 0.05-0.5% v / v IGEPAL CA-630, as described herein. In some cases, the lysis buffer can include one or more terminal electrolytes. In some cases, the lysis buffer can include a terminal electrolyte buffer containing a tissue disruption or cell lysis additive described herein. In some cases, the lysis buffer can include one or more leading electrolytes. In some cases, the lysis buffer can include a leading electrolyte buffer containing a tissue disruption or cell lysis additive described herein. In some cases, the lysis buffer can include one or more leading electrolytes and one or more terminal electrolytes. In some cases, the lysis buffer can include one or more leading electrolytes and one or more terminal electrolytes containing a tissue disruption or cell lysis additive described herein.

[0191] In some cases, the methods or processes herein may include lysing cells or tissue samples using a lysis buffer that minimizes mechanical disruption of DNA and / or RNA during the lysis reaction. For example, cells or tissues may be lysed in a buffer solution containing HCl (e.g., 1 mM, 5 mM, 10 mM HCl) and Tris (e.g., 5 mM, 10 mM, 20 mM, 30 mM Tris), including a non-ionic detergent. The non-ionic detergent (e.g., IGEPAL CA-630) may be present in the lysis buffer at about 1%, about 2%, about 3%, about 4%, or more, or less than about 1%. Cells or tissues may be lysed in the lysis buffer by gentle mixing, such as by inversion and low speed (automated pipette). Enzymes such as proteinase K may be included in the lysate or lysis buffer in some cases. In some cases, lysis is performed without centrifugation. In some cases, centrifugation is used in the lysis method. The lysate can be introduced into an isotachophoresis device to purify desired analytes, such as high molecular weight DNA fragments.

[0192] Proteins in a sample can be digested, for example, by enzymatic digestion with a protease. Proteases include, but are not limited to, proteinase K, proteases, endoproteinases (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Other protein or polynucleotide digesting or degrading agents, such as lysozyme, can be used. Protein digestion can remove crosslinked proteins from crosslinked nucleic acids, converting them to uncrosslinked nucleic acids. Protein digestion can be performed at room temperature or at elevated temperatures (e.g., greater than about 25°C) as described herein.

[0193] The sample can be processed in a device (e.g., an electrokinetic device or system with at least one reservoir connected to at least one channel) such that the sample volume passes through the reservoir and into the channel, and less than 20% of the sample volume remains in the reservoir, and then an ionic current can be applied to the sample volume in the channel. The ionic current can be substantially absent from 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.

[0194] The sample can be processed in a device (e.g., an electrokinetic device or system with at least one reservoir connected to at least one channel) such that the sample volume passing through the reservoir and entering the channel is at least 50% of the sample volume loaded into the reservoir, and then an ionic current can be applied to 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 is transferred from the reservoir to the channel. In some examples, the total volume loaded into the reservoir is less than or equal to the internal volume of the reservoir. The ionic current can substantially not pass through the channel. In some cases, applying the ionic current comprises performing isotachophoresis.

[0195] isotachophoresis device

[0196] Isotachophoresis and / or sample preparation (e.g., deparaffinization, digestion, lysis) can be performed in a fluidic device, such as a microfluidic chip. For example, FIG. 5A shows a schematic diagram of a channel having a sample preparation (e.g., deparaffinization) zone 500 with a sample inlet 501 and a final 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 be provided between the sample fluid and the leading electrolyte buffer prior to application of a voltage. A capillary barrier can be provided between the sample preparation zone 500 and the final electrolyte reservoir 502 to limit, reduce, or prevent mixing or pressure-driven flow of the sample fluid and the final electrolyte buffer. A capillary barrier can be provided between the purification zone 510 and the leading electrolyte reservoir 511 to limit, reduce, or prevent mixing or pressure-driven flow of the contents of zone 510 and the leading electrolyte reservoir 511. In another example, deparaffinization can be performed off-chip initially or can be unnecessary depending on the starting material, in which case the channel can include a lysis and digestion zone (e.g., pH 7, 56°C) and a decrosslinking and purification (e.g., isotachophoresis) zone (e.g., pH 7, 80°C). In another example, deparaffinization can be performed off-chip initially or can be unnecessary depending on the starting material, in which case the channel can include a lysis and digestion zone (e.g., pH 7, temperature T1) and a decrosslinking and / or purification (e.g., isotachophoresis) zone (e.g., pH 7, temperature T2). In another example, deparaffinization can be performed off-chip initially or can be unnecessary depending on the starting material, in which case the channel can include a disruption and / or lysis zone (e.g., pH 7, temperature T1) and a digestion and / or purification (e.g., isotachophoresis) zone (e.g., pH 7, temperature T2).In another example, deparaffinization can be performed off-chip initially or can be unnecessary depending on the starting material, in which case the channel can include a disruption and / or lysis zone (e.g., pH 7, temperature T1) and an isothermal enzyme amplification zone (e.g., pH 7, temperature T2). In another example, deparaffinization can be performed off-chip initially or can be unnecessary depending on the starting material, in which case the channel can include a disruption and / or lysis zone (e.g., pH 7, temperature T1) and an isothermal enzyme digestion zone (e.g., pH 7, temperature T2). In some cases, the channel can include three zones, for example, a disruption and / or lysis zone (e.g., pH 7 and temperature T1), an isothermal enzyme amplification zone (e.g., pH 7, temperature T2), and a purification (e.g., isotachophoresis) zone (e.g., pH 7 and temperature T3). Figure 5B shows an exemplary fluidic device cartridge, each containing eight parallel channels shown in Figure 5A. Figure 5C shows a schematic top view of the fluidic device shown in Figure 5B, while Figures 5D and 5E show side and end views, respectively. The device can include a sample inlet or reservoir 530, a reservoir 531 for ITP electrolyte buffer, and a sample elution outlet or reservoir 532. The channels and / or reservoirs may be connected to one or more pneumatic ports. Each of the eight parallel channels of the fluidic device can be operated independently of each of the other channels. In some cases, each channel has a dedicated set of electrodes and electrical circuitry for driving the ITP. Electrodes may be positioned, for example, in the final electrolyte reservoir 502 and the leading electrolyte reservoir 511, so that the electrodes do not directly contact the sample material.

[0197] In some cases, there is little or no fluid or ion flow between the parallel channels. In some cases, the parallel channels may not be in fluid communication with each other. The fluid leak rate between the 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 per hour.

[0198] In some examples, the parallel channels are electrically isolated from one another, with little or no electrical communication between them. Each of the parallel channels can be independently electrically controlled to apply an independent electric field to each of the channels. In some examples, the leakage current between the channels is less than about 0.1 microamperes (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 examples, the impedance between the channels can be greater than 0.1 megaohms (MOhm), 0.2 MOhm, 0.3 MOhm, 0.4 MOhm, 0.5 MOhm, 0.6 MOhm, 0.7 MOhm, 0.8 MOhm, 0.9 MOhm, 1 MOhm, 5 MOhm, 10 MOhm, 20 MOhm, 30 MOhm, 40 MOhm, or 50 MOhm.

[0199] In some cases, each zone in an isokinetic fluidic device can be heated. In some cases, the zones are heated to the same temperature. In some cases, individual zones are heated to different temperatures. In some cases, a first zone can be heated to a temperature greater than 37°C, for example, within a range of about 60°C to about 100°C. In some cases, a second zone can be heated to a temperature greater than 37°C, for example, within a range of about 40°C to about 60°C.

[0200] Isotachophoresis fluidic devices can contain one or more reservoirs, including, but not limited to, a buffer loading reservoir, a sample loading reservoir (including reservoirs containing solids, multiphase or other heterogeneous liquids, or solutions such as tissue, whole blood, or unlysed cell suspensions), a leading electrolyte reservoir, a final electrolyte reservoir, a reagent reservoir, an elution reservoir (e.g., for removing processed samples), and a gas or air reservoir. In some cases, a single physical reservoir can be used for multiple purposes, such as buffer loading and sample loading. Liquid or air reservoirs can be used to apply external pressure (e.g., positive pressure to liquid wells or vacuum to gas-only reservoirs) to load the liquid.

[0201] The reservoir can be in thermal communication with a heat or cold source, allowing for control of the temperature of the reservoir and any material therein (e.g., reagents, samples, products). For example, an elution reservoir can be thermally controlled to control the temperature of the eluted product while it is in the fluidic device (to preserve its structural integrity).

[0202] A reagent reservoir can be used to load one or more reagents for processing the sample before, during, or after isotachophoresis. Reagents can include digestion reagents, amplification reagents, reverse transcription reagents, linear polymer solutions, probes for hybridization reactions, ligation reagents, dyes, tracers, labels, and other reagents for size separation. The reagent reservoir can be connected to a reaction channel or a reaction zone of another channel where a reaction can occur. Heat or cooling can be applied (e.g., using a thermal control device discussed herein) to catalyze reactions (such as enzymatic reactions using nucleic acids or proteins), hybridize or dissolve nucleic acids, or remove intercalating dyes from nucleic acids (e.g., before elution). Heating and cooling can also be used to control a fixed operating temperature for ITP (e.g., cooling can 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 stable storage of purified nucleic acids. Light can be applied (eg, using the light sources discussed herein) for purposes including optical interrogation, fluorescence excitation, and reaction energy or catalysis.

[0203] Gas or air reservoirs or gas or air outlets may be connected via gas channels to liquid channels within the fluidic device to allow for purging air or other gases from the fluidic device (e.g., while filling the fluidic device with liquid). Gas or air reservoirs or pneumatic ports may be connected via gas channels to liquid channels to allow for pumping of fluids on or within the fluidic device (e.g., to pump fluid from a reservoir into a channel).

[0204] The device can include multiple purification (e.g., isotachophoresis) zones connected to one another. For example, a second isotachophoresis zone can be separated from and connected in parallel with a first isotachophoresis zone, allowing for separation of sample bands at a specific ratio (e.g., based on the current ratio between the two zones) for parallel processing.

[0205] A fluidic device can include multiple purification zones in parallel (e.g., see FIG. 5C). For example, a fluidic device can include more than one set of purification zones, each with an associated reservoir, inlet, outlet, channel, and any other components described herein (e.g., sample preparation zone, electrode, heater, detector), in parallel and separate from one another, each capable of independently processing a sample. A fluidic device can include 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 purification zones in parallel. A fluidic device can include multiple channels in parallel. A fluidic device can include 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 channels in parallel. Components such as purification zones or channels arranged in parallel can be arranged side-by-side in different device layers (e.g., horizontal or vertical layers) or can be arranged in different arrangements. Parallel components can be identical or designed differently but function equivalently or nearly equivalently. For example, parallel channels can have different shapes to allow for a smaller overall fluidic device footprint, but still function similarly. Alternatively, parallel components can be designed to function differently, for example, to process different types of samples in parallel or to perform different operations on samples. In some cases, parallel components can be designed to perform different operations in parallel on different sample types. In some cases, parallel components can be designed to perform different operations in parallel on the same sample type. In some cases, parallel components can be designed to perform the same operation in parallel on different sample types. In some cases, parallel components can be designed to perform the same operation in parallel on the same sample type. In some cases, parallel components can be designed to perform one or more operations simultaneously and / or independently on two or more samples in parallel.In some cases, the leak rate between two or more channels (or between two or more purification zones) is less than 0.5 μl / hr, less than 1 μl / hr, less than 5 μl / hr, or less than 10 μl / hr. In some embodiments, the amount of leak current between two or more channels (or between two or more purification zones) is 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 zones is greater than 0.5 megaohms, greater than 1 megaohm, greater than 5 megaohms, or greater than 10 megaohms.

[0206] As discussed herein, fluidic devices can be designed to process different sample volumes. For example, Figures 6A, 6B, 6C, and 6D show top, side, bottom, and top perspective views, respectively, of a rapid purification ITP fluidic device 600 for a sample volume greater than or equal to approximately 200 μL. The device includes a channel 600 connected to a sample input well 601, an ITP buffer well 602, and a sample output (elution) well 603. The ITP buffer well 602 can include an elution buffer reservoir 605, a leading electrolyte reservoir 606, a leading electrolyte buffer reservoir 607, and a final electrolyte reservoir 608. The elution reservoir 603 may be connected to the elution buffer reservoir 605 by an elution buffer channel 609. A capillary barrier 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. Leading electrolyte reservoir 606 may be connected to leading electrolyte buffer reservoir 607 by leading electrolyte buffer channel 610. A capillary barrier may be provided in leading electrolyte buffer channel 610 to reduce or prevent mixing or pressure-driven flow between the contents of leading electrolyte buffer reservoir 607 and leading electrolyte reservoir 606. Buffer reservoir 605 may contain an elution buffer electrolyte of higher ionic strength than that in elution reservoir 603, while buffer reservoir 607 may contain a leading electrolyte of higher ionic strength than that in leading electrolyte reservoir 606. The device may further include a pneumatic port 604 along its edge configured to connect to a pneumatic device, for example, a vacuum source in a benchtop instrument. Pneumatic port 604 may be connected to channel 600 and the reservoir by a gas channel as described herein. Applying suction to pneumatic port 604 allows sample, leading electrolyte, and elution buffer to be loaded into channel 600. In some cases, the final electrolyte buffer fluid remains in final electrolyte reservoir 608.Suction can be applied simultaneously or sequentially to pneumatic ports 604 to simultaneously or stepwise load channel 600, respectively. The sample can be loaded into channel 600 in a first zone or subchannel of channel 600 extending from final electrolyte reservoir 608 to capillary barrier 611 at a small divergent turn of 180°. A capillary barrier 611 may be provided at the interface between the sample and leading electrolyte buffer during loading to limit, reduce, or prevent mixing or pressure-driven flow. A capillary barrier may be provided between final electrolyte reservoir 608 and the first zone or subchannel to limit, reduce, or prevent mixing or pressure-driven flow between the contents of final electrolyte reservoir 608 and the sample. The leading electrolyte can be loaded into a second zone or subchannel of channel 600 extending from capillary barrier 611 to capillary barrier 612. A capillary barrier 612 may be provided at the interface between the leading electrolyte buffer and the elution buffer. The elution buffer may be loaded into a third zone, or subchannel, of the channel 600 extending from the capillary barrier 612 to the elution reservoir 603. In some embodiments, the ITP buffer well 602 may further include a final electrolyte buffer reservoir (not shown) containing a final electrolyte of higher ionic strength than that in the final electrolyte reservoir 608. The final electrolyte buffer reservoir may be connected to the final electrolyte reservoir 608 by a final electrolyte buffer channel (not shown). The final electrolyte buffer channel may include a capillary barrier to restrict, reduce, or prevent mixing or pressure-driven flow between the contents of the final electrolyte buffer reservoir and the contents of the final electrolyte reservoir 608.

[0207] The electrodes may be positioned, for example, in the final electrolyte reservoir 608, the final electrolyte buffer reservoir (not shown), the leading electrolyte reservoir 606, and / or the leading electrolyte buffer reservoir 607, so that the electrodes do not directly contact the sample material. The electrodes may be actuated to modify or control the applied electric field in response to feedback from a sensor, such as a voltage, current, conductivity, or temperature sensor described herein. For example, the passage of nucleic acid in the ITP zone from the second zone of the channel 600 to the third zone of the channel 600 may be detected, and feedback from the detector may be actuated to vary the applied current. The current may be increased, decreased, or terminated, for example, according to the instrument's protocol. The current may be paused (e.g., temporarily reduced to zero) to allow on-chip quantitation of the nucleic acid. Alternatively, or in combination, the current may be reduced to slow down the isotachophoresis in the third zone, allowing the nucleic acids that can disperse to become more concentrated before reaching the elution well 603 as the time passes from the leading electrolyte buffer to the elution buffer (or second leading electrolyte buffer).

[0208] The methods and processes provided herein include methods and processes using any of the devices provided herein. The devices provided herein, which have multiple channels for processing multiple samples in parallel, can be used in a variety of situations. In some cases, the method can include using the device to process multiple samples that share certain characteristics (e.g., solid tissue lysate, cell lysate, solid tissue, fixed tissue) (e.g., by performing isotachophoresis on such samples). In some cases, the multiple samples can be different samples. For example, the method can include performing isotachophoresis on a tissue sample in one zone of the device, and simultaneously but independently performing isotachophoresis on various samples, such as a cell sample or a sample containing crosslinked nucleic acids.

[0209] In some cases, the methods or multiplexing processes provided herein can include performing isotachophoresis on a sample in a channel in parallel with performing isotachophoresis on a second sample in a second channel using the same or similar leading electrolyte and / or final electrolyte buffers. In some cases, a sample in one of the channels is processed using a first leading electrolyte buffer, and samples in a different channel are processed using a second leading electrolyte buffer that is different from the first leading electrolyte buffer. For example, the first leading electrolyte buffer can contain one or more leading electrolyte ions that are different from those contained in the second leading electrolyte buffer. In another example, the first leading electrolyte buffer can contain the same one or more leading electrolyte ions as those contained in the second leading electrolyte buffer, but the concentration of such leading electrolyte ions in the first leading electrolyte buffer is different from the concentration of such ions in the second leading electrolyte buffer. In some cases, the methods or processes provided herein may include performing isotachophoresis on a sample in one channel in parallel with performing isotachophoresis on a second sample in a second channel using the same or similar final electrolyte or final electrolyte buffer. In some cases, the sample in one channel is processed using a first final electrolyte buffer, and the samples in the different channels are processed using a second final electrolyte buffer that is different from the first final electrolyte buffer. For example, the first final electrolyte buffer may contain one or more final electrolyte ions that are different from those contained in the second final electrolyte buffer. In another example, the first final electrolyte buffer may contain the same one or more final electrolyte ions as those contained in the second final electrolyte buffer, with the concentration of such final electrolyte ions in the first final electrolyte buffer being different from the concentration in the second final electrolyte buffer.

[0210] In some embodiments, one or more reservoirs may be connected to two channels or subchannels. For example, elution reservoir 603 may be connected to both channel 600 and elution buffer channel 609. Alternatively, or in combination, leading electrolyte reservoir 606 may be connected to both channel 600 and leading electrolyte buffer channel 610. Alternatively, or in combination, final electrolyte reservoir 608 may be connected to both 600 and final electrolyte buffer channel 610. Alternatively, or in combination, sample input well 601 may be connected to the midpoint of channel 600 such that channel 600 extends to the left (as a first subchannel) and right (as a second subchannel) of input well 601. The two channels or subchannels may be connected to one or more reservoirs at an angle between the two channels (extending on a major surface of the fluidic device) of 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°. The two channels or subchannels may be connected to one or more reservoirs at an angle between the two channels (extending on a major face of the fluidic device) of at most about 5°, 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 135°, 140°, 150°, 160°, 170° or 180°.

[0211] The device can include, for example, eight channels as shown. Each channel can hold a sample volume of about 50 μL to about 275 μL, and a total volume of about 500 μL. The small dispersion angle of 180° in each channel can facilitate such large sample volumes in an eight-channel multichannel plate with a standard SLAS footprint.

[0212] 7A, 7B, 7C, and 7D show top, side, bottom, and bottom oblique views, respectively, of a rapid purification ITP fluidic device 700 for a sample volume less than or equal to about 100 μL. The device includes a sample input well 701, an ITP buffer well 702, and a sample output (elution) well 703. Device 700 can be substantially similar to device 600, although with various channel geometries (and corresponding reservoir geometries) that do not include 180° turns in the channels.

[0213] The device can include, for example, eight channels as shown. Each channel can hold a sample volume of about 10 μL to about 100 μL. Devices with smaller sample volumes can be useful for PCR cleanup or other reaction cleanup applications, or for smaller sample sizes (e.g., samples with low cell counts or small amounts of tissue).

[0214] 8A, 8B, 8C, and 8D show top, side, bottom, and oblique views, respectively, of another rapid purification ITP fluidic device 800 for sample volumes less than or equal to about 100 μL. The device includes a sample input well 801, an ITP buffer well 802, and a sample output (elution) well 803. Device 800 can be substantially similar to devices 600 and 700, although it includes multiple different channel geometries on a single chip.

[0215] A fluidic device can include one or more electrodes that apply an electric field to the fluidic device or a portion of the fluidic device. The applied electric field can be used to perform isotachophoresis. A fluidic device can include one or more electrodes that apply a single electric field to all channels of the fluidic device. A fluidic device can include one or more electrodes that apply more than one electric field to the fluidic device, for example, one electric field per channel of the device. In some examples, the first and second electric fields are generated from a single electrode pair. In some examples, the first and second electric fields are generated from different electrode pairs. The electric fields can be applied simultaneously, sequentially, and / or independently or mutually. The electrodes can be external, such as wires encased in a reservoir. The electrodes can be internal, such as microfabricated, printed, or other embedded elements included in the fabrication of a fluidic device. Electrode materials can include, but are not limited to, metals (e.g., platinum, titanium) and carbon.

[0216] One or more electrodes of a fluidic device may be part of one or more electrical circuits that apply an electric field to the fluidic device or a portion of the fluidic device. The fluidic device may include one or more electrical circuits that apply a single electric field to all channels of the fluidic device or the isotachoporesis region of that zone. The fluidic device may include one or more electrical circuits that apply more than one electric field to the fluidic device, for example, one electric field per channel of the device. In some examples, the first and second electric fields can be generated from a single electrical circuit. In some examples, the first and second electric fields can be generated from different electrical circuits. The electric fields can be applied simultaneously, sequentially, and / or independently or mutually by one or more electrical circuits. In some examples, the device (or benchtop instrument) can be configured to control a first electrical circuit simultaneously and independently from a second electrical circuit.

[0217] The electrodes can be positioned in reservoirs, such as a final electrolyte reservoir and a leading electrolyte reservoir, which can be separated from the sample reservoir by a buffer channel. In some cases, the electrodes are located in the buffer channel or buffer reservoir. The location of the electrodes in the electrolyte reservoir or electrolyte buffer reservoir can isolate the electrodes from analytes, such as nucleic acids, to reduce or eliminate contamination of the electrodes by sample materials. This approach allows for reuse of electrodes without cross-contamination between samples. In one example, the final electrolyte reservoir or final electrolyte channel is connected by a buffer channel to a buffer reservoir containing final electrolyte ions and electrodes, which is also connected to a sample reservoir or sample channel, which is in turn connected to the leading electrolyte reservoir by a leading electrolyte channel. The leading electrolyte reservoir is also connected by a buffer channel to a buffer reservoir, which also contains a leading electrolyte and electrodes. In another example, or continuing from the previous example, an elution reservoir containing an elution buffer is connected by an elution channel to a leading electrolyte reservoir, which is in turn connected to a buffer reservoir containing an elution buffer electrolyte and electrodes. The buffer channel between a buffer reservoir and its corresponding reservoir can include capillary barriers and / or small cross-sectional areas to restrict, reduce, or prevent mixing and pressure-driven flow, as described herein. A buffer reservoir can contain electrolytes of the same or higher ionic strength as its corresponding reservoir. For example, an elution reservoir can be connected to a buffer reservoir containing elution buffer electrolytes of the same or higher ionic strength or concentration as the elution reservoir. A final electrolyte reservoir can be connected to a buffer reservoir containing final electrolytes of the same or higher ionic strength or concentration as the final electrolyte reservoir.The leading electrolyte reservoir may be connected to a buffer reservoir containing a leading electrolyte of the same ionic strength or concentration as the leading electrolyte reservoir, or of a higher ionic strength or concentration. If the buffer reservoir is dedicated to connecting to the elution reservoir, the final electrolyte reservoir and / or the leading electrolyte reservoir with a higher ionic strength can provide additional ion reservoirs to maintain the pH and conductivity in the channel as the sample moves through the channel.

[0218] Fluidic devices can be used with one or more thermal control devices. For example, FIG. 9A shows a schematic diagram of an octaplex sample preparation and isotachophoresis device including eight parallel channels 900 of the design shown in FIG. 5A. FIG. 9B shows a schematic diagram of first and second thermal control devices 901, 902. A first thermal control device 901 at temperature T1 (e.g., 80°C) is aligned with the sample preparation zone of the channel, and a second thermal control device 902 at temperature T2 (e.g., 50°C) is aligned with the isotachophoresis zone of the channel. In some cases, additional thermal control devices may be aligned with additional zones of the channel (not shown); for example, a third thermal control device at temperature T3 may be aligned with the third zone at temperature T3. In some cases, each zone of each channel can have its own individual thermal control device rather than sharing a common thermal control device with individual zones of other channels. In other cases, the zones or channels can all share one thermal control device. In other cases, more than one but fewer than the total number of zones or channels can share a single thermal control device. Thermal control devices can include components including, but not limited to, resistive heaters, fluid-based heating or cooling systems, and Pelletier devices. Thermal control devices can be fabricated from materials including, but not limited to, metals (e.g., platinum, titanium, copper, gold), carbon, and indium tin oxide (ITO). Thermal control devices can include temperature sensors that can be used to monitor the controlled temperature and provide temperature feedback for thermal control. Thermal control devices can be used with computer control systems, as discussed further in this disclosure. In some cases, the thermal control devices are operated without temperature feedback. Thermal control devices can be integrated into the fluidic device or located externally, such as in a benchtop system.

[0219] The fluidic device can be used with one or more light sources. The light source can be integrated into the fluidic device or can be located external to the fluidic device, such as in a benchtop system or in a separate device. The light source can provide light for optical interrogation, fluorescence excitation, temperature sensing, reaction energy or catalysis, and other purposes.

[0220] Fluidic devices can be designed so that their outermost framework or dimensions meet microtiter plate standards (e.g., SLAS microtiter plate standards). Fluidic devices can be designed to use defined ports of a microtiter plate (e.g., a SLAS standard microtiter plate) as liquid reservoirs, with pneumatic actuation ports located on the outer, unused surface of the liquid reservoirs. Pneumatic ports can be arranged on the edge of a fluidic device with a microtiter plate-compatible layout to avoid cross-contamination due to pneumatic actuation on the liquid reservoirs and to allow easy access to pneumatic hardware. Defined secondary ports can also be used for pneumatic actuation in addition to their other functions. In some cases, the fluidic device can be designed and manufactured into two interlocking parts: a channel unit (e.g., a layer with a flat surface that allows for easy film bonding), a first insertion port containing wells and pneumatic ports, and a second outer ring that conforms to microtiter plate standards (e.g., SLAS microtiter plate dimensional standards), including an alignment mechanism for aligning the fluidic device to a benchtop system and a mating mechanism that interlocks with the first part. The wells can be connected to form protrusions that can be compatible with injection molding.

[0221] Fluidic devices can be made from a variety of materials, including, but not limited to, glass (e.g., borosilicate glass), silicon, plastics, and elastomers. Plastics can include polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polyethylene, polyethylene terephthalate (PET), high density polyethylene (HDPE), and low density polyethylene (LDPE). Elastomers can include polydimethylsiloxane (PDMS).

[0222] Materials used in the fabrication of fluidic devices can be selected for their optical properties. For example, materials can be used that exhibit low autofluorescence, low scattering, and high transmittance at wavelengths of interest (e.g., excitation and emission wavelengths for nucleic acid labels or dyes). A variety of materials can be used in a single fluidic device. For example, the detection region can be fabricated using a material that exhibits useful optical properties, while other regions of the device can comprise other materials.

[0223] Materials for use in fabricating fluidic devices can be selected for their thermal properties. For example, materials can be selected for their high thermal conductivity. Alternatively, materials can be selected for their low thermal conductivity (e.g., to insulate the fluidic device or a region of the fluidic device). A variety of materials can be used in a single fluidic device. For example, a heating region can have a material with a high thermal conductivity to improve thermal communication with a thermal control unit, but the heating region can be surrounded by a material with a low thermal conductivity to thermally isolate it from other regions of the device.

[0224] Materials used in fabricating fluidic devices or microchannels therein can be selected for their elastomeric or deformation properties. For example, materials can be selected for low elasticity to allow for channel closure by plasticity, as described herein. Alternatively, materials can be selected for high elasticity. A variety of materials can be used in a single fluidic device. For example, poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), etc. can be used in a single fluidic device. The material can have an elastic modulus of at least 1 GPa, 1.5 GPa, 2 GPa, 2.5 GPa, 3 GPa, 3.5 GPa, 4 GPa, 4.5 GPa, or 5 GPa. The material can have an elastic modulus of at most 1 GPa, 1.5 GPa, 2 GPa, 2.5 GPa, 3 GPa, 3.5 GPa, 4 GPa, 4.5 GPa, or 5 GPa. The material may have a tensile strength of at least 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa, 100 MPa, 110 MPa, 120 MPa, 130 MPa, 140 MPa, 150 MPa, 160 MPa, 170 MPa, 180 MPa, 190 MPa, 200 MPa. The material may have a tensile strength of at most 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa, 100 MPa, 110 MPa, 120 MPa, 130 MPa, 140 MPa, 150 MPa, 160 MPa, 170 MPa, 180 MPa, 190 MPa, 200 MPa.

[0225] In some cases, the surfaces of a fluidic device can be used without a surface treatment or coating. In other cases, the surfaces of a fluidic device can be used with a hydrophobic treatment, a hydrophilic treatment, or a surface coating such as a selective binding agent (e.g., an antibody). Different regions of a fluidic device can include different surface treatments (or the absence thereof). For example, some channels, reservoirs, or portions thereof can be hydrophobic, while others are hydrophilic.

[0226] Fluidic devices can include a wide range of flow control units and techniques, including but not limited to capillary barriers, reservoirs for air evacuation, gas / air lines, fill level monitors (e.g., by electrode measurements), specific reservoir shapes, specific fluidic resistances of channels, and fluid loading sequences.

[0227] The capillary barrier can be paired with an air evacuation reservoir for purging air (e.g., to prevent bubbles), thereby positioning and successfully establishing liquid-liquid interfaces between (i) the leading and final electrolyte solutions required for isotachophoresis, and (ii) between the buffer reservoir and the leading or final electrolyte solution and / or sample solution. The capillary barrier can be designed in a preferred order in combination with the channel shape to automate channel filling. The channel resistance can be selected, for example, by designing the channel dimensions, to create differences in fluid resistance. The liquid loading order allows for the precise formation of liquid-liquid interfaces without bubbles for electrokinetic processes. In one example, the final ion reservoir is directly connected to the analyte or sample channel.

[0228] Gas (e.g., air) channels or lines can be used to provide pneumatic actuation to capillary barriers or other regions of a fluidic device. Gas channels can be connected to an external gas pressure source via a pneumatic port. Gas channels can have higher fluidic resistance than the liquid channels they supply pressure to, for example, reduce or prevent liquid flow into the gas channel. For example, a gas channel can have less than half the cross-sectional area of ​​the isotachophoresis channel body. Multiple gas channels can be connected to a single gas reservoir or port (e.g., with a branching channel). A capillary valve with a branching air line can be used to prevent upstream liquid migration. Figure 10A shows an exemplary gas channel 1001, which can include a capillary barrier 1002 connected to the liquid channel interface 1003 between the sample 1004 and leading electrolyte buffer 1005 subchannels. FIG. 10B is an enlarged schematic view of gas channel 1001 highlighting capillary barrier 1002 that prevents upstream liquid migration toward pneumatic port 1006.

[0229] To load the fluid channels, negative pressure or vacuum can be applied to the gas channels through the gas ports. The fluid channels of a microfluidic device can be loaded simultaneously or independently (e.g., sequentially). Within a channel, fluids can be loaded simultaneously or independently. For example, before, simultaneously with, or after loading the sample, a leading electrolyte buffer, a high-concentration leading electrolyte buffer, a final electrolyte buffer, a high-concentration final electrolyte buffer, an elution buffer, a high-concentration elution buffer, or any combination thereof can be loaded. For example, negative pressure can be applied to a gas port at one end of the chip to load one or more fluids (e.g., a final electrolyte buffer, an elution buffer, etc.). Subsequently, negative pressure can be applied to a gas port at the other end of the chip to load additional fluids (e.g., a leading electrolyte buffer). Alternatively, negative pressure can be applied simultaneously to all of the gas ports connected to a channel. The sample can be loaded by applying negative pressure or vacuum before, during, or after loading the isotachophoresis buffer. The sample can be loaded without applying negative pressure or vacuum, for example, by wetting or by gravity.

[0230] Sensors (e.g., electrodes) can be used to detect liquid fill levels or air bubbles (e.g., via current or voltage sensing) and provide feedback. Geometric features (e.g., occlusion, expansion, or tortuosity) can be used in combination with electrodes to monitor channel impedance and, therefore, the time progression of isotachophoresis. For example, during ITP, nucleic acids collect, and voltage can be used to track the position of the collected band in the channel from start to finish. In one example, monitoring the expansion of fluid from a connecting channel with a smaller cross-sectional area into a reservoir (such as an elution reservoir) can be used to determine the time at which the analyte elutes, allowing for automated elution and process termination control. In another example, channel occlusions can be designed to allow for detection of the timing (or actuation) of steps in an electrokinetic process, such as when an analyte of interest enters a channel zone where a reaction or optical detection event occurs, allowing for control of the reaction timing or detection actuation.

[0231] Reservoirs and channel features can be designed to control or prevent pressure-driven flow. For example, reservoirs (e.g., sample and elution reservoirs) can have internal geometries designed so that large changes in liquid height result in only small variations in internal volume at the intended head height, as shown in Figure 11. This allows for more precise control of the volume of liquid contained in the reservoir. For other reservoirs, the liquid volume can be varied without disrupting the separation process. Such reservoirs can be designed to have large volume changes in response to small liquid height changes, which can help stabilize the liquid height throughout the fluidic device. Low fluidic resistance between reservoirs can be used to allow rapid head pressure equilibration times and minimize liquid flow in the channel before, during, or after the electrokinetic process.

[0232] Reservoirs can be designed to minimize evaporation by, for example, controlling the surface area within the reservoir to maintain a constant or fixed volume. Reservoirs can be designed to maximize liquid recovery from the reservoir, for example, by using angled wall designs designed to minimize dead zones. Reservoirs can be designed to prevent liquid flow when connecting channels to the reservoir during withdrawal, which can help maintain the purity or separation of materials (e.g., nucleic acids) being withdrawn. Reservoirs can be designed for easy loading or withdrawal by pipetting, for example, by having appropriate dimensions to accommodate pipette tips or by having volumes within the range of typical pipetting. For example, an elution reservoir can be configured to accommodate pipette tips for extraction of nucleic acids. Reservoirs can be designed or spaced (e.g., with an approximately 9 mm pitch) to accommodate multichannel pipettors.

[0233] Reservoirs (e.g., sample reservoirs) can be located directly above the channels to be filled, thereby minimizing liquid loss in the connecting channels between the reservoirs and the channels that fill them. Reservoirs (e.g., sample reservoirs) can have conical bottoms and cylindrical through-holes. The large inner diameter at the top of such reservoirs allows the liquid meniscus at the bottom of the reservoir to have a smaller inner diameter while maintaining a large volume, reducing the amount of liquid left behind after dispensing. Such designs can also reduce or prevent wicking of wetting fluid into concave corners. In some cases, the through-holes from the reservoirs (e.g., sample reservoirs) to the channels are less than or equal to about 2 millimeters (mm).

[0234] 11 shows a sample reservoir 1100 configured to reduce the amount of sample remaining in (or lost from) the reservoir 1100 after transferring the sample to a connecting channel 1101. The low-loss sample reservoir 1100 can reduce the amount of sample remaining in the reservoir 1100 after transferring the sample to a connected channel 1101 without adding or pumping additional volume (sample or other fluid) into the sample reservoir 1100 after or during sample delivery to the connected channel 1101. The low-loss sample reservoir 1100 can include an upper or top side 1102 having an inner hydraulic diameter D1 configured to contain a sample volume before loading the channel 1101, a lower or bottom side 1103 having an inner hydraulic diameter or through-hole D2 and a height H1 configured to contain the sample volume after loading the channel 1101, and a tapered or conical portion 1104 therebetween. In some cases, the upper portion 1102 and / or the lower portion 1103 are asymmetrical, in which case the dimension D1 to D2 may be the maximum dimension across the upper portion and / or the lower portion 1102, 1103, respectively.

[0235] The sample reservoir 1100 can be configured to create a residual sample head height H2 that is equal to or approximately equal to the buffer head height H3 in other reservoirs 1110 connected to the channel 1101 to restrict, prevent, or reduce pressure-driven flow and mixing in the channel 1101. A standard buffer reservoir 1110 can include an upper portion 1112 having an inner hydraulic diameter D3 and a lower portion 1113 having an inner hydraulic diameter D4. Unlike in a sample well, D3 can be substantially similar to D4 such that a larger volume of fluid is maintained in the buffer well 1110 compared to the sample well 1100 when the head heights H2 and H3 are equal or approximately equal.

[0236] The sample reservoir 1100 can be configured to hold a sample volume (with or without buffer) of 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, the sample reservoir 1100 can be configured to hold a sample volume in the range of about 1 nL to about 10 nL.

[0237] The hydraulic inner diameter D1 may be greater than the through-hole hydraulic diameter D2. The hydraulic inner diameter D1 of the upper portion can be at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm. The hydraulic inner diameter D1 of the upper portion can be at most about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm. The hydraulic inner diameter D2 of the lower portion can be at least about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm. The inner hydraulic diameter D2 of the lower portion may be at most about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0238] The ratio of D1 to D2 can determine the amount of sample remaining in the sample reservoir after the sample has been transferred to the channel. In some cases, the ratio of D1 to D2 is at least about 2:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1. In some cases, the ratio of D1 to D2 is at most about 2:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1. The ratio of D1 to D2 can be greater than 2:1 to facilitate transferring at least 50% of the sample volume from the low-loss sample reservoir 1100 to the channel 1101.

[0239] The cross-sectional area of ​​the upper portion is at least about 3 mm 2 , 5mm 2 , 10mm 2 , 15mm 2 , 20mm 2 , 25mm 2 , 30mm 2 , 35mm 2 , 40mm 2 , 45mm 2 , 50mm 2 , 55mm 2 , 60mm 2 , 65mm 2 , 70mm 2 , 75mm 2 The cross-sectional area of ​​the upper portion may be at most about 3 mm 2 , 5mm 2 , 10mm 2 , 15mm 2 , 20mm 2 , 25mm 2 , 30mm 2 , 35mm 2 , 40mm 2 , 45mm 2 , 50mm 2 , 55mm 2 , 60mm 2 , 65mm 2 , 70mm 2 , 75mm 2 The cross-sectional area of ​​the lower portion may be at least about 0.2 mm 2 , 0.3mm2 , 0.4mm 2 , 0.5mm 2 , 1mm 2 , 1.5mm 2 , 2mm 2 , 2.5mm 2 , 3mm 2 , 3.5mm 2 , 4mm 2 , 4.5mm 2 , 5mm 2 , 6mm 2 , 7mm 2 , 8mm 2 , 9mm 2 , 10mm 2 , 11mm 2 , 12mm 2 The cross-sectional area of ​​the lower portion may be at most about 0.2 mm 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 1mm 2 , 1.5mm 2 , 2mm 2 , 2.5mm 2 , 3mm 2 , 3.5mm 2 , 4mm 2 , 4.5mm 2 , 5mm 2 , 6mm 2 , 7mm 2 , 8mm 2 , 9mm 2 , 10mm 2 , 11mm 2 , 12mm 2 It can be said that:

[0240] The ratio of the cross-sectional area of ​​the upper portion to the cross-sectional area of ​​the lower portion can determine the amount of sample remaining in the sample reservoir after the sample has migrated into the channel. In some cases, the ratio of the cross-sectional area of ​​the upper portion to the cross-sectional area of ​​the lower portion is at least about 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1500:1, 2000:1, or 2500:1. In some cases, the ratio of the cross-sectional area of ​​the upper portion to the cross-sectional area of ​​the lower portion is at most about 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1500:1, 2000:1, or 2500:1.

[0241] The tapered section between the upper and lower sections can include an angle that promotes wetting of the sample into the lower section and transfer of the sample from the low-loss sample well to the channel. In some cases, the tapered section of the low-loss sample reservoir can include a half angle between the upper and lower sections that is less than about 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, or 90°. In some cases, the tapered section of the low-loss sample reservoir can include a half angle between the upper and lower sections that is greater than about 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, or 90°.

[0242] In some cases, the height H1 of the lower portion can be configured to produce a head height of the remaining sample equal to or approximately equal to the head height of the buffer in other reservoirs connected to the channel to limit, prevent, or reduce pressure-driven flow and mixing in the channel. The height H1 of the lower portion can be at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm. The height H2 of the lower portion can be at most about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm.

[0243] The reservoir (e.g., elution reservoir) can have a large diameter compared to the diffusion length scale of the analyte (e.g., nucleic acid) to reduce diffusion of the analyte out of the reservoir. In some cases, the length scale of the reservoir diameter can be on the order of millimeters, and the resulting diffusion time of the analyte out of the reservoir can be on the order of hours. The connections between the channel and the reservoir (e.g., elution reservoir) can be designed without sharp angles, thereby reducing the occurrence of high electric field regions at these connections and increasing the residence time of the analyte in the reservoir. In some cases, the cross section of the reservoir (e.g., elution reservoir) perpendicular to the electric field can be much larger than the cross section of the channel perpendicular to the electric field, thereby reducing the electric field strength at the reservoir and increasing the residence time of the analyte in the reservoir.

[0244] In some cases, the elution channel and / or elution reservoir can contain a second leading electrolyte buffer of a different type or concentration than the first leading electrolyte buffer used in the channel body. This allows the purified substance to be eluted in the second leading electrolyte buffer (e.g., elution buffer or discharge solution). The magnitude of the effective mobility of the second leading electrolyte ions in the second leading electrolyte buffer can be greater than the magnitude of the effective mobility of nucleic acids. The second leading electrolyte buffer can have a low ionic strength, e.g., an ionic strength compatible with downstream assays (e.g., qPCR, next-generation sequencing). In some cases, the second leading electrolyte buffer is identical to the first leading electrolyte buffer but exists at a different concentration or ionic strength (e.g., a lower ionic strength than that of the first leading electrolyte buffer). For example, the first leading electrolyte buffer can have an electrolyte ion concentration of 70-100 mM (e.g., 70-100 mM Tris HCl), while the second leading electrolyte buffer can have an electrolyte ion concentration of less than 70 mM, less than 60 mM, or less than 50 mM (e.g., less than 50 mM Tris HCl).

[0245] A channel of a fluidic device can be closed. For example, a mechanical actuator coupled to a mechanical member can be used to apply pressure to completely or partially close the channel (e.g., by deforming the channel). An elution reservoir can be blocked from the ITP channel to define a fixed elution volume. Channel closure can reduce flow or completely block flow. Channel closure can increase resistance to fluid flow. In some examples, channel closure can increase fluid resistance by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0246] FIG. 12A shows an exemplary mechanical member 1200 that can be used to apply pressure to close or at least partially close a channel 1201 of a fluidic device 1210, which includes multiple parallel channels (e.g., the device of FIG. 6C includes eight independent parallel channels). The mechanical member 1200 can include a comb-like structure with teeth 1202 aligned with two locations 1204, 1205 in each of the eight channels of the chip shown in FIG. 12B. Mechanical pressure can be applied by the teeth 1202 to permanently or plastically close or at least partially close the channel 1201 to restrict, reduce, or prevent fluid flow into or out of the elution reservoir 1203 and control the elution volume. At least partially closing the channel 1201 can increase resistance to fluid flow between the channel 1201 and the elution reservoir 1203. The mechanical member 1200 may be coupled to a mechanical driver by the teeth 1202 of the mechanical member 1200, which generates a force applied to the channel 1201. The mechanical member 1200 may comprise a material having a Young's modulus higher than that of the channel 1201. One or more teeth 1202 of the mechanical member 1200 may be configured to heat the channel 1201. One or more teeth 1202 of the mechanical member 1200 may be thermally coupled to a heater or a heating element. The mechanical member 1200 may optionally include a heater or a heating element. Heat can optionally be applied by the teeth 1202 to permanently or plastically close the channel 1201. The one or more teeth 1202 may be heated to a temperature higher than the glass transition temperature of at least one wall of the one or more channels 1201. 12C shows how 16 teeth 1202 of mechanical member 1200 align with 16 locations 1204, 1205 on tip 1210 (two per channel). Each tooth 1202 can be configured to apply mechanical pressure to channel 1201 to plastically deform at least one wall of channel 1201.Each channel 1201 is contacted by a mechanical member 1200 that plastically deforms in a first closed position 1204 and a second closed position 1205 to isolate the elution reservoir volume and increase fluid resistance between the channel 1201 and the reservoir 1203. In some examples, the teeth 1202 can apply mechanical pressure to a channel position 1204 upstream of the reservoir 1203. In some examples, the teeth 1202 can apply mechanical pressure to a junction 1205 where the reservoir 1203 and the channel 1201 meet to prevent fluid communication between the reservoir 1203 and the buffer reservoir.

[0247] In some cases, the mechanical member 1200 may include one tooth 1202 per channel that is aligned with the first closed position 1204. For example, the channel shown in FIG. 5A does not include a buffer channel or reservoir connected to an elution reservoir, and therefore may not require a second closed position 1205 after the elution reservoir. In some cases, the mechanical member 1200 is configured to close each of the channels 1201 on the chip 1210 in one or more positions. In some cases, the mechanical member 1200 is configured to leave one or more channels 1201 on the chip 1210 open, such that only a portion of the channels 1201 on the chip 1210 are closed.

[0248] The mechanical member 1200 can apply at least 0.25 lb of force per channel via the teeth 1202. Each tooth 1202 of the mechanical member 1200 can apply at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, or 5 lbs of force to the channel 1201.

[0249] A channel of a fluidic device (e.g., a sample preparation zone, an isotachophoresis zone) can have a width, height, or diameter large enough to allow contaminants such as embedding materials (e.g., paraffin) to accumulate on the channel walls while still leaving adequate room for fluid flow within the channel. In some cases, a channel of a fluidic device has a width, height, or diameter that is less than or equal to 20 millimeters (mm), 19 mm, 18 mm, 17 mm, 16 mm, 15 mm, 14 mm, 13 mm, 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm. In some cases, the channels of the fluidic device have a width, height, or diameter of at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, or 20 mm. In some cases, the channels of the fluidic device have a width in the range of about 1 mm to about 3.8 mm. In some cases, the channels of the fluidic device have a height in the range of about 0.1 mm to about 1.2 mm.

[0250] In some cases, the channels of the fluidic device may be at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 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, 200 mm, 210 mm, 220 mm, 230 mm, 240 mm, 250 mm, 260 mm, 270 mm, 280 mm, 290 mm, 300 mm, 310 mm, 320 mm, 330 mm, 340 mm, 350 mm, 360 mm, 370 mm, 380 mm, 390 mm, 410 mm, 420 mm, 430 mm, 440 mm, 450 mm, 460 mm, 470 mm, 480 mm, 490 mm, 500 mm, 510 mm, 520 mm, 530 mm, 540 mm, 550 mm, 560 mm, 570 mm, 580 mm, 590 mm, 610 mm, 620 mm, 630 mm, 640 mm, 650 mm, 660 mm, 670 mm, 680 mm, 690 mm, 700 mm, The length may be 0mm, 180mm, 190mm, 200mm, 210mm, 220mm, 230mm, 240mm, 250mm, 260mm, 270mm, 280mm, 290mm, 300mm, 310mm, 320mm, 330mm, 340mm, 350mm, 360mm, 370mm, 380mm, 390mm, 400mm, 410mm, 420mm, 430mm, 440mm, 450mm, 460mm, 470mm, 480mm, 490mm or 500mm. In some cases, the channels of the fluidic device may be approximately 500 mm, 490 mm, 480 mm, 470 mm, 460 mm, 450 mm, 440 mm, 430 mm, 420 mm, 410 mm, 400 mm, 390 mm, 380 mm, 370 mm, 360 mm, 350 mm, 340 mm, 330 mm, 320 mm, 310 mm, 300 mm, 290 mm, 280 mm, 270 mm, 260 mm, 250 mm, 240 mm, 230 mm, 220 mm, 210 mm, 200 mm, 190 mm, 180 mm, 190 mm, 200 mm, 210 mm, 220 mm, 230 mm, 240 mm, 250 mm, 260 mm, 270 mm, 280 mm, 290 mm, 280 mm, 290 mm, 290 mm, 280 mm, 290 mm, 260 mm, 250 mm, 240 mm, 230 mm, 220 mm, 210 mm, 200 mm, 250 mm, 260 mm, 270 mm, 280 mm, 290 mm, 290 mm, 300 mm, 300 mm, 300 mm, 320 mm, 330 mm, 340 mm, 350 mm, 360 mm, 370 mm, 380 mm, 390 mm, 400 mm, 430 mm, 440 mm, 450 mm, 460 mm, 470 mm, 480 mm, 490 mm, 500 mm, 510 mm The length may be less than or equal to 0mm, 170mm, 160mm, 150mm, 140mm, 130mm, 120mm, 110mm, 100mm, 90mm, 80mm, 70mm, 60mm, 50mm, 45mm, 40mm, 35mm, 30mm, 25mm, 20mm, 19mm, 18mm, 17mm, 16mm, 15mm, 14mm, 13mm, 12mm, 11mm, 10mm, 9mm, 8mm, 7mm, 6mm, 5mm, 4mm, 3mm, 2mm or 1mm.

[0251] The channels of the fluidic device can have a width, height, or diameter large enough to accommodate a large sample volume. In some cases, the channels of the fluidic device have a width greater than its height to reduce Joule heating temperature rise in the channel. In some cases, the channels of the fluidic device have a width-to-height ratio of at least 2:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1. In some cases, the channels of the fluidic device have a width to height ratio of at most 2:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1. In some cases, the channels of the fluidic device have a width to height ratio of at most about 0.1 mm 2 , 0.2mm 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , 1mm 2 , 1.1mm 2 , 1.2mm 2 , 1.3mm 2 , 1.4mm 2 , 1.5mm 2 , 1.6mm 2 , 1.7mm 2 , 1.8mm 2 , 1.9mm 2 , 2mm 2 , 2.1mm 2 , 2.2mm 2 , 2.3mm 2 , 2.4mm 2 , 2.5mm 2 , 2.6mm 2 , 2.7mm 2 , 2.8mm 2 , 2.9mm 2 , 3mm 2 , 3.1mm 2, 3.2mm 2 , 3.3mm 2 , 3.4mm 2 , 3.5mm 2 , 3.6mm 2 , 3.7mm 2 , 3.8mm 2 , 3.9mm 2 , 4mm 2 , 4.1mm 2 , 4.2mm 2 , 4.3mm 2 , 4.4mm 2 , 4.5mm 2 , 4.6mm 2 , 4.7mm 2 , 4.8mm 2 , 4.9mm 2 , 5mm 2 , 6mm 2 , 7mm 2 , 8mm 2 , 9mm 2 , 10mm 2 , 11mm 2 , 12mm 2 , 13mm 2 , 14mm 2 or 15mm 2 In some cases, the channels of the fluidic device have a cross-sectional area of ​​less than about 0.1 mm 2 , 0.2mm 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , 1mm 2 , 1.1mm 2 , 1.2mm 2 , 1.3mm 2 , 1.4mm 2 , 1.5mm 2 , 1.6mm 2 , 1.7mm 2 , 1.8mm 2 , 1.9mm 2 , 2mm 2 , 2.1mm 2 , 2.2mm 2 , 2.3mm 2, 2.4mm 2 , 2.5mm 2 , 2.6mm 2 , 2.7mm 2 , 2.8mm 2 , 2.9mm 2 , 3mm 2 , 3.1mm 2 , 3.2mm 2 , 3.3mm 2 , 3.4mm 2 , 3.5mm 2 , 3.6mm 2 , 3.7mm 2 , 3.8mm 2 , 3.9mm 2 , 4mm 2 , 4.1mm 2 , 4.2mm 2 , 4.3mm 2 , 4.4mm 2 , 4.5mm 2 , 4.6mm 2 , 4.7mm 2 , 4.8mm 2 , 4.9mm 2 , 5mm 2 , 6mm 2 , 7mm 2 , 8mm 2 , 9mm 2 , 10mm 2 , 11mm 2 , 12mm 2 , 13mm 2 , 14mm 2 or 15mm 2 In some cases, the channels of the fluidic device have a minimum length scale for heat dissipation that is less than about 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, or 600 μm. In some cases, the channels of the fluidic device have a minimum length scale for heat dissipation greater than about 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, or 600 μm.

[0252] In some cases, the channels of the fluidic device may be at least about 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 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 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 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 1 ... Liters (mL), 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 13mL, 14mL, 15mL, 16mL, 17mL, 18mL, 19mL, 20mL, 25mL, 30mL, 35mL, 40mL, 50mL, 55mL, 60mL, 65mL, 70mL, 75mL, 80mL, 85mL, 90mL, 95mL or 100mL total volume. In some cases, the channels of the fluidic device may have a volume of at most about 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 μL, Liters (mL), 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 13mL, 14mL, 15mL, 16mL, 17mL, 18mL, 19mL, 20mL, 25mL, 30mL, 35mL, 40mL, 50mL, 55mL, 60mL, 65mL, 70mL, 75mL, 80mL, 85mL, 90mL, 95mL or 100mL total volume.

[0253] In some cases, the fluidic device includes more than one channel. The channels may be spaced apart within the fluidic device at a given density. In some cases, the edge-to-edge distance between channels is at least about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.25 mm, 1.5 mm, 1.75 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm. In some cases, the edge-to-edge distance between channels is at most about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.25 mm, 1.5 mm, 1.75 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm. Channel density can be defined as the ratio of the width of the channels to the space (or distance) between the channels. In some cases, the ratio of channel width to distance between channels is at least about 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1.

[0254] In some cases, the total volume of all channels in a microfluidic device (e.g., chip) can be in the range of 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, 1000 μL, 1500 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, 1000 μL, 1500 μL, 1750 μL, 2000 μL, 2250 μL, 2500 μL, 2750 μL, 3000 μL, 3500 μL, 4000 μL, 4500 μL, 5000 μL, 6000 μL, 7000 μL, 8000 μL, 10000 μL, 15000 μL, 17500 μL, 2000 μL, 2250 μL, 2500 μL, 2750 μL, 3000 μL, 3500 μL, 400 L, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL or 100 mL. In some cases, the total volume of all channels in a microfluidic device (e.g., a chip) is at most about 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, 1000 μL, 1500 μL, 1750 μL, 2000 μL, 225 μL, 250 μL, 275 μL, 3000 μL, 3500 μL, 4000 μL, 450 μL, 500 μL, 6000 μL, 7000 μL, 8000 μL, 9000 μL, 10000 μL, 15000 μL, 17500 μL, 2000 μL, 2250 μL, 2500 μL, 2750 μL, 3000 μL, 3500 μL, 4000 μL, 4500 μL, 5000 μL, 6000 μL, 7000 μL, 8000 μL, 9000 μL, 10000 μL, 10000 μL, 10000 μL, 10000 μL, 10000 μL, 10000 μL 00 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL or 100 mL.

[0255] The inlets and / or outlets of a fluidic device can be arranged and spaced so that they are compatible with standard fluid handling formats. For example, the inlets and / or outlets can be spaced to align with the wells on a 5" x 3.33" titer plate. The device can include a standard 8-tip pipettor and / or 8 inlets and / or outlets spaced to correspond to 8 wells in a standard 24-, 48-, or 96-well plate. The device can include a standard 12-tip pipettor and / or 12 inlets and / or outlets spaced to correspond to 12 wells in a standard 96-well plate. The device can include a standard 16-tip pipettor and / or 16 inlets and / or outlets spaced to correspond to 16 wells in a standard 384-well plate. The device can include a standard 24-tip pipettor and / or 24 inlets and / or outlets spaced to correspond to 24 wells in a standard 384-well plate. This may allow for easy handling of fluids from such plates onto the device, for example, by a robotic pipetting system or other multiple pipettes.

[0256] Isotachophoresis can be performed using a benchtop system or a base station. For example, Figure 13A shows a benchtop system 1300 for performing sample preparation and isotachophoresis on a fluidic device cartridge 1301. The fluidic device cartridge can be loaded into the benchtop system shown, and a lid with a matching cover and controls 1302 can be lowered onto the fluidic device cartridge. The benchtop system can also include a control panel 1303 with a user interface (e.g., a touch screen) for operation of the system.

[0257] The benchtop system can include a pressure control that provides pressure for handling fluids (e.g., samples, buffers, reagents, enzyme solutions, electrolyte solutions) on the fluidic device. The benchtop system can receive a pressure feedback signal to regulate or control the fluid handling. The fluid handling can be used to load fluids (e.g., reagents, buffers, samples) into the fluidic device. The fluid handling can be used to inject fluids (e.g., reagent solutions) into dry channels of the fluidic device. The pressure can be regulated, for example, using a solenoid valve.

[0258] The benchtop system can include electrodes or electrical contacts. The electrodes can be part of an electrical circuit and are inserted into a reservoir or other opening in the fluidic device, with the completed circuit allowing for the application of an electric field within the fluidic device. The electrical contacts can be connected to corresponding contacts on the fluidic device, for example, a fluidic device with integrated electrodes.

[0259] The benchtop system can include one or more detectors or sensors, such as optical detectors, reflectance sensors, infrared (IR) detectors, electrical detectors, thermal sensors, flow sensors, and pressure sensors, including those further described in this disclosure. Optical detectors can include, but are not limited to, three-axis point detectors, complementary metal-oxide semiconductor (CMOS) detectors, charge-coupled device (CCD) detectors, photodiode photosensors, photoresistors, photomultiplier tubes, and phototransistors. Electrical detectors can include electrodes or other detectors capable of detecting voltage, voltage difference, current, charge, or other electrical properties. For example, electrical detectors can be used to detect the path of extracted or purified nucleic acid bands by detecting changes in conductivity at the interface between the destination electrolyte and the leading electrolyte. Thermal sensors can include infrared (IR) sensors, probe temperature sensors, thermistors, negative temperature coefficient (NTC) thermistors, resistance temperature detectors (RTDs), thermocouples, semiconductor-based sensors, and the like.

[0260] One or more detectors or sensors can be operated or controlled simultaneously or independently. In some cases, a single channel has a dedicated sensor, such as a thermal or voltage sensor, that operates independently of other sensors dedicated to other channels of the microfluidic device. Feedback from the independent sensor can be used to independently control one or more electric fields of the device. For example, a sensor can detect voltage changes over time in a well, as described herein, and feedback from that sensor can be used to control the current in the channel. A second sensor can similarly, but independently, act on a second channel. In some cases, a sensor can detect current changes over time in a well, and feedback from that sensor can be used to control the voltage in the channel.

[0261] The benchtop system can include one or more thermal control devices that control the temperature of the fluidic device or portions of the fluidic device. Thermal control devices can include components including, but not limited to, resistive heaters, fluid-based heating or cooling systems, and Peltier elements. Thermal control devices can be fabricated from materials including, but not limited to, metals (e.g., platinum, titanium, copper, gold), carbon, and indium tin oxide (ITO). Thermal control devices can include temperature sensors that can be used to monitor the controlled temperature and provide temperature feedback for thermal control. Thermal control devices can be used with computer control systems, which are discussed further in this disclosure. For example, temperature sensors (e.g., infrared sensors) can be used to monitor temperature changes in the channels of the chip. Such temperature changes can be indicative of the location of ITP bands (e.g., nucleic acid bands) during the ITP process, and the temperature difference can be attributed to conductivity changes between the leading and trailing electrolytes. In some cases, the thermal control devices are operated without temperature feedback.

[0262] The techniques of the present disclosure (e.g., including the use of the fluidic devices and / or benchtop systems discussed herein) can achieve rapid processing times. For example, samples containing nucleic acids can be prepared (e.g., by removing embedding material, disrupting tissue, lysing cells, and decrosslinking nucleic acids), with the sample having extracted or purified nucleic acids for subsequent analysis, use, or storage.

[0263] Detection and quantification

[0264] The techniques of the present disclosure can use one or more detectors. The detector can be integrated into the fluidic device or can be located external to the fluidic device. The detector can be used to quantify the nucleic acid in a sample or to measure the quality of the nucleic acid in a sample, for example, by fluorescence measurement or ultraviolet (UV) irradiation (e.g., for measuring quantity or purity, such as by measuring A260 / A280). The nucleic acid can be detected while it is located on the fluidic device, for example, while it is in a purification zone (e.g., an ITP channel) or a reservoir (e.g., an elution reservoir). The concentration of the nucleic acid can be detected (or calculated based on a measured amount in a known volume, such as in an elution well, as described herein). The nucleic acid can be labeled, such as with a dye, and the fluorescence intensity of the nucleic acid can be measured by a detector and used to quantify the nucleic acid present (see, for example, Figure 14). The nucleic acid can be labeled before loading into the fluidic device, while in the fluidic device, or after recovery from the fluidic device.

[0265] The use of detector can enable the quantification of nucleic acid from sample with high sensitivity or lower limit of detection.For example, nucleic acid can be detected (for example, in-line in isotachophoresis channel) with a detection limit of less than about 1000 picograms / microliter (pg / μL), 100pg / μL, 10pg / μL, 1pg / μL, 0.9pg / μL, 0.8pg / μL, 0.7pg / μL, 0.6pg / μL, 0.5pg / μL, 0.4pg / μL, 0.3pg / μL, 0.2pg / μL or 0.1pg / μL or equal thereto.Nucleic acid can be detected (for example, in-line in isotachophoresis channel) with a detection limit of less than about 1000 picograms (pg), 100pg, 10pg, 1pg or 0.1pg or equal thereto.

[0266] The use of detector can make it possible to identify or quantify the nucleic acid in sample.For example, techniques such as nucleic acid amplification (including for example PCR, real-time PCR and reverse transcription PCR), hybridization (including for example fluorescent in situ hybridization (FISH) and Q-FISH) and sequencing can be used to identify the existence or absence of specific sequence in the nucleic acid in sample, and optionally can be quantified.

[0267] The detector can be used to control the nucleic acid extraction or purification operation. For example, the detector can detect bands of concentrated nucleic acids by isotachophoresis. Once the concentrated nucleic acids reach a certain location within the device, the process is terminated (e.g., the electric field can be turned off), and the extracted or purified sample can be recovered from the device.

[0268] Detectors can include, but are not limited to, optical and electrical detectors, thermal sensors, and pressure sensors (e.g., pressure transducers). Optical detectors can include, but are not limited to, triaxial point detectors, complementary metal-oxide semiconductor (CMOS) detectors, charge-coupled device (CCD) detectors, photodiode photosensors, photoresistors, photomultiplier tubes, and phototransistors. Optical detection can be achieved by LED illumination coupled with photodiode detection. Electrical detectors can include electrodes or other detectors capable of detecting voltage, voltage difference, current, charge, or other electrical properties. For example, electrical detectors can be used to detect the path of bands of extracted or purified nucleic acids.

[0269] End of execution operation

[0270] When purifying a sample using an ITP, it can be important to precisely stop applying current when the sample ITP zone is at the elution location (e.g., channel or reservoir). The present disclosure provides techniques for assessing the position of the ITP zone, which can be used to trigger the end of a purification run. These techniques can include measuring drive voltage, conductivity, and temperature.

[0271] FIG. 15 shows a schematic diagram of an ITP channel 1500, with a drive electrode located in a buffer elution electrode (EH) reservoir 1501 and a buffer leading electrolyte (LEH) reservoir 1502, and a ground electrode located in a buffer final electrolyte (TEH) reservoir 1503. As shown on the left side of the figure, a conductivity detector (e.g., a capacitively coupled contactless conductivity detector (C4D)) electrode 1504 can be located outside the chip, such as near the elution reservoir 1505. The channel can also include a leading electrolyte reservoir 1506 and a sample reservoir or injection point 1507. Gas ports are indicated by small circles on the left and right edges of the channel, quite far from the channel. The gas ports can be used to inject fluid into the channel from attached reservoirs, either automatically or using, for example, vacuum or applied pressure.

[0272] One method for measuring the position of the ITP band is to measure the voltage or resistance of the channel, such as between the driving electrode and the ground electrode. In systems with more than two electrodes, this measurement can be made between any pair of electrodes. This measurement is easy to make because the voltage-driven electrophoresis is also the measured voltage. Throughout the purification process, the voltage may increase as terminal ions fill the channel. However, the elution reservoir may have a large cross-sectional area and therefore contribute little to the overall resistance. Therefore, changes in the buffer conductivity in this region do not strongly affect the overall resistance of the channel, and the voltage may stop increasing once the ITP zone enters the elution reservoir. This can be used as a signal to stop the current application and stop the run.

[0273] To evaluate this voltage change, the voltage derivative can be calculated, for example, as shown in FIG. 16. High-frequency noise can be suppressed using the Lanzcos derivative method. A threshold can be set for the derivative, and when the derivative value exceeds the threshold, an actuation is performed. In some cases, introducing additional actuations can improve the robustness of the control. For example, FIG. 16 shows four actuation points. In some cases, only two of these actuations are used to change the drive current (e.g., actuations 1 and 4), while the others (e.g., actuations 2 and 3) are used to mark time points during runtime, thereby improving the timing of actuation 4. FIG. 17 shows the voltage derivative analysis in FIG. 16, with the arrows representing the derivative thresholds used to select actuation points.

[0274] FIG. 16 shows example data from a drive voltage measurement. Each vertical line represents an actuation point. Two lines represent two electrodes, namely, the electrodes at the EH and LEH reservoirs, relative to the ground electrode. Points A, B, C, and D indicate the time at which the ITP zone is at the corresponding positions marked in FIG. 15 (A, B, C, and D; labeled 1508, 1509, 1510, and 1511, respectively). In some cases, the conductivity anywhere in the channel can affect the total drive voltage, making it more difficult to assess what is occurring near the elution reservoir.

[0275] A second method for detecting the location of the ITP band is to perform a local measurement of conductivity. This can be done using a capacitively coupled contactless conductivity detector (C4D). This method uses a high-frequency alternating current to flow through the channel wall and bind the electrolyte. This local measurement can be performed in the elution reservoir itself. This technique can reduce or eliminate the ambiguity associated with measurements performed across the entire channel. With this technique, the end of the run can be selected as soon as a change in conductivity is observed in the elution reservoir conductivity detector, for example, as shown in FIG. 18.

[0276] C4D detection can be performed using electrodes placed below the elution channel. Maximizing the electrode area can reduce the required drive frequency. For example, drive frequencies from about 100 kHz to about 10 MHz can be used, with the electrode contact pads being approximately 0.2 mm apart. 2 ~approx. 50mm 2 The C4D sensor can be implemented using electrical components including resistors, capacitors, diode bridges, and high frequency operational amplifiers, and the high frequency signal source can be from a direct digital synthesizer or the like. Figure 19 shows an example schematic diagram of a C4D sensor implementation.

[0277] A third method for detecting the location of the ITP band is to perform local temperature measurements near the elution reservoir. This measurement can be performed using a temperature sensor, including a thermocouple or infrared temperature sensor. This sensor can be placed under the channel near the elution reservoir and monitor the temperature over time. When lower-mobility terminal ions are displaced by higher-mobility leading ions (e.g., at the LE-TE interface of the ITP zone), the electric field in the channel can increase and the temperature can rise. During isotachophoresis, the lower-mobility terminal electrolyte ions and the higher-mobility leading electrolyte ions can meet at the isotachophoresis interface. The ITP interface can contain concentrated sample nucleic acids between the leading and terminal electrolyte ions. A temperature increase can detect the presence of an ITP interface between the higher-mobility leading ions and the lower-mobility terminal ions, thus also indicating the presence of nucleic acids between them. This temperature increase can be between 1 and 10°C.

[0278] Figures 20A and 20B show exemplary temperature measurements using a thermal imaging camera. These images show a clear increase in temperature as terminal ions enter the channel. Figure 20A shows a temperature map of the ITP channel taken using a thermal imaging camera. The channel orientation is the same as in Figure 15. Figure 20B shows a plot of temperature over time at the location of cursor 1 in Figure 20A. At approximately 450 seconds, the ITP interface and terminal ions enter this region, causing a temperature increase. This temperature increase can be detected and used as an actuation signal to modify the current applied to the channel.

[0279] The temperature can be measured at a detection location at or near the elution reservoir (e.g., as shown in Figure 21). In some examples, the detection location can be located at least about 5 mm from the elution reservoir. In some examples, the detection location can be located at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, or 25 mm from the elution reservoir. In some examples, the detection location may be located at most about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, or 25 mm from the elution reservoir. In some examples, the temperature sensor may be located at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, or 25 mm from the elution reservoir. In some examples, the temperature sensor may be located at most about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, or 25 mm from the elution reservoir.

[0280] The temperature sensor can activate a change in current when a change in temperature is sensed. In some examples, the detected change in temperature is in the range of about 0.2°C to about 5°C. In some examples, the detected change in temperature is at least about 0.2°C, 0.3°C, 0.4°C, 0.5°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. In some examples, the detected change in temperature is at most about 0.2°C, 0.3°C, 0.4°C, 0.5°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C.

[0281] In some cases, detection of an ITP zone at one or more actuation points, for example, by voltage monitoring, conductivity measurement, or temperature sensing, can cause the benchtop controller to modify the current applied to the microfluidic chip. This modification can be applied immediately upon detection or after a predetermined delay. Detection of an ITP zone can activate a decrease, increase, or removal of the current. For example, detection of an ITP zone at point C1510 can activate a decrease in current to increase the residence time of the ITP zone in the channel following the elution reservoir. Alternatively, or in combination, detection of an ITP zone at point D1511, located at or near the elution reservoir, can activate removal of the current to position the ITP zone (and nucleic acid) or a portion thereof within the elution reservoir, well, or region of the channel or chip. In some examples, detection of an ITP zone can activate a change in current after a predetermined amount of time. For example, the detection location (e.g., 1504 or cursor 1 location) can be located at or near the elution reservoir at a known distance so that the time required for the ITP zone to travel between the detection location and the elution reservoir can be calculated for a given current. The controller can predetermine the travel time, and upon detection of the ITP zone at the detection location, a delay in current removal can be activated after a predetermined amount of time. In some examples, detection of the ITP zone at a particular detection location can provide a spatial-temporal relationship of the ITP zone, which can provide more accurate activation than other sensing methods.

[0282] In some cases, detection of an ITP zone at an activation point can change the direction or path of the current applied to the microfluidic chip. For example, the ITP zone can activate a current reversal to reverse the direction of travel within the channel. In another example, the system can be activated to stop applying current between a first pair of electrodes and start applying current to a second pair of electrodes to propel ion flow along a different path. For example, the channel can be "y-shaped," with the first channel being directed into two side channels that branch off from the first channel in different directions. Current can be initially driven between first and second electrodes connected to the first channel and the first side channel, respectively. The ITP zone can move from the first channel to the first side channel without interruption of the current. When an ITP zone is detected at the connection point between the first channel and the two side channels, the first and second electrodes can be activated to stop the drive current, and the third and fourth electrodes connected to the first channel and the second side channel can be activated to start the drive current, respectively. The ITP zone then moves from the first channel to the second side channel. In some cases, the first and third electrodes are the same electrode. This actuation can thus change the current so that the path of the ITP zone changes along the channel.

[0283] Further Processing and Use of the Purified Sample

[0284] The extracted or purified nucleic acids can be used for sequencing, genotyping, mutation or polymorphism analysis, analysis of gene expression levels, disease diagnosis, disease prediction, cytological classification, origin or ancestry analysis, or the application of suggested treatment modalities.

[0285] The extracted or purified nucleic acids can be used in amplification reactions, including, but not limited to, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), rolling circle amplification (RCA), nicking enzyme amplification reaction (NEAR), PCR, reverse transcription PCR, real-time PCR, quantitative PCR (qPCR), digital PCR, and methylation-specific PCR.

[0286] The extracted or purified nucleic acids can be used in sequencing reactions including Maxam-Gilbert sequencing, chain termination sequencing (e.g., Sanger sequencing), shotgun sequencing, pyrosequencing, bridge PCR, colony sequencing, polony sequencing, sequencing by synthesis, ion semiconductor sequencing, nanopore sequencing, nanoball sequencing, sequencing by ligation, sequencing by hybridization, and single molecule real-time sequencing.

[0287] The extracted or purified nucleic acids can be used in protein binding assays, such as DNA footprinting assays. For example, DNase (e.g., DNase I) can be used to indiscriminately cleave target DNA molecules. The techniques of the present disclosure can be used to separate digested DNA from the DNase enzyme and prevent further digestion. In some cases, DNase digestion can be performed outside the fluidic device, and then the sample can be loaded into the fluidic device for purification. In other cases, DNase digestion can be performed on the fluidic device, and once digestion is complete, the nucleic acids can be purified on the fluidic device.

[0288] Samples such as fixed or embedded samples (e.g., FFPE samples) can be used in longitudinal studies, genome-wide association studies, and other large-scale analyses across populations.

[0289] Vertical or column ITP

[0290] Planar ITP device designs, such as those discussed herein, can utilize horizontal space for the moving ITP band. For high-throughput sample processing, such as in a 96-well plate format, it can be advantageous to fit the entire ITP separation system onto a given footprint, such as a 9 mm x 9 mm footprint. One way to do this is to increase the height of the system to accommodate larger sample volumes. This provides the option of increasing the total sample volume into the milliliter range and still be able to process samples with reasonable run times.

[0291] In some cases, it may be important to reduce or prevent gravity-driven and / or buoyancy-driven flows with such systems. It may also be important to assemble the electrolyte zones required for ITP without mixing the electrolytes.

[0292] A vertical or columnar ITP system can include several ITP stages, where each stage comprises a column (e.g., plastic) with a gel (e.g., agarose) or similar material at the bottom. The gel can have high conductivity. Each stage can be prepared by introducing an electrolyte to the top of the gel. The gel can slow or prevent liquid flow. To create a column, the stages can be stacked with a terminal electrolyte at the top and a leading electrolyte at the bottom. An electric current can then be driven through the system. The purified analyte can be recovered by unstacking the column and removing it with a pipette.

[0293] Figure 22A shows an exemplary schematic diagram of a vertical (or column-type) ITP setup. The gel in each stage can support the weight of water (e.g., aqueous electrolyte solution) above. The cross-sectional area of ​​the column can be approximately 9 mm x 9 mm. Such a system can process samples with a column cross-sectional area of ​​approximately 9 mm x 9 mm. This design can be sized, for example, for 96 samples (columns), with the overall dimensions of the device fitting into a standard microtiter plate. Figure 22B shows an exemplary image of a vertical ITP setup with DNA ITP bands. The stages are the terminal electrolyte (high) (TEH), sample, leading electrolyte (LE), and leading electrolyte (high) (LEH). The ITP zones move downward through the system. This image does not show the elution stage (E, shown in Figure 22A), which is the final destination of the analytes.

[0294] Computer Control System

[0295] The present disclosure provides a computer control system that can be programmed to implement the methods of the present disclosure. Figure 13B shows a computer system 1304 that is programmed or otherwise configured to control sample preparation, sample extraction or purification, or detection. The computer system 1304 can regulate various aspects of the extraction, purification, and detection processes of the present disclosure, such as, for example, application of pressure or electric fields, thermal control, detection, quantification, feedback, and process initiation or termination. The computer system 1304 can be a user's electronic device or computer system that is located remotely relative to the electronic device. The electronic device can be a portable electronic device.

[0296] The computer system 1304 includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") 1305, which can be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 1304 also includes memory or memory locations 1310 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1315 (e.g., a hard disk), a communication interface 1320 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 1325, such as cache, other memory, data storage, and / or electronic display adapters. The memory 1310, the storage unit 1315, the interface 1320, and the peripheral devices 1325 communicate with the CPU 1305 by a communication bus (solid lines), such as a motherboard. The storage unit 1315 can be a data storage unit (or data repository) for storing data. The computer system 1304 can be operably coupled to a computer network ("network") 1330 with the aid of the communication interface 1320. The network 1330 can be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. In some cases, the network 1330 is a telecommunications and / or data network. The network 1330 can include one or more computer servers, which can enable distributed computing such as cloud computing. In some cases, with the assistance of the computer system 1304, the network 1330 can implement a peer-to-peer network, which can enable devices coupled to the computer system 1304 to act as clients or servers.

[0297] The CPU 1305 can execute a series of machine-readable instructions, which may be embedded in a program or software. The instructions may be stored in a memory location, such as the memory 1310. The instructions may instruct the CPU 1305, which then runs a program or otherwise configures the CPU 1305 to imp...

Claims

1. 1. A method for concentrating nucleic acids, comprising: (a) a fluidic device; (i) a sample containing nucleic acids; (ii) a final electrolyte buffer comprising a final electrolyte ion having a first effective mobility that has a magnitude that is smaller than the magnitude of the effective mobility of the nucleic acid; (iii) a first leading electrolyte buffer solution in a first leading electrolyte reservoir, the first leading electrolyte buffer solution including a first leading electrolyte ion having a second effective mobility, the second effective mobility having an order of magnitude greater than the order of magnitude of the effective mobility of the nucleic acid; and (iv) a second leading electrolyte buffer in a second leading electrolyte reservoir, the second leading electrolyte buffer comprising a second leading electrolyte ion having a third effective mobility, the third effective mobility being greater than the magnitude of the effective mobility of the nucleic acid, the first leading electrolyte buffer being different from the second leading electrolyte buffer, and the first leading electrolyte buffer having an ionic strength greater than the ionic strength of the second leading electrolyte buffer. charging the (b) applying a first electric field within the fluidic device using a first electrical circuit to perform a first isotachophoresis using the final electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby concentrating the nucleic acid from contaminants in the sample; (c) performing a second isotachophoresis run using the final electrolyte ions, the nucleic acid, and the second leading electrolyte ions by applying a second electric field within the fluidic device using a second electric circuit, wherein the first electric circuit is different from the second electric circuit; and (d) collecting the concentrated nucleic acid in the second leading electrolyte reservoir and removing the concentrated nucleic acid from the second leading electrolyte reservoir. A method comprising:

2. The method of claim 1 , wherein the fluidic device comprises a first fluid channel that branches into a first side fluid channel and a second side fluid channel.

3. 3. The method of claim 2, wherein the first electric field is applied to the first fluid channel and the step of performing the second isotachophoresis comprises varying an applied current from the first side fluid channel to the second side fluid channel.

4. 4. The method of claim 3, wherein the altering of the applied current comprises ceasing application of a first current between the first fluid channel and the first side fluid channel and then commencing application of a second current between the first fluid channel and the second side fluid channel.

5. 5. The method of claim 1, wherein the first leading electrolyte ions are the same as the second leading electrolyte ions, and the concentration of the first leading electrolyte ions in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ions in the second leading electrolyte buffer.

6. 5. The method of claim 1, wherein a first leading electrolyte ion is the same as the second leading electrolyte ion, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer or the second leading electrolyte buffer contains a third leading electrolyte ion.

7. 7. The method of claim 1, wherein the concentration of the second leading electrolyte ion in the second leading electrolyte buffer is less than 50 mM.

8. 8. The method of any one of claims 1 to 7, wherein the second lead electrolyte buffer comprises 50 mM Tris HCl.

9. 9. The method of claim 1, wherein the first electrical circuit comprises a first pair of electrodes and the second electrical circuit comprises a second pair of electrodes, the first pair of electrodes being different from the second pair of electrodes.

10. 1. A system comprising: (a) Below: (i) a sample reservoir in fluid communication with the first fluid channel; (ii) a reservoir for a final electrolyte buffer in fluid communication with the first fluid channel; (iii) a first leading electrolyte reservoir in fluid communication with the first fluid channel; and (iv) a second leading electrolyte reservoir in fluid communication with the first fluid channel; a fluidic device comprising: (b) a first electrical circuit configured to perform a first isotachophoresis run within the fluidic device by applying a first electric field along a first path including the final electrolyte buffer reservoir, the first fluid channel, and the first leading electrolyte reservoir, wherein the sample reservoir is located along the first path and between the final electrolyte buffer reservoir and the first leading electrolyte reservoir; and (c) a second electrical circuit configured to perform a second isotachophoresis run by applying a second electric field within the fluidic device along a second path including the final electrolyte buffer reservoir, the first fluid channel, and the second leading electrolyte reservoir. wherein the first electrical circuit is different from the second electrical circuit and the first path is different from the second path.

11. 11. The system of claim 10, wherein the first electrical circuit includes a first pair of electrodes and the second electrical circuit includes a second pair of electrodes, the first pair of electrodes being different from the second pair of electrodes.

12. 12. The system of claim 10 or claim 11, wherein the first electrical circuit includes a first electrode in electrical communication with the final electrolyte buffer reservoir and a second electrode in electrical communication with the first leading electrolyte reservoir, and the second electrical circuit includes a third electrode in electrical communication with the final electrolyte buffer reservoir and a fourth electrode in electrical communication with the second leading electrolyte reservoir.

13. The system of claim 12 , wherein the first electrode and the third electrode comprise the same electrode.

14. 14. The system of claim 10, wherein the first electrical circuit includes a first power supply configured to apply the first electric field and the second electrical circuit includes a second power supply configured to apply the second electric field.

15. 15. The system of claim 10, wherein the first leading electrolyte reservoir contains a first leading electrolyte buffer and the second leading electrolyte reservoir contains a second leading electrolyte buffer, the second leading electrolyte buffer having an ionic strength lower than the ionic strength of the first leading electrolyte buffer.

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