Isoelectric focusing for nucleic acid purification

Abstract

The present disclosure relates to fluidic systems and devices for processing, extracting, or purifying one or more analytes. These systems and devices can be used to process samples and extract nucleic acids, for example, by isoelectric focusing. In particular, the systems and related methods can allow for the extraction of nucleic acids, including non-crosslinked nucleic acids, from samples such as tissues or cells. The systems and devices can also be used for multiplexed parallel sample processing.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780022050.1, filed on January 28, 2017, entitled "Isotachycoelography for Nucleic Acid Purification" (the corresponding PCT application was filed on January 28, 2017, and has the application number PCT / US2017 / 015519).

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 288,930, filed January 29, 2016, entitled “Isotachophoresis for Purification of Nucleic Acids,” the entire contents of which are incorporated herein by reference. Background Technology

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

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

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

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

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

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

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

[0011] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a fluid device: (i) a tissue sample containing lysed solid tissue, wherein the lysed solid tissue contains nucleic acids and contaminants; (ii) a trailing electrolyte buffer containing a first trailing electrolyte ion having an effective migration rate less than the effective migration rate of the nucleic acid; and (iii) a leading electrolyte buffer containing a first leading electrolyte ion having a second effective migration rate greater than the effective migration rate of the nucleic acid; and (b) applying an electric field within the fluid device to perform isovelocity electrophoresis with the first trailing electrolyte ion, the nucleic acid, and the first leading electrolyte ion, thereby purifying the nucleic acid from the contaminants in the tissue sample.

[0012] In some embodiments of the aspects provided herein, the effective mobility of the first trailing electrolyte ion is greater than the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the fluidic device is a microfluidic chip, and the tissue sample, the trailing electrolyte buffer, and the leading electrolyte buffer are loaded into a first region of the microfluidic chip. Some embodiments of the aspects provided herein may further include performing at least one sample preparation procedure selected from the group consisting of the first region of the microfluidic chip: (1) removing embedding material, (2) disrupting tissue, (3) lysing cells, (4) uncrosslinking the nucleic acids, (5) digesting proteins, and (6) digesting the nucleic acids. In some embodiments of the aspects provided herein, the isovelocity electrophoresis is performed in a second region of the microfluidic chip, wherein the second region is separated from and fluidly connected to the first region. In some embodiments of the aspects provided herein, the solid tissue is derived from a solid organ. In some embodiments of the aspects provided herein, the lysed solid tissue contains a chemical fixative. In some embodiments of the aspects provided herein, the chemical fixative is formalin. In some embodiments of the aspects provided herein, the solid tissue is formalin-fixed paraffin-embedded tissue (FFPE). In some embodiments of the aspects provided herein, the lysed solid tissue comprises urea or thiourea. In some embodiments of the aspects provided herein, the lysed solid tissue further includes disrupting cell-cell junctions, extracellular matrix, or connective tissue to obtain the lysed solid tissue. In some embodiments of the aspects provided herein, the lysed solid tissue comprises solid particles. In some embodiments of the aspects provided herein, the nucleic acid comprises dispersed or solvated nucleic acid. In some embodiments of the aspects provided herein, the contaminant is selected from cross-linked nucleic acids, embedding materials, tissue fragments, fixation chemicals, proteins, inhibitors, and combinations thereof. In some embodiments of the aspects provided herein, the contaminant comprises cross-linked nucleic acids. In some embodiments of the aspects provided herein, the tissue sample is combined with the trailing electrolyte buffer prior to loading. In some embodiments of the aspects provided herein, the tissue sample is combined with the leading electrolyte buffer prior to loading. In some embodiments of the aspects provided herein, the loading of the leading electrolyte buffer is performed prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the solid tissue is lysed in the leading electrolyte buffer prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the solid tissue is lysed in the trailing electrolyte buffer prior to the loading of the tissue sample.In some embodiments of the aspects provided herein, the sample preparation procedure includes removing embedding material by incubating the tissue sample in the fluid device at a temperature of at least about 37°C for a duration of at least about 1 minute prior to the application of the electric field. In some embodiments of the aspects provided herein, the temperature is from about 40°C to about 80°C. In some embodiments of the aspects provided herein, the duration is from about 1 minute to about 120 minutes. In some embodiments of the aspects provided herein, the sample preparation procedure includes destroying tissue or lysing cells by applying mechanical stress to the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure includes destroying tissue or lysing cells by applying heat to the tissue sample. In some embodiments of the aspects provided herein, the application of heat results in a temperature of the tissue sample of about 30°C to about 80°C. In some embodiments of the aspects provided herein, the sample preparation procedure includes destroying tissue or lysing cells by contacting the tissue sample with a solution having a pH of at least 10 or by digesting the tissue sample with protein hydrolysis. In some embodiments of the aspects provided herein, the protein hydrolysis is performed at a temperature greater than about 25°C. In some embodiments of the aspects provided herein, the sample preparation procedure includes disrupting tissue or lysing cells by applying at least one surfactant to the tissue sample. In some embodiments of the aspects provided herein, the sample preparation procedure includes disrupting tissue or lysing cells by applying a solution containing urea to the tissue or cell sample. In some embodiments of the aspects provided herein, the solution further contains thiourea. In some embodiments of the aspects provided herein, the concentration of urea in the solution is in the range of about 4 M to about 9 M, and the concentration of thiourea in the solution is in the range of about 0.5 M to about 3.5 M. In some embodiments of the aspects provided herein, the concentration of urea in the solution is about 6.5 M to about 7.5 M, and the concentration of thiourea in the solution is about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the sample preparation procedure includes uncrosslinking the nucleic acid by digesting the crosslinked protein with proteinase K. In some embodiments of the aspects provided herein, the sample preparation procedure includes digesting the nucleic acid with a DNase or RNase. Some embodiments of the aspects provided herein further include eluting an output solution containing the purified nucleic acid from an outlet reservoir of the fluid device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two times higher than the concentration of the nucleic acid in the tissue sample.In some embodiments of the aspects provided herein, the purified nucleic acid in the tissue sample and the output solution comprises cross-linked nucleic acid, and the concentration of the cross-linked nucleic acid in the output solution is at least two-fold lower than the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the contaminant is present in the output solution at a concentration at least two-fold lower than the concentration of the contaminant in the tissue sample. In some embodiments of the aspects provided herein, the first trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the first leading electrolyte ion comprises chloride. In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a second trailing electrolyte ion having a different effective mobility than the first trailing electrolyte ion. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) or MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the first trailing electrolyte ion comprises MOPS. In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a second trailing electrolyte ion having a second effective mobility, wherein the magnitude of the second effective mobility is substantially the same as or lower than the magnitude of the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the tissue sample loaded into the fluidic device has a volume of at least 50 μl. Some embodiments of the aspects provided herein further include performing a first sample processing procedure on the tissue sample in the first region of the microfluidic chip, and performing an enzymatic reaction on the tissue sample in the second region of the microfluidic chip. In some embodiments of the aspects provided herein, the first sample processing procedure includes removing embedding material, disrupting tissue or cell lysis, and the enzymatic reaction includes decrosslinking the nucleic acid, digesting proteins, or digesting nucleic acids. In some embodiments of the aspects provided herein, the first region and the second region are each heated to a temperature above 37°C.In some embodiments of the aspects provided herein, the first region is heated to a temperature of about 60°C to 100°C during the first sample processing procedure, and the second region is heated to a temperature of 40°C to 60°C.

[0013] One aspect of this disclosure provides a method for simultaneously purifying nucleic acids from at least two different samples, the method comprising: (a) loading a first sample containing: (i) a first sample comprising a first nucleic acid and a first contaminant; (ii) a first tailing electrolyte buffer containing a first tailing ion, wherein the effective mobility of the first tailing ion is less than the effective mobility of the first nucleic acid; and (iii) a first leading electrolyte buffer containing a first leading ion, wherein the effective mobility of the first leading ion is greater than the effective mobility of the first nucleic acid; and (b) loading a second sample containing: (i) a second sample comprising a second nucleic acid and a second contaminant; and (ii) a first tailing electrolyte buffer containing: (i) a first sample comprising a first nucleic acid and a first contaminant; and (iii) a first leading ...iv) a first leading electrolyte buffer containing: (i) a first sample comprising a first nucleic acid and a first contaminant; and (ii) a first leading electrolyte buffer containing: (i) a first sample comprising a first nucleic acid and a first contaminant; and (iii) a first leading electrolyte buffer containing: (i) a first sample comprising a first nucleic acid and a first contaminant; and (iv) a first leading electrolyte buffer containing: (i) a first sample comprising a first nucleic acid and a first contaminant; and (iii) a first leading electrolyte buffer containing: (i) a first sample comprising a first nucleic acid and a first contaminant; and (iv) a first leading electrolyte buffer containing: (i) a first sample comprising (iii) a second tailing electrolyte buffer containing a second tailing ion, wherein the amount of the second tailing ion is less than the amount of the effective mobility of the second nucleic acid; and (c) a second leading electrolyte buffer containing a second leading ion, wherein the amount of the effective mobility of the second leading ion is greater than the amount of the effective mobility of the second nucleic acid; and (d) applying a first electric field within the microfluidic chip to perform isotachophoresis in the first channel using the first tailing 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 tailing ion, the second nucleic acid, and the second leading ion, thereby simultaneously purifying the first nucleic acid from the first contaminant and purifying the second nucleic acid from the second contaminant.

[0014] 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 trailing electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or tissue disrupting agent. In some embodiments of the aspects provided herein, the lysing agent or the tissue disrupting agent comprises one or more reagents selected from solutions with a pH greater than about 12, proteases, urea, thiourea, and surfactants. 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, during the isotachophoresis, the first sample does not contact the second sample. Some embodiments of the aspects provided herein further include loading the following substances into a third channel of the microfluidic chip: (i) a third sample containing a third nucleic acid and a third contaminant, (ii) a third tailing electrolyte buffer containing a third tailing ion, wherein the effective mobility of the third tailing ion is less than the effective mobility of the third nucleic acid, and (iii) a third leading electrolyte buffer containing a third leading ion, wherein the effective mobility of the third leading ion is greater than the effective mobility of the third nucleic acid, wherein an electric field is applied within the microfluidic chip to perform isovelocity electrophoresis in the third channel using the third tailing ion, the third nucleic acid, and the third leading ion, thereby simultaneously purifying the first nucleic acid from the first contaminant, purifying the second nucleic acid from the second contaminant, and purifying the third nucleic acid from the third contaminant. In some embodiments of the aspects provided herein, the first and second electric fields are generated by a single electrode pair. In some embodiments of the aspects provided herein, the first and second electric fields are generated by different electrode pairs. In some embodiments of the aspects provided herein, the first and second channels are coupled to independent sensors. In some embodiments of the aspects provided herein, feedback from the independent sensor is used to independently control the first and second electric fields. In some embodiments of the aspects provided herein, the independent sensor senses a voltage, and the feedback is used to control the current (or resistance) within 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.

[0015] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances onto a fluid device: (i) a sample containing fixed cells, fixed tissue, or embedded tissue, wherein the sample contains nucleic acids; (ii) a tail electrolyte buffer containing a tail electrolyte having a lower effective migration rate than the nucleic acids; and (iii) a lead electrolyte buffer containing a lead electrolyte having a higher effective migration rate than the nucleic acids; and (b) applying an electric field to the fluid device to perform isovelocity electrophoresis using the tail electrolyte, the nucleic acids, and the lead electrolyte, thereby purifying the nucleic acids from contaminants in the sample.

[0016] In some embodiments of the aspects provided herein, the contaminant is selected from cross-linked nucleic acids, embedding materials, immobilization chemicals, enzymes, and inhibitors. In some embodiments of the aspects provided herein, the sample comprises the fixed cells, the fixed tissue, or both the fixed cells and the fixed tissue. In some embodiments of the aspects provided herein, the sample is formalin-fixed. In some embodiments of the aspects provided herein, the sample comprises the embedded tissue. In some embodiments of the aspects provided herein, the sample comprises the tissue embedded in paraffin. In some embodiments of the aspects provided herein, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In some embodiments of the aspects provided herein, the sample comprises tissue biopsy. In some embodiments of the aspects provided herein, the sample is an anatomically dissected formalin-fixed paraffin-embedded (FFPE) sample. Some embodiments of the aspects provided herein further include comparing the characteristics of the nucleic acid with nucleic acids from other samples, wherein the characteristics are expression level, nucleic acid sequence, molecular weight, nucleic acid integrity, nucleic acid strand type (e.g., double-stranded vs. single-stranded), or nucleic acid purity. In some embodiments of the aspects provided herein, the sample is a tumor sample. In some embodiments of the aspects provided herein, the trailing electrolyte buffer has a pH greater than about 7. Some embodiments of the aspects provided herein further include incubating the tissue sample in the fluid device at a temperature of at least about 37°C for a duration of at least about 1 minute prior to applying the electric field. In some embodiments of the aspects provided herein, the temperature is from about 40°C to about 80°C. In some embodiments of the aspects provided herein, the duration is from about 1 minute to about 120 minutes. In some embodiments of the aspects provided herein, the leading electrolyte buffer contains proteinase K. Some embodiments of the aspects provided herein further include removing protein cross-links from the nucleic acid using the proteinase K. Some embodiments of the aspects provided herein further include removing protein cross-links from the nucleic acid by heating after applying the electric field. Some embodiments of the aspects provided herein further include eluting an output solution containing the purified nucleic acid from an outlet reservoir of the fluid device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two times higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the concentration of the cross-linked nucleic acid in the output solution is at least about two times lower than the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the output solution has a volume 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 trailing electrolyte has a higher effective migration rate than the contaminant. In some embodiments of the aspects provided herein, the trailing electrolyte comprises (i) a first ion having a higher effective migration rate than the contaminant, and (ii) a second ion having a migration rate substantially the same as or lower than that of the contaminant. In some embodiments of the aspects provided herein, the isotachophoresis quenches the pH of the tissue sample to approximately 7.5. Some embodiments of the aspects provided herein further include dewaxing the sample prior to loading. Some embodiments of the aspects provided herein further include detecting the concentration of the nucleic acid. In some embodiments of the aspects provided herein, the concentration is less than or equal to about 1 picogram per microliter (pg / μL).

[0017] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a fluid device: (i) a tissue sample comprising lysed solid tissue and nucleic acids; (ii) a trailing electrolyte buffer containing trailing electrolyte ions having a first effective mobility, wherein the magnitude of the first effective mobility is less than the magnitude of the effective mobility of the nucleic acids; and (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir containing first leading electrolyte ions having a second effective mobility, wherein the magnitude of the second effective mobility is greater than the magnitude of the effective mobility of the nucleic acids. (iv) a second lead electrolyte buffer in a second lead electrolyte reservoir, the second lead electrolyte buffer containing a second lead electrolyte ion having a third effective mobility, wherein the magnitude of the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acid, wherein the first lead electrolyte buffer is different from the second lead electrolyte buffer; (b) isotachophoresis is performed for the first time using the trailing electrolyte ion, the nucleic acid and the first lead electrolyte ion to purify the nucleic acid from the contaminant in the tissue sample; and (c) isotachophoresis is performed for the second time using the trailing electrolyte ion, the nucleic acid and the second lead electrolyte ion.

[0018] In some embodiments of the aspects provided herein, the second isotachophoresis includes changing the applied current from the first channel to the second channel. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the magnitude of the second effective mobility is greater than the magnitude of the third effective mobility. In some embodiments of the aspects provided herein, the first leading electrolyte ion is different from the second leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the first leading electrolyte buffer contains a third leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer contains a third leading electrolyte ion. Some embodiments of the aspects provided herein further include collecting the nucleic acid in the second leading electrolyte reservoir and removing the nucleic acid from the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the first isotachyphoresis and the second isotachyphoresis are performed by applying a single electric field. In some embodiments of the aspects provided herein, the first isotachyphoresis and the second isotachyphoresis are performed by applying more than one electric field. In some embodiments of the aspects provided herein, the concentration of the second leading electrolyte ion in the second leading electrolyte buffer is less than 50 mM. In some embodiments of the aspects provided herein, the second leading electrolyte buffer contains 50 mM Tris HCl.

[0019] One aspect of this disclosure provides a microfluidic device comprising: (a) a first isotachymeter electrophoresis region in a microfluidic chip, the first isotachymeter electrophoresis region comprising: (i) a first sample reservoir in fluid communication with a first fluid channel, (ii) a first buffer reservoir in fluid communication with the first fluid channel, and (iii) a second buffer reservoir in fluid communication with the first channel; and (b) a second isotachymeter electrophoresis region in the microfluidic chip, the second isotachymeter electrophoresis region comprising: (i) a second sample reservoir in fluid communication with a second fluid channel, (ii) a third buffer reservoir in fluid communication with the second fluid channel, and (iii) a fourth buffer reservoir in fluid communication with the second channel, wherein the first isotachymeter electrophoresis region is not in fluid communication with the second isotachymeter electrophoresis region, and wherein the microfluidic device is configured to independently control a first circuit for applying current to the first isotachymeter electrophoresis region and a second circuit for applying current to the second isotachymeter electrophoresis region.

[0020] In some embodiments of the aspects provided herein, the leakage rate between the first and second isotachycardia electrophoresis zones is less than 1 μl / hour. In some embodiments of the aspects provided herein, the current leakage between the first and second isotachycardia electrophoresis zones is less than 1 μA. In some embodiments of the aspects provided herein, the impedance is greater than 1 megohm. In some embodiments of the aspects provided herein, the first fluid channel contains a liquid volume greater than 100 μl. In some embodiments of the aspects provided herein, the first fluid channel and the second fluid channel are separated by a distance at least 5 folds less than the width of the first channel. In some embodiments of the aspects provided herein, the microfluidic device is configured to simultaneously control the first circuit with the second circuit. Some embodiments of the aspects provided herein further include an elution reservoir in fluid communication with the first channel, wherein a temperature sensor is located within 5 mm of the elution reservoir.

[0021] One aspect of this disclosure provides a method comprising: (a) providing an electrofluidic apparatus including a sample input reservoir in fluid communication with a channel; (b) loading a sample volume into the sample input reservoir; (c) moving at least 50% of the sample volume from the sample input reservoir to the channel without adding additional volume to the sample input reservoir; and (d) applying an ion current through the channel.

[0022] In some embodiments of the aspects provided herein, the movement is carried out by means of gravity. In some embodiments of the aspects provided herein, the ion current substantially does not pass through the channel. In some embodiments of the aspects provided herein, the at least 50% of the sample volume comprises at least 80% of the sample volume. In some embodiments of the aspects provided herein, the sample volume contains nucleic acids. In some embodiments of the aspects provided herein, the sample volume contains tissue samples or formalin-fixed paraffin-embedded (FFPE) samples. In some embodiments of the aspects provided herein, the application of the ion current comprises isovelocity electrophoresis. In some embodiments of the aspects provided herein, the total sample volume loaded into the sample input reservoir is less than or equal to the internal volume of the input reservoir. In some embodiments of the aspects provided herein, the sample input reservoir comprises a top region connected to a bottom region via a conical region, wherein the top region has a first diameter and the bottom region has a second diameter, wherein the first diameter is at least twice as long as the second diameter to facilitate the movement of at least 50% of the sample volume from the sample input reservoir to the channel. In some embodiments of the aspects provided herein, the sample volume is at least 25 μl. In some embodiments of the 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.

[0023] One aspect of this disclosure provides a microfluidic chip comprising: a first sample input reservoir, wherein the first sample input reservoir includes a top region connected to a bottom region via a tapered region, wherein the top region has a first internal hydraulic diameter and the bottom region has a second internal hydraulic diameter, wherein the first internal hydraulic diameter is at least twice as long as the second internal hydraulic diameter, and wherein the first sample input reservoir is in fluid communication with a first channel; a first buffer reservoir in fluid communication with the first channel, wherein the first sample reservoir is configured such that the free surface of 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.

[0024] In some embodiments of the aspects provided herein, the first internal hydraulic diameter is in the range of about 1 mm to about 15 mm. In some embodiments of the aspects provided herein, the second internal hydraulic diameter is in the range of about 0.5 mm to about 5 mm. In some embodiments of the aspects provided herein, the first sample reservoir is configured to contain a sample volume of at least 100 μl. In some embodiments of the aspects provided herein, the microfluidic chip is configured to move at least 50% of the sample volume from the first sample reservoir to the first channel when a vacuum is applied thereto. In some embodiments of the aspects provided herein, the microfluidic chip is configured to perform isovelocity electrophoresis on the sample entering the first channel.

[0025] One aspect of this disclosure provides a method for extracting nucleic acids, the method comprising: (a) exposing a biological sample containing cells or tissue to a solution containing urea or thiourea to lyse the cells or tissue within the biological sample and produce cell lysates; (b) introducing the cell lysates into an apparatus; and (c) performing isovelocity electrophoresis using the apparatus to separate nucleic acids from the cell lysates.

[0026] Some embodiments of the aspects provided herein further include digesting the sample with proteinase K. In some embodiments of the aspects provided herein, the solution comprises urea and thiourea. In some embodiments of the aspects provided herein, the solution comprises urea and thiourea in a ratio of about 2:1. In some embodiments of the aspects provided herein, the concentration of urea in the solution is about 4 M to about 9 M, and the concentration of thiourea in the solution is about 0.5 M to about 3.5 M. In some embodiments of the aspects provided herein, the concentration of urea in the solution is about 6.5 M to about 7.5 M, and the concentration of thiourea in the solution is about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the solution comprises a trailing electrolyte ion or a leading electrolyte ion, or both a trailing electrolyte ion and a leading electrolyte ion.

[0027] One aspect of this disclosure provides a method for purifying high molecular weight nucleic acids from a tissue sample, the method comprising: (a) loading a fluid device with: (i) a cell sample containing genomic DNA and contaminants, wherein the cell sample is contacted with a lysis buffer before or after loading the cell sample into the fluid device; (ii) a trailing electrolyte buffer containing trailing electrolyte ions having a first effective migration rate, wherein the first effective migration rate is less than the effective migration rate of the high molecular weight nucleic acid and greater than the amount of the contaminants; and (iii) (a) a first lead electrolyte buffer containing a first lead electrolyte ion having a second effective mobility, wherein the value of the second effective mobility is greater than the value of the effective mobility of the high molecular weight nucleic acid; (b) isotachophoresis using the trailing electrolyte ion, the high molecular weight nucleic acid, and the first lead electrolyte ion to separate the high molecular weight nucleic acid from the contaminant and enrich the high molecular weight nucleic acid in the isotachophoresis region; and (c) eluting the genomic DNA into a solution in an output reservoir, wherein more than 50% by mass of the nucleic acid in the solution is greater than 30 kilobases.

[0028] In some embodiments of the aspects provided herein, the lysis buffer does not include an alkaline buffer. In some embodiments of the aspects provided herein, the lysis buffer contains octylphenol ethoxylate. In some embodiments of the aspects provided herein, the solution contains more than 50% by mass of nucleic acid greater than 50 kilobases.

[0029] One aspect of this disclosure provides a method for performing isotachophoresis, the method comprising: (a) providing a fluid apparatus including a first channel in fluid communication with a sample input reservoir containing a tissue sample containing lysed solid tissue, a first buffer reservoir containing a first leading electrolyte buffer, and a second buffer reservoir containing a trailing electrolyte buffer; (b) contacting a first electrode with the first leading electrolyte buffer in the first buffer reservoir; (c) contacting a second electrode with the trailing electrolyte buffer in the second buffer reservoir; and (d) applying an electric field within the fluid apparatus to perform isotachophoresis, wherein the isotachophoresis occurs without direct contact between the tissue sample and the first and second electrodes.

[0030] In some embodiments of the aspects provided herein, the fluid device further includes a third buffer reservoir in fluid communication with the first channel and the first buffer reservoir, the third buffer reservoir containing a lower concentration of the first lead electrolyte buffer than the first buffer reservoir. In some embodiments of the aspects provided herein, the third buffer reservoir and the first buffer reservoir are connected via a second channel comprising one or more capillary barriers to restrict pressure-driven flow within the second channel and between the third buffer reservoir and the first buffer reservoir. In some embodiments of the aspects provided herein, the fluid 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.

[0031] One aspect of this disclosure provides a microfluidic system comprising: (a) a microfluidic chip including a first channel and a first reservoir in fluid communication with the first channel, wherein the first channel and the first reservoir meet at a first connection; and (b) a mechanical member including a first tooth, wherein the mechanical member is configured to apply mechanical pressure to the first channel via the first tooth to at least partially close the first channel and increase the fluid resistance between the first channel and the first reservoir through plastic deformation of at least one wall of the first channel.

[0032] 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 wherein the mechanical component further includes a second tooth configured to apply mechanical pressure to the second channel to plastically close the second channel and prevent fluid communication between the first reservoir and the second reservoir. In some embodiments of the aspects provided herein, the first tooth is configured to deliver mechanical pressure to the first connection 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 tooth is configured to heat the first channel. In some embodiments of the aspects provided herein, the mechanical component comprises a material with a Young's modulus greater than that of the first channel. In some embodiments of the aspects provided herein, the microfluidic system is configured to perform isovelocity electrophoresis. In some embodiments of the aspects provided herein, the first tooth is thermally coupled to a heating element. In some embodiments of the aspects provided herein, the first tooth is heated to a temperature higher than the glass transition temperature of the at least one wall of the first channel. Some embodiments of the aspects provided herein include a method for completing a process in a fluid system, the method comprising using the microfluidic system to at least partially close the first channel by plastic deformation, thereby increasing the resistance to fluid flow between the first channel and the first reservoir. In some embodiments of the aspects provided herein, the first tooth of the mechanical member applies a force of at least 0.25 lbs to the first channel. In some embodiments of the aspects provided herein, the process in the fluid system is isokinetic electrophoresis.

[0033] One aspect of this disclosure provides a method for isotachyphoresis of a sample containing nucleic acids, the method comprising: (a) loading the sample containing nucleic acids into a first reservoir of a microfluidic chip; (b) loading a trailing electrolyte buffer into a second reservoir of the microfluidic chip, wherein the trailing electrolyte buffer contains a first trailing electrolyte ion having an effective mobility less than the effective mobility of the nucleic acid; (c) loading a leading electrolyte buffer into a third reservoir of the microfluidic chip, wherein the third reservoir contains a first leading electrolyte ion having a second effective mobility greater than the effective mobility of the nucleic acid; (d) applying an electric field within the microfluidic chip to perform isotachyphoresis with the first trailing electrolyte ion, the nucleic acid, and the first leading electrolyte ion, thereby confining the nucleic acid or a portion thereof within an isotachyphoresis region; and (e) using a temperature sensor to sense temperature changes in or near the isotachyphoresis region, wherein feedback from the temperature sensor is used to control the electric field.

[0034] In some embodiments of the aspects provided herein, the control of the electric field causes the nucleic acid or a portion thereof to be located in the elution reservoir or region of the microfluidic chip. In some embodiments of the aspects provided herein, the temperature sensor is located within at most 8 mm of the elution reservoir. In some embodiments of the aspects provided herein, the temperature variation is in the range of about 0.2 °C to 5 °C. In some embodiments of the aspects provided herein, the applied electric field causes the leading electrolyte and the trailing electrolyte to meet at an isotachophoretic interface, and the temperature sensor senses the isotachophoretic interface.

[0035] One aspect of this disclosure provides a microfluidic device comprising: (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, wherein the first and second buffer reservoirs are separated by a capillary barrier; and (iii) an elution reservoir in fluid communication with the first fluid channel; (b) a sensor configured to detect temperature changes in the first fluid channel within the first isotachophoresis region; and (c) means positioned to provide current within the first channel within the first isotachophoresis region.

[0036] Some embodiments of the aspects provided herein further include a controller configured to trigger a reduction or elimination of the current when the sensor receives a thermal signal. In some embodiments of the aspects provided herein, the temperature change is an increase in temperature within the range of about 0.2°C to 5°C. In some embodiments of the aspects provided herein, the microfluidic device is further configured to separate nucleic acid samples in the elution reservoir after the sensor detects a temperature change. In some embodiments of the aspects provided herein, the sensing of the nucleic acid is performed using a sensor located at most 8 mm within the elution reservoir. In some embodiments of the aspects provided herein, the first channel comprises a single sensor.

[0037] One aspect of this disclosure provides a kit comprising: (a) the microfluidic device of claim 111, the microfluidic device of claim 165, or the microfluidic chip of claim 128; (b) a tailing electrolyte buffer comprising a tailing electrolyte; and (c) a leading electrolyte buffer comprising a leading electrolyte.

[0038] In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a mixture of at least two electrolytes having different effective migration rates. In some embodiments of the aspects provided herein, the mixture comprises (i) a first electrolyte having an effective migration rate value lower than that of nucleic acids and a higher effective migration rate value than that of contaminants, and (ii) a second electrolyte having an effective migration rate value lower than that of said contaminants. In some embodiments of the aspects provided herein, the first electrolyte comprises hexanoic acid. In some embodiments of the aspects provided herein, the second electrolyte comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid). In some embodiments of the aspects provided herein, the kit further comprises a sample buffer, wherein the sample buffer comprises any combination of a lead electrolyte buffer, a trailing electrolyte buffer, or urea. In some embodiments of the aspects provided herein, the kit further comprises a sample buffer containing urea and thiourea.

[0039] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a fluid device: (i) a tissue sample containing nucleic acids and contaminants, wherein the tissue sample is not an unlysaturated whole blood sample; (ii) a trailing electrolyte buffer containing trailing electrolyte ions having an effective migration rate greater than the effective migration rate of the contaminants and less than the effective migration rate of the nucleic acids; and (iii) a leading electrolyte buffer containing leading electrolyte ions having a second effective migration rate, wherein the second effective migration rate is greater than the effective migration rate of the nucleic acids; and (b) applying an electric field within the fluid device to perform isovelocity electrophoresis with the trailing electrolyte ions, the nucleic acids, and the leading electrolyte ions, thereby purifying the nucleic acids from the contaminants in the tissue sample.

[0040] 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 trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the leading electrolyte ion comprises chloride. In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a second trailing electrolyte ion having a different effective mobility than the first trailing electrolyte ion. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid). In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid. In some embodiments of the aspects provided herein, the second trailing electrolyte ion comprises HEPES, and the first trailing electrolyte ion comprises MOPS. In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a second trailing electrolyte ion having a second effective mobility, wherein the magnitude of the second effective mobility is substantially the same as or less than the magnitude of the effective mobility of the contaminant. In some embodiments of the aspects provided herein, the contaminant is selected from cross-linked nucleic acids, embedding materials, immobilization chemicals, proteins, inhibitors, and combinations thereof. In some embodiments of the aspects provided herein, the contaminant comprises cross-linked nucleic acids. In some embodiments of the aspects provided herein, the tissue sample is combined with the trailing electrolyte buffer prior to loading. In some embodiments of the aspects provided herein, the tissue sample is combined with the lead electrolyte buffer prior to loading. In some embodiments of the aspects provided herein, the loading of the lead electrolyte buffer is performed prior to the loading of the tissue sample. In some embodiments of the aspects provided herein, the method further comprises eluting an output solution containing the purified nucleic acid from an outlet reservoir of the fluid device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about two times higher than the concentration of the nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the concentration of the cross-linked nucleic acid in the output solution is at least about two times lower than the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the output solution does not contain the contaminant.In some embodiments of the aspects provided herein, the tissue sample is fresh tissue. In some embodiments of the aspects provided herein, the tissue sample is fresh frozen (FF) tissue. In some embodiments of the aspects provided herein, the tissue sample is formalin-fixed paraffin-embedded tissue (FFPE). In some embodiments of the aspects provided herein, the method further includes lysing or destroying the tissue sample prior to the loading. In some embodiments of the aspects provided herein, the lysis or destruction is performed using urea or thiourea.

[0041] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading a first channel of a fluid device with: (i) a first tissue sample containing a first nucleic acid and a first contaminant; (ii) a first tailing electrolyte buffer containing a first tailing ion, wherein the effective mobility of the first tailing ion is less than the effective mobility of the first nucleic acid; and (iii) a first leading electrolyte buffer containing a first leading ion, wherein the effective mobility of the first leading ion is greater than the effective mobility of the first nucleic acid; (b) loading a second channel of the fluid device with: (iv) a second tissue sample containing a second nucleic acid and a second contaminant; and (v) [other contaminants]. The device comprises (vi) a second tailing electrolyte buffer containing a second tailing ion, wherein the amount of the second tailing ion is less than the amount of the effective mobility of the second nucleic acid; and (vi) a second leading electrolyte buffer containing a second leading ion, wherein the amount of the effective mobility of the second leading ion is greater than the amount of the effective mobility of the second nucleic acid; and (c) applying an electric field within the fluid device to perform isotachophoresis in the first channel using the first tailing ion, the first nucleic acid, and the first leading ion, and in the second channel using the second tailing ion, the second nucleic acid, and the second leading ion, thereby purifying the first nucleic acid from the first contaminant and purifying the second nucleic acid from the second contaminant.

[0042] In some embodiments of the aspects provided herein, the first trailing electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or a tissue disrupting agent. In some embodiments of the aspects provided herein, the second trailing electrolyte buffer or the second leading electrolyte buffer further comprises a lysing agent or a tissue disrupting agent. In some embodiments of the aspects provided herein, the lysing agent or the tissue disrupting agent comprises one or more agents selected from solutions with a pH greater than about 12, proteases, urea, thiourea, and surfactants.

[0043] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a first zone of a fluid device: (i) a tissue sample containing nucleic acids and contaminants; (ii) a tailing electrolyte buffer containing tailing ions, wherein the effective mobility of the tailing ions is less than the effective mobility of the nucleic acids; and (iii) a leading electrolyte buffer containing leading ions, wherein the effective mobility of the leading ions is greater than the effective mobility of the nucleic acids; and (b) applying an electric field to the fluid device to perform isovelocity electrophoresis in a second zone of the fluid device using the tailing ions, the nucleic acids, and the leading ions, thereby purifying the nucleic acids from the contaminants, wherein during the application of the electric field, the first zone is maintained at a first temperature, and the second zone is maintained at a second temperature different from the first temperature.

[0044] In some embodiments of the aspects provided herein, the trailing electrolyte buffer or the leading electrolyte buffer further comprises a lysing agent or a tissue disrupting agent. In some embodiments of the aspects provided herein, the lysing agent or the tissue disrupting agent comprises one or more agents selected from solutions with a pH greater than about 12, proteases, urea, thiourea, and surfactants. In some embodiments of the aspects provided herein, the first temperature is from about 4°C to about 40°C. In some embodiments of the aspects provided herein, the first temperature is from about 40°C to about 80°C.

[0045] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a first zone of a fluid device: (i) a tissue sample containing nucleic acids; (ii) a tailing electrolyte buffer containing tailing ions, wherein the effective mobility of the tailing ions is less than the effective mobility of the nucleic acids; and (iii) a leading electrolyte buffer containing leading ions, wherein the effective mobility of the leading ions is greater than the effective mobility of the nucleic acids; (b) in the first zone, performing at least one sample preparation selected from the group consisting of: (1) removing embedding material; (2) disrupting tissue; (3) lysing cells; (4) decrosslinking nucleic acids; (5) digesting proteins; and (6) digesting nucleic acids; and (c) applying an electric field within the fluid device to perform isovelocity electrophoresis in a second zone of the fluid device using the tailing ions, the nucleic acids, and the leading ions, thereby purifying the nucleic acids from contaminants in the tissue sample.

[0046] In some embodiments of the aspects provided herein, the removal of the embedding material or the lysed cells comprises incubating the tissue sample in the fluid apparatus at a temperature of at least about 37°C for a duration of at least about 1 minute prior to applying the electric field. In some embodiments of the aspects provided herein, the temperature is from about 40°C to about 80°C. In some embodiments of the aspects provided herein, the duration is from about 1 minute to about 60 minutes. In some embodiments of the aspects provided herein, the destruction of tissue or the lysed cells comprises applying mechanical stress to the sample. In some embodiments of the aspects provided herein, the destruction of tissue or the lysed cells comprises applying heat to the sample. In some embodiments of the aspects provided herein, the application of heat results in a temperature of the tissue sample of about 30°C to about 65°C. In some embodiments of the aspects provided herein, the destruction of tissue or the lysed cells comprises a solution with a pH of at least 12. In some embodiments of the aspects provided herein, the destruction of tissue or the lysed cells comprises proteolytic digestion. In some embodiments of the aspects provided herein, the proteolytic digestion is performed at a temperature greater than about 25°C. In some embodiments of the aspects provided herein, the temperature is from about 30°C to about 65°C. In some embodiments of the aspects provided herein, the destruction of tissue or the lysis of cells comprises applying at least one surfactant to the tissue or the cells. In some embodiments of the aspects provided herein, the destruction of tissue or the lysis of cells comprises applying a solution containing urea to the tissue or the 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 from about 4 M to about 9 M, and the concentration of thiourea in the solution is from about 0.5 M to about 3.5 M. In some embodiments of the aspects provided herein, the concentration of urea in the solution is from about 6.5 M to about 7.5 M, and the concentration of thiourea in the solution is from about 1.5 M to about 2.5 M. In some embodiments of the aspects provided herein, the decrosslinking of nucleic acids comprises digesting crosslinked proteins with proteinase K. In some embodiments of the aspects provided herein, the digestion of nucleic acids is performed using a DNase or an RNase.

[0047] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances onto a fluid device: (i) a tissue sample containing nucleic acids, wherein the tissue sample is embedded or immobilized; (ii) a tailing electrolyte buffer containing a tailing electrolyte having a lower effective migration rate than the nucleic acids; and (iii) a leading electrolyte buffer containing a leading electrolyte having a higher effective migration rate than the nucleic acids; and (b) applying an electric field to the fluid device to perform isovelocity electrophoresis using the tailing electrolyte, the nucleic acids, and the leading electrolyte, thereby purifying the nucleic acids from contaminants in the tissue sample.

[0048] In some embodiments of the aspects provided herein, the contaminant is selected from cross-linked nucleic acids, embedding materials, immobilization chemicals, enzymes, and inhibitors. In some embodiments of the aspects provided herein, the 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 dissected tissue sample. In some embodiments of the aspects provided herein, the dissected tissue sample is a dissected FFPE sample. In some embodiments of the aspects provided herein, the method further includes the step of comparing the characteristics of the nucleic acid with nucleic acids from other samples. In some embodiments of the aspects provided herein, the characteristic is expression level. In some embodiments of the aspects provided herein, the characteristic is 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 includes the step of administering a drug based on the characteristics of the nucleic acid. In some embodiments of the aspects provided herein, the tissue sample is a tumor sample. In some embodiments of the aspects provided herein, the trailing electrolyte buffer has a pH of about 7. In some embodiments of the aspects provided herein, the trailing electrolyte buffer has a pH greater than about 7. In some embodiments of the aspects provided herein, the method further includes incubating the tissue sample in the fluid device at a temperature of at least about 37°C for a duration of at least about 1 minute before applying the electric field. In some embodiments of the aspects provided herein, the temperature is from about 40°C to about 80°C. In some embodiments of the aspects provided herein, the duration is from about 1 minute to about 60 minutes. In some embodiments of the aspects provided herein, the leading electrolyte buffer contains proteinase K. In some embodiments of the aspects provided herein, the method further includes removing protein cross-links from the nucleic acid using the proteinase K. In some embodiments of the aspects provided herein, the method further includes removing protein cross-links from the nucleic acid using heating after applying the electric field. In some embodiments of the aspects provided herein, the method further includes eluting an output solution containing the purified nucleic acid from an outlet reservoir of the fluid device. In some embodiments of the aspects provided herein, the concentration of the purified nucleic acid in the output solution is at least about twice as high as the concentration of the nucleic acid in the tissue sample.In some embodiments of the aspects provided herein, the concentration of the cross-linked nucleic acid in the output solution is at least about two times lower than the concentration of the cross-linked nucleic acid in the tissue sample. In some embodiments of the aspects provided herein, the output solution does not contain the contaminant. In some embodiments of the aspects provided herein, the output solution has a volume equal to or less than about 50 μL. In some embodiments of the aspects provided herein, the tissue sample has a mass of at least about 1 ng. In some embodiments of the aspects provided herein, the tissue sample has a volume less than about 500 μL. In some embodiments of the aspects provided herein, the trailing electrolyte has a higher effective migration rate than the contaminant. In some embodiments of the aspects provided herein, the trailing electrolyte comprises (i) a first ion having a higher effective migration rate than the contaminant, and (ii) a second ion having a migration rate substantially the same as or lower than that of the contaminant. In some embodiments of the aspects provided herein, the isotachophoresis quenches the pH of the tissue sample to about 7. In some embodiments of the aspects provided herein, the method further includes dewaxing the tissue sample prior to loading. In some embodiments of the aspects provided herein, the tissue sample is a historical formalin-fixed paraffin-embedded (FFPE) sample, and the method further includes comparing the characteristics of the nucleic acid with the characteristics of different nucleic acids from different tissue samples. In some embodiments of the aspects provided herein, the method further includes the step of detecting the concentration of the nucleic acid. In some embodiments of the aspects provided herein, the concentration is less than or equal to about 1 picogram per microliter (pg / μL). In some embodiments of the aspects provided herein, the concentration is less than or equal to about 0.5 pg / μL. In some embodiments of the aspects provided herein, the concentration is at least about 1 picogram per microliter (pg / μL).

[0049] One aspect of this disclosure provides a fluid apparatus comprising: a sample purification region including: (a) a first region; (b) a sample inlet located in the first region; (c) a trailing electrolyte reservoir in fluid communication with the first region; (d) a second region in fluid communication with the first region; (e) a leading electrolyte reservoir in fluid communication with the second region; (f) a sample outlet in fluid communication with the second region; (g) a first heater in thermal communication with the first region; and (h) a second heater configured to transfer heat to the second region, wherein the first region and the second region are substantially thermally isolated.

[0050] One aspect of this disclosure provides a fluid apparatus comprising: a sample purification region including: (a) a first region; (b) a sample inlet located in the first region; (c) a trailing electrolyte reservoir in fluid communication with the first region; (d) a second region in fluid communication with the first region; (e) a leading electrolyte reservoir in fluid communication with the second region; (f) a sample outlet in fluid communication with the second region; and (g) a heater in thermal communication with the first region and the second region.

[0051] 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 region is a dewaxing region. In some embodiments of the aspects provided herein, the first region is a destruction region. In some embodiments of the aspects provided herein, the second region is an isotachophoresis region. In some embodiments of the aspects provided herein, the first region or the second region has a width of less than about 1 mm. In some embodiments of the aspects provided herein, the first region or the second region has a width of less than about 0.5 mm.

[0052] One aspect of this disclosure provides a kit comprising the device provided herein, a tailing electrolyte buffer containing a tailing electrolyte, and a leading electrolyte buffer containing a leading electrolyte.

[0053] In some embodiments of the aspects provided herein, the trailing electrolyte buffer comprises a mixture of at least two electrolytes having different effective migration rates. In some embodiments of the aspects provided herein, the mixture comprises (i) a first electrolyte having a lower effective migration rate than nucleic acids and a higher effective migration rate than contaminants, and (ii) a second electrolyte having a lower effective migration rate than said contaminants. In some embodiments of the aspects provided herein, the contaminant comprises cross-linked nucleic acids. In some embodiments of the aspects provided herein, the first electrolyte comprises hexanoic acid. In some embodiments of the aspects provided herein, the second electrolyte comprises HEPES.

[0054] One aspect of this disclosure provides a method for sample purification, the method comprising: (a) loading the following substances into a fluid device: (i) a tissue sample containing nucleic acids; (ii) a trailing electrolyte buffer containing trailing electrolyte ions having a first effective mobility, wherein the magnitude of the first effective mobility is lower than the magnitude of the effective mobility of the nucleic acids; (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir containing first leading electrolyte ions having a second effective mobility, wherein the magnitude of the second effective mobility is greater than the magnitude of the effective mobility of the nucleic acids; and ( (iv) a second lead electrolyte buffer in a second lead electrolyte reservoir, the second lead electrolyte buffer containing a second lead electrolyte ion having a third effective mobility, wherein the magnitude of the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acid, wherein the first lead electrolyte buffer is different from the second lead electrolyte buffer; (b) isotachophoresis is performed for the first time using the trailing electrolyte ion, the nucleic acid and the first lead electrolyte ion to purify the nucleic acid from the contaminant in the tissue sample; and (c) isotachophoresis is performed for the second time using the trailing electrolyte ion, the nucleic acid and the second lead electrolyte ion.

[0055] In some embodiments of the aspects provided herein, the second isotachophoresis includes changing the applied current from a first channel to a second channel. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, and wherein the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is different from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer. In some embodiments of the aspects provided herein, the concentration of the first leading electrolyte ion in the first leading electrolyte buffer differs from the concentration of the second leading electrolyte ion in the second leading electrolyte buffer by at least 1.5 times. In some embodiments of the aspects provided herein, the first leading electrolyte ion is different from the second leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, and wherein 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 wherein the first leading electrolyte buffer contains a third leading electrolyte ion. In some embodiments of the aspects provided herein, the first leading electrolyte ion is the same as the second leading electrolyte ion, and the concentration of the first leading electrolyte ion in the first leading electrolyte buffer is the same as the concentration of the second leading electrolyte ion in the second leading electrolyte buffer, and the second leading electrolyte buffer contains a third leading electrolyte ion. In some embodiments of the aspects provided herein, the method further includes collecting the nucleic acid in the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the method further includes removing the nucleic acid from the second leading electrolyte reservoir. In some embodiments of the aspects provided herein, the trailing electrolyte buffer is loaded into a trailing electrolyte reservoir, which is separated from the first and second leading electrolyte reservoirs. In some embodiments of the aspects provided herein, the first and second isotachyphoresis are performed by applying an electric field. In some embodiments of the aspects provided herein, the first and second isotachyphoresis are performed by applying more than one electric field.

[0056] One aspect of this disclosure provides a fluid apparatus comprising: a sample purification region including: (a) a channel comprising a first region and a second region in fluid communication with the first region; (b) a sample inlet, a tail electrolyte reservoir comprising a tail electrolyte buffer and a first lead electrolyte reservoir comprising a first lead electrolyte buffer, each in fluid communication with the first region; and (c) a second lead electrolyte reservoir comprising a second lead electrolyte buffer in fluid communication with the second region, wherein the second lead electrolyte buffer is different from the first lead electrolyte buffer.

[0057] 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 pre-lead electrolyte buffer contains a pre-lead electrolyte co-ion different from the first pre-lead electrolyte buffer. In some embodiments of the aspects provided herein, the first pre-lead electrolyte buffer contains a first pre-lead electrolyte ion, and the second pre-lead electrolyte buffer contains a second pre-lead electrolyte ion identical to the first pre-lead electrolyte ion, wherein the concentration of the first pre-lead electrolyte ion in the first pre-lead electrolyte buffer is different from the concentration of the second pre-lead electrolyte ion in the second pre-lead electrolyte buffer. In some embodiments of the aspects provided herein, the first pre-lead electrolyte buffer contains a first pre-lead electrolyte ion, and the second pre-lead electrolyte buffer contains a second pre-lead electrolyte ion, wherein the concentration of the first pre-lead electrolyte ion in the first pre-lead electrolyte buffer differs from the concentration of the second pre-lead electrolyte ion in the second pre-lead electrolyte buffer by at least 1.5 times. In some embodiments of the aspects provided herein, the first pre-lead electrolyte buffer contains a first pre-lead electrolyte ion, and the second pre-lead electrolyte buffer contains a second pre-lead electrolyte ion different from the first pre-lead electrolyte ion. In some embodiments of the aspects provided herein, the first pre-lead electrolyte buffer contains a first pre-lead electrolyte ion, and the second pre-lead electrolyte buffer contains a second pre-lead electrolyte ion that is the same as the first pre-lead electrolyte ion, wherein the concentration of the first pre-lead electrolyte ion in the first pre-lead electrolyte buffer is the same as the concentration of the second pre-lead electrolyte ion in the second pre-lead electrolyte buffer, and wherein the first pre-lead electrolyte buffer contains a third pre-lead electrolyte ion.

[0058] One aspect of this disclosure provides a method comprising: (a) providing an electrofluidic apparatus including a reservoir in fluid communication with a channel; (b) loading a sample volume into the reservoir; (c) moving at least 50% of the sample volume from the reservoir to the channel; and (d) applying an ion current through the channel.

[0059] In some embodiments of the aspects provided herein, the movement is carried out by means of gravity. In some embodiments of the aspects provided herein, the ion current substantially does not pass through the reservoir. In some embodiments of the aspects provided herein, the at least 50% of the sample volume includes at least 80% of the sample volume. In some embodiments of the aspects provided herein, the sample volume contains nucleic acids. In some embodiments of the aspects provided herein, the sample volume contains tissue samples. In some embodiments of the aspects provided herein, the sample volume contains formalin-fixed paraffin-embedded (FFPE) samples. In some embodiments of the aspects provided herein, the application of the ion current includes performing isotachyplastic electrophoresis (ITP).

[0060] Other aspects and advantages of this disclosure will become apparent to those skilled in the art based on the following detailed description, which only shows and describes illustrative embodiments of the disclosure. It will be appreciated that this disclosure is capable of having other different embodiments and that modifications can be made to certain details in various obvious aspects, all without departing from this disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0061] Incorporation

[0062] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by reference. Attached Figure Description

[0063] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings, which illustrate embodiments utilizing the principles of the invention, in which:

[0064] Figure 1A An exemplary protocol for sample processing and nucleic acid extraction or purification is shown.

[0065] Figure 1B An exemplary scheme for automating sample processing and nucleic acid extraction or purification is shown.

[0066] Figure 2A An exemplary schematic diagram shows the separation and purification of DNA and RNA from contaminants by medium-speed electrophoresis.

[0067] Figure 2B An exemplary schematic diagram illustrates the isochronous electrophoresis separation and purification of nucleic acids from paraffin and other possible sample contaminants, simultaneously with protease-mediated tissue disruption and decrosslinking of nucleic acids.

[0068] Figure 3 Exemplary results of DNA extraction and purification by automating isotachophoresis in a fluid apparatus are shown, compared to exemplary results from a typical solid-phase column extraction kit.

[0069] Figure 4A Exemplary results of unbiased (e.g., relative to sequence) extraction of GC-rich and AT-rich synthetic DNA oligonucleotides mixed in proportion to sample concentrations using isotachophoresis are shown.

[0070] Figure 4B Exemplary results of DNA molecular weight gradient purification before and after purification using isovelocity electrophoresis with no bias (e.g., relative to size or molecular weight) are shown; a comparison with two solid-phase column-based nucleic acid purification methods is also shown.

[0071] Figure 5A An exemplary schematic diagram of a channel with a sample preparation area and an isotachophoresis purification area is shown.

[0072] Figure 5B An exemplary fluid device cassette is shown, comprising eight parallel fluid channels and a reservoir for simultaneously processing up to eight samples (e.g., ...). Figure 5A (As shown).

[0073] Figure 5C It shows how to use such Figure 5B An exemplary top view of a channel of the fluid device box and its connected reservoir is shown, further illustrating the channel through which external pressure or vacuum is applied to the fluid device box using a gas port.

[0074] Figure 5D As shown Figure 5B An exemplary side view of the fluid device box shown.

[0075] Figure 5E As shown Figure 5B An exemplary end view of the fluid device box shown.

[0076] Figure 6A An exemplary top view of a fluid device box is shown.

[0077] Figure 6B An exemplary side view of a fluid device box is shown.

[0078] Figure 6C An exemplary bottom view of the fluid device box is shown.

[0079] Figure 6D An exemplary three-dimensional top-view schematic diagram of a fluid device box is shown.

[0080] Figure 7A An exemplary top view of a fluid device box is shown.

[0081] Figure 7B An exemplary side view of a fluid device box is shown.

[0082] Figure 7C An exemplary bottom view of the fluid device box is shown.

[0083] Figure 7D An exemplary three-dimensional bottom-view full-view schematic diagram of a fluid device box is shown.

[0084] Figure 8A An exemplary top view of a fluid device box is shown.

[0085] Figure 8B An exemplary side view of a fluid device box is shown.

[0086] Figure 8C An exemplary bottom view of the fluid device box is shown.

[0087] Figure 8D An exemplary three-dimensional bottom-view full-view schematic diagram of a fluid device box is shown.

[0088] Figure 9A As shown Figure 5B The diagram shown is an exemplary schematic of a fluid device housing containing eight parallel channels.

[0089] Figure 9B An exemplary schematic diagram of two thermal controllers is shown, each thermal controller being coupled with, as shown in the diagram. Figure 9A The regions of the eight parallel channels shown are aligned.

[0090] Figure 10A An exemplary gas channel is shown, which may include a capillary barrier.

[0091] Figure 10B yes Figure 10A An enlarged schematic diagram of the gas channel.

[0092] Figure 11 An exemplary low-loss sample reservoir is shown.

[0093] Figure 12A An exemplary mechanical component is shown, which can be used to apply pressure to close the passage of a fluid device.

[0094] Figure 12B An exemplary comb-shaped mechanical component is shown.

[0095] Figure 12C The alignment of channels in comb-shaped mechanical components and fluid devices is shown.

[0096] Figure 13A An exemplary benchtop device for automating sample preparation and isotachophoresis on a fluid device box is shown.

[0097] Figure 13B An exemplary computer control system is shown that is programmed or otherwise configured to implement the methods provided herein.

[0098] Figure 14 Exemplary results are shown of fluorescence-based measurement and quantification of a titration series of nucleic acids using isotachophoresis.

[0099] Figure 15 A schematic diagram of an exemplary design is shown, comprising a connected reservoir, a non-contact electrode (which can be used as a conductivity sensor), and a fluid channel for automatically loading fluid into the channel / device and for automating isokinetic electrophoresis.

[0100] Figure 16 A graph showing the voltage measurements in the ITP channel over time during operation is shown.

[0101] Figure 17 Two from Figure 16 A graph showing the derivative analysis of the voltage measurements.

[0102] Figure 18 An example of conductivity measurements over time in the ITP channel near the elution reservoir is shown.

[0103] Figure 19 An exemplary schematic diagram of a C4D sensor implementation is shown.

[0104] Figure 20A An exemplary temperature map of an ITP channel, captured using a thermal imaging camera, is shown.

[0105] Figure 20B It shows in Figure 20A A graph showing the temperature at the cursor position 1 over time.

[0106] Figure 21 The graph shows the temperature measurements and temperature derivatives over time during ITP operation.

[0107] Figure 22A An exemplary schematic diagram of a vertical (or columnar) ITP setup is shown.

[0108] Figure 22B An exemplary image of a vertical ITP setup with DNA ITP bands is shown.

[0109] Figure 23Exemplary images and corresponding fluorescence intensity traces are shown of the extraction and separation of amplifiable (e.g., uncrosslinked) DNA from crosslinked DNA in FFPE samples using isotachophoresis.

[0110] Figure 24A An exemplary image is shown showing DNA extraction and purification from an FFPE sample using isodynamic electrophoresis.

[0111] Figure 24B Exemplary DNA yields, measured by quantitative PCR of DNA extracted and purified from FFPE samples using isospeed electrophoresis, are shown compared to exemplary results from a typical solid-phase column extraction kit.

[0112] Figure 25A An image of a single-channel ITP chip loaded with nucleic acids (RNA extracted and digested from human cells) stained with useful dyes is shown for visualization.

[0113] Figure 25B An image of a single-channel ITP chip loaded with nucleic acids (RNA extracted and digested from human cells) stained with useful dyes is shown for visualization.

[0114] Figure 26A Images of RNA ITP bands in the chip channels during purification are shown.

[0115] Figure 26B Images of total nucleic acid ITP bands in the chip channels during the purification process are shown.

[0116] Figure 26C It shows Figure 26A The image shows an electrophoresis diagram of the RNA mass of the sample.

[0117] Figure 26D It shows Figure 26B The image shows an electrophoresis diagram of the RNA mass of the sample.

[0118] Figure 27A Results for DNA yield (ng) of ITP (square) as a function of the percentage of whole blood volume in the starting sample are shown compared to column (rhomboid, Qiagen QiaAmp) extraction from mouse whole blood.

[0119] Figure 27B An image of total nucleic acids in the ITP bands during ITP purification of lysed mouse whole blood on a chip is shown.

[0120] Figure 27C and Figure 27DImages of the ITP chip channels under white light and fluorescence overlays are shown, illustrating the physical separation of heme in the sample / lead electrolyte channels from the elution channels and reservoirs before and after ITP purification of 50% volume of whole blood lysate. Nucleic acids were stained with green dye for visualization in the elution wells. Figure 27C The chip prior to ITP is shown (blood lysate and ITP buffer loaded in the chip; buffer only in the elution wells). Figure 27D The image shows the chip after ITP (blood lysate and ITP buffer loaded on the chip; purified DNA in the elution wells).

[0121] Figure 27E The chip is shown after ITP purification (50% volume of blood).

[0122] Figure 27F The chip is shown after ITP purification (25% volume of blood).

[0123] Figure 28 The results of high molecular weight DNA purification by ITP are shown compared to solid-phase extraction.

[0124] Figure 29A A fluid device containing eight closed channels is shown.

[0125] Figure 29B An enlarged microscopic view of the second channel closure position adjacent to the elution reservoir of each channel is shown.

[0126] Figure 29C The calculated percentage of closure is shown as a function of the force applied to the fluid device.

[0127] Figure 29D The conductivity measurements with the channel closed are shown.

[0128] Figure 30 The graph shows the voltage measurements and voltage derivatives over time during ITP operation.

[0129] Figure 31A The image shows a micrograph of an ITP band with focused DNA in each of eight samples in the sample channel area of ​​the device.

[0130] Figure 31B The independent voltage signal data of each of the eight channels over time under a fixed current are shown.

[0131] Figure 31C Photomicrographs of the same eight ITP bands of focused DNA from a sample eluted in an elution reservoir are shown.

[0132] Figure 31D It is used for Figure 31B The image shows an amplified portion of the triggered voltage tracking (monitoring). Detailed Implementation

[0133] Overview

[0134] Sample preparation is the first step in almost all genomics and transcriptomics analyses and can be a major source of variability. Sample preparation can also be manual-intensive, especially when the samples are formalin-fixed paraffin-embedded (FFPE) samples containing cross-linked proteins.

[0135] This disclosure provides methods and apparatus for improving the efficiency of nucleic acid extraction and purification from tissue and cell samples, including samples that have been treated in some way, such as paraffin-embedded samples or chemically fixed samples (e.g., FFPE samples, samples containing solid tissue). The methods provided herein include preparing such treated samples on or off a chip prior to isotachyphoresis using methods incorporating leading and trailing electrolyte ions. In some cases, the method includes treating (e.g., by removing embedding material, lysis, enzymatic destruction) the fixed solid tissue in a trailing or leading electrolyte buffer prior to isotachyphoresis of the sample. The method may also include using a second leading electrolyte buffer of lower ionic strength to produce a sample compatible with downstream processes such as amplification or other enzymatic assays. The apparatus and systems provided herein include devices suitable for isotachyphoresis of tissue-derived samples, including microfluidic devices with parallel processing features and an automated feedback control mechanism that may include a thermal sensor detecting temperature changes within the sample processing channel.

[0136] The methods and apparatus of this disclosure provide improved nucleic acid recovery from samples, particularly from low-abundance samples (e.g., less than 100 ng of nucleic acids), samples with relatively large volumes (e.g., total volume greater than 25 μl, 50 μl, 100 μl, or more), or liquid samples containing solid particles. The methods and apparatus provided herein also offer high reproducibility and reduced bias from short nucleic acids. The apparatus provided herein integrates sample preparation (e.g., removal of crosslinking or embedding materials) and nucleic acid extraction operations into a single device. The apparatus and methods of this disclosure also provide compatibility with process automation, integration with downstream processes, integration with online quantification (e.g., at single-pico resolution), and / or integration with nucleic acid length and sequence distribution analysis.

[0137] The methods described herein are generally isotachophoresis methods performed under conditions suitable for extracting nucleic acids from certain samples, particularly FFPE samples. In some cases, the disclosed methods include isotachophoresis using a trailing electrolyte buffer containing at least two ions with different effective mobility values. The method may also include isotachophoresis using two different leading electrolyte buffers (one of which can be used as a sample elution buffer). The method may include process automation and parallel processing of multiple samples.

[0138] This disclosure also includes protocols for using buffer and spacer chemicals. These buffer and spacer chemicals may include the use of a variety of electrolytes for ITP. For example, a trailing electrolyte may comprise a mixture of electrolyte types capable of separating non-crosslinked nucleic acids from crosslinked nucleic acids, while simultaneously separating non-crosslinked nucleic acids or both crosslinked and non-crosslinked nucleic acids from contaminants within the sample.

[0139] The apparatus described herein includes injection fluidization apparatus having parallel sample processing channels capable of multiplexing ITP and ITP apparatus having two or more regions connected to a thermal device. The techniques of this disclosure can employ ITP to simultaneously collect, purify, and focus extracted RNA and DNA to quantify total nucleic acids extracted on a chip (e.g., by online ITP-assisted concentration to very small volumes or labeling with inserted fluorescent dyes) and deliver nucleic acids downstream to parallel output reservoirs compatible with robotic pipetting.

[0140] The techniques disclosed herein enable the purification of sample materials (e.g., nucleic acids) without binding the sample material to a solid support. The techniques disclosed herein also enable the purification of sample materials (e.g., nucleic acids) without using liquid-phase extraction. This allows purification to be performed without relying on differences in solubility.

[0141] The operation of the devices disclosed herein can be automated, largely automated, or partially automated. In some cases, the methods of this disclosure involve only a single out-of-chip mixing step of dispensing a sample (e.g., the FFPE portion) into a solution (e.g., an alkaline solution, a lysis solution, or a buffer solution containing urea and / or thiourea), followed by loading the sample into a reservoir of a fluid device for further on-device sample preparation (e.g., dewaxing, tissue disruption and cell lysis, protease digestion, protein hydrolysis, or other treatments, including protein denaturation or nuclease digestion) and nucleic acid extraction, purification, enrichment, online quantification, and sorting or grading by size (e.g., size selection). In some cases, the methods of this disclosure include dispensing samples (e.g., FFPE fractions or other tissue samples) into reservoirs or channels of a fluid device (e.g., a cartridge) pre-filled with solutions (e.g., alkaline solutions, lysis solutions, or buffer solutions containing urea and / or thiourea) for on-board sample preparation (e.g., dewaxing, tissue disruption and cell lysis, protease digestion or other treatments, including protein denaturation or nuclease digestion) and nucleic acid extraction, purification, enrichment, online quantification, and size sorting or grading (e.g., size selection). In some cases, the methods of this disclosure include disrupting tissue and / or lysing cells in samples outside the chip, followed by loading samples (which may be homogeneous or may be a heterogeneous mixture of lysed solid tissue and nucleic acids) into reservoirs of a fluid device for further on-board sample preparation (e.g., dewaxing, protease digestion or other treatments, including protein denaturation or nuclease digestion) and nucleic acid extraction, purification, enrichment, online quantification, and size sorting or grading (e.g., size selection). Nuclease digestion may include removing DNA for DNA-free RNA extraction or removing RNA for RNA-free DNA extraction. The fluid apparatus described herein can be used with benchtop systems to automate electric field-based methods for extracting DNA and RNA from samples.

[0142] The devices disclosed herein include systems that automate and integrate on-chip heating (e.g., temperatures from 37°C to 80°C), sample preparation (e.g., dewaxing, tissue disruption, and cell lysis), buffer replacement, nucleic acid extraction and purification, enrichment of non-crosslinked or amplifiable nucleic acids (e.g., by separating them and delivering them separately from crosslinked nucleic acids), and delivery of purified nucleic acids to output reservoirs (such as arrays compatible with manual or robotic pipetting). For example, this disclosure includes a standard, robotically automated, microtiter-compatible eight-channel box and an integrated benchtop controller prototype that provides automated control of the loading of buffers and other fluids, application of temperature and electric fields to the device, and automated start and end of parallel sample processing. The system can be readily modified in the future to provide higher throughput for larger diagnostic or clinical laboratories (e.g., 96-well sample formats), as needed.

[0143] For example, Figure 1A A schematic diagram of an exemplary process for sample processing and nucleic acid extraction using the techniques of this disclosure is shown. A sample (101) can be provided and subjected to any pretreatment step 102, such as mixing with a buffer, lysis, or removal of embedding material (if present). The sample (and, for example, buffer) can then be loaded onto a fluid device (103). Sample preparation steps (104) can then be performed on the fluid device, such as removal of embedding material (if present and if not previously removed during pretreatment), tissue disruption, cell lysis, protein or proteolytic digestion, and, for example, nuclease digestion. Isotachyphoresis (105) can then be performed to separate and purify nucleic acids from contaminants within the sample, such as cell debris, embedding material, cross-linked nucleic acids, fixatives such as formalin, inhibitors, enzymes such as digestive enzymes, or restriction enzymes. Other steps can occur concurrently with isotachyphoresis, such as decrosslinking of cross-linked nucleic acids (e.g., using heating or protease digestion). Nucleic acids can be detected and quantified during or after isotachyphoresis (106). Once extracted or purified, nucleic acids can be eluted and recovered from the device (107).

[0144] Figure 1BAn exemplary processing workflow for automating ITP is illustrated. In step 110, a scheme can be selected, such as by using a graphical user interface on a benchtop device. This user interface software can enable ease of use or manual operation-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., an optical barcode, RFID chip) associated with the sample or fluid device chip, which can indicate the scheme to be performed. In step 111, the instrument cover can be opened (e.g., manually or automatically by a motor). The motorized cover opening is compatible with robotic laboratory automation. In step 112, the user can load the chip (e.g., a fluid device) onto the benchtop instrument. The chip may contain a monolithic, multichannel SLAS standard microtiter plate (MTP) footprint for automated ITP. In step 113, the ITP liquid can be loaded into the chip wells. Reservoirs for the ITP fluid and user samples can be designed for easy loading, such as via a multichannel pipette (e.g., in the form of a 9mm pitch SLAS standard microtiter plate). The geometry of the channel connecting the reservoir to the ITP channel (e.g., a capillary barrier) can resist gravity flow or wetting of liquid into the channel prior to operation. These structures prevent fluid from defining locations within the ITP channel (including establishing the leading electrolyte / tailing electrolyte interface) and enable bubble-free loading. In some cases, pneumatic actuation can be applied to activate the channel prior to operation. Chip materials can be selected to prevent or resist fluid wetting or wicking into the channel (e.g., plastics with hydrophobic properties or high contact angles). The user can load ITP reagents and buffers onto the chip (e.g., five different fluids); alternatively, the chip can be pre-loaded with reagents. In step 114, the user or device can close the device cap. Sample loading can be actuated via a gas or air port on the chip. Wetting and / or gravity flow can be used to fill the channel with liquid, for example, without active pressure application.

[0145] In step 115, the instrument can apply pressure to load fluid into the chip, thereby activating the channel. In step 116, the device can check whether the channel has been properly activated. For example, optical (e.g., reflective), electrical, pressure, and / or flow rate sensors can be used to check whether the fluid has been loaded into the correct position within the chip. Sensors and device software enable real-time monitoring and control of liquid loading. ITP reagent and buffer loading can be performed before loading the sample onto the chip to avoid wasting sample material in case of incorrect loading. If the channel is not properly activated, the device can issue an error report (130). In step 117, the device cap can be opened. In step 118, the sample can be loaded onto the device. Sample loading can be performed manually by the user or can be performed automatically, such as by a laboratory automation robot. Other sample preparation steps can also be performed. For example, paraffin-embedded samples (e.g., FFPE) can be loaded, and the device can then control the temperature within the sample reservoir to dewax the sample. In step 119, the device cap can be closed. In step 120, the device can perform a self-test. For example, electrical feedback from device electrodes coupled to on-chip storage can be used for self-testing of successful liquid initiation (e.g., bubble detection). Optical sensors can be used to provide feedback on the liquid initiation status (e.g., whether the liquid has reached a designated capillary barrier). Other sensing mechanisms, such as those disclosed herein, can also be used. If the self-test determines that the device has not been properly initiated, the device can issue an error report (131).

[0146] In step 121, ITP-based purification can be performed. Feedback control and process timing using sensors (e.g., triggers) as described herein can be used to control ITP purification and / or automate it. The device can determine whether purification has been successfully performed, and if not, it can issue an error report (132). In step 122, sensors on the device (e.g., optical sensors) can be used to quantify the sample, for example by fluorescence, UV, or other optical detection. Sample size classification can also be performed. If the device determines that the sample has not been properly quantified or detects other problems, it can issue an error report (133). In step 123, changes in conductivity can be detected, which can be used to indicate the timing for ending the ITP run (e.g., when nucleic acids reach a designated elution location or reservoir). Other detection methods described herein (such as temperature or drive voltage) can also be used to determine the run end timing or other triggers. For example, temperature or voltage sensors can be used to control the electric field applied to channels within the device to automate the ITP process. As an example, an electric field can be applied to a channel to initiate ITP purification. Sensed voltage changes can be used to trigger the initiation of temperature or other sensing at a fixed location within the channel (such as at or near the elution reservoir). The voltage can change as the ITP region containing confined nucleic acids moves. Changes in the ITP region, indicating a decrease in the cross-sectional area of ​​the channel, can be sensed by a voltage sensor, and feedback can be used to alter the electric field, for example, by reducing the applied current. Temperature changes can be detected when the ITP region passes at or near the elution reservoir by a temperature sensor, and feedback from the sensor can be used to control the electric field, for example, by removing it to terminate the ITP run. In step 124, the device can terminate the run, for example, based on a trigger signal. When the ITP run terminates, nucleic acids can be located or isolated within the elution reservoir or region. In step 125, the device can close the channel, which can fix the elution volume to maintain a constant elution volume (e.g., by resisting or preventing flow into the elution reservoir or outlet reservoir during removal of the elution volume). Fixing the elution volume can improve ease of use and can help in reporting the concentration of eluted sample material. In step 126, the device cover may be opened (e.g., by the user or automatically).

[0147] In step 127, the purified sample can be extracted from the device. As discussed herein, the chip and / or device can be designed for a given elution volume. Removal of the purified material from the device can be performed by pipetting or otherwise removing the material from the chip. Alternatively, sample extraction can be performed by engaging the ITP chip with another fluidic chip or system (e.g., without an elution reservoir). The purified sample material can then be subjected to further operations using other fluidic systems, such as next-generation sequencing (NGS) library preparation, sample analysis (e.g., PCR, ddPCR), other sequencing operations, or other downstream processes. In step 128, the device can report quantitative data about the sample, such as sample volume and / or sample concentration. The device may contain algorithms or other software for converting measurements (e.g., fluorescence signals) into sample quantification and can report this data to the user. In step 129, the process ends.

[0148] These issues may be particularly important for resolving precious, hard-to-collect, or low-abundance samples (e.g., samples containing less than 100 ng of nucleic acids or containing low abundance of undamaged or uncrosslinked nucleic acids). For such samples, current protocols may lack reproducibility, introduce sample material loss, introduce bias towards short or long nucleic acid targets, introduce bias towards nucleic acid target sequences, and / or lack reproducibility. Such protocols may also lack compatibility with process automation or downstream analysis. Current protocols for nucleic acid preparation may include liquid phase extraction (LPE) such as phenol-chloroform extraction or Trizol extraction, and solid phase extraction (SPE). SPE-type methods may use structures including filled beads, monolithic porous structures, and / or magnetic beads. In some cases, LPE and SPE-type methods can lead to mechanical shearing during processing, which may cause fragmentation and / or reduce the yield of long or high molecular weight nucleic acids.

[0149] The isotachophoresis method and apparatus described herein are particularly well-suited for extracting nucleic acids from lysates of solid or semi-solid tissues. Solid-phase extraction (SPE) techniques typically process lysates by pumping the entire lysate sample volume through a column to selectively adsorb nucleic acids onto the column surface. This pumping of complex lysates (which may contain liquid-particle mixtures) through a porous column can lead to column clogging or fouling, thereby reducing the efficiency of nucleic acid extraction. In contrast, the isotachophoresis method and apparatus described herein typically do not involve pumping or “filtering” the entire lysate sample volume through a column. Instead, an electric field can be applied to the lysate to cause charged, solvated nucleic acids dispersed throughout the composite sample lysate to migrate through and out of the continuous liquid phase of the sample. Nucleic acids can contain relatively high electrophoretic mobility values ​​relative to other solutes, debris, or contaminants in the sample lysate. Solutes in the sample may have relatively low electrophoretic mobility and be too low to focus on the isotachophoretic region located at the interface between the leading and trailing electrolytes. Applying an electric field can induce nucleic acid migration while leaving behind particles and / or other tissue debris (including, for example, cell debris, unlysed cells, or tissues that can connect cells to other cells). Therefore, the isotachophoresis method and apparatus presented herein are well-suited for extracting charged solvated nucleic acids from complex lysed solid tissue samples without having to process the entire mixture through a column as in SPE.

[0150] As used herein, “particle” can refer to a component of a sample mixture or a mixture of sample lysates that is a phase distinct from the continuous liquid phase of the sample (e.g., an aqueous solution). Particles can be non-liquid components of a sample mixture. Particles can be, for example, suspended solid particles or colloids suspended in the sample. Such particles can have a variety of characteristic length scales ranging from about 1 nanometer (nm) to about 1 millimeter (mm). In some cases, particles may not be single-celled organisms or cells.

[0151] Compared to typical SPE methods, the isotachophoresis method and apparatus presented in this paper can provide a reduced strain rate as the sample moves through the channel. In some cases, the strain rate of the method and apparatus presented in this paper is less than about 250 s⁻¹. -1 500s -1 750s -1 1000s -1 2000s -1 3000s -1 4000s -1 5000s -1 6000s -1 7000s -1 8000s -1 9000s -1 Or 10,000s -1In some cases, the strain rate of the methods and apparatus presented in this paper is greater than approximately 250 s. -1 500s -1 750s -1 1000s -1 2000s -1 3000s -1 4000s -1 5000s -1 6000s -1 7000s -1 8000s -1 9000s -1 Or 10,000s -1 In some cases, the method presented in this paper can be performed without centrifugation.

[0152] Isotachyphoresis chemical process and operation

[0153] Figure 2A An exemplary schematic diagram of an isotachophoresis (ITP) process for purifying nucleic acids is shown. A sample 201 (e.g., a lysed solid tissue sample) containing nucleic acids (DNA and RNA) 202 and contaminants 203 is loaded with trailing electrolyte (TE) 204 and introduced into an isotachophoresis channel 200 containing a leading electrolyte (LE) 205. Under the influence of an electric field 220 applied to the isotachophoresis channel 210, nucleic acids 212 migrate away from contaminants 213. The electric field also causes trailing electrolyte 214 to migrate through the channel, which is normally located behind the nucleic acids, and causes leading electrolyte 215 to migrate through the channel, which is normally in front of the nucleic acids. The effective migration rate of the leading electrolyte is greater than that of the nucleic acids, and in turn, the effective migration rate of the nucleic acids is greater than that of the trailing electrolyte, while the effective migration rate of the trailing electrolyte is greater than that of the contaminants.

[0154] Figure 2BAn exemplary schematic diagram illustrates a process of simultaneously decrosslinking nucleic acids and separating them from crosslinked nucleic acids and contaminants (e.g., paraffin) using isotachyphoresis (ITP) on a fluid apparatus. In some cases, the contaminants may contain crosslinked nucleic acids. Paraffin-embedded samples are loaded onto the fluid apparatus in an alkaline buffer and incubated for 10–30 minutes at approximately pH 10 and approximately 50°C to approximately 80°C for tissue lysis and initial dewaxing. Incubation can be performed before or simultaneously with isotachyphoresis under an applied electric field. Alternatively, the sample can be loaded into a lead electrolyte buffer. After incubation, at a first time point 240, the sample containing crosslinked nucleic acids 236 and paraffin 237 is located in an ITP channel with a trailing electrolyte 232. In front of the ITP channel, in the lead electrolyte (LE) region 238, are lead electrolyte 231 and proteinase K enzyme 233. At a second time point 250, at 50°C, ITP-driven pH quenching lowers the pH, and proteinase K contacts and decrosslinks the crosslinked nucleic acids, producing uncrosslinked nucleic acids 235, which focus in the ITP region 239 between the trailing and leading electrolytes. The decrease in pH (e.g., in the range of about 10-12 to about 7 (or about 6.5 to about 8.5)) provides an environment suitable for enzyme activity and improved nucleic acid chemical stability. At a third time point 260, proteinase K decrosslinks more nucleic acids, producing free proteins 234, and the decrosslinked nucleic acids further migrate upstream from paraffin, free proteins, and other contaminants. This process can be operated by a fluidic apparatus or automated by a benchtop system.

[0155] In some cases, samples may be loaded in a sample buffer containing a concentration of lead electrolytes 205 and 231, different from the concentration of lead electrolytes 205 and 231 used for isotachophoresis. In other cases, samples may be loaded in a sample buffer containing a second lead electrolyte, different from lead electrolyte 215. The second lead electrolyte may have an effective migration rate greater than the effective migration rate of nucleic acids. The second lead electrolyte may have an effective migration rate less than the effective migration rate of lead electrolyte 215.

[0156] In some cases, the pH of a sample can be quenched by performing isotachophoresis. In some cases, the pH of the sample can be quenched to a range of approximately 6.5 to approximately 8.5 (e.g., approximately 7 or 7.5).

[0157] Various lead and follow-up electrolytes can be used for ITP. Lead electrolytes can be selected to have a higher effective migration rate than the extraction target (e.g., nucleic acid), and follow-up electrolytes can be selected to have a lower effective migration rate than the extraction target. Lead and / or follow-up electrolytes can be present at concentrations from about 10 mM to about 200 mM. Lead and / or follow-up electrolytes can be present at concentrations of about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The leading electrolyte and / or trailing electrolyte may be present at concentrations of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM. The leading electrolyte and / or trailing electrolyte may be present at concentrations of up to about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The leading electrolyte used in a specific ITP condition may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different ion species. The trailing electrolyte used in a specific ITP condition may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different ion species. Different types of ions in the leading electrolyte and / or trailing electrolyte may be present at different concentrations. Different concentrations of ions (e.g., in tailing electrolytes or leading electrolytes) can be selected to manipulate the size of the spacer region. The spacer region can be used to further separate one type of target from another, such as separating uncrosslinked nucleic acids from protein-crosslinked nucleic acids.

[0158] The trailing electrolyte can contain a mixture of ions with different effective migration values. Using a first trailing electrolyte ion with a first effective migration value and a second trailing electrolyte ion with a second effective migration value lower than that of the first ion can be used to separate non-crosslinked nucleic acids from protein-crosslinked nucleic acids, while simultaneously separating both nucleic acids (or at least uncrosslinked nucleic acids) from contaminants. In this case, non-crosslinked nucleic acids may have a larger effective migration value than the first trailing electrolyte ion, the first trailing electrolyte ion may have a larger effective migration value than the crosslinked nucleic acid, and conversely, crosslinked nucleic acids may have a larger effective migration value than the second trailing electrolyte ion, and conversely, the second trailing electrolyte ion may have a larger effective migration value than the contaminant. For example, crosslinked and non-crosslinked nucleic acids can be enriched separately by isovelocity electrophoresis using a leader electrolyte and two trailing electrolytes (such as hexanoic acid as the first ion and HEPES as the second ion).

[0159] Electrolyte ions can also be selected based on acidity (e.g., pKa). Ions with specific pKas can be selected, for example, to induce pH changes along ITP channels. Ions can also be selected for non-electrophoretic reasons, such as compatibility with downstream processes (e.g., enzymatic processes like PCR or next-generation sequencing library preparation). For example, hexanoic acid, MOPS, and HEPES can be selected to achieve good downstream enzymatic compatibility.

[0160] Exemplary leading electrolyte ions include, but are not limited to, hydrochloric acid, acetic acid, 2-chloroisocrotonic acid, salicylic acid, clotrimazole, nicotinic acid, gallic acid, trichlorolactic acid, butyric acid, p-aminobenzenesulfonic acid, benzoic acid, crotonic acid, trichloroacrylate, propionic acid, levulinic acid, sorbic acid, orotic acid, valeric acid, picric acid, 2-naphthalenesulfonic acid, saccharin, dinitrophenol, p-toluenesulfonic acid, aspartic acid, trimethacrylic acid, isohexanoic acid, hexanoic acid, octylsulfonic acid, nitrophenol, GABA, dimethylarsonic acid, trimethylpyruvic acid, ethylmaleic acid, ethylfumaric acid, benzoic acid, heptanoic acid, mandelic acid, cinnamic acid, cresol, glutamic acid, MES, their isomers, and combinations thereof.

[0161] Exemplary trailing electrolyte ions include, but are not limited to, caprylic acid, gluconic acid, vanillic acid, decyl sulfonic acid, aspirin, glucuronic acid, nonanoic acid, benzyl aspartic acid, ascorbic acid, dodecyl sulfonic acid, MOPS (3-(N-morpholino)propanesulfonic acid), dichlorophenol, hexanoic acid, decanoic acid, tyrosine, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), their isomers, and combinations thereof.

[0162] Using mixtures of different trailing electrolyte ions can be used to achieve in-bracket separations (e.g., separation of non-crosslinked nucleic acids from crosslinked nucleic acids and contaminants), improved mobility, compatibility with downstream assays, favorable surface energy or contact angle between fluid and fluid device materials, buffering capacity, and total ion solubility.

[0163] Isotachymeter electrophoresis can quench the pH of a sample to neutral or near-neutral. Ions that affect local pH (e.g., sodium ions (Na+)) can migrate from the sample region during isotachymeter electrophoresis, thereby shifting the pH of the sample region to neutral.

[0164] Isokinetic electrophoresis can be performed within a certain range of voltage, current, and electric field strength. For example, it can be performed at voltages from approximately 100V to approximately 1500V. Isokinetic electrophoresis can be performed at voltages of approximately 100V, 200V, 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1100V, 1200V, 1300V, 1400V, or 15000V. Isokinetic electrophoresis can be performed at voltages of at least approximately 100V, 200V, 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1100V, 1200V, 1300V, 1400V, or 15000V. Isotachyphoresis can be performed at voltages of up to approximately 100V, 200V, 300V, 400V, 500V, 600V, 700V, 800V, 900V, 1000V, 1100V, 1200V, 1300V, 1400V, or 15000V. Isotachyphoresis can be performed at currents of approximately 10nA to approximately 10mA. Isotachyphoresis can be performed at currents of approximately 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA, or 10 mA. Isotachyphoresis can be performed at currents of at least approximately 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA, or 10 mA. Isotachyphoresis can be performed at currents of up to approximately 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA, or 10 mA. Isotachyphoresis can be performed at electric field strengths ranging from approximately 10 V / cm to approximately 100 V / cm.Isotachymetry can be performed at electric field strengths of approximately 10 V / cm, 15 V / cm, 20 V / cm, 25 V / cm, 30 V / cm, 35 V / cm, 40 V / cm, 45 V / cm, 50 V / cm, 55 V / cm, 60 V / cm, 65 V / cm, 70 V / cm, 75 V / cm, 80 V / cm, 85 V / cm, 90 V / cm, 95 V / cm, or 100 V / cm. Isotachymetry can be performed at field strengths of at least approximately 10 V / cm, 15 V / cm, 20 V / cm, 25 V / cm, 30 V / cm, 35 V / cm, 40 V / cm, 45 V / cm, 50 V / cm, 55 V / cm, 60 V / cm, 65 V / cm, 70 V / cm, 75 V / cm, 80 V / cm, 85 V / cm, 90 V / cm, 95 V / cm, or 100 V / cm. Isovelocity electrophoresis can be performed at field strengths of up to approximately 10 V / cm, 15 V / cm, 20 V / cm, 25 V / cm, 30 V / cm, 35 V / cm, 40 V / cm, 45 V / cm, 50 V / cm, 55 V / cm, 60 V / cm, 65 V / cm, 70 V / cm, 75 V / cm, 80 V / cm, 85 V / cm, 90 V / cm, 95 V / cm, or 100 V / cm.

[0165] Isotachyphoresis can be used to concentrate nucleic acids in a sample. The concentration of nucleic acids in the sample can be increased by at least approximately 2, 5, 10, 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, or 1,000,000,000. The operation time for concentrating nucleic acids using isotachyphoresis can be less than or equal to approximately 5 hours, 4.5 hours, 4 hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 10 seconds, or 1 second. In some cases, isotachophoresis can be used to increase the concentration of nucleic acids in a sample by 1,000,000-fold in less than or equal to about 2 minutes. In some cases (e.g., samples from 25 μL of blood lysate), isotachophoresis can be used to increase the concentration of nucleic acids in a sample by 100,000-fold in less than or equal to about 5 minutes.

[0166] The technology disclosed herein can be used to reduce the concentration of cross-linked nucleic acids in a sample. The concentration of cross-linked nucleic acids in the sample can be reduced by at least approximately 2, 5, 10, 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, or 1,000,000,000. Isotachyphoresis can also be used to reduce the concentration of contaminants in a sample. The concentration of contaminants in the sample can be reduced by at least approximately 2, 5, 10, 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, or 1,000,000,000.

[0167] Nucleic acid samples can contain from about 0.1 picograms (pg) to about 25 micrograms (μg). For example, a nucleic acid sample can contain from about 5 pg to about 5 μg. Nucleic acid samples may contain approximately 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 nanogram (ng), 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8ng, 9ng, 10ng, 20ng, 30ng, 40ng, 50ng, 60ng, 70ng, 80ng, 90ng, 100ng, 200ng, 300ng, 400ng, 500ng, 600ng, 700ng, 800ng, 900ng, 1μg , 2μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12μg, 13μg, 14μg, 15μg, 16μg, 17μg, 18μg, 19μg, 20μg, 21μg, 22μg, 23μg, 24μg or 25μg.

[0168] Nucleic acid samples may contain deoxyribonucleic acid (DNA), single-stranded DNA, double-stranded DNA, genomic DNA, complementary DNA, ribonucleic acid (RNA), ribosomal RNA, transfer RNA, messenger RNA, microRNA, etc., or any combination thereof. Nucleic acid samples may contain lengths of at least about 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kB or longer. The techniques of this disclosure can be used to extract different sample types in different channels of a fluidic apparatus. For example, different channels can be used to extract nucleic acids of different lengths and / or different types.

[0169] In some cases, the characteristics of a nucleic acid sample can be compared with one or more nucleic acids from another sample. These characteristics can be, for example, expression levels, nucleic acid sequence, molecular weight, nucleic acid integrity, nucleic acid chain type, or nucleic acid purity.

[0170] Nucleic acid samples may have specific quality before and / or after extraction or other processing. Nucleic acid quality can be assessed by a variety of metrics, including but not limited to RNA integrity number (RIN), DNA integrity number (DIN), size distribution (e.g., using electrophoresis), and amplification capacity (e.g., by PCR) or by other enzymatic treatments (e.g., fragmentation, ligation, α-tailing, or hybridization for next-generation sequencing library preparation). The technology disclosed herein can be used to extract or process nucleic acids and provides RINs of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, Nucleic acids extracted or processed at levels 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. The technology disclosed herein can be used to extract or process nucleic acids, and provides RINs of up to approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, Nucleic acids extracted or processed at levels 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0.The technology disclosed herein can be used to extract or process nucleic acids, and provides DIN values ​​of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, Nucleic acids extracted or processed at levels 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. The technology disclosed herein can be used to extract or process nucleic acids, and provides DIN values ​​of up to about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, Nucleic acids extracted or processed at levels 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0.The technology disclosed herein can be used to extract or process nucleic acids, and provides extracted or processed nucleic acids such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% by mass of nucleic acids in a sample have a molecular weight of at least about 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kB or higher. In some cases, about 90% to about 100% by mass of the processed nucleic acid is about 10 bp to about 1000 bp, about 200 bp to about 2000 bp, or about 200-5000 bp.

[0171] Isotropic electrophoresis can be used to extract nucleic acids at a certain extraction efficiency or yield (described as the percentage of nucleic acids produced from a given starting amount of nucleic acid). The techniques of this disclosure can provide extracted nucleic acids at yields of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%. Even for low input amounts of nucleic acids (including less than or equal to about 10... 4 10 ng 3 ng, 10 2 ng, 10 1 ng, 10 0 ng, 10 -1 ng or 10 -2 The technology disclosed herein can also provide high yields. For example, Figure 3 Exemplary nucleic acid yields are shown for a range of different input amounts and nucleic acid sources. High nucleic acid yields and / or low losses can be important for next-generation sequencing library preparation. Nucleic acid recovery rates can be 100% or close to 100%.

[0172] The techniques disclosed herein can extract nucleic acids with low or no sequence bias. That is, the sequence composition of the extracted and purified nucleic acids (e.g., the ratio of GC-rich nucleic acids to AT-rich nucleic acids) can be similar to or identical to the sequence composition of the input nucleic acids (see, for example, Figure 4A The difference between the sequence composition of the extracted nucleic acid and the sequence composition of the input nucleic acid can be less than or equal to approximately 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0173] The technology disclosed herein can extract nucleic acids with low or no length bias. That is, the length distribution of the extracted nucleic acids (e.g., the proportion of nucleic acids of different sizes) can be similar to or the same as the length distribution of the input nucleic acids (see, for example, ...). Figure 4B The length distribution of the extracted nucleic acids may differ from that of the input nucleic acids by less than or equal to about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, short nucleic acids (e.g., about 10 bp to about 300 bp), long nucleic acids (e.g., about 10 kB, 20 kB, 30 kB, 40 kB, 50 kB, 60 kB, 70 kB, 80 kB, 90 kB, 100 kB or longer), or both short and long nucleic acids can be extracted with reduced shedding or bias. In some cases, solid-phase columns may lose up to 100% of the short and / or long nucleic acid material. The techniques of this disclosure can recover nucleotides ranging in size from a single base to hundreds of kilobases. The technology disclosed herein can recover at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of short and / or long nucleic acids present in a sample.

[0174] The techniques disclosed herein can lead to the removal of contaminants from samples. Contaminants may include, but are not limited to, embedding materials, cell debris, extracellular matrix components, tissue debris, embedding fragments, lipids, carbohydrates, enzymes, ligation byproducts, primers, unbound probes or ligators, divalent metals, detergents, preservatives, fixatives, anticoagulants, collagen fibers, and PCR inhibitors. Contaminants may originate from tissues or cells in the sample, from preservatives or embedding materials used on the sample, or from previous preparations, reactions, or assays performed on the sample. For example, enzymes such as restriction nucleases can be used to prepare DNA for fingerprint analysis assays, and the DNA can be separated from the enzyme after digestion (e.g., DNase digestion).

[0175] sample

[0176] The techniques disclosed herein can be used to process various sample types, including but not limited to biological samples, solid tissues, biopsies, tissue biopsies, liquid biopsies, organs, tumors, fresh tissues, solid organs, preserved tissues (e.g., FFPE), dissected FFPE, fresh frozen tissues, fixed samples, fixed tissues, embedded samples, lysed samples, unlysed samples, samples containing intercellular junctions (e.g., gap junctions, tight junctions, adhesion junctions), samples containing lysed solid tissues and nucleic acids, multiphase samples, heterogeneous liquids or solutions (e.g., tissues, whole blood, or unlysed cell suspensions), biological samples containing genomic DNA, lysed and unlysed whole blood, plasma, and serum, oral swabs, dried blood spots, and other forensic samples, fresh or fresh frozen (FF) tissues, cells cultured or harvested from blood or tissues (lysed and unlysed), fixed cells, feces and bodily fluids (e.g., saliva, urine), or any combination thereof. Non-limiting examples of solid organs include the liver, pancreas, brain, heart, gallbladder, colon, lungs, and reproductive organs. For both eukaryotes and prokaryotes, samples may include cellular nucleic acids and cell-free nucleic acids. Fixed samples may be chemically or physically fixed (e.g., heated or frozen). For example, samples may be chemically fixed using fixatives such as formalin, neutral buffered formalin (NBF), formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, mercuric chloride, zinc salts, Bouin's fluid, ethanol-formaldehyde-acetic acid (AFA or FAA), citrate-acetone-formaldehyde (CAF), acetone, methanol, ethanol, Clarke's solution, Carnoy's fluid, or Puchtler's methicarn. Embedded samples may be embedded in materials including, but not limited to, waxes (e.g., paraffin), agar, gelatin, or plastic resins. Formalin-fixed paraffin-embedded (FFPE) samples can be processed using the techniques described in this disclosure. Samples may include oral swabs, bloodstains, and other forensic samples. Samples may include clinical samples, fine-needle aspirates, biopsies, whole blood, lysed blood, serum, plasma, urine, cell culture lysates or freshly harvested cell lysates (e.g., blood cells, isolated fresh tissue, stem cells), blood cells, circulating cells (e.g., circulating tumor cells (CTCs)), nucleic acids from blood or other body fluids, and other sample categories. Cell-free nucleic acids (e.g., cfDNA or cfRNA) can be recovered using the techniques described in this disclosure, such as from unlysaturated whole blood; typically, cell-free nucleic acids are circulating cell-free nucleic acids. Samples may come from a variety of sources, including but not limited to normal tissues, benign tumors, malignant tumors, stem cells, human tissues, animal tissues, plant tissues, bacteria, viruses, and environmental sources (e.g., water).Human or animal tissues may include, but are not limited to, epithelial tissue, connective tissue (e.g., blood, bone), muscle tissue (e.g., smooth muscle, skeletal muscle, cardiac muscle), and nervous tissue (e.g., brain, spinal cord).

[0177] The sample may contain one or more suspended particles. These particles can range in size from colloidal to visible. The particles may have a particle size of at least about 1 nanometer (nm), 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 950 nm, 1 micrometer (μm), 10 μm, 20 μm, 3 0μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 150μm, 175μm, 200μm, 225μm, 250μm, 275μm, 300μm, 350 μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm or 1 millimeter (mm) size. The one or more particles may have a maximum of about 1 nanometer (nm), 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 600nm, 700nm, 800nm, 900nm, 950nm, 1 micrometer (μm), 10μm, 20μm, 3 The particle sizes are 0 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). The one or more particles can be the same size or different sizes. A sample can, for example, contain multiple particles ranging in size from 1 nm to 500 μm.

[0178] Samples of various volumes can be processed on fluid devices (e.g., for the extraction and purification of nucleic acids). For example, the sample volume (with or without buffer) can be at least about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 175 μL, 200 μL, 225 μL, 250 μL, 275 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. Sample volumes (with or without buffer) can be up to approximately 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. In some cases, sample volumes can be from approximately 1 nL to approximately 10 nL. Sample volumes (with or without buffer) can be at least about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. In some cases, sample volumes can be from about 1 nL to about 10 nL.

[0179] Samples with varying cell numbers can be processed on fluidic devices (e.g., for nucleic acid extraction and purification). For example, samples may contain less than or equal to approximately 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, etc. 00 cells, 1,000 cells, 900 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 samples contained at least approximately 10,000,000 cells, 5,000,000 cells, 1,000,000 cells, 500,000 cells, 100,000 cells, 50,000 cells, 20,000 cells, 15,000 cells, 10,000 cells, 9,000 cells, 8,000 cells, 7,000 cells, 6,000 cells, etc. 1 cell, 5,000 cells, 4,500 cells, 4,000 cells, 3,500 cells, 3,000 cells, 2,500 cells, 2,000 cells, 1,500 cells, 1,000 cells, 900 cells, 800 cells, 700 cells, 600 cells, 500 cells, 400 cells, 300 cells, 200 cells, or 100 cells.

[0180] Samples of varying qualities can be processed on fluidic devices (e.g., for the extraction and purification of nucleic acids). For example, a sample may contain approximately 0.001 mg to approximately 10 mg of tissue. Samples may contain up to approximately 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue. The sample may contain at least about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue. The sample may contain approximately 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue.

[0181] Samples containing varying amounts of nucleic acids can be processed on fluidic instruments (e.g., for the extraction and purification of nucleic acids). For example, samples may contain less than or equal to about 1 microgram (1 μg), 100 nanograms (ng), 10 ng, 1 ng, 100 picograms (pg), 10 pg, or 1 pg of nucleic acid. In some cases, samples may contain greater than or equal to about 1 microgram (1 μg), 100 nanograms (ng), 10 ng, 1 ng, 100 picograms (pg), 10 pg, or 1 pg of nucleic acid.

[0182] Samples can be loaded into a buffer containing a trailing electrolyte or a leading electrolyte. Samples can be loaded into a buffer containing a second leading electrolyte, which is different from the leading electrolyte used for ITP. Samples can be loaded into a buffer such as an alkaline aqueous buffer or a neutral aqueous buffer. Exemplary alkaline solutions or buffers (e.g., for DNA extraction) may contain 30-120 mM NaOH (in some cases, 40-80 mM NaOH) at a pH of about 10-13 (in some cases, with at least one other component). In some cases, when samples are lysed by treatment with an alkaline solution or buffer before being loaded onto the chip, the lysed sample can subsequently be quenched by adding an acidic solution or buffer to bring the pH of the lysed sample to the range of about 7.5 to about 8.5 before isotachophoresis. Exemplary aqueous buffers (e.g., for DNA or RNA extraction) may contain 2-150 mM Tris-HCl (pH about 7 to about 8) or BisTris-HCl (pH about 5.8 to about 7.3), having at least one other component. Other components used in the buffer may include nonionic surfactants or detergents, ionic or zwitterionic surfactants or detergents, dissociation agents, disulfide bond reducing agents, proteases, nucleases, and other additives or components that are digested, denatured, destroyed, or degraded for the purpose of extracting, purifying, enriching, or otherwise isolating nucleic acids.

[0183] Nonionic surfactants or detergents may include, but are not limited to, surfactants from the following categories: octylphenol ethoxylates, polysorbates, poloxamers, or polyoxyethylene. Octylphenol ethoxylate surfactants may include, but are not limited to, branched octylphenoxy polyethylene glycol (IGEPAL CA-630) and tert-octylphenoxy polyethylene glycol (Triton). TM X-100) or other polyethylene oxide chains having aromatic lipophilic or hydrophobic groups. Polysorbate surfactants may include, but are not limited to, polyethylene glycol dehydrated sorbitol monolaurate. Polyethylene glycol dehydrated sorbitan monooleate Or dehydrated sorbitan monooleate Poloxamer surfactants (i.e., block copolymers based on ethylene oxide and propylene oxide) may include, but are not limited to, polyoxyethylene-polyoxypropylene block copolymers. Or polyethylene glycol-polypropylene glycol block copolymer Polyoxyethylene surfactants may include, but are not limited to, nonylphenoxy polyethylene glycol (NP-40).

[0184] Nonionic surfactants or detergents may include, but are not limited to, (For example, Triton TMX-100 NP-40, including Other block copolymers of (e.g., F-68 or F-127), Polyethylene glycol-modified polymers or copolymers. Nonionic surfactants or detergents can be used to reduce or prevent biomolecule adsorption onto channel walls, or to control the wetting and / or surface tension properties of fluids to control sample loading into fluid devices. Nonionic surfactants or detergents can be present at concentrations of about 0.0005-5% v / v or w / v. For example, IGEPAL CA-630 can be used at about 0.05-0.5% v / v. Ionic surfactants or detergents may include, but are not limited to, sodium dodecyl sulfate (e.g., at 0.01-2% w / v), sodium dodecylbenzenesulfonate (e.g., at 0.01-2% w / v), sodium cholesterol sulfate (e.g., at 0.01%-2% w / v), and sodium deoxycholate (e.g., at about 10-1000 mM). The dissociation agent may include, but is not limited to, urea (e.g., about 0.5-9.5M, or in some cases, 5-9.5M), thiourea, butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, lithium chloride, magnesium chloride, phenol, and propanol. For example, 7.0M urea and 2.0M thiourea can be used in a 5-50mM Tris-HCl (in some cases, 10-20mM Tris-HCl) buffer solution for RNA or DNA extraction, or for total nucleic acid extraction. The ratio of urea to thiourea can be at least about 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 6.5:1, 7:1, 7.5:1, or 8:1. Disulfide bond reducing agents may include, but are not limited to, DTT (e.g., at about 0.1-40 mM, or in some cases about 10 mM) and β-mercaptoethanol (e.g., at about 0.5-2%, or in some cases about 1%). Proteases may include, but are not limited to, proteinase K, proteases, endopeptides (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Nucleases may include, but are not limited to, nonspecific nucleases such as DNases including DNase I (e.g., for preparing DNA-free RNA extracts) and RNases (e.g., RNase A, RNase T) or combinations thereof (e.g., for preparing RNA-free DNA extracts). Nucleases may also include specific nucleases (e.g., restriction enzymes) that can cleave specific nucleic acid sequences and produce predictable fragment sizes and fragment size distributions. In some cases, one or more methods or processes provided herein are performed without the use of nucleases, DNases, or RNases. For example, methods provided herein include RNA extraction without the use of DNases.

[0185] Restriction enzymes may include, but are not limited to, type I to V restriction enzymes, BamHI, EcoP15I, EcoRI, EcoRII, EcoRV, HaeIII, HgaI, HindIII, HinFI, KpnI, NotI, PstI, PvuII, SacI, SalI, SmaI, SpeI, SphI, XbaI, and StuI. Nucleases can be used at concentrations ranging from 50 to 400 μg / mL. Nuclease digestion can be performed at temperatures ranging from approximately 20°C to approximately 37°C. Other nucleic acid modifying enzymes, such as transposases, ligases, polymerases, and phosphatases, may be used. Other protein or polynucleotide digestive agents or degrading agents, such as lysozyme, may be used.

[0186] Samples may undergo varying degrees of pretreatment before being loaded onto a fluidic device. In some cases, samples may simply be loaded into a buffer solution before being loaded onto the fluidic device, and any other necessary or required sample preparation steps may be performed on the device. In other cases, samples may be added to a sample reservoir pre-filled with a processing fluid, such as a solution or buffer solution. In still other cases, samples may undergo embedding material removal, tissue disruption, cell lysis, or digestion before being loaded onto the fluidic device. In one example, the sample is dewaxed before being loaded onto the fluidic device, and nucleic acid decrosslinking is performed on the fluidic device. In another example, the sample is dewaxed, disrupted, and lysed before being loaded onto the fluidic device, and optionally, nucleic acid decrosslinking is performed on the fluidic device. In yet another example, the sample is dewaxed before being loaded onto the fluidic device, and tissue disruption and cell lysis are performed on the fluidic device. In yet another example, the sample is loaded onto the fluidic device, and dewaxing, tissue disruption, cell lysis, and nucleic acid decrosslinking are all performed on the fluidic device. Sample preparation steps are further discussed in this disclosure.

[0187] Sample preparation

[0188] Sample preparation can be performed prior to isotachophoresis. Sample preparation may involve steps including, but not limited to, removal of embedding materials, tissue disruption, cell lysis, protein digestion, removal of nucleic acid crosslinks, isothermal enzymatic processes, enzymatic amplification, enzymatic digestion, disruption of cell-cell junctions, disruption of the extracellular matrix, disruption of connective tissue, and combinations thereof. Sample preparation may involve techniques such as polymerase chain reaction (PCR) or other nucleic acid amplification, isolation or purification of materials of interest (e.g., cells, nucleic acids), probe hybridization, and antibody hybridization (e.g., antibody-nucleosome hybridization). In some cases, samples can be prepared for further analysis by isolating a portion of cellular material from the sample. For example, circulating tumor cells can be isolated from heterogeneous cell populations using cell sorting equipment such as flow cytometry or magnetized columns. In another instance, peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs) can be isolated from blood samples. Sample preparation can be performed on-device or off-device. In some cases, some sample preparation steps are performed off-device, and the sample is subsequently loaded onto a fluidic device for further sample preparation steps.

[0189] Biological material (e.g., cells, tissues, nucleic acids) in embedded samples can be removed from the embedding material. For example, paraffin-embedded samples can be dewaxed. Removal of the embedding material can be performed using techniques including, but not limited to, heat treatment, chemical treatment (e.g., acid or alkali), enzymatic treatment, and combinations thereof. Dewaxing can be performed by chemically treating the sample, by heat treatment, by enzymatic treatment, or by other methods. For example, dewaxing can be performed at elevated temperatures (e.g., from about 50°C to about 80°C) in the presence of a neutral buffer or a slightly acidic buffer (e.g., as low as about pH 5.5), a slightly alkaline buffer (up to about pH 9), or an alkaline solution (e.g., pH from about 12 to about 13). Removal of the embedding material can be performed outside or on the device. In one example, the embedded sample can be incubated in a container at an elevated temperature and then loaded onto a fluid device. In another example, the embedded sample can be loaded onto a fluid device and incubated on the device (e.g., in a channel or reservoir) at an elevated temperature.

[0190] The embedded material can be removed by heat treatment. The incubation for removing the embedded material can be carried out at temperatures of at least about 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 96°C, 97°C, 98°C, 99°C, 99.5°C, or 100°C. The incubation for removing the embedded material can also be carried out at temperatures of about 40°C to about 80°C, about 50°C to about 80°C, about 50°C to about 99.9°C, or about 95°C to about 99.5°C. Incubation for removing embedded material can be performed for a duration of at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, or 120 minutes. Incubation for removing embedded material can also be performed for a duration of about 1 minute to about 20 minutes, about 1 minute to about 30 minutes, about 1 minute to about 60 minutes, about 1 minute to about 120 minutes, or about 5 minutes to about 20 minutes. Incubation for removing embedded material can be performed, for example, at a temperature of at least about 37°C for a duration of at least about 1 minute. Incubation for removing embedded material can be performed in the presence of an alkaline buffer or a neutral buffer (e.g., lysis buffer). Alkaline buffers (e.g., lysis buffers) may have a pH of at least about 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, or 13.5. Neutral buffers may have a pH of about 7.0 (e.g., about 7 to about 8).

[0191] Tissues or cells can be disrupted or lysed to release nucleic acids for isolation, purification, or extraction. Tissue disruption or cell lysis can be achieved using techniques including, but not limited to, mechanical stress, acoustic treatment, electroporation, osmotic pressure, chemical treatment (e.g., acid or alkali), enzymatic treatment, thermal treatment, and combinations thereof. For example, pressure can be used to drive tissue through a structure (e.g., a channel, a resin such as a glass frit or porous resin or glass material) to mechanically disrupt the tissue or lyse cells. In some cases, the trailing electrolyte buffer may contain one or more tissue disruptors and / or cell lysants. In some cases, the leading electrolyte buffer may contain one or more tissue disruptors and / or cell lysants. In some cases, the removal of the embedding material can be achieved through the same process as tissue disruption or cell lysis. For example, incubation at elevated temperatures (e.g., about 30°C to about 80°C, about 50°C to about 80°C, or about 30°C to about 65°C) can achieve the removal of the embedding material, tissue disruption, and cell lysis. Tissue disruption or cell lysis can be performed off-device or on-device. In one instance, the tissue sample is disrupted in a container and subsequently loaded onto a fluid device. In another instance, a tissue sample previously loaded onto a fluid device was destroyed on the device.

[0192] Samples containing tissues or cells can be lysed using a lysis solution or buffer compatible with isotachyphoresis before or after loading onto a fluidic apparatus. Isotachyphoresis-compatible lysis buffers may include nonionic surfactants or detergents, ionic or zwitterionic surfactants or detergents, dissociative agents, disulfide reducing agents, proteases, nucleases, and other additives or components digested, denatured, destroyed, or degraded for the purpose of extracting, purifying, enriching (concentrating), or otherwise isolating nucleic acids. In some cases, the lysis buffer may contain an alkaline buffer. In some cases, the lysis buffer may not contain an alkaline buffer. Exemplary lysis buffers may include 0.5M to 9.5M, 4M to 9M, or 6.5M to 7M urea as described herein. Exemplary lysis buffers may include 0.5M to 3.5M or 1.5M to 2.5M thiourea as described herein. Exemplary lysis buffers may include 0.5-9.5M urea and thiourea, such as 7M urea and 2M thiourea having nonionic surfactants as described herein. Urea, used alone or in combination with thiourea, can be used to lyse cells for nucleic acid purification. In combination, urea and thiourea work synergistically to lyse cells and provide an uncharged, isotropic electrophoresis-compatible buffer for nucleic acid purification.

[0193] Exemplary lysis buffers may include nonionic surfactants, such as 0.05-0.5% v / v IGEPAL CA-630 as described herein. In some cases, the lysis buffer may contain one or more trailing electrolytes. In some cases, the lysis buffer may contain a trailing electrolyte buffer with additives for tissue destruction or cell lysis as described herein. In some cases, the lysis buffer may contain one or more leading electrolytes. In some cases, the lysis buffer may contain a leading electrolyte buffer with additives for tissue destruction or cell lysis as described herein. In some cases, the lysis buffer may contain one or more leading electrolytes and one or more trailing electrolytes. In some cases, the lysis buffer may contain one or more leading electrolytes and one or more trailing electrolytes with additives for tissue destruction or cell lysis as described herein.

[0194] In some cases, the methods or procedures described herein may involve lysing cell or tissue samples using a lysis buffer that minimizes mechanical damage to DNA and / or RNA during the lysis reaction. For example, cells or tissues may be lysed in a buffer solution containing HCl (e.g., 1 mM, 5 mM, 10 mM HCl), Tris (e.g., 5 mM, 10 mM Tris), and a nonionic surfactant. A nonionic detergent (e.g., IGEPAL CA-630) may be present in the lysis buffer at about 1%, about 2%, about 3%, about 4%, or higher, or less than about 1%. Cells or tissues may be lysed in the lysis buffer by gentle mixing (e.g., by inversion and low speed (automatic pipette)). In some cases, enzymes such as proteinase K may be included in the lysate or lysis buffer. In some cases, lysis is performed without centrifugation. In some cases, centrifugation is used in the lysis method. The lysate may be introduced into an isotachophoresis apparatus to purify desired analytes such as high molecular weight DNA fragments.

[0195] Proteins in a sample can be digested, for example, by enzymatic digestion with proteases. Proteases may include, but are not limited to, proteinase K, proteases, endopeptides (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Other protein or polynucleotide digestive agents or degrading agents, such as lysozyme, may be used. Protein digestion can remove cross-linked proteins from cross-linked nucleic acids, thereby converting them into non-cross-linked nucleic acids. Protein digestion can occur at room temperature or at elevated temperatures (e.g., greater than about 25°C) as described herein.

[0196] The sample can be processed on a device (e.g., an electric device or system having at least one reservoir connected to at least one channel) such that the sample volume passes through the reservoir into the channel, wherein less than 20% of the sample volume remains in the reservoir, and an ion current can subsequently be applied through the sample volume in the channel. The ion current may substantially not pass through the channel. In some cases, less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the sample volume remains in the reservoir.

[0197] The sample can be processed on an apparatus (e.g., an electric device or system having at least one reservoir connected to at least one channel) such that the sample volume entering the channel through the reservoir is at least 50% of the sample volume loaded into the reservoir, and an ion current can subsequently be applied through the sample volume in the channel. In some cases, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of the sample volume moves from the reservoir to the channel. In some cases, the total volume loaded into the reservoir is less than or equal to the internal volume of the reservoir. The ion current may substantially not pass through the channel. In some cases, applying the ion current includes performing isovelocity electrophoresis.

[0198] Isokinetic electrophoresis equipment

[0199] Isotachyphoresis and / or sample preparation (e.g., dewaxing, digestion, lysis) can be performed in fluidic devices (e.g., microfluidic chips). Figure 5AA schematic diagram of a channel is shown, comprising a sample preparation (e.g., dewaxing) zone 500 with a sample inlet 501 and a trailing electrolyte reservoir 502, a purification (e.g., isotachophoresis) zone 510 with a leading electrolyte reservoir 511, and an elution outlet 520. A capillary barrier can provide an interface between the sample fluid and the leading electrolyte buffer before voltage is applied. A capillary barrier can be provided between the sample preparation zone 500 and the trailing electrolyte reservoir 502 to limit, reduce, or prevent mixing or pressure-driven flow of the sample fluid and the trailing electrolyte buffer. A capillary barrier can be provided between the purification zone 510 and the leading electrolyte reservoir 511 to limit, reduce, or prevent mixing or pressure-driven flow of the contents of zone 510 and the leading electrolyte reservoir 511. In another example, dewaxing may be performed outside the chip initially, or since the starting material may be unnecessary. In this case, the channel may include a lysis and digestion zone (e.g., pH 7, 56°C) and a crosslink removal and purification zone (e.g., isotachophoresis) zone (e.g., pH 7, 80°C). In another example, dewaxing may be performed outside the chip initially, or since the starting material may be unnecessary. In this case, the channel may include a lysis and digestion zone (e.g., pH 7, temperature T1) and a crosslink removal and / or purification zone (e.g., isotachophoresis) zone (e.g., pH 7, temperature T2). In another example, dewaxing may be performed outside the chip initially, or since the starting material may be unnecessary. In this case, the channel may include a destruction and / or lysis zone (e.g., pH 7, temperature T1) and a digestion and / or purification zone (e.g., isotachophoresis) zone (e.g., pH 7, temperature T2). In another example, dewaxing may be performed outside the chip initially, or since the starting material may be unnecessary, in which case the channel may include a disruption and / or lysis zone (e.g., pH 7, temperature T1) and an isothermal enzymatic amplification zone (e.g., pH 7, temperature T2). In some cases, the channel may include three zones, such as a disruption and / or lysis zone (e.g., pH 7, temperature T1), an isothermal enzymatic amplification zone (e.g., pH 7, temperature T2), and a purification (e.g., isotachophoresis) zone (e.g., pH 7, temperature T3). Figure 5B An exemplary fluid device housing with eight parallel channels is shown, each channel as follows: Figure 5A As shown. Figure 5C It shows Figure 5B The diagram shows a top view of the fluid device, while Figure 5D and Figure 5ESide and end views are shown respectively. The device may include a sample inlet or reservoir 530, an ITP electrolyte buffer reservoir 531, and a sample elution outlet or reservoir 532. Channels and / or reservoirs may be coupled to one or more pneumatic ports. Each of the eight parallel channels of the fluidic device can be operated independently from each of the other channels. In some cases, each channel has a dedicated set of electrodes and circuitry to drive the ITP. The electrodes may, for example, be located in the trailing electrolyte reservoir 502 and the leading electrolyte reservoir 511, such that the electrodes do not directly contact the sample material.

[0200] 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 fluidly connected to each other. The fluid leakage 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 / hour.

[0201] In some cases, there is little or no electrical communication between the parallel channels, making them electrically isolated from each other. Each parallel channel can be electrically controlled independently to apply an independent electric field to each channel. In some cases, the current leakage between channels is less than about 0.1 μA, 0.2 μA, 0.3 μA, 0.4 μA, 0.5 μA, 0.6 μA, 0.7 μA, 0.8 μA, 0.9 μA, or 1 μA. In some cases, the impedance between channels can be greater than 0.1 MΩ, 0.2 MΩ, 0.3 MΩ, 0.4 MΩ, 0.5 MΩ, 0.6 MΩ, 0.7 MΩ, 0.8 MΩ, 0.9 MΩ, 1 MΩ, 5 MΩ, 10 MΩ, 20 MΩ, 30 MΩ, 40 MΩ, or 50 MΩ.

[0202] In some cases, each zone of a constant-velocity fluid device can be heated. In some cases, these zones are heated to the same temperature. In some cases, the individual zones are heated to different temperatures. In some cases, the first zone can be heated to a temperature above 37°C, for example, in the range of about 60°C to about 100°C. In some cases, the second zone can be heated to a temperature above 37°C, for example, in the range of about 40°C to about 60°C.

[0203] Isotachyphoresis fluid apparatus may include one or more reservoirs, including but not limited to buffer loading reservoirs, sample loading reservoirs (including reservoirs that accept solid, multiphase, or other heterogeneous liquids or solutions such as tissue, whole blood, or unlysed cell suspensions), lead electrolyte reservoirs, tail electrolyte reservoirs, reagent reservoirs, elution reservoirs (e.g., for unloading processed samples), and gas or air reservoirs. In some cases, a single physical reservoir may be used for multiple purposes, such as buffer loading and sample loading. Liquid or air reservoirs may be used to apply external pressure for liquid loading (e.g., applying positive pressure to the liquid orifice or applying a vacuum only to the gas reservoir).

[0204] The reservoir can be thermally connected to a heating or cooling source, thereby allowing temperature control of the reservoir and any materials within it (e.g., reagents, samples, products). For example, when within a fluid device, the elution reservoir can be thermally controlled to control the temperature of the eluted products (e.g., to maintain structural integrity).

[0205] Reagent reservoirs can be used to load one or more reagents to process samples before, during, or after isotachyphoresis. Reagents may include digestion reagents, amplification reagents, reverse transcription reagents, linear polymer solutions for size-based separation, probes for hybridization reactions, ligation reagents, dyes, tracers, labels, and other reagents. Reagent reservoirs may be connected to a reaction channel or a reaction section of another channel where reactions can occur. Heating or cooling (e.g., using a thermal controller as discussed herein) may be applied to catalyze reactions (e.g., enzymatic reactions with nucleic acids or proteins), to hybridize or unwind nucleic acids, or to remove inserted dyes from nucleic acids (e.g., prior to elution). Heating and cooling may also be used to control a fixed operating temperature for ITP (e.g., cooling may be applied to reduce the effects of Joule heating) or to maintain a reservoir (e.g., an elution reservoir) at a fixed temperature (e.g., cooler than room temperature), such as for the stable storage of purified nucleic acids. Light (e.g., using a light source as discussed herein) may be applied for purposes including optical interrogation, fluorescence excitation, and reaction energy or catalysis.

[0206] A gas or air reservoir or gas or air outlet may be connected via a gas passage to a liquid passage within the fluid device to allow purging of air or other gases from the fluid device (e.g., during liquid filling of the fluid device). A gas or air reservoir or pneumatic pressure port may be connected via a gas passage to a liquid passage to allow pumping of fluid onto or into the fluid device (e.g., for pumping fluid from the reservoir into the passage).

[0207] The device may contain multiple purification (e.g., isotachophoresis) zones connected to each other. For example, a second isotachophoresis zone may be separated from and run in parallel with a first isotachophoresis zone, thereby allowing sample bands to be separated in a specific ratio (e.g., based on the current ratio between the two zones) for parallel processing.

[0208] Fluid apparatus may contain multiple parallel purification zones (see, for example, Figure 5C For example, a fluidic apparatus may contain more than one set of purification zones, each set having associated reservoirs, inlets, outlets, channels, and any other components described herein (e.g., sample preparation zones, electrodes, heaters, detectors), these components being parallel, separate from each other, and each capable of independently processing samples. A fluidic apparatus may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48, 96 or more parallel purification zones. A fluidic apparatus may contain multiple parallel channels. A fluidic apparatus may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48, 96 or more parallel channels. Parallel components, such as purification zones or channels, can be side-by-side, located in different equipment layers (e.g., horizontal or vertical layers), or arranged differently. Parallel components can be identical or can be designed differently but functionally equivalent or nearly equivalent. For example, parallel channels can have different geometries to allow for a smaller overall fluidic apparatus footprint while still having similar functionality. Alternatively, parallel components can be designed to function differently, such as processing different types of samples in parallel or subjecting samples to different operations. In some cases, parallel components can be designed to subject different sample types to different operations in parallel. In some cases, parallel components can be designed to subject the same sample type to different operations in parallel. In some cases, parallel components can be designed to subject different sample types to the same operations in parallel. In some cases, parallel components can be designed to subject the same sample type to the same operations in parallel. In some cases, parallel components can be designed to subject two or more samples to one or more operations in parallel simultaneously and / or independently. In some cases, the leakage rate between two or more channels (or between two or more purification zones) is less than 0.5 μl / hour, less than 1 μl / hour, less than 5 μl / hour, or less than 10 μl / hour. In some embodiments, the current leakage rate between two or more channels (or between two or more purification zones) 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 megohms, greater than 1 megohm, greater than 5 megohms, or greater than 10 megohms.

[0209] As discussed in this article, fluid devices can be designed to handle different sample volumes. For example, Figure 6A , Figure 6B , Figure 6C and Figure 6DTop, side, bottom, and three-quarter top views of a rapid purification ITP fluid apparatus 600 for sample volumes greater than or equal to about 200 μL are shown. The apparatus includes channels 600 connecting to a sample inlet 601, an ITP buffer well 602, and a sample outlet (elution) well 603. The ITP buffer well 602 may include an elution buffer reservoir 605, a lead electrolyte reservoir 606, a lead electrolyte buffer reservoir 607, and a trailing electrolyte reservoir 608. An elution reservoir 603 can be connected to an elution buffer reservoir 605 via 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. The lead electrolyte reservoir 606 can be connected to the lead electrolyte buffer reservoir 607 via a lead electrolyte buffer channel 610. A capillary barrier can be provided in the lead electrolyte buffer channel 610 to reduce or prevent mixing or pressure-driven flow between the contents of the lead electrolyte buffer reservoir 607 and the lead electrolyte reservoir 606. Buffer reservoir 605 may contain an elution buffer electrolyte with an ionic strength higher than that of the electrolyte in elution reservoir 603, while buffer reservoir 607 may contain a lead electrolyte with an ionic strength higher than that of the electrolyte in lead electrolyte reservoir 606. The device may further include a pneumatic port 604 along its edge, configured to couple to a pneumatic device, such as a vacuum source on a benchtop instrument. Pneumatic port 604 can be coupled to channel 600 and the reservoir via a gas channel as described herein. Applying suction at pneumatic port 604 can load the sample, lead electrolyte, and elution buffer into channel 600. In some cases, the trailing electrolyte buffer fluid is retained in the trailing electrolyte reservoir 608. Aspiration can be applied simultaneously or sequentially to the pneumatic port 604 to load channel 600 simultaneously or in stages. A sample can be loaded into a first region or subchannel of channel 600, which extends from the trailing electrolyte reservoir 608 to the capillary barrier 611 within channel 600 with a 180° low-dispersion turn. The capillary barrier 611 provides an interface between the sample and the lead electrolyte buffer during loading to limit, reduce, or prevent mixing or pressure-driven flow. A capillary barrier can be provided between the trailing electrolyte reservoir 608 and the first region or subchannel to limit, reduce, or prevent mixing or pressure-driven flow between the contents of the trailing electrolyte reservoir 608 and the sample. A lead electrolyte can be loaded into a second region or subchannel of channel 600, which extends from capillary barrier 611 to capillary barrier 612. The capillary barrier 612 provides an interface between the lead electrolyte buffer and the elution buffer. Elution buffer can be loaded into the third region or sub-channel of channel 600, which extends from capillary barrier 612 to elution reservoir 603.In some embodiments, the ITP buffer orifice 602 may further include a trailing electrolyte buffer reservoir (not shown) containing a trailing electrolyte with an ionic strength higher than that of the electrolyte in the trailing electrolyte reservoir 608. The trailing electrolyte buffer reservoir may be connected to the trailing electrolyte reservoir 608 via a trailing electrolyte buffer channel (not shown). The trailing electrolyte buffer channel may include a capillary barrier to limit, reduce, or prevent mixing or pressure-driven flow between the contents of the trailing electrolyte buffer reservoir and the contents of the trailing electrolyte reservoir 608.

[0210] Electrodes may be located, for example, in a trailing electrolyte reservoir 608, a trailing electrolyte buffer reservoir (not shown), a leading electrolyte reservoir 606, and / or a leading electrolyte buffer reservoir 607, such that the electrodes do not directly contact the sample material. In response to feedback from a sensor (e.g., a voltage, current, conductivity, or temperature sensor as described herein), the electrodes may be triggered to alter or control the applied electric field. For example, a pathway of nucleic acids within the ITP region from the second region of channel 600 to the third region of channel 600 may be detected, and feedback from the detector may trigger a change in the applied current. For example, the current may be increased, decreased, or stopped according to the instrument's protocol. For example, the current may be paused (e.g., temporarily reduced to zero) to enable on-chip quantification of nucleic acids. Alternatively or in combination, the current may be reduced to slow isovelocity electrophoresis within the third region, thereby further concentrating nucleic acids that may have been dispersed during the transition from the leading electrolyte buffer to the elution buffer (or the second leading electrolyte buffer) before reaching the elution well 603.

[0211] The methods and procedures provided herein include those using any of the devices provided herein. The devices provided herein, having multiple channels for parallel processing of multiple samples, can be used in a variety of environments. In some cases, the method may include using the device to process multiple samples that share a common characteristic (e.g., solid tissue lysate, cell lysate, solid tissue, fixed tissue) (e.g., by performing isotachyphoresis on these samples). In some cases, the multiple samples may be different samples. For example, the method may involve performing isotachyphoresis on a tissue sample in one region of the device while simultaneously, but independently, performing isotachyphoresis on different samples (e.g., cell samples or samples containing cross-linked nucleic acids).

[0212] In some cases, the methods or multiplexing procedures described herein may involve isotachophoresis of a sample in a channel, parallel to isotachophoresis of a second sample in a second channel using the same or similar leading electrolyte and / or trailing electrolyte buffer. In some cases, a sample in one channel is treated with a first leading electrolyte buffer, and samples in different channels are treated with a second leading electrolyte buffer different from the first. For example, the first leading electrolyte buffer may contain one or more leading electrolyte ions that are different from the leading electrolyte ions contained in the second leading electrolyte buffer. In another example, the first leading electrolyte buffer may contain one or more leading electrolyte ions that are the same as the leading electrolyte ions contained in the second leading electrolyte buffer, but the concentration of such leading electrolyte ions in the first leading electrolyte buffer differs from the concentration of such ions in the second leading electrolyte buffer. In some cases, the methods or procedures described herein may involve isotachophoresis of a sample in a channel, parallel to isotachophoresis of a second sample in a second channel using the same or similar trailing electrolyte or trailing electrolyte buffer. In some cases, a first trailing electrolyte buffer is used to treat a sample in one channel, and a second trailing electrolyte buffer, different from the first, is used to treat samples in different channels. For example, the first trailing electrolyte buffer may contain one or more trailing electrolyte ions that are different from those contained in the second trailing electrolyte buffer. In another example, the first trailing electrolyte buffer may contain one or more trailing electrolyte ions that are the same as those contained in the second trailing electrolyte buffer, but the concentration of these trailing electrolyte ions in the first trailing electrolyte buffer differs from the concentration in the second trailing electrolyte buffer.

[0213] In some implementations, one or more reservoirs may be connected to two channels or sub-channels. For example, elution reservoir 603 may be connected to both channel 600 and elution buffer channel 609. Alternatively or in combination, lead electrolyte reservoir 606 may be connected to both channel 600 and lead electrolyte buffer channel 610. Alternatively or in combination, trailing electrolyte reservoir 608 may be connected to both channel 600 and trailing electrolyte buffer channel. Alternatively or in combination, sample inlet port 601 may be connected to the midpoint of channel 600, such that channel 600 extends to the left (as a first sub-channel) and right (as a second sub-channel) of inlet port 601. Two channels or sub-channels can be connected to one or more reservoirs, wherein the angle between the two channels (scanned in the main plane of the fluid device) is at least about 5°, 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 135°, 140°, 150°, 160°, 170°, or 180°. Two channels or sub-channels can be connected to one or more reservoirs, wherein the angle between the two channels (scanned in the main plane of the fluid device) is 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°.

[0214] For example, the device may include eight channels as shown in the figure. Each channel can hold a sample volume of approximately 50 μL to approximately 275 μL and a total volume of approximately 500 μL. The 180° low-dispersion steering in each channel facilitates such a large sample volume in an 8-channel multichannel plate with a standard SLAS footprint.

[0215] Figure 7A , Figure 7B , Figure 7C and Figure 7D Top, side, bottom, and three-quarter bottom views of a rapid purification ITP fluid device 700 for sample volumes less than or equal to about 100 μL are shown. The device includes a sample inlet port 701, an ITP buffer port 702, and a sample outlet (elution) port 703. Device 700 may be substantially similar to device 600, but has a different channel geometry (and corresponding reservoir geometry) excluding 180° turns within the channels.

[0216] For example, the device may include eight channels as shown in the figure. Each channel can hold a sample volume of approximately 10 μL to approximately 100 μL. Devices with smaller sample volumes can be used for PCR cleaning or other reaction cleaning applications or for smaller sample sizes (e.g., samples with a small number of cells or tissues).

[0217] Figure 8A , Figure 8B , Figure 8C and Figure 8D Top, side, bottom, and three-quarter bottom views of another rapid purification ITP fluid device 800 for sample volumes less than or equal to approximately 100 μL are shown. This device includes a sample inlet well 801, an ITP buffer well 802, and a sample outlet (elution) well 803. Device 800 may be substantially similar to devices 600 and 700, but contains multiple different channel geometries on a single chip.

[0218] A fluid device may include one or more electrodes that apply an electric field to the fluid device or a portion thereof. The applied electric field can be used to perform isovelocity electrophoresis. A fluid device may include one or more electrodes that apply a single electric field to all channels of the fluid device. A fluid device may include one or more electrodes that apply more than one electric field to the fluid device, such as one electric field per channel on the device. In some cases, the first and second electric fields are generated by a single electrode pair. In some cases, the first and second electric fields are generated by different electrode pairs. The electric fields may be applied simultaneously, sequentially, and / or independently, or applied to each other. Electrodes may be external, such as wires falling into a reservoir. Electrodes may be internal, such as microfabricated, printed, or other embedded elements included in the fabrication of the fluid device. Electrode materials may include, but are not limited to, metals (e.g., platinum, titanium) and carbon.

[0219] One or more electrodes of a fluid apparatus may be part of one or more circuits that apply an electric field to the fluid apparatus or a portion thereof. A fluid apparatus may contain one or more circuits that apply a single electric field to all channels or isokinetic electrophoresis zones of a region of the fluid apparatus. A fluid apparatus may contain one or more circuits that apply more than one electric field to the fluid apparatus, such as one electric field per channel on the apparatus. In some cases, the first and second electric fields may be generated by a single circuit. In some cases, the first and second electric fields may be generated by different circuits. Electric fields may be applied simultaneously, sequentially, and / or independently, or to each other, by one or more circuits. In some cases, the apparatus (or benchtop instrument) may be configured to control a first circuit simultaneously with and independently of a second circuit.

[0220] Electrodes can be located in reservoirs such as trailing and leading electrolyte reservoirs, which can be separated from the sample reservoir via buffer channels. In some cases, electrodes are located in buffer channels or buffer reservoirs. The location of electrodes in electrolyte reservoirs or electrolyte buffer reservoirs can isolate the electrodes from analytes such as nucleic acids to reduce or eliminate contamination of the electrodes by sample materials. This method can allow for the reuse of electrodes without cross-contamination between samples. In one example, a trailing electrolyte reservoir or trailing electrolyte channel is connected via a buffer channel to a buffer reservoir containing trailing electrolyte ions and an electrode, and the trailing electrolyte reservoir is also connected to a sample reservoir or sample channel, which in turn is connected to a leading electrolyte reservoir via a leading electrolyte channel; the leading electrolyte reservoir is also connected via a buffer channel to a buffer reservoir containing a leading electrolyte and an electrode. In another example, or as a continuation of the previous example, an elution reservoir containing elution buffer is connected via an elution channel to a leading electrolyte reservoir, and is also connected to a buffer reservoir containing elution buffer electrolyte and an electrode. Buffer channels between a buffer reservoir and its corresponding reservoir may include capillary barriers and / or low cross-sectional areas to limit, reduce, or prevent mixing and pressure-driven flow as described herein. Buffer reservoirs may contain electrolytes with the same or higher ionic strength as their corresponding reservoirs. For example, an elution reservoir may be connected to a buffer reservoir containing an elution buffer electrolyte with the same or higher ionic strength or concentration as the elution reservoir. A trailing electrolyte reservoir may be connected to a buffer reservoir containing a trailing electrolyte with the same or higher ionic strength or concentration as the trailing electrolyte reservoir. A leading electrolyte reservoir may be connected to a buffer reservoir containing a leading electrolyte with the same or higher ionic strength or concentration as the leading electrolyte reservoir. Providing dedicated buffer reservoirs for connection to elution reservoirs, trailing electrolyte reservoirs, and / or leading electrolyte reservoirs with higher ionic strengths can provide additional ion pools to maintain pH and conductivity in the channels as the sample moves through them.

[0221] Fluid equipment can be used with one or more thermal controllers. For example, Figure 9A A schematic diagram of an eight-fold sample preparation and isotropic electrophoresis apparatus is shown, which includes Figure 5A The design shown has eight parallel channels 900. Figure 9BSchematic diagrams of first and second thermal controllers 901, 902 are shown. The first thermal controller 901 at temperature T1 (e.g., 80°C) is aligned with the sample preparation area of ​​the channel, and the second thermal controller 902 at temperature T2 (e.g., 50°C) is aligned with the isovelocity electrophoresis area of ​​the channel. In some cases, additional thermal controllers may be aligned with other areas of the channel (not shown), for example, a third thermal controller at temperature T3 may be aligned with a third area at temperature T3. In some cases, each area of ​​each channel may have its own separate thermal controller, rather than sharing a common thermal controller with corresponding areas of other channels. In other cases, all areas or channels may share a single thermal controller. In still other cases, multiple, but fewer than all areas or channels may share a single thermal controller. The thermal controller may include components including, but not limited to, resistance heaters, fluid-based heating or cooling systems, and Peltier devices. The thermal controller may be made of materials including, but not limited to, metals (e.g., platinum, titanium, copper, gold), carbon, and indium tin oxide (ITO). A thermal controller may include a temperature sensor that can be used to monitor the controlled temperature and provide temperature feedback for thermal control. As further discussed in this disclosure, the thermal controller may be used in conjunction with a computer control system. In some cases, the thermal controller operates without temperature feedback. The thermal controller may be integrated into the fluid device or located externally, such as within a benchtop system.

[0222] Fluid apparatuses can be used with one or more light sources. The light source can be integrated into the fluid apparatus or located externally, such as within a benchtop system or in a separate device. The light source can provide illumination for optical interrogation, fluorescence excitation, temperature sensing, reaction energy or catalysis, and other purposes.

[0223] Fluid devices can be designed such that their outermost frame or dimensions conform to microtiter plate standards (e.g., SLAS microtiter plate standards). Fluid devices can be designed to use defined ports of a microtiter plate (e.g., SLAS standard microtiter plate) as liquid reservoirs, with pneumatically actuated ports located on unused surfaces outside the liquid reservoir. The pneumatic ports can be positioned at the edges of the fluid device with a layout compatible with microtiter plates, thereby avoiding cross-contamination via pneumatic actuation of the liquid reservoir and facilitating access to the ports with pneumatic hardware. A subset of the defined ports may also be used for pneumatic actuation, among other functions. In some cases, the fluid device can be designed and manufactured with two interlocking components: first, an insert comprising a channel unit (e.g., a layer with a flat surface conducive to film adhesion), holes, and pneumatic ports; and second, an outer ring conforming to microtiter plate standards (e.g., SLAS microtiter plate size standards), including alignment features for aligning the fluid device with a benchtop system and mating features for interlocking with the first component. These holes can be connected to form bosses, which makes them more compatible with injection molding.

[0224] Fluid devices can be made of a variety of materials, including but not limited to glass (e.g., borosilicate glass), silicon, plastics, and elastomers. Plastics may include polymethyl methacrylate (PMMA), cyclic olefin copolymers (COC), cyclic olefin polymers (COP), polyethylene, polyethylene terephthalate (PET), high-density polyethylene (HDPE), and low-density polyethylene (LDPE). Elastomers may include polydimethylsiloxane (PDMS).

[0225] Materials used to manufacture fluid devices can be selected based on their optical properties. For example, materials that exhibit low autofluorescence, low scattering, and high transmittance at wavelengths of interest (e.g., the excitation and emission wavelengths of nucleic acid markers or dyes) can be used. Different materials can be used in a single fluid device; for example, the detection region can be made of a material exhibiting useful optical properties, while other regions of the device can contain different materials.

[0226] Materials used in manufacturing fluid devices can be selected based on their thermal properties. For example, materials with high thermal conductivity can be chosen. Alternatively, materials with low thermal conductivity can be chosen (e.g., to thermally insulate the fluid device or areas thereof). Different materials can be used in a single fluid device; for example, the heating zone can have a material with high thermal conductivity to improve thermal communication with the thermal controller, while the heating zone is surrounded by a material with low thermal conductivity to thermally insulate it from other areas of the device.

[0227] Materials used to manufacture fluid devices or microchannels can be selected based on their elasticity or deformability. For example, a low-elasticity material can be chosen to allow the closure of plastic channels as described herein. Alternatively, a high-elasticity material can be selected. Different materials can be used in a single fluid device; for example, poly(methyl methacrylate) can be used in a single fluid device.

[0228] Materials can be made of PMMA, cyclic olefin copolymers (COC), cyclic olefin polymers (COP), etc. The materials may 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 materials may have an elastic modulus of up to 1 GPa, 1.5 GPa, 2 GPa, 2.5 GPa, 3 GPa, 3.5 GPa, 4 GPa, 4.5 GPa, or 5 GPa. The materials 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, or 200 MPa. The material may have tensile strengths of up to 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, and 200 MPa.

[0229] In some cases, the surface of a fluid device may be used without surface treatment or coating. In other cases, the surface of a fluid device may be used with surface coatings such as hydrophobic treatments, hydrophilic treatments, or selective binders (e.g., antibodies). Different areas of a fluid device may contain different surface treatments (or lack surface treatments). For example, some channels, reservoirs, or components thereof may be hydrophobic, while others may be hydrophilic.

[0230] Fluid devices may include a range of flow control units and technologies, including but not limited to capillary barriers, air outlet reservoirs, gas / air lines, fill level monitors (e.g., measured by electrodes), specific reservoir geometries, specific channel fluid resistances, and fluid loading sequences.

[0231] Capillary barriers can be paired with air outlet reservoirs to purge air (e.g., to prevent air bubbles), thereby positioning and successfully establishing (i) the liquid-liquid interface between the lead electrolyte solution and the trailing electrolyte solution required for isotachophoresis, and (ii) the liquid-liquid interface between the buffer reservoir and the lead or trailing electrolyte and / or sample solution. Capillary barriers can be designed in conjunction with channel geometry to automatically fill channels in a preferred order. Channel resistance can be selected, such as through the design of channel dimensions, to provide different fluid resistances. The sequencing of liquid loading can allow for the proper formation of a bubble-free liquid-liquid interface for electrodynamic processes. In one example, the trailing ion reservoir is directly connected to the analyte or sample channel.

[0232] Gas (e.g., air) passages or lines can be used to provide actuated pneumatic pressure to other areas of a capillary barrier or fluid device. Gas passages can be connected to an external gas pressure source via pneumatic ports. Gas passages can have higher fluid resistance than liquid passages, providing pressure to, for example, reduce or prevent liquid inflow into the gas passage. For example, a gas passage may be less than half the cross-sectional area of ​​the main isokinetic electrophoresis channel. Multiple gas passages can be connected to a single gas reservoir or port (e.g., with branched passages). Capillary valves can be used with branched air lines to prevent upstream liquid movement. Figure 10A An exemplary gas channel 1001 is shown, which may include a capillary barrier 1002 connected to a liquid channel interface 1003 between a sample 1004 and a lead electrolyte buffer 1005 subchannel. Figure 10B An enlarged schematic diagram of the gas passage 1001 of the capillary barrier 1002 is shown to highlight the capillary barrier 1002, which prevents upstream liquid from moving toward the pneumatic port 1006.

[0233] Negative pressure or vacuum can be applied to gas channels through gas ports to load fluid channels. Each fluid channel on a microfluidic device can be loaded simultaneously or independently of each other (e.g., sequentially). Within the channels, fluids can be loaded simultaneously or independently of each other. For example, a lead electrolyte buffer, a high-concentration lead electrolyte buffer, a trailing electrolyte buffer, a high-concentration trailing electrolyte buffer, an elution buffer, a high-concentration elution buffer, or any combination thereof can be loaded before, simultaneously with, or after sample loading. For example, negative pressure can be applied to a gas port on one side of the chip to load one or more fluids (e.g., trailing electrolyte buffer, elution buffer, etc.). Subsequently, negative pressure can be applied to a gas port on the other side of the chip to load another fluid (e.g., lead electrolyte buffer). Alternatively, negative pressure can be applied simultaneously to all gas ports connected to the channels. Samples can be loaded by applying negative pressure or vacuum before, during, or after loading isotachophoresis buffer. Samples can be loaded without applying negative pressure or vacuum, for example, by wetting or gravity.

[0234] Sensors (e.g., electrodes) can be used to detect liquid fill levels or air bubbles (e.g., by current or voltage sensing) and provide feedback. Geometric features (e.g., contraction, expansion, or directional changes) can be combined with electrodes to monitor channel impedance, thereby monitoring the time progression of isokinetic electrophoresis. For example, in the ITP process, nucleic acids are focused, and voltage can be used to track the position of the focused band in the channel from start to finish. In one instance, the time for analyte elution can be determined by monitoring fluid expansion into a reservoir (such as an elution reservoir) from a connecting channel with a small cross-sectional area, allowing for automated elution and termination process control. In another instance, channel contraction can be designed to allow for the timing (or triggering) of steps in the electrokinetic process, such as when the focused analyte enters the channel region where a reaction occurs or an optical detection event takes place, thereby allowing for control of reaction timing or detector triggering.

[0235] Reservoir and channel features can be designed to control or prevent pressure-driven flow. For example, reservoirs (e.g., sample and elution reservoirs) can have designed internal shapes such that large changes in liquid height occur only when... Figure 11 The expected head height, as shown, produces a small change in internal volume. This allows for more precise control over the volume of liquid contained in the reservoir. For other reservoirs, the liquid volume can vary without impairing the separation process; such reservoirs can be designed to have a large volume change in response to small changes in liquid height and can help stabilize the liquid height throughout the fluid device. The low fluid resistance between reservoirs allows for rapid head pressure equalization and enables minimization of liquid flow in the channels before, during, or after the electrodynamic process.

[0236] Reservoirs can be designed to minimize evaporation, for example, by 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 minimizing dead zones through the use of stretched cornerwall designs. Reservoirs can be designed to prevent liquid from flowing into the reservoir from the connecting channels during unloading, which can help maintain the purity or separation of the unloaded material (e.g., nucleic acids). Reservoirs can be designed for easy loading or unloading by pipetting, for example, by making the dimensions suitable for receiving pipette tips or having a volume in typical pipetting operations. For example, an elution reservoir can be configured to receive pipette tips for nucleic acid extraction. Reservoirs can be designed or spaced to accept multichannel pipettes (e.g., with a spacing of approximately 9 mm).

[0237] A reservoir (e.g., a sample reservoir) can be positioned directly above the channel to be filled, minimizing liquid loss in the connecting channel between the reservoir and the channel it is filling. The reservoir (e.g., a sample reservoir) may have a conical bottom and a cylindrical through-hole; the large inner diameter at the top of such a reservoir allows it to accommodate a large volume, while the smaller inner diameter of the liquid meniscus at the bottom reduces the amount of liquid left after dispensing. This design also reduces or prevents the wetting fluid wick from being drawn into the concave corner. In some cases, the through-hole from the reservoir (e.g., a sample reservoir) into the channel is less than or equal to about 2 millimeters (mm).

[0238] Figure 11 A sample reservoir 1100 is shown, configured to reduce the amount of sample remaining (or lost) in the reservoir 1100 after a sample has been moved to the connection channel 1101. The low-loss sample reservoir 1100 can reduce the amount of sample remaining in the reservoir 1100 after a sample has been moved to the connection channel 1101 without adding or pumping additional volume (of the sample or other fluid) into the sample reservoir 1100 after or during sample delivery to the connection channel 1101. The low-loss sample reservoir 1100 may include an upper or top portion 1102 having an internal hydraulic diameter D1 configured to contain a sample volume before the sample is loaded into the channel 1101, a lower or bottom portion 1103 having an internal hydraulic diameter or through-hole D2 configured to contain a sample volume after the sample is loaded into the channel 1101 and a height H1, and a tapered or conical portion 1104 between them. In some cases, the upper part 1102 and / or the lower part 1103 are asymmetrical. In such cases, dimensions D1 to D2 can represent the maximum dimensions of the upper and / or lower parts 1102 and 1103, respectively.

[0239] Sample reservoir 1100 may be configured to produce a head height H2 of the retained sample, which is equal to or nearly equal to the head height H3 of the buffer solution in another reservoir 1110 connected to channel 1101, to limit, prevent, or reduce pressure-driven flow and mixing in channel 1101. Standard buffer solution reservoir 1110 may include an upper portion 1112 having an internal hydraulic diameter D3 and a lower portion 1113 having an internal hydraulic diameter D4. Unlike the sample well, D3 may be substantially similar to D4, such that when head heights H2 and H3 are equal or nearly equal, a larger volume of fluid is retained within the buffer solution well 1110 compared to the sample well 1100.

[0240] Sample reservoir 1100 may be configured to hold sample volumes 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 (with or without buffer). In some cases, sample reservoir 1100 may be configured to hold sample volumes ranging from about 1 nL to about 10 nL.

[0241] The internal hydraulic diameter D1 can be larger than the through-hole hydraulic diameter D2. The internal hydraulic diameter D1 of the upper part can be at least approximately 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, or 15mm. The internal hydraulic diameter D1 of the upper part can be at most approximately 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, or 15mm. The internal hydraulic diameter D2 of the lower part can be at least approximately 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm, or 5mm. The internal hydraulic diameter D2 of the lower part can be up to about 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm or 5mm.

[0242] The ratio of D1 to D2 determines the amount of sample remaining in the sample reservoir after the sample has been moved 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 other 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 the movement of at least 50% of the sample volume from the low-loss sample reservoir 1100 into the channel 1101.

[0243] The cross-sectional area of ​​the upper part can be at least about 3 mm. 2 5mm 2 10mm 2 15mm 2 20mm 2 25mm2 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 part can be at most about 3 mm². 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 lower cross-sectional area can be at least approximately 0.2 mm². 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 The cross-sectional area of ​​the lower part can be at most about 0.2 mm. 2 0.3mm 2 0.4mm 2 0.5mm 2 1mm 2 1.5mm 2 2mm2 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 .

[0244] The ratio of the upper cross-sectional area to the lower cross-sectional area determines the amount of sample remaining in the sample reservoir after the sample has been moved into the channel. In some cases, the ratio of the upper cross-sectional area to the lower cross-sectional area 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 upper cross-sectional area to the lower cross-sectional area 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.

[0245] The tapered portion between the upper and lower sections may include an angle to facilitate sample wetting to the lower section and sample movement from the low-loss sample orifice to the channel. In some cases, the tapered portion of the low-loss sample reservoir may include a half-angle between the upper and lower sections of less than about 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, or 90°. In other cases, the tapered portion of the low-loss sample reservoir may include a half-angle between the upper and lower sections of greater than about 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, or 90°.

[0246] In some cases, the lower height H1 can be configured to produce a head height of the remaining sample, equal to or nearly equal to the head height of the buffer solution in other reservoirs connected to the channel, to limit, prevent, or reduce pressure-driven flow and mixing within the channel. The lower height H1 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 lower height H2 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.

[0247] The reservoir (e.g., an elution reservoir) may have a diameter larger than the diffusion length scale of the analyte (e.g., nucleic acid) to reduce analyte diffusion from the reservoir. In some cases, the reservoir diameter can be on the order of millimeters, and the diffusion time of the analyte obtained from the reservoir can be on the order of hours. The connection between the channel and the reservoir (e.g., an elution reservoir) can be designed without sharp corners, thereby reducing the prevalence of high electric field regions at these connections and increasing the residence time of the analyte within the reservoir. In some cases, the cross-section of the reservoir (e.g., an elution reservoir) perpendicular to the electric field can be significantly larger than the cross-section of the channel perpendicular to the electric field, thereby reducing the electric field strength in the reservoir and increasing the residence time of the analyte within the reservoir.

[0248] In some cases, the elution channel and / or elution reservoir may contain a second lead electrolyte buffer, which may be of a different type or concentration than the first lead electrolyte buffer used in the main channel. This allows purified material to be eluted in the second lead electrolyte buffer (e.g., elution buffer or output solution). The effective mobility of the second lead electrolyte ions within the second lead electrolyte buffer may be greater than the effective mobility of the nucleic acid. The second lead electrolyte buffer may have a low ionic strength, such as an ionic strength compatible with downstream assays (e.g., qPCR, next-generation sequencing). In some cases, the second lead electrolyte buffer is the same as the first lead electrolyte buffer but exists at a different concentration or ionic strength (e.g., a lower ionic strength than the first lead electrolyte buffer). For example, the first lead electrolyte buffer may have an electrolyte ion concentration of 70–100 mM (e.g., 70–100 mM Tris HCl), while the second lead electrolyte buffer may 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).

[0249] Channels on fluid equipment can be closed. For example, a mechanical actuator coupled to a mechanical component can be used to apply pressure to completely or partially close the channel (e.g., by deformation of the channel). An elution reservoir can be closed from an ITP channel to define a fixed elution volume. Channel closure can result in reduced flow or complete flow blockage. Channel closure can result in increased fluid flow resistance. In some cases, channel closure can increase fluid resistance by at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times.

[0250] Figure 12A An exemplary mechanical component 1200 is shown, which can be used to apply pressure to close or at least partially close the channel 1201 of a fluid device 1210, the fluid device 1210 comprising a plurality of parallel channels (e.g., Figure 6C (A device comprising eight independent parallel channels). Mechanical component 1200 may include a comb-like structure with teeth 1202 aligned with two positions 1204, 1205 in each of the eight channels of the chip, such as... Figure 12B As shown. Mechanical pressure can be applied by the teeth 1202 to permanently or plastically close or at least partially close the channel 1201, thereby limiting, reducing, or preventing the inflow or outflow of liquid into the eluent reservoir 1203 and controlling the eluent volume. At least partially closing the channel 1201 can increase the resistance to fluid flow between the channel 1201 and the eluent reservoir 1203. The mechanical component 1200 can be coupled to a mechanical actuator that generates a force applied to the channel 1201 by the teeth 1202 of the mechanical component 1200. The mechanical component 1200 may contain a material with a Young's modulus greater than that of the channel 1201. One or more teeth 1202 of the mechanical component 1200 may be configured to heat the channel 1201. One or more teeth 1202 of the mechanical component 1200 may be thermally coupled to a heater or heating element. The mechanical component 1200 may optionally include a heater or heating element. Optionally, heat can be applied through the teeth 1202 to permanently or plastically close the channel 1201. One or more teeth 1202 can be heated to a temperature greater than the glass transition temperature of at least one wall of one or more channels 1201. Figure 12CThe diagram illustrates how sixteen teeth 1202 of the mechanical component 1200 are aligned with sixteen positions 1204, 1205 (two positions per channel) on the chip 1210. Each tooth 1202 can be configured to deliver mechanical pressure to the channel 1201 to plastically deform at least one wall of the channel 1201. Each channel 1201 contacts the mechanical component 1200 and plastically deforms at 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 cases, the teeth 1202 can apply mechanical pressure to the channel position 1204 upstream of the reservoir 1203. In some cases, the teeth 1202 can apply mechanical pressure to the contact point where the reservoir 1203 and the channel 1201 meet. In some cases, the teeth 1202 can apply mechanical pressure to the contact point 1205, where the reservoir meets a buffer channel to prevent fluid communication between the reservoir 1203 and the buffer reservoir.

[0251] In some cases, the mechanical component 1200 may include a tooth 1202 for each channel, aligned with the first closed position 1204. For example, Figure 5A The channels shown do not include buffer channels or reservoirs connected to the elution reservoir, and therefore a second closure position 1205 outside the elution reservoir may not be required. In some cases, the mechanical member 1200 is configured to close each channel 1201 on the chip 1210 at one or more locations. In some cases, the mechanical member 1200 is configured to open one or more channels 1201 on the chip 1210, such that only a portion of the channels 1201 on the chip 1210 are closed.

[0252] Mechanical component 1200 can apply a force of at least 0.25 lbs per channel via teeth 1202. Each tooth 1202 of mechanical component 1200 can apply a force of 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 pounds to channel 1201.

[0253] Channels in fluid apparatus (e.g., sample preparation zones, isotachophoresis zones) may have a sufficiently large width, height, or diameter to allow contaminants such as embedding materials (e.g., paraffin) to deposit on the channel walls while still providing enough space for fluid to flow within the channel. In some cases, the width, height, or diameter of channels in fluid apparatus is less than or equal to 20 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 width, height, or diameter of the channels in the fluid apparatus is 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 width of the channels in the fluid apparatus ranges from about 1 mm to about 3.8 mm. In some cases, the height of the channels in the fluid apparatus ranges from about 0.1 mm to about 1.2 mm.

[0254] In some cases, the length of the channels in fluid equipment is at least approximately 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, or 160 mm. 170mm, 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 length of the channels in fluid equipment is less than or equal to approximately 500mm, 490mm, 480mm, 470mm, 460mm, 450mm, 440mm, 430mm, 420mm, 410mm, 400mm, 390mm, 380mm, 370mm, 360mm, 350mm, 340mm, 330mm, 320mm, 310mm, 300mm, 290mm, 280mm, 270mm, 260mm, 250mm, 240mm, 230mm, 220mm, 210mm, 2 00mm, 190mm, 180mm, 170mm, 160mm, 150mm, 140mm, 130mm, 120mm, 110mm, 100mm, 90mm, 80mm, 70mm, 60mm, 50mm, 45mm, 40mm, 35m m, 30mm, 25mm, 20mm, 19mm, 18mm, 17mm, 16mm, 15mm, 14mm, 13mm, 12mm, 11mm, 10mm, 9mm, 8mm, 7mm, 6mm, 5mm, 4mm, 3mm, 2mm or 1mm.

[0255] Channels in fluid apparatus may have sufficiently large widths, heights, or diameters to accommodate large sample volumes. In some cases, channels in fluid apparatus have widths greater than their heights to reduce temperature rise due to Joule heating within the channels. In some cases, the width-to-height ratio of channels in fluid apparatus is 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 width-to-height ratio of channels in fluid equipment is 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 cross-sectional area of ​​channels in fluid equipment is less than about 0.1 mm². 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 3.4mm2 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 cross-sectional area of ​​the channels in fluid equipment is greater than approximately 0.1 mm². 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 22.7mm 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, channels on fluid devices have a minimum length dimension for heat dissipation of less than about 1 micrometer (μm), 5μm, 10μm, 20μm, 30μm, 40μm, 50μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, or 600μm. In other cases, channels on fluid devices have a minimum length dimension for heat dissipation of greater than about 1 micrometer (μm), 5μm, 10μm, 20μm, 30μm, 40μm, 50μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, or 600μm.

[0256] In some cases, the total volume of channels in fluid devices is 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, or 900 μL. , 1 milliliter (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. In some cases, the total volume of channels in fluid devices 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, or 900 μL. , 1 milliliter (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.

[0257] In some cases, fluid devices include more than one channel. The channels may be spaced apart in the fluid 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 approximately 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 1.25mm, 1.5mm, 1.75mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm, 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, or 10mm. Channel density can be defined as the ratio of channel width to the space (or distance) between channels. In some cases, the ratio of channel width to the 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.

[0258] In some cases, the total volume of all channels within a microfluidic device (e.g., a chip) is 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, 90 μL, etc. 0μL, 1 milliliter (mL), 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 13mL, 14mL, 15mL, 16mL, 17mL, 18m L, 19mL, 20mL, 25mL, 30mL, 35mL, 40mL, 50mL, 55mL, 60mL, 65mL, 70mL, 75mL, 80mL, 85mL, 90mL, 95mL or 100mL. In some cases, the total volume of all channels within 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. 900μL, 1 milliliter (mL), 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 13mL, 14mL, 15mL, 16mL, 17mL, 18 mL, 19mL, 20mL, 25mL, 30mL, 35mL, 40mL, 50mL, 55mL, 60mL, 65mL, 70mL, 75mL, 80mL, 85mL, 90mL, 95mL or 100mL.

[0259] The inlets and / or outlets of fluid handling devices can be arranged and spaced to be compatible with standard fluid handling configurations. For example, the inlets and / or outlets can be spaced to align with the wells on a 5” x 3.33” titration plate. A device may contain eight inlets and / or outlets spaced to correspond to eight wells in a standard eight-tip pipette and / or a standard 24-, 48-, or 96-well plate. A device may contain twelve inlets and / or outlets spaced to correspond to twelve wells in a standard twelve-tip pipette and / or a standard 96-well plate. A device may contain sixteen inlets and / or outlets spaced to correspond to sixteen wells in a standard sixteen-tip pipette and / or a standard 384-well plate. A device may contain twenty-four inlets and / or outlets spaced to correspond to twenty-four wells in a standard twenty-four-tip pipette and / or a standard 384-well plate. This allows for easier handling of fluid from such a plate onto the device, for example, via a robotic pipette system or other multi-pipettes.

[0260] Isokinetic electrophoresis can be performed using a desktop system or a base station. For example, Figure 13A A benchtop system 1300 for sample preparation and isotachophoresis on a fluid device cartridge 1301 is shown. The fluid device cartridge can be loaded onto the benchtop system as shown, and a lid with a matching cap and a controller 1302 can be lowered onto the fluid device cartridge. The benchtop system may also include a control panel 1303 having a user interface (e.g., a touchscreen) for an operating system.

[0261] The benchtop system may include a pressure controller that provides pressure to process fluids (e.g., samples, buffers, reagents, enzyme solutions, electrolyte solutions) on a fluid handling device. The benchtop system may receive pressure feedback signals to regulate or control fluid handling. Fluid handling can be used to load fluids (e.g., reagents, buffers, samples) onto a fluid handling device. Fluid handling can be used to introduce fluids (e.g., reagent solutions) into a drying channel on a fluid handling device. Pressure can be regulated using, for example, a solenoid valve.

[0262] A benchtop system may include electrodes or electrical contacts. Electrodes may be part of a circuit and may be inserted into a reservoir or other opening on a fluid device to allow an electric field to be applied within the fluid device through the completed circuitry. Electrical contacts may be coupled to corresponding contacts on the fluid device (e.g., a fluid device with integrated electrodes).

[0263] The benchtop system may include one or more detectors or sensors, such as photodetectors, reflective sensors, infrared (IR) detectors, electrical detectors, thermal sensors, flow sensors, and pressure sensors, including the detectors further described in this disclosure. Photodetectors may 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. Electrical detectors may include electrodes or other detectors capable of detecting voltage, voltage difference, current, charge, or other electrical properties. Electrical detectors can be used to detect pathways of extracted or purified nucleic acid bands, for example, by detecting changes in conductivity at the interface between the trailing electrolyte and the leading electrolyte. Thermal sensors may include infrared (IR) sensors, probe temperature sensors, thermistors, negative temperature coefficient (NTC) thermistors, resistance temperature detectors (RTDs), thermocouples, semiconductor-based sensors, etc.

[0264] The one or more detectors or sensors can be operated and controlled simultaneously or independently. In some cases, a single channel may have a dedicated sensor, such as a thermal sensor or a voltage sensor, which operates independently of other sensors dedicated to other channels on the microfluidic device. Feedback from the independent sensor can be used to independently control one or more electric fields on the device. For example, a sensor may detect changes in the voltage within the orifice over time, as described herein, and feedback from that sensor can be used to control the current within the channel. A second sensor may act on a second channel in a similar but independent manner. In some cases, a sensor may detect changes in the current within the orifice over time, and feedback from that sensor can be used to control the voltage within the channel.

[0265] A benchtop system may include one or more thermal controllers that control the temperature on or a portion of a fluid device. The thermal controller may include components, including but not limited to resistance heaters, fluid-based heating or cooling systems, and Peltier devices. The thermal controller may be made of materials including but not limited to metals (e.g., platinum, titanium, copper, gold), carbon, and indium tin oxide (ITO). The thermal controller may include temperature sensors that can be used to monitor the controlled temperature and provide temperature feedback for thermal control. As further discussed in this disclosure, the thermal controller may be used in conjunction with a computer control system. For example, a temperature sensor (e.g., an infrared sensor) may be used to monitor temperature changes in channels on a chip. Such temperature changes can indicate the location of ITP bands (e.g., nucleic acid bands) during ITP processes, and the temperature difference can be attributed to changes in conductivity between the leading electrolyte and the trailing electrolyte. In some cases, the thermal controller operates without temperature feedback.

[0266] The techniques disclosed herein (including, for example, the use of fluidic devices and / or benchtop systems discussed herein) can provide rapid processing times. For example, samples containing nucleic acids can be prepared (e.g., removal of embedding materials, tissue disruption, cell lysis, nucleic acid decrosslinking) and have extracted or purified nucleic acids for subsequent analysis, use, or storage.

[0267] Detection and quantification

[0268] The techniques disclosed herein can employ one or more detectors. The detectors can be integrated into a fluidic device or located externally to the fluidic device. The detectors can be used for the quantification of nucleic acids in a sample, for example by fluorescence measurement or ultraviolet (UV) radiation (e.g., for measuring quantity or purity, such as by measuring A260 / A280), or for providing qualitative measurements of nucleic acids in a sample. Nucleic acids can be detected when located on a fluidic device (e.g., in a purification zone (e.g., an ITP channel) or in a reservoir (e.g., an elution reservoir). The concentration of nucleic acids can be detected (or calculated based on a known volume (e.g., in an elution well as described herein)). Nucleic acids can be labeled with dyes, and the fluorescence intensity of the nucleic acids can be measured by the detector and the presence of nucleic acids can be quantified using the fluorescence intensity (see, for example,...). Figure 14 Nucleic acids can be labeled before loading onto a fluid device, during the fluid device, or after recovery from the fluid device.

[0269] Detectors can be used to achieve nucleic acids in samples with high sensitivity or low detection limits. For example, nucleic acids can be detected at detection limits less than or equal to about 1000 picograms per microliter (pg / μL), 100 pg / μL, 10 pg / μL, 1 pg / μL, 0.9 pg / μL, 0.8 pg / μL, 0.7 pg / μL, 0.6 pg / μL, 0.5 pg / μL, 0.4 pg / μL, 0.3 pg / μL, 0.2 pg / μL, or 0.1 pg / μL (e.g., online detection in an isotachymeter electrophoresis channel). Nucleic acids can also be detected at detection limits less than or equal to about 1000 picograms (pg), 100 pg, 10 pg, 1 pg, or 0.1 pg (e.g., online detection in an isotachymeter electrophoresis channel).

[0270] Detectors can be used to identify or quantify nucleic acids in a sample. For example, techniques such as nucleic acid amplification (including, for example, PCR, real-time PCR, and reverse transcription PCR), hybridization (including, for example, fluorescence in situ hybridization (FISH) and Q-FISH), and sequencing can be used to identify the presence or absence of specific sequences within nucleic acids in a sample, and optionally to quantify them.

[0271] Detectors can be used to control nucleic acid extraction or purification procedures. For example, a detector can detect nucleic acid bands concentrated by isodynamic electrophoresis. When the concentrated nucleic acid reaches a certain point within the device, the process can be terminated (e.g., the electric field can be turned off), and the extracted or purified sample can be recovered from the device.

[0272] Detectors may include, but are not limited to, photodetectors and electrical detectors, thermal sensors and pressure sensors (e.g., pressure sensors). Photodetectors may 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. Photodetection can be achieved by LED illumination paired with photodiode detection. Electrical detectors may 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 pathways in extracted or purified nucleic acid bands.

[0273] Triggered upon completion of execution

[0274] When purifying samples using ITP, it may be important to accurately stop the applied current when the sample's ITP region is in the elution position (e.g., in a channel or reservoir). This disclosure provides techniques for assessing the ITP region position, which can be used to trigger the termination of a purification run. These techniques may include measurements of the driving voltage, conductivity, and temperature.

[0275] Figure 15 A schematic diagram of an ITP channel 1500 is shown, in which a drive electrode is placed in a buffered elution electrode (EH) reservoir 1501 and a buffered lead electrolyte (LEH) reservoir 1502, and a ground electrode is placed in a buffered trailing electrolyte (TEH) reservoir 1503. A conductivity detector (e.g., a capacitively coupled non-contact conductivity detector (C4D)) electrode 1504 may be placed externally to the chip, such as near the elution reservoir 1505, as shown on the left side of the figure. The channel may also include a lead electrolyte reservoir 1506 and a sample reservoir or injection point 1507. Gas ports are indicated by small circles on the leftmost and rightmost edges of the channel. Gas ports can be used to automatically load or direct fluid from the attached reservoirs into the channel, for example, using a vacuum or applied pressure.

[0276] One method for measuring the location 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 performed between any pair of electrodes. Since the voltage driving the electrophoresis is also the measurement voltage, this measurement can be easily performed. Throughout the purification process, the voltage can increase as the trailing ions fill the channel. However, the elution reservoir can have a large cross-section, so its contribution to the overall resistance may be small. Therefore, changes in the buffer conductivity in this region may not strongly affect the overall channel resistance, and the voltage may stop rising when the ITP zone enters the elution reservoir. This can be used as a signal to stop applying current and end the operation.

[0277] To evaluate this voltage change, the derivative of the voltage can be calculated, for example, as... Figure 16 As shown. High-frequency noise can be suppressed using the Lanzcos derivative method. A threshold can be set for the derivative, and a trigger can be activated when the derivative exceeds the threshold. In some cases, introducing additional triggers can improve the robustness of the control. For example, Figure 16 Four trigger points are shown. In some cases, only two of these triggers are used to change the drive current (e.g., triggers 1 and 4), while the others (e.g., triggers 2 and 3) are used to mark points in the operation, which can improve the timing of trigger 4. Figure 17 It shows Figure 16 The derivative analysis of the voltage in the figure, where the arrows indicate the derivative thresholds used to select the trigger point.

[0278] Figure 16 Example data from measured drive voltage is shown. Each vertical line represents a trigger point. Two lines represent two electrodes, namely, the electrodes in the EH and LEH reservoirs relative to the ground electrode. Points A, B, C, and D indicate that the ITP zone is in... Figure 15 The times at the corresponding positions marked in the middle (A, B, C, and D; marked as 1508, 1509, 1510, and 1511, respectively). In some cases, the conductivity at each position in the channel can affect the overall drive voltage, which can make it more difficult to assess what is happening near the elution reservoir.

[0279] A second method for detecting the location of the ITP band is to perform a localized conductivity measurement. This can be accomplished using a capacitively coupled non-contact conductivity detector (C4D). This method uses a high-frequency alternating current through the channel wall and coupled to the electrolyte. This localized measurement can be performed within the elution reservoir itself. This technique can reduce or eliminate the ambiguity associated with measurements performed across the entire channel. In this technique, once a change in conductivity is observed in the elution reservoir conductivity detector, a run-end trigger can be selected, for example, as... Figure 18 As shown.

[0280] C4D detection can be performed using electrodes positioned below the elution channel. Maximizing the electrode area reduces the required drive frequency. For example, a drive frequency of approximately 100 kHz to approximately 10 MHz can be used, with the electrode contact pad being approximately 0.2 mm. 2 approximately 50mm 2 C4D sensors can be implemented using electronic components (including resistors, capacitors, diode bridges, and high-frequency operational amplifiers) and high-frequency signal sources (such as those from direct digital synthesizers). Figure 19 An exemplary schematic diagram of a C4D sensor implementation is shown.

[0281] A third method for detecting the location of the ITP band is to locally measure the temperature near the elution reservoir. This measurement can be performed using a temperature sensor, including a thermocouple or an infrared temperature sensor. The sensor can be placed below the channel near the elution reservoir and the temperature can be monitored over time. When a lower-mobility trailing ion displaces a higher-mobility leading ion (e.g., the LE-TE interface of the ITP region), the electric field in the channel may increase, and the temperature may rise. During isotachophoresis, the lower-mobility trailing electrolyte ion and the higher-mobility leading electrolyte ion may meet at the isotachophoresis interface. The ITP interface may contain concentrated sample nucleic acids between the leading and trailing electrolyte ions. The temperature rise can detect the presence of the ITP interface between the higher-mobility leading ion and the lower-mobility trailing ion, and therefore also indicate the presence of nucleic acids between them. This temperature rise can be 1–10 °C.

[0282] Figure 20A and Figure 20B Exemplary temperature measurements using a thermal imaging camera are shown. These images demonstrate a significant increase in temperature as trailing ions enter the channel. Figure 20A This shows a temperature map of the ITP channel taken using a thermal imaging camera; the channel orientation is... Figure 15 The same as in. Figure 20B It shows Figure 20A The graph shows the temperature at the location of cursor 1 over time. At approximately 450 seconds, the ITP interface and trailing ions enter the region, causing a temperature increase. This temperature rise can be detected and used as a trigger signal to change the current applied to the channel.

[0283] Temperature can be measured at or near the washing reservoir (e.g., as shown in the image). Figure 21(As shown in the diagram). In some cases, the detection location may be at least approximately 5 mm from the eluent reservoir. In some cases, the detection location may be at least approximately 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 eluent reservoir. In some cases, the detection location may be at most approximately 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 eluent reservoir. In some cases, the temperature sensor may be located at a distance of at least approximately 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 eluent reservoir. In other cases, the temperature sensor may be located at a distance of at most approximately 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 eluent reservoir.

[0284] When a temperature change is sensed, the temperature sensor can trigger a change in current. In some cases, the detected temperature change is in the range of approximately 0.2°C to approximately 5°C. In some cases, the detected temperature change is at least approximately 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 cases, the detected temperature change is at most approximately 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.

[0285] In some cases, detecting the ITP region at one or more trigger points (e.g., via voltage monitoring, conductivity measurement, or temperature sensing) can cause a benchtop controller to alter the current applied to the microfluidic chip. This alteration can be applied at the time of detection or immediately after a predetermined delay. Detection of the ITP region can trigger a decrease, increase, or elimination of the current. For example, detecting the ITP region at point C 1510 can trigger a decrease in current to increase the residence time of the ITP region in the channel leading to the elution reservoir. Alternatively or in combination, detecting the ITP region at point D 1511, located at or near the elution reservoir, can trigger the elimination of current to position the ITP region (and nucleic acid) or a portion thereof within the region of the elution reservoir, well, or channel, or the chip. In some cases, detection of the ITP region can trigger a change in current after a predetermined amount of time. For example, the detection location (e.g., location 1504 or cursor 1) can be located at or near the elution reservoir at a known distance, allowing the time required for the ITP region to travel between the detection location and the elution reservoir to be calculated for a given current. The controller can predetermine the travel time, and detecting the ITP region at the detection location can trigger current delay cancellation after a predetermined amount of time. In some cases, detecting the ITP region at a specific detection location can provide the spatiotemporal relationship of the ITP region, which can lead to more accurate triggering than other sensing methods.

[0286] In some cases, detecting the ITP region at a trigger point can change the direction or path of the current applied to the microfluidic chip. For example, current reversal can be triggered, causing the ITP region to reverse its direction of travel within the channel. In another instance, the system can be triggered to stop applying current between a first pair of electrodes and begin applying current to a second pair of electrodes to drive the ion flow along a different path. For example, the channel can be "Y-shaped," where a first channel leads to two side channels separated from the first channel in different directions. Initially, current can be driven between a first electrode connected to the first channel and a second electrode connected to the first side channel, respectively. Without interrupting the current, the ITP region can travel from the first channel to the first side channel. Detecting the ITP region at the junction between the first channel and the two side channels triggers the first and second electrodes to stop driving the current, and the third and fourth electrodes connected to the first and second side channels, respectively, to begin driving the current. The ITP region then travels from the first channel to the second channel. In some cases, the first and third electrodes are the same electrode. In this way, triggering can change the current, causing the path of the ITP region to change along the channel.

[0287] Further processing and use of purified samples

[0288] Extracted or purified nucleic acids can be used for sequencing, genotyping, mutation or polymorphism analysis, gene expression level analysis, disease diagnosis, disease prediction, cytological classification, paternal or pedigree analysis, or as indications of recommended treatment methods.

[0289] Extracted or purified nucleic acids can be used for amplification reactions, including but not limited to loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), rolling circle amplification (RCA), nickase amplification reaction (NEAR), PCR, reverse transcription PCR, real-time PCR, quantitative PCR (qPCR), digital PCR, and methylation-specific PCR.

[0290] Extracted or purified nucleic acids can be used for sequencing reactions, including Maxam-Gilbert sequencing, chain termination sequencing (e.g., Sanger sequencing), shotgun sequencing, pyrosequencing, bridge PCR, colony sequencing, polymerase cloning (polony) sequencing, synthetic sequencing, ion semiconductor sequencing, nanopore sequencing, nanosphere sequencing, ligation sequencing, hybridization sequencing, and single-molecule real-time sequencing.

[0291] Extracted or purified nucleic acids can be used for protein binding assays, such as DNA footprinting assays. For example, DNases (e.g., DNase I) can be used to randomly cleave DNA molecules of interest. The techniques of this disclosure can be used to separate digested DNA from DNases, thereby preventing further digestion. In some cases, DNase digestion can be performed outside of a fluidic apparatus, after which the sample can be loaded onto a fluidic apparatus for purification. In other cases, DNase digestion can be performed on a fluidic apparatus, and once digestion has occurred, the nucleic acids can be purified on the fluidic apparatus.

[0292] Samples such as fixed or embedded samples (e.g., FFPE samples) can be used for longitudinal studies, genome-wide association studies, and other large-scale analyses across populations.

[0293] Vertical or columnar ITP

[0294] Planar ITP device designs, such as those discussed in this paper, can utilize horizontal space to allow the ITP belt to travel. For high-throughput sample processing, such as in a 96-well plate format, mounting the entire ITP separation system within a given footprint (e.g., a 9mm × 9mm footprint) may be advantageous for the sample. One approach to do this is to increase the height of the system to accommodate larger sample volumes. This provides the option to increase the total sample volume to the milliliters range while still processing samples with reasonable run times.

[0295] In some cases, reducing or preventing gravity-driven and / or buoyant flows through such a system may be important. Assembling the electrolyte zone required for ITP without mixing the electrolyte may also be important.

[0296] Vertical or columnar ITP systems may comprise several ITP steps, each step consisting of a column (e.g., plastic) with a gel (e.g., agarose) or a similar material at the bottom. The gel may have high electrical conductivity. Each step may be prepared by introducing an electrolyte on top of the gel. The gel may slow down or impede liquid flow. To form a column, the steps can be stacked with a trailing electrolyte at the top and a leading electrolyte at the bottom. A current can then be driven through the system. The purified analyte can be recovered by destacking and removing the column.

[0297] Figure 22A An exemplary schematic diagram of a vertical (or columnar) ITP setup is shown. The gel at each step can support the weight of the aforementioned water (e.g., an electrolyte solution). The cross-sectional area of ​​the column can be approximately 9 mm × 9 mm. Such a system can process samples with a cross-sectional column area of ​​approximately 9 mm × 9 mm. This design can be scaled up, for example, to 96 samples (columns), with the overall device dimensions conforming to a standard microtiter plate. Figure 22B An exemplary image of a vertical ITP structure established using DNA ITP bands is shown. The steps are: Tail Electrolyte High (TEH), Sample, Lead Electrolyte (LE), and Lead Electrolyte High (LEH). The ITP region is moving downwards through the system. The image does not show the elution phase (E, ) as the final destination of the analyte. Figure 22A (as shown in the image).

[0298] Computer control system

[0299] This disclosure provides a computer control system programmed to implement the methods of this disclosure. Figure 13B A computer system 1304 is shown, which 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 this disclosure, such as applying pressure or an electric field, thermal control, detection, quantification, feedback, and starting or ending the process. The computer system 1304 can be a user's electronic device or a computer system located remotely relative to an electronic device. The electronic device can be a mobile electronic device.

[0300] Computer system 1304 includes a central processing unit (CPU, also referred to herein as a “processor” and “computer processor”) 1305, which may be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 1304 also includes memory or memory location 1310 (e.g., random access memory, read-only memory, flash memory), electronic storage unit 1315 (e.g., hard disk), communication interface 1320 for communicating with one or more other systems (e.g., network adapter), and peripheral devices 1325, such as cache memory, other memory, data storage, and / or electronic display adapters. Memory 1310, storage unit 1315, interface 1320, and peripheral devices 1325 communicate with CPU 1305 via a communication bus (solid line) such as a motherboard. Storage unit 1315 may be a data storage unit (or data repository) for storing data. Computer system 1304 may be operatively coupled to computer network (“network”) 1330 by means of communication interface 1320. Network 1330 may be the Internet, the Internet and / or an extranet, or an intranet and / or extranet communicating with the Internet. In some cases, network 1330 is a telecommunications and / or data network. Network 1330 may include one or more computer servers that support distributed computing, such as cloud computing. In some cases, network 1330 may be a peer-to-peer network via computer system 1304, which allows devices coupled to computer system 1304 to act as clients or servers.

[0301] CPU 1305 can execute a series of machine-readable instructions, which may be embodied in a program or software. These instructions may be stored in a storage location such as memory 1310. The instructions may be directed to CPU 1305, which can then be programmable or otherwise configured to implement the methods of this disclosure. Examples of operations performed by CPU 1305 may include fetching, decoding, executing, and writing back.

[0302] CPU 1305 may be part of a circuit such as an integrated circuit. One or more other components of system 1304 may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0303] Storage unit 1315 may store files, such as drivers, libraries, and saved programs. Storage unit 1315 may store user data, such as user preferences and user programs. In some cases, computer system 1304 may include one or more additional data storage units located outside of computer system 1304, such as on a remote server communicating with computer system 1304 via an intranet or the Internet.

[0304] Computer system 1304 can communicate with one or more remote computer systems via network 1330. For example, computer system 1304 can communicate with a user's remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), tablets, or tablet PCs (e.g., tablet PCs). Galaxy Tab), telephone, smartphone (e.g., iPhone, Android-compatible devices (or personal digital assistant). Users can access computer system 1304 via network 1330.

[0305] The methods described herein may be implemented via machine-executable code (e.g., a computer processor) stored in an electronic storage location of the computer system 1304, such as in memory 1310 or electronic storage unit 1315. The machine-executable code or machine-readable code may be provided in software form. During use, the code may be executed by processor 1305. In some cases, the code may be retrieved from storage unit 1315 and stored in memory 1310 for access by processor 1305. In some cases, electronic storage unit 1315 may be excluded, and the machine-executable instructions may be stored in memory 1310.

[0306] The code may be pre-compiled and configured for use with a machine having a processor suitable for executing the code, or it may be compiled at runtime. The code may be provided in a programming language, which may be selected to enable the code to be executed in a pre-compiled or just-in-time (JIT) compiled manner.

[0307] Various aspects of the systems and methods provided herein, such as computer system 1304, can be embodied in programming. Several aspects of this technology can be considered "products" or "articles of art," generally in the form of machine (or processor) executable code and / or associated data carried or embodied on a machine-readable medium of a certain type. Machine-executable code can be stored on electronic storage units such as memory (e.g., read-only memory, random access memory, flash memory) or hard disks. "Storage" media can include any or all of a computer's tangible memory, processor, etc., or associated modules such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage for software programming at any time. All or part of the software can sometimes be communicated via the Internet or various other telecommunications networks. Such communication, for example, enables the software to be loaded from one computer or processor to another, for example, from a management server or host computer platform to an application server. Therefore, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as those used through physical interfaces between local devices, via wired and optical terrestrial networks, and via various air links. Physical elements carrying such waves, such as wired or wireless links, optical links, etc., can also be considered as media carrying software. As used herein, unless limited to non-transitory tangible "storage" media, the term "readable medium" for a computer or machine refers to any medium that participates in providing instructions to a processor for execution.

[0308] Therefore, machine-readable media, such as computer-executable code, can take many forms, including but not limited to: tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical discs or disks, any storage device such as any computer, which can be used to implement, for example, a database as shown in the attached figure. Volatile storage media include dynamic memory, such as the main memory of a computer platform. Tangible transmission media include coaxial cables; copper wires and optical fibers, including conductors that comprise buses within a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals, or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communication. Therefore, common forms of computer-readable media include, for example: floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card tapes, any other physical storage media with a perforated pattern, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chips or cartridges, carrier waves for transmitting data or instructions, cables or links for transmitting such carrier waves, or any other media from which a computer can read programming code and / or data. Many of these forms of computer-readable media can participate in loading one or more sequences of one or more instructions to a processor for execution.

[0309] Computer system 1304 may include or communicate with an electronic display 535, the electronic display 535 including a user interface (UI) 1340 for providing, for example, operating parameters (e.g., processing time, temperature, field strength), nucleic acid quantification information, or other information. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0310] The methods and systems disclosed herein can be implemented using one or more algorithms. These algorithms can be implemented in software when executed by the central processing unit 1305. For example, the algorithm can adjust the thermal controller, calculate nucleic acid quantification, control process functions, and start or stop the process.

[0311] Reagent test kit

[0312] This disclosure provides kits for performing isotachophoresis. Typically, such kits contain the microfluidic device described herein and one or more buffers. Buffers may include any combination of one or more sample buffers, one or more lead electrolyte buffers, one or more trailing electrolyte buffers, one or more lysis buffers, and / or one or more elution buffers. In some cases, the kit may include one or more enzymes (e.g., RNase, DNA, nuclease, proteases, proteinases, polymerases). Buffers may be provided in separate tubes. In some cases, the microfluidic device is pre-loaded with one or more buffers. The kit may include a set of instructions on operating the device and / or processing samples.

[0313] This invention provides, but is not limited to, the following embodiments:

[0314] 1. A method for purifying a sample, the method comprising:

[0315] (a) Loading the following substances into a fluid device:

[0316] (i) A tissue sample containing lysed solid tissue, wherein the lysed solid tissue contains nucleic acids and contaminants.

[0317] (ii) a trailing electrolyte buffer containing a first trailing electrolyte ion having an effective migration rate, wherein the effective migration rate of the first trailing electrolyte ion is less than the effective migration rate of the nucleic acid, and

[0318] (iii) a lead electrolyte buffer containing a first lead electrolyte ion having a second effective mobility, wherein the magnitude of the second effective mobility of the first lead electrolyte ion is greater than the magnitude of the effective mobility of the nucleic acid; and

[0319] (b) An electric field is applied within the fluid apparatus to perform isovelocity electrophoresis using the first trailing electrolyte ions, the nucleic acid, and the first leading electrolyte ions, thereby purifying the nucleic acid from the contaminants in the tissue sample.

[0320] 2. According to the method of embodiment 1, the effective migration rate of the first trailing electrolyte ion is greater than the effective migration rate of the pollutant.

[0321] 3. The method according to Embodiment 1, wherein the fluid device is a microfluidic chip, and the tissue sample, the trailing electrolyte buffer, and the leading electrolyte buffer are loaded into a first region of the microfluidic chip.

[0322] 4. The method according to any one of embodiments 1-3, further comprising performing at least one sample preparation procedure selected from the group consisting of the following in the first region of the microfluidic chip: (1) removing embedding material, (2) destroying tissue, (3) lysing cells, (4) decrosslinking the nucleic acid, (5) digesting protein, and (6) digesting the nucleic acid.

[0323] 5. The method according to any one of embodiments 1-4, wherein the isokinetic electrophoresis is performed in a second region of the microfluidic chip, wherein the second region is separated from the first region and fluidly connected to the first region.

[0324] 6. The method according to any one of embodiments 1-5, wherein the solid tissue is derived from a solid organ.

[0325] 7. The method according to any one of embodiments 1-6, wherein the lysed solid tissue comprises a chemical fixative.

[0326] 8. The method according to embodiment 7, wherein the chemical fixative is formalin.

[0327] 9. The method according to embodiment 8, wherein the solid tissue is formalin-fixed paraffin-embedded tissue (FFPE).

[0328] 10. The method according to any one of embodiments 1-9, wherein the lysed solid tissue comprises urea or thiourea.

[0329] 11. The method according to any one of embodiments 1-10 further includes disrupting cell-cell junctions, extracellular matrix, or connective tissue to obtain the lysed solid tissue.

[0330] 12. The method according to any one of embodiments 1-10, wherein the ruptured solid tissue comprises solid particles.

[0331] 13. The method according to any one of embodiments 1-12, wherein the nucleic acid comprises dispersed or solvated nucleic acid.

[0332] 14. The method according to any one of embodiments 1-13, wherein the contaminant is selected from cross-linked nucleic acids, embedding materials, tissue fragments, fixation chemicals, proteins, inhibitors, and combinations thereof.

[0333] 15. The method according to any one of embodiments 1-14, wherein the contaminant comprises cross-linked nucleic acid.

[0334] 16. The method according to any one of embodiments 1-15, wherein the tissue sample is combined with the trailing electrolyte buffer before the loading.

[0335] 17. The method according to any one of embodiments 1-16, wherein the tissue sample is combined with the lead electrolyte buffer before the loading.

[0336] 18. The method according to any one of embodiments 1-17, wherein the loading of the lead electrolyte buffer is performed prior to the loading of the tissue sample.

[0337] 19. The method according to any one of embodiments 1-18, wherein the solid tissue is lysed in the lead electrolyte buffer prior to the loading of the tissue sample.

[0338] 20. The method according to any one of embodiments 1-19, wherein the solid tissue is lysed in the trailing electrolyte buffer prior to the loading of the tissue sample.

[0339] 21. The method according to any one of embodiments 4-20, wherein the sample preparation procedure comprises: removing the embedding material by incubating the tissue sample in the fluid device at a temperature of at least about 37°C for a duration of at least about 1 minute prior to the application of the electric field.

[0340] 22. The method according to embodiment 21, wherein the temperature is about 40°C to about 80°C.

[0341] 23. The method according to embodiment 21, wherein the duration is from about 1 minute to about 120 minutes.

[0342] 24. The method according to any one of embodiments 4-23, wherein the sample preparation procedure includes destroying tissue or lysing cells by applying mechanical stress to the tissue sample.

[0343] 25. The method according to any one of embodiments 4-24, wherein the sample preparation procedure includes destroying tissue or lysing cells by applying heat to the tissue sample.

[0344] 26. The method according to any one of embodiments 4-25, wherein the applied heat causes the temperature of the tissue sample to be in the range of about 30°C to about 80°C.

[0345] 27. The method according to any one of embodiments 4-26, wherein the sample preparation procedure includes destroying tissue or lysing cells by contacting the tissue sample with a solution having a pH of at least 10 or by digesting the tissue sample by protein hydrolysis.

[0346] 28. The method according to embodiment 27, wherein the protein hydrolysis is carried out at a temperature greater than about 25°C.

[0347] 29. The method according to any one of embodiments 4-28, wherein the sample preparation procedure includes destroying tissue or lysing cells by applying at least one surfactant to the tissue sample.

[0348] 30. The method according to any one of embodiments 4-29, wherein the sample preparation procedure includes destroying tissue or lysing cells by applying a solution containing urea to the tissue or cell sample.

[0349] 31. The method according to embodiment 30, wherein the solution further comprises thiourea.

[0350] 32. The method according to embodiment 31, wherein the concentration of urea in the solution is in the range of about 4M to about 9M, and the concentration of thiourea in the solution is in the range of about 0.5M to about 3.5M.

[0351] 33. The method according to embodiment 31, wherein the concentration of urea in the solution is about 6.5M to about 7.5M, and the concentration of thiourea in the solution is about 1.5M to about 2.5M.

[0352] 34. The method according to any one of embodiments 4-33, wherein the sample preparation procedure includes decrosslinking the nucleic acid by digesting the crosslinked protein with proteinase K.

[0353] 35. The method according to any one of embodiments 4-34, wherein the sample preparation procedure includes digesting the nucleic acid with a DNA enzyme or an RNA enzyme.

[0354] 36. The method according to any one of embodiments 1-35, further comprising eluting an output solution containing the purified nucleic acid from the outlet reservoir of the fluid device.

[0355] 37. The method according to embodiment 36, wherein the concentration of the purified nucleic acid in the output solution is at least about twice as high as the concentration of the nucleic acid in the tissue sample.

[0356] 38. The method according to embodiment 36, wherein the purified nucleic acid in the tissue sample and the output solution comprises cross-linked nucleic acid, and the concentration of the cross-linked nucleic acid in the output solution is at least about twice the concentration of the cross-linked nucleic acid in the tissue sample.

[0357] 39. The method according to embodiment 36, wherein the contaminant is present in the output solution at a concentration at least twice lower than the concentration of the contaminant in the tissue sample.

[0358] 40. The method according to any one of embodiments 1-39, wherein the first trailing electrolyte ion comprises hexanoic acid.

[0359] 41. The method according to any one of embodiments 1-40, wherein the first leading electrolyte ion comprises chloride.

[0360] 42. The method according to any one of embodiments 1-41, wherein the trailing electrolyte buffer contains a second trailing electrolyte ion having an effective mobility different from that of the first trailing electrolyte ion.

[0361] 43. The method according to embodiment 42, wherein the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) or MOPS (3-(N-morpholino)propanesulfonic acid).

[0362] 44. The method according to embodiment 42, wherein the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid.

[0363] 45. The method according to embodiment 42, wherein the second trailing electrolyte ion comprises MOPS (3-(N-morpholino)propanesulfonic acid), and the first trailing electrolyte ion comprises hexanoic acid.

[0364] 46. ​​The method according to embodiment 42, wherein the second trailing electrolyte ion comprises HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the first trailing electrolyte ion comprises MOPS.

[0365] 47. The method according to any one of embodiments 1-46, wherein the trailing electrolyte buffer comprises a second trailing electrolyte ion having a second effective mobility, wherein the amount of the second effective mobility is substantially the same as or lower than the amount of the effective mobility of the contaminant.

[0366] 48. The method according to any one of embodiments 1-47, wherein the tissue sample loaded into the fluid device has a volume of at least 50 μl.

[0367] 49. The method according to any one of embodiments 1-48, further comprising performing a first sample processing procedure on the tissue sample in the first region of the microfluidic chip, and performing an enzymatic reaction on the tissue sample in the second region of the microfluidic chip.

[0368] 50. The method according to embodiment 49, wherein the first sample processing procedure includes removing embedding material, disrupting tissue or cell lysis, and the enzymatic reaction includes decrosslinking the nucleic acid, digesting protein, or digesting nucleic acid.

[0369] 51. The method according to embodiment 49, wherein the first region and the second region are each heated to a temperature of 37°C or higher.

[0370] 52. The method according to embodiment 49, wherein the first region is heated to a temperature of about 60°C to 100°C during the first sample processing procedure, and wherein the second region is heated to a temperature of 40°C to 60°C.

[0371] 53. A method for simultaneously purifying nucleic acids from at least two different samples, the method comprising:

[0372] (a) The following substances are loaded into a first channel of a microfluidic chip: (i) a first sample containing a first nucleic acid and a first contaminant, (ii) a first tailing electrolyte buffer containing a first tailing ion, wherein the effective mobility of the first tailing ion is less than the effective mobility of the first nucleic acid, and (iii) a first leading electrolyte buffer containing a first leading ion, wherein the effective mobility of the first leading ion is greater than the effective mobility of the first nucleic acid.

[0373] (b) Loading the following substances into the second channel of the microfluidic chip: (i) a second sample containing a second nucleic acid and a second contaminant; (ii) a second tailing electrolyte buffer containing a second tailing ion, wherein the amount of the second tailing ion is less than the amount of the effective mobility of the second nucleic acid; and (iii) a second leading electrolyte buffer containing a second leading ion, wherein the amount of the effective mobility of the second leading ion is greater than the amount of the effective mobility of the second nucleic acid; and

[0374] (c) A first electric field is applied within the microfluidic chip to perform isotachophoresis in the first channel using the first trailing ion, the first nucleic acid, and the first leader ion, and a second electric field is applied to perform isotachophoresis in the second channel using the second trailing ion, the second nucleic acid, and the second leader ion, thereby simultaneously purifying the first nucleic acid from the first contaminant and purifying the second nucleic acid from the second contaminant.

[0375] 54. The method according to embodiment 53, wherein the first sample and the second sample are different sample types.

[0376] 55. The method according to embodiment 53, wherein the first nucleic acid and the second nucleic acid are nucleic acids of different types or lengths.

[0377] 56. The method according to any one of embodiments 1-53, wherein the first trailing electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or a tissue disrupting agent.

[0378] 57. The method according to embodiment 56, wherein the lysing agent or the tissue disrupting agent comprises one or more agents selected from solutions with a pH greater than about 12, proteases, urea, thiourea, and surfactants.

[0379] 58. The method according to any one of embodiments 53-57, wherein the first sample comprises lysed solid tissue.

[0380] 59. The method according to embodiment 58, wherein the second sample comprises lysed cells.

[0381] 60. The method according to any one of embodiments 53-59, wherein during the isotachophoresis process, the first sample does not contact the second sample.

[0382] 61. The method according to any one of embodiments 53-60, further comprising loading the following substances into a third channel of the microfluidic chip: (i) a third sample containing a third nucleic acid and a third contaminant, (ii) a third tailing electrolyte buffer containing a third tailing ion, wherein the effective mobility of the third tailing ion is less than the effective mobility of the third nucleic acid, and (iii) a third leading electrolyte buffer containing a third leading ion, wherein the effective mobility of the third leading ion is greater than the effective mobility of the third nucleic acid, wherein an electric field is applied within the microfluidic chip to perform isovelocity electrophoresis in the third channel using the third tailing ion, the third nucleic acid, and the third leading ion, thereby simultaneously purifying the first nucleic acid from the first contaminant, purifying the second nucleic acid from the second contaminant, and purifying the third nucleic acid from the third contaminant.

[0383] 62. The method according to any one of embodiments 53-61, wherein the first electric field and the second electric field are generated by a single electrode pair.

[0384] 63. The method according to any one of embodiments 53-62, wherein the first electric field and the second electric field are generated by different electrode pairs.

[0385] 64. The method according to embodiment 63, wherein the first channel and the second channel are coupled to independent sensors.

[0386] 65. The method according to embodiment 64, wherein feedback from the independent sensor is used to independently control the first electric field and the second electric field.

[0387] 66. The method according to embodiment 64, wherein the independent sensor detects voltage, and the feedback is used to control the current in the first channel and the second channel.

[0388] 67. The method according to any one of embodiments 1-66, wherein the nucleic acid comprises DNA.

[0389] 68. The method according to any one of embodiments 1-66, wherein the nucleic acid comprises RNA.

[0390] 69. A method for purifying a sample, the method comprising:

[0391] (a) Loading the following substances onto fluid equipment:

[0392] (i) A sample comprising fixed cells, fixed tissue, or embedded tissue, wherein the sample contains nucleic acids.

[0393] (ii) a tailing electrolyte buffer containing a tailing electrolyte, wherein the tailing electrolyte has a lower effective migration rate than the nucleic acid, and

[0394] (iii) a lead electrolyte buffer containing a lead electrolyte, wherein the lead electrolyte has a higher efficient migration rate than the nucleic acid; and

[0395] (b) An electric field is applied to the fluid apparatus to perform isovelocity electrophoresis using the trailing electrolyte, the nucleic acid, and the leading electrolyte, thereby purifying the nucleic acid from contaminants in the sample.

[0396] 70. The method according to embodiment 69, wherein the contaminant is selected from cross-linked nucleic acids, embedding materials, immobilization chemicals, enzymes, and inhibitors.

[0397] 71. The method according to embodiment 69, wherein the sample comprises the fixed cells, the fixed tissue, or both the fixed cells and the fixed tissue.

[0398] 72. The method according to embodiment 69, wherein the sample is formalin fixed.

[0399] 73. The method according to embodiment 69, wherein the sample comprises the embedded tissue.

[0400] 74. The method according to embodiment 69, wherein the sample comprises the tissue embedded in paraffin.

[0401] 75. The method according to embodiment 69, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

[0402] 76. The method according to embodiment 69, wherein the sample comprises a tissue biopsy.

[0403] 77. The method according to embodiment 75, wherein the sample is a dissected formalin-fixed paraffin-embedded (FFPE) sample.

[0404] 78. The method according to embodiment 69 further includes comparing the characteristics of the nucleic acid with nucleic acids from other samples, wherein the characteristics are expression level, nucleic acid sequence, molecular weight, nucleic acid integrity, nucleic acid purity, or nucleic acid chain type.

[0405] 79. The method according to embodiment 69, wherein the sample is a tumor sample.

[0406] 80. The method according to embodiment 69, wherein the trailing electrolyte buffer has a pH greater than about 7.

[0407] 81. The method according to embodiment 69 further includes, prior to applying the electric field, incubating the tissue sample in the fluid device at a temperature of at least about 37°C for a duration of at least about 1 minute.

[0408] 82. The method according to embodiment 81, wherein the temperature is about 40°C to about 80°C.

[0409] 83. The method according to embodiment 81, wherein the duration is from about 1 minute to about 120 minutes.

[0410] 84. The method according to embodiment 60, wherein the lead electrolyte buffer contains proteinase K.

[0411] 85. The method according to embodiment 84, further comprising using the proteinase K to remove protein crosslinks from the nucleic acid.

[0412] 86. The method according to embodiment 60 further includes removing protein cross-links from the nucleic acid by heating after the electric field is applied.

[0413] 87. The method according to embodiment 60 further comprises eluting an output solution containing the purified nucleic acid from the outlet reservoir of the fluid device.

[0414] 88. The method according to embodiment 87, wherein the concentration of the purified nucleic acid in the output solution is at least about twice as high as the concentration of the nucleic acid in the tissue sample.

[0415] 89. The method according to embodiment 87, wherein the concentration of the cross-linked nucleic acid in the output solution is at least about twice the concentration of the cross-linked nucleic acid in the tissue sample.

[0416] 90. The method according to embodiment 87, wherein the output solution has a volume equal to or less than about 50 μL.

[0417] 91. The method according to embodiment 60, wherein the tissue sample has a mass of at least about 1 ng.

[0418] 92. The method according to embodiment 60, wherein the tissue sample has a volume greater than 25 μL.

[0419] 93. The method according to embodiment 60, wherein the trailing electrolyte has a higher effective migration rate than the contaminant.

[0420] 94. The method according to embodiment 60, wherein the trailing electrolyte comprises (i) a first ion having an effective mobility value higher than that of the contaminant, and (ii) a second ion having an effective mobility value substantially the same as or lower than that of the contaminant.

[0421] 95. The method according to embodiment 60, wherein the isotachophoresis is performed to quench the pH of the tissue sample to about 7.5.

[0422] 96. The method according to embodiment 60 further includes dewaxing the sample prior to the loading.

[0423] 97. The method according to embodiment 60 further includes detecting the concentration of the nucleic acid.

[0424] 98. The method according to embodiment 88, wherein the concentration is less than or equal to about 1 picogram per microliter (pg / μL).

[0425] 99. A method for purifying a sample, the method comprising:

[0426] (a) Loading the following substances into a fluid device:

[0427] (i) Tissue samples containing lysed solid tissue and nucleic acids.

[0428] (ii) a trailing electrolyte buffer containing trailing electrolyte ions having a first effective mobility, wherein the magnitude of the first effective mobility is lower than the magnitude of the effective mobility of the nucleic acid.

[0429] (iii) A first leader electrolyte buffer in a first leader electrolyte reservoir, the first leader electrolyte buffer containing a first leader electrolyte ion having a second effective mobility, wherein the magnitude of the second effective mobility is greater than the magnitude of the effective mobility of the nucleic acid, and

[0430] (iv) A second leader electrolyte buffer in a second leader electrolyte reservoir, the second leader electrolyte buffer containing second leader electrolyte ions having a third effective mobility, wherein the magnitude of the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acid.

[0431] The first leading electrolyte buffer is different from the second leading electrolyte buffer.

[0432] (b) Purifying the nucleic acid from the contaminants in the tissue sample by first performing isovelocity electrophoresis with the trailing electrolyte ions, the nucleic acid, and the first leading electrolyte ions; and

[0433] (c) A second isotachophoresis is performed using the trailing electrolyte ions, the nucleic acid, and the second leading electrolyte ions.

[0434] 100. The method according to embodiment 99, wherein the second isokinetic electrophoresis includes changing the applied current from the first channel to the second channel.

[0435] 101. The method according to embodiment 99 or 100, wherein the first precursor electrolyte ion is the same as the second precursor electrolyte ion, and wherein the concentration of the first precursor electrolyte ion in the first precursor electrolyte buffer is different from the concentration of the second precursor electrolyte ion in the second precursor electrolyte buffer.

[0436] 102. The method according to embodiment 99, 100 or 101, wherein the magnitude of the second effective mobility is greater than the magnitude of the third effective mobility.

[0437] 103. The method according to any one of embodiments 99-102, wherein the first leading electrolyte ion is different from the second leading electrolyte ion.

[0438] 104. The method according to any one of embodiments 99-103, wherein the first precursor electrolyte ion is the same as the second precursor electrolyte ion, and wherein the concentration of the first precursor electrolyte ion in the first precursor electrolyte buffer is the same as the concentration of the second precursor electrolyte ion in the second precursor electrolyte buffer, and wherein the first precursor electrolyte buffer contains a third precursor electrolyte ion.

[0439] 105. The method according to any one of embodiments 99-104, wherein the first precursor electrolyte ion is the same as the second precursor electrolyte ion, wherein the concentration of the first precursor electrolyte ion in the first precursor electrolyte buffer is the same as the concentration of the second precursor electrolyte ion in the second precursor electrolyte buffer, and wherein the second precursor electrolyte buffer contains a third precursor electrolyte ion.

[0440] 106. The method according to any one of embodiments 99-105, further comprising collecting the nucleic acid in the second lead electrolyte reservoir and removing the nucleic acid from the second lead electrolyte reservoir.

[0441] 107. The method according to any one of embodiments 99-106, wherein the first isokinetic electrophoresis and the second isokinetic electrophoresis are performed by applying a single electric field.

[0442] 108. The method according to any one of embodiments 99-107, wherein the first isotropic electrophoresis and the second isotropic electrophoresis are performed by applying more than one electric field.

[0443] 109. The method according to any one of embodiments 99-108, wherein the concentration of the second precursor electrolyte ion in the second precursor electrolyte buffer is less than 50 mM.

[0444] 110. The method according to any one of embodiments 99-108, wherein the second lead electrolyte buffer contains 50 mM Tris HCl.

[0445] 111. A microfluidic device comprising:

[0446] (a) A first isovelocity electrophoresis region in a microfluidic chip, the first isovelocity electrophoresis region comprising:

[0447] i. A first sample reservoir in fluid communication with a first fluid channel.

[0448] ii. A first buffer reservoir in fluid communication with the first fluid channel, and

[0449] iii. A second buffer reservoir in fluid communication with the first channel; and

[0450] (b) A second isokinetic electrophoresis region in the microfluidic chip, the second isokinetic electrophoresis region comprising:

[0451] i. A second sample reservoir in fluid communication with the second fluid channel.

[0452] ii. A third buffer reservoir in fluid communication with the second fluid channel, and

[0453] iii. A fourth buffer reservoir in fluid communication with the second channel.

[0454] The first isokinetic electrophoresis zone is not in fluid communication with the second isokinetic electrophoresis zone, and the microfluidic device is configured to independently control a first circuit that applies current to the first isokinetic electrophoresis zone and a second circuit that applies current to the second isokinetic electrophoresis zone.

[0455] 112. The microfluidic device according to embodiment 111, wherein the leakage rate between the first isokinetic electrophoresis zone and the second isokinetic electrophoresis zone is less than 1 μl / hour.

[0456] 113. The microfluidic device according to embodiment 111 or 112, wherein the current leakage between the first region and the second region is less than 1 μA.

[0457] 114. The microfluidic device according to embodiment 111, 112 or 113, wherein the impedance is greater than 1 megohm.

[0458] 115. The microfluidic device according to any one of the foregoing embodiments, wherein the first fluid channel contains a liquid volume greater than 100 μl.

[0459] 116. The microfluidic device according to any one of the foregoing embodiments, wherein the first fluid channel and the second fluid channel are separated by a distance at least five times smaller than the width of the first channel.

[0460] 117. The microfluidic device according to any one of the foregoing embodiments, wherein the microfluidic device is configured to control the first circuit simultaneously with and independently of the second circuit.

[0461] 118. The microfluidic device according to any one of the foregoing embodiments further includes an elution reservoir in fluid communication with the first channel, wherein a temperature sensor is located within 5 mm of the elution reservoir.

[0462] 119. A method comprising:

[0463] (a) Provides an electrofluidic device comprising a sample input reservoir in fluid communication with a channel;

[0464] (b) Load the sample volume into the sample input reservoir;

[0465] (c) Moving at least 50% of the sample volume from the sample input reservoir to the channel without adding additional volume to the sample input reservoir; and

[0466] (d) Apply an ion current through the channel.

[0467] 120. The method according to embodiment 119, wherein the movement is carried out by means of gravity.

[0468] 121. The method according to embodiment 119 or 120, wherein the ion current substantially does not pass through the channel.

[0469] 122. The method according to embodiment 119, 120 or 121, wherein the at least 50% of the sample volume includes at least 80% of the sample volume.

[0470] 123. The method according to any one of the foregoing embodiments, wherein the sample volume comprises nucleic acid.

[0471] 124. The method according to any one of the foregoing embodiments, wherein the sample volume comprises a tissue sample or a formalin-fixed paraffin-embedded (FFPE) sample.

[0472] 125. The method according to any one of the foregoing embodiments, wherein applying the ion current includes performing isotachophoresis.

[0473] 126. The method according to any one of the foregoing embodiments, wherein the total sample volume loaded into the sample input reservoir is less than or equal to the internal volume of the input reservoir.

[0474] 127. The method according to any one of the foregoing embodiments, wherein the sample input reservoir includes a top region connected to a bottom region via a conical region, wherein the top region has a first diameter and the bottom region has a second diameter, wherein the first diameter is at least twice as long as the second diameter to facilitate the movement of at least 50% of the sample volume from the sample input reservoir to the channel.

[0475] 128. The method according to any one of the foregoing embodiments, wherein the volume of the sample loaded into the sample input reservoir is at least 25 μl.

[0476] 129. A microfluidic chip comprising:

[0477] A first sample input reservoir, wherein the first sample input reservoir includes a top region connected to a bottom region via a conical region, wherein the top region has a first internal hydraulic diameter and the bottom region has a second internal hydraulic diameter, wherein the first internal hydraulic diameter is at least twice as large as the second internal hydraulic diameter, and wherein the first sample input reservoir is in fluid communication with a first channel.

[0478] A first buffer reservoir in fluid communication with the first channel, wherein the first sample reservoir is configured such that the free surface of the liquid in the first sample reservoir has a negligible buffer head height difference relative to the liquid in the first buffer reservoir; and

[0479] A second buffer reservoir in fluid communication with the first channel.

[0480] 130. The microfluidic chip according to embodiment 129, wherein the first internal hydraulic diameter is in the range of about 1 mm to about 15 mm.

[0481] 131. The microfluidic chip according to embodiment 129 or embodiment 130, wherein the second internal hydraulic diameter is in the range of about 0.5 mm to about 5 mm.

[0482] 132. The microfluidic chip according to any one of the foregoing embodiments, wherein the first sample reservoir is configured to contain a sample volume of at least 100 μl.

[0483] 133. The microfluidic chip according to any one of the foregoing embodiments, wherein the microfluidic chip is configured to move at least 50% of the sample volume from the first sample reservoir to the first channel when a vacuum is applied thereto.

[0484] 134. The microfluidic chip according to any one of the foregoing embodiments, wherein the microfluidic chip is configured to perform isotachophoresis on a sample entering the first channel.

[0485] 135. A method for extracting nucleic acids, the method comprising:

[0486] (a) Exposing a biological sample containing cells or tissue to a solution containing urea or thiourea, thereby lysing the cells or tissue within the biological sample and producing cell lysates;

[0487] (b) Introducing the cell lysate into the device; and

[0488] (c) Isotachymeter electrophoresis is performed using the device to separate nucleic acids from the cell lysate.

[0489] 136. The method according to any one of the foregoing embodiments further comprises digesting the sample with proteinase K.

[0490] 137. The method according to any one of the foregoing embodiments, wherein the solution comprises urea and thiourea.

[0491] 138. The method according to any one of the foregoing embodiments, wherein the solution comprises urea and thiourea in a ratio of about 2:1.

[0492] 139. The method according to any one of the foregoing embodiments, wherein the concentration of urea in the solution is from about 4M to about 9M, and the concentration of thiourea in the solution is from about 0.5M to about 3.5M.

[0493] 140. The method according to any one of the foregoing embodiments, wherein the concentration of urea in the solution is about 6.5M to about 7.5M, and the concentration of thiourea in the solution is about 1.5M to about 2.5M.

[0494] 141. The method according to any one of the foregoing embodiments, wherein the solution contains either a trailing electrolyte ion or a leading electrolyte ion, or both a trailing electrolyte ion and a leading electrolyte ion.

[0495] 142. A method for purifying high molecular weight nucleic acids from a tissue sample, the method comprising:

[0496] (a) Loading the following substances into a fluid device:

[0497] (i) A cell sample containing genomic DNA and contaminants, wherein the cell sample is contacted with a lysis buffer before or after loading the cell sample into the fluid device.

[0498] (ii) a trailing electrolyte buffer containing trailing electrolyte ions having a first effective mobility, wherein the first effective mobility is less than the effective mobility of the high molecular weight nucleic acid and greater than the amount of the contaminant.

[0499] (iii) A first leader electrolyte buffer, the first leader electrolyte buffer containing a first leader electrolyte ion having a second effective mobility, wherein the value of the second effective mobility is greater than the value of the effective mobility of the high molecular weight nucleic acid;

[0500] (b) Isokinetic electrophoresis is performed using the trailing electrolyte ions, the high molecular weight nucleic acid, and the first leading electrolyte ions to separate the high molecular weight nucleic acid from the contaminants and enrich the high molecular weight nucleic acid in the isokinetic electrophoresis region; and

[0501] (c) The genomic DNA is eluted into a solution in an output reservoir, wherein more than 50% of the mass of the nucleic acid is greater than 30 kilobases.

[0502] 143. The method according to embodiment 142, wherein the lysis buffer does not include an alkaline buffer.

[0503] 144. The method according to any one of the foregoing embodiments, wherein the lysis buffer comprises octylphenol ethoxylate.

[0504] 145. The method according to any one of the foregoing embodiments, wherein the nucleic acid in the solution is greater than 50% by mass and greater than 50 kilobases.

[0505] 146. A method for performing isovelocity electrophoresis, the method comprising:

[0506] (a) Provides a fluid device including a first channel, the first channel being in fluid communication with a sample input reservoir containing a tissue sample containing lysed solid tissue, a first buffer reservoir containing a first leading electrolyte buffer and a second buffer reservoir containing a trailing electrolyte buffer.

[0507] (b) Contact the first electrode with the first leader electrolyte buffer in the first buffer reservoir;

[0508] (c) bringing the second electrode into contact with the trailing electrolyte buffer in the second buffer reservoir; and

[0509] (d) An electric field is applied within the fluid apparatus to perform isokinetic electrophoresis, wherein the isokinetic electrophoresis occurs without direct contact between the tissue sample and the first and second electrodes.

[0510] 147. The method according to embodiment 146, wherein the fluid device further comprises a third buffer reservoir in fluid communication with the first channel and the first buffer reservoir, the third buffer reservoir comprising a lower concentration of the first lead electrolyte buffer than the first buffer reservoir.

[0511] 148. The method according to any one of the foregoing embodiments, wherein the third buffer reservoir and the first buffer reservoir are connected via a second channel comprising one or more capillary barriers to restrict pressure-driven flow within the second channel and between the third buffer reservoir and the first buffer reservoir.

[0512] 149. The method according to any one of the foregoing embodiments, wherein the fluid apparatus further comprises an elution reservoir.

[0513] 150. The method according to any one of the foregoing embodiments, wherein the elution reservoir is in fluid communication with the fourth buffer reservoir.

[0514] 151. A microfluidic system, the microfluidic system comprising:

[0515] (a) A microfluidic chip comprising 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 connection; and

[0516] (b) A mechanical component including a first tooth, wherein the mechanical component is configured to apply mechanical pressure to the first channel via the first tooth to at least partially close the first channel and increase the fluid resistance between the first channel and the first reservoir by plastic deformation of at least one wall of the first channel.

[0517] 152. The microfluidic system according to embodiment 151, wherein the microfluidic chip further includes a second reservoir in fluid communication with the first reservoir and a second channel connecting the first reservoir and the second reservoir, and wherein the mechanical component further includes a second tooth configured to apply mechanical pressure to the second channel to plastically close the second channel and prevent fluid communication between the first reservoir and the second reservoir.

[0518] 153. The microfluidic system according to any one of the foregoing embodiments, wherein the first tooth is configured to deliver mechanical pressure to the first connection to close the first channel by plastic deformation of at least one wall of the first channel.

[0519] 154. The microfluidic system according to any one of the foregoing embodiments, wherein the first tooth is configured to heat the first channel.

[0520] 155. The microfluidic system according to any one of the foregoing embodiments, wherein the mechanical component comprises a material having a Young's modulus greater than that of the first channel.

[0521] 156. The microfluidic system according to any one of the foregoing embodiments, wherein the microfluidic system is configured to perform isotachophoresis.

[0522] 157. The microfluidic system according to any one of the foregoing embodiments, wherein the first tooth is thermally coupled to a heating element.

[0523] 158. The microfluidic system according to any one of the foregoing embodiments, wherein the first tooth is heated to a temperature higher than the glass transition temperature of the at least one wall of the first channel.

[0524] 159. A method for completing a process in a fluid system, the method comprising using the microfluidic system of embodiment 151 to at least partially close the first channel by plastic deformation, thereby increasing the resistance to fluid flow between the first channel and the first reservoir.

[0525] 160. The method according to embodiment 159, wherein the first tooth of the mechanical component applies a force of at least 0.25 lbs to the fi...

Claims

1. A method for simultaneously purifying nucleic acids from at least two different samples, 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 trailing electrolyte buffer comprising a first trailing ion, wherein the magnitude of the effective mobility of the first trailing ion is less than the magnitude of the effective mobility of the first nucleic acid, (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, and (iv) an elution buffer; (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 trailing electrolyte buffer comprising a second trailing ion, wherein the magnitude of the effective mobility of the second trailing ion is less than the magnitude of the effective mobility of the second nucleic acid, (iii) a second leading electrolyte buffer comprising 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 (iv) an elution buffer, wherein the first channel and the second channel are not in fluid communication with each other; (c) applying a first electric field within the microfluidic chip to perform isoelectric electrophoresis in the first channel with the first trailing ion, the first nucleic acid, and the first leading ion, and applying a second electric field to perform isoelectric electrophoresis in the second channel with the second trailing ion, the second nucleic acid, and the second leading ion, thereby simultaneously purifying the first nucleic acid from the first contaminant and purifying the second nucleic acid from the second contaminant; and (d) after independently controlling the first electric field and the second electric field, applying a third electric field different from the first electric field to perform isoelectric electrophoresis in a third region of the first channel comprising elution buffer, and applying a fourth electric field different from the second electric field in a third region of the second channel comprising elution buffer.

2. The method of claim 1, wherein the first sample and the second sample are different sample types.

3. The method of claim 1, wherein the first nucleic acid and the second nucleic acid are different types or lengths of nucleic acids.

4. The method of claim 1, wherein the first trailing electrolyte buffer or the first leading electrolyte buffer further comprises a lysing agent or a tissue disrupting agent.

5. The method of claim 4, wherein the lysing agent or the tissue disrupting agent comprises one or more agents selected from the group consisting of a solution having a pH greater than 12, a protease, urea, thiourea, and a surfactant.

6. The method of claim 1, wherein the first sample comprises lysed solid tissue.

7. The method of claim 6, wherein the second sample comprises lysed cells.

8. The method of claim 1, wherein during the performing isoelectric electrophoresis, the first sample does not contact the second sample.

9. The method of claim 1, further comprising 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 trailing electrolyte buffer comprising a third trailing ion, wherein an effective mobility of the third trailing ion has a magnitude that is less than an effective mobility of the third nucleic acid, and (iii) a third leading electrolyte buffer comprising a third leading ion, wherein an effective mobility of the third leading ion has a magnitude that is greater than the magnitude of the effective mobility of the third nucleic acid, wherein a third electric field is applied within the microfluidic chip to perform isoelectric electrophoresis in the third channel with the third trailing 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.

10. The method of claim 1, wherein the first and second electric fields are generated by a single pair of electrodes.

11. The method of claim 1, wherein the first and second electric fields are generated by different pairs of electrodes.

12. The method of claim 11, wherein the first and second channels are coupled to independent sensors.

13. The method of claim 12, wherein feedback from the independent sensors is used to independently control the first and second electric fields.

14. The method of claim 13, wherein the independent sensors detect voltage, and the feedback is used to control current within the first and second channels.

15. The method of claim 1, wherein the nucleic acid comprises DNA.

16. The method of claim 1, wherein the nucleic acid comprises RNA.

17. The method of claim 1, wherein step (c) further comprises: performing a second separate isoelectric electrophoresis with the first trailing electrolyte buffer comprising the first trailing ion, the first nucleic acid, and a fourth leading electrolyte buffer comprising a fourth leading ion having a fourth effective mobility, wherein a magnitude of the fourth effective mobility is greater than the magnitude of the effective mobility of the first nucleic acid, wherein the first leading electrolyte buffer is different from the fourth leading electrolyte buffer.

18. The method of claim 1, wherein, the microfluidic chip further comprises an electrode in a buffer reservoir in fluid communication with the first channel.

19. A method for enriching a nucleic acid, the method comprising: (a) loading into a fluidic device: (i) a sample comprising a nucleic acid, (ii) a trailing electrolyte buffer comprising a trailing electrolyte ion having a first effective mobility, wherein a magnitude of the first effective mobility is lower than a magnitude of an effective mobility of the nucleic acid, (iii) a first leading electrolyte buffer in a first leading electrolyte reservoir, the first leading electrolyte buffer comprising a first leading electrolyte ion having a second effective mobility, wherein a magnitude of the second effective mobility is greater than the magnitude of the effective mobility of the nucleic acid, and (iv) a second leading electrolyte buffer in a second leading electrolyte reservoir, the second leading electrolyte buffer comprising a second leading electrolyte ion having a third effective mobility, wherein a magnitude of the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acid. (iv) a second pre-run electrolyte buffer in a second pre-run electrolyte reservoir, the second pre-run electrolyte buffer comprising a second pre-run electrolyte ion having a third effective mobility, wherein the magnitude of the third effective mobility is greater than the magnitude of the effective mobility of the nucleic acid, wherein the first pre-run electrolyte buffer is different from the second pre-run electrolyte buffer, (b) first performing isoelectric electrophoresis with the trailing electrolyte ion, the nucleic acid, and the first pre-run electrolyte ion by applying a first electric field within the fluidic device with a first circuit, thereby enriching the nucleic acid from contaminants in the sample; and (c) second performing isoelectric electrophoresis with the trailing electrolyte ion, the nucleic acid, and the second pre-run electrolyte ion by applying a second electric field within the fluidic device with a second circuit, wherein the first circuit is different from the second circuit.

20. The method of claim 19, wherein the fluidic device comprises a first fluidic channel, the first fluidic channel being partitioned into a first side fluidic channel and a second side fluidic channel.

21. The method of claim 20, wherein the first electric field is applied to the first fluidic channel, and wherein the second performing isoelectric electrophoresis comprises changing an applied current from the first side fluidic channel to the second side fluidic channel.

22. The method of claim 21, wherein changing the applied current comprises stopping a first electric current applied between the first fluidic channel and the first side fluidic channel, and then starting a second electric current applied between the first fluidic channel and the second side fluidic channel.

23. The method of any one of claims 19-22, wherein the first pre-run electrolyte ion is the same as the second pre-run electrolyte ion, and wherein a concentration of the first pre-run electrolyte ion in the first pre-run electrolyte buffer is different from a concentration of the second pre-run electrolyte ion in the second pre-run electrolyte buffer.

24. The method of any one of claims 19-23, wherein an ionic strength of the first pre-run electrolyte buffer is higher than an ionic strength of the second pre-run electrolyte buffer.

25. The method of any one of claims 19-22, wherein the first pre-run electrolyte ion is the same as the second pre-run electrolyte ion, and wherein a concentration of the first pre-run electrolyte ion in the first pre-run electrolyte buffer is the same as a concentration of the second pre-run electrolyte ion in the second pre-run electrolyte buffer, and wherein the first pre-run electrolyte buffer or second pre-run electrolyte buffer comprises a third pre-run electrolyte ion.

26. The method of any one of claims 19-25, further comprising collecting the enriched nucleic acid in the second pre-run electrolyte reservoir, and removing the enriched nucleic acid from the second pre-run electrolyte reservoir.

27. The method of any one of claims 19-26, wherein a concentration of the second pre-run electrolyte ion in the second pre-run electrolyte buffer is less than 50 mM.

28. The method of any one of claims 19-27, wherein the second lead electrolyte buffer comprises 50 mM Tris HC1.

29. The method of any one of claims 19-28, wherein the first electrical circuit comprises a first electrode pair, wherein the second electrical circuit comprises a second electrode pair, and wherein the first electrode pair is different from the second electrode pair.

30. A system comprising: (a) a fluidic device comprising: (i) a sample reservoir in fluid communication with a first fluidic channel; (ii) a trailing electrolyte buffer reservoir in fluid communication with the first fluidic channel; (iii) a first lead electrolyte reservoir in fluid communication with the first fluidic channel; (iv) a second lead electrolyte reservoir in fluid communication with the first fluidic channel; (b) a first electrical circuit configured to perform a first isoelectric focusing by applying a first electric field within the fluidic device along a first path, the first path comprising the trailing electrolyte buffer reservoir, the first fluidic channel, and the first lead electrolyte reservoir; and (c) a second electrical circuit configured to perform a second isoelectric focusing by applying a second electric field within the fluidic device along a second path, the second path comprising the trailing electrolyte buffer reservoir, the first fluidic channel, and the second lead electrolyte reservoir, wherein the first electrical circuit is different from the second electrical circuit, and wherein the first path is different from the second path.

31. The system of claim 30, wherein the first electrical circuit comprises a first electrode pair, wherein the second electrical circuit comprises a second electrode pair, and wherein the first electrode pair is different from the second electrode pair.

32. The system of any one of claims 30-31, wherein the first electrical circuit comprises a first electrode in electrical communication with the trailing electrolyte buffer reservoir and a second electrode in electrical communication with the first lead electrolyte reservoir, and wherein the second electrical circuit comprises a third electrode in electrical communication with the trailing electrolyte buffer reservoir and a fourth electrode in electrical communication with the second lead electrolyte reservoir.

33. The system of claim 32, wherein the first electrode and the third electrode comprise the same electrode.

34. The system of any one of claims 30-33, wherein the first electrical circuit comprises a first current source configured to apply the first electric field, and wherein the second electrical circuit comprises a second current source configured to apply the second electric field.

35. The system of any one of claims 30-34, wherein the first lead electrolyte reservoir contains a first lead electrolyte buffer and the second lead electrolyte reservoir contains a second lead electrolyte buffer, wherein the second lead electrolyte buffer has a lower ionic strength than the first lead electrolyte buffer.

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