Apparatus for volume bonding in epitachophoresis

Abstract

Epitachorheological (ETP) methods and apparatus for improving concentration of a sample and / or separation of components of a sample. The ETP methods and apparatus allow for electromigration in two dimensions. Electromigration of a sample may occur initially in a first dimension along a single plane. Electromigration may then continue in a second dimension, which may be different from the first dimension. The volume in which electromigration occurs may decrease significantly from the first dimension to the second dimension. This smaller dimension may allow for an increase in the concentration of the sample or an improvement in the separation of components of the sample.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 177,637, filed April 21, 2021, which is incorporated herein by reference for all purposes.

Technical Field

[0002] This disclosure relates to the field of electrophoresis for sample analysis, and to the analysis of biological samples by selective separation, detection, extraction, isolation, purification, and / or (pre -)concentration of samples by means of an apparatus and method for epitacoforesis.

Background Art

[0003] Background Electrophoresis techniques have been used to separate and analyze samples for various purposes, such as to identify specific substances or to determine the size and type of molecules in a solution. For example, various molecular biology applications use electrophoresis to separate proteins or nucleic acids, determine molecular weights, and / or prepare samples for further analysis. In these and other applications, electrophoresis generally involves the movement of charged substances (e.g., molecules or ions) under the influence of an electric field. This movement can facilitate the separation of a sample from other samples or substances. Once separated, the sample can be easily analyzed using optical or other techniques.

[0004] Various electrophoresis - based techniques are typically used in different applications depending on the specific needs of the analysis being performed. For example, isotachophoresis ("ITP") is a concentration - separation technique that utilizes electrolytes with different electrophoretic mobilities to focus and, in some cases, separate ionic analytes into distinct regions ("focusing regions"). In ITP, analytes simultaneously focus and separate between high - effective - mobility leading electrolyte ("LE") ions and low - effective - mobility trailing electrolyte ("TE") ions. The balance between electromigration and diffusion at the region boundaries in ITP typically results in sharp migration boundaries.

[0005] Conventionally, ITP is performed using instruments and methods characterized by capillary or microfluidic channel designs. Such instruments and methods can only handle small amounts (μl scale) of sample for analysis, which can make the analysis of biological samples, such as nucleic acid extraction from blood and / or plasma, difficult. Therefore, further development of instruments and methods for analyzing samples that may have large volumes would likely be beneficial. Furthermore, it may be advantageous to concentrate the sample in a small volume. Epitachophoresis (ETP) methods and instruments that offer these and other improvements are described herein. [Overview of the project]

[0006] Brief Overview This disclosure describes an epitachophoresis (ETP) method and apparatus for improving the concentration of a sample and / or the separation of components of a sample. The ETP method and apparatus enable two-dimensional electromigration. Electromigration of a sample may first occur in a first dimension along a single plane. Electromigration may then continue in a second dimension which may differ from the first dimension. The volume over which electromigration occurs may decrease significantly from the first dimension to the second dimension. This smaller dimension may allow for increased concentration of the sample or improved separation of components of the sample.

[0007] Some embodiments include a method for concentrating components from a sample. The system may apply a voltage difference between a first electrode and a second electrode. The first electrode is placed in a first mixture containing a first electrolyte and the sample. The second electrode is placed in a second electrolyte. The first electrolyte is discontinuous with the second electrolyte. The first mixture is in contact with the second electrolyte. The system may use the voltage difference to flow components in a first channel in a first direction. The first direction is away from the first electrode and toward the second channel. The components are focused into a band. The first mixture is characterized by having a first thickness perpendicular to the first direction. The system may flow the band-focused components in a second channel in a second direction. The second electrolyte is located in the second channel. The second direction is from the first channel toward the orifice of the container. The second channel is an annular space. The second mixture, containing the components in the second channel and the second electrolyte, is characterized by having a second thickness perpendicular to the second direction. The first thickness is greater than the second thickness. The system can collect the second mixture in the container while applying a voltage difference. The concentration of the components in the second mixture in the container is higher than the concentration of the components in the sample.

[0008] In some embodiments, a system for concentrating components in a sample includes a base defining a first channel and cavity, a first electrode positioned in the first channel, and a second electrode. The first channel is in fluid communication with the cavity. The outer diameter of the first channel is greater than the first outer diameter at the top of the cavity. The cavity may have a conical shape with a first outer diameter greater than the second outer diameter at the bottom of the cavity. The second electrode is configured to be in more close electrical communication with the cavity than the first channel, if the first channel and cavity contain an electrolyte. The first channel is characterized by a first volume. The cavity may be configured to accept a container and form a second channel when the container is placed inside the cavity. The second channel may be an annular space. The annular space may be defined by the surface of the base and the surface of the container when the container is placed inside the cavity. The second channel is characterized by a second volume. The first volume is larger than the second volume.

[0009] A good understanding of the characteristics and advantages of the embodiments of this disclosure can be obtained by referring to the following detailed description and accompanying drawings. [Brief explanation of the drawing]

[0010] [Figure 1] A schematic diagram of an exemplary apparatus for performing epitaphoresis is provided. [Figure 2A] A schematic plan view of an exemplary apparatus for performing epitaphoresis is provided. In Figure 2A, reference numerals 1-7 refer to: 1. outer circular electrode; 2. terminal electrolyte reservoir; 3. leading electrolyte, optionally contained in a gel or hydrodynamically separated from the terminal electrolyte; 4. leading electrolyte electrode / collection reservoir; 5. central electrode; 6. power supply; and 7. boundary between the leading and terminal electrolytes where sample ions are focused; the symbols r and d are used to represent the leading electrolyte reservoir radius and distance moved by the LE / TE boundary, respectively. [Figure 2B]A schematic side view of an exemplary apparatus for performing epitacophoresis is provided. In Figure 2B, reference numerals 1-8 refer to: 1. outer circular electrode; 2. terminal electrolyte reservoir; 3. leading electrolyte, optionally contained in a gel or hydrodynamically separated from the terminal electrolyte; 4. leading electrolyte electrode / collection reservoir; 5. central electrode; 6. power supply; 7. boundary between the leading and terminal electrolytes where sample ions are focused; and 8. bottom support; the symbols r and d are used to represent the leading electrolyte reservoir radius and distance moved by the LE / TE boundary, respectively. [Figure 3] A schematic diagram of an exemplary apparatus for performing epitaphoresis is provided. [Figure 4] A schematic diagram of an exemplary apparatus for performing epitacophoresis is provided. In Figure 4, reference numerals 1 to 10 indicate: 1. Outer circular electrode; 2. Terminal electrolyte reservoir; 3. Preceding electrolyte, optionally contained in a gel or hydrodynamically separated from the terminal electrolyte; 4. Opening to the preceding electrolyte / collection reservoir; 5. Central electrode; 6. Power supply; 7. Boundary between the preceding electrolyte and the terminal electrolyte where sample ions are focused; 8. Bottom support; 9. Tube connection device to the preceding electrolyte reservoir; 10. Preceding electrolyte reservoir. [Figure 5] A schematic diagram of an exemplary apparatus for performing epitachophoresis is provided, in which the sample is packed between the packing of the leading electrolyte and the packing of the terminal electrolyte. [Figure 6] A schematic diagram of the apparatus for performing epitaphoresis is provided, and the equations described are mentioned. [Figure 7] This invention illustrates a system for concentrating components in a sample using epitaphoresis, according to an embodiment of the present invention. [Figure 8] This diagram shows an exploded view of a system for concentrating components in a sample using epitaphoresis, according to an embodiment of the present invention. [Figure 9] This diagram shows a cross-sectional view of a system for concentrating components in a sample using epitaphoresis, according to an embodiment of the present invention. [Figure 10A]This shows a support for holding a pipette tip according to an embodiment of the present invention (bottom view). [Figure 10B] This shows a support for holding a pipette tip according to an embodiment of the present invention (plan view). [Figure 10C] This shows a support for holding a pipette tip according to an embodiment of the present invention (plan view). [Figure 10D] This shows a support for holding a pipette tip according to an embodiment of the present invention (plan view). [Figure 10E] This shows a support for holding a pipette tip according to an embodiment of the present invention (plan view). [Figure 11] This is a flowchart of an exemplary process for concentrating components from a sample according to an embodiment of the present invention. [Figure 12] This shows a measurement system according to an embodiment of the present invention. [Figure 13] This shows a computer system according to an embodiment of the present invention.

[0011] term As used herein, the term “isophasic electrophoresis” generally refers to the separation of charged particles by using an electric field to form a boundary or interface between substances (e.g., between charged particles and other substances in solution). ITP generally uses multiple electrolytes, where the electrophoretic mobility of the sample ions is lower than that of the leading electrolyte (LE) placed in the instrument for ITP, and higher than that of the trailing electrolyte (TE). The leading electrolyte (LE) generally contains ions with relatively high mobility, and the trailing electrolyte (TE) generally contains ions with relatively low mobility. The TE and LE ions are selected to have lower and higher effective mobilities, respectively, than the target analyte ions of interest. That is, the effective mobility of the analyte ions is higher than that of the TE and lower than that of the LE. These target analytes have the same charge sign as the LE and TE ions (i.e., coions). The applied electric field pushes the LE ions away from the TE ions and attracts the TE ions backward. A migration interface is formed between adjacent and continuous TE and LE regions. This forms a region of electric field gradient (typically from the low electric field of LE to the high electric field of TE). Analyte ions in TE can overtake TE ions but not LE ions and can accumulate at the interface between TE and LE (forming a "focusing" or "focusing region"). Alternatively, target ions in LE can be overtaken by LE ions and also accumulate at the interface. With a sensible selection of LE and TE chemistry, ITP is fairly generally applicable and can be achieved by a sample initially dissolved in either or both of the TE and LE electrolytes, without requiring a background electrolyte with very low electrical conductivity.

[0012] As used herein, the term “epitachophoresis” generally refers to a method of electrophoretic separation performed using circular or spheroidal and / or concentric apparatus and / or circular and / or concentric electrode configurations, such as the use of circular / concentric and / or polygonal apparatuses as described herein. Due to the circular / concentric or other polygonal configurations used in epitachophoresis, unlike conventional isokinetic electrophoresis apparatuses, the cross-sectional area changes during ion and region movement, and the rate of region movement is not constant over time due to the change in cross-sectional area. Thus, epitachophoresis configurations do not strictly adhere to the principle of conventional isokinetic electrophoresis, where regions move at a constant speed. Despite these significant differences shown herein, epitachophoresis can be used to efficiently separate and focus charged particles by using an electric field to form a boundary or interface between materials that may have different electrophoretic mobilities (e.g., between charged particles and other materials in solution). LE and TE can also be used in epitachophoresis, as described for use with ITP. In some embodiments, epitacholesis may be performed using a constant current, a constant voltage, and / or a constant power. In some embodiments, epitacholesis may be performed using a changing current, a changing voltage, and / or a changing power. In some embodiments, epitacholesis may be performed in the context of an arrangement of apparatus and / or electrodes whose shape can generally be described as circular or spherical, so that the basic principle of epitacholesis can be achieved as described herein. In some embodiments, epitacholesis may be performed in the context of an arrangement of apparatus and / or electrodes whose shape can generally be described as polygonal, so that the basic principle of epitacholesis can be achieved as described herein. In some embodiments, epitacholesis may be performed by any nonlinear continuous arrangement of electrodes, such as electrodes arranged in a circular shape and / or electrodes arranged in a polygonal shape.

[0013] As used herein, terms such as “in vitro diagnostic application (IVD application),” “in vitro diagnostic method (IVD method),” and “in vitro diagnostic assay” generally refer to any application and / or method and / or apparatus that can evaluate a sample for diagnostic and / or monitoring purposes, such as disease identification in a subject or optionally in a human subject. In some embodiments, the sample may include nucleic acids and / or target nucleic acids from a subject and / or sample, and optionally further, the nucleic acids may be derived from a urine sample. In some embodiments, an epitacophoresis apparatus may be used as an in vitro diagnostic apparatus. In some embodiments, a target analyte enriched / enriched / isolated / purified by epitacophoresis may be used in a downstream in vitro diagnostic assay. In some embodiments, an in vitro diagnostic assay may include nucleic acid sequencing, e.g., DNA sequencing, e.g., RNA sequencing. In some embodiments, an IVD assay may include gene expression profiling.In some embodiments, the in vitro diagnostic method can be any one or more of the following, but is not limited thereto: staining, immunohistochemical staining, flow cytometry, FACS, fluorescence-activated droplet sorting, image analysis, hybridization, DASH, molecular beacon, primer extension, microarray, CISH, FISH, fiber FISH, quantitative FISH, flow FISH, comparative genomic hybridization, blotting, western blotting, southern blotting, eastern blotting, far-western blotting, southwestern blotting, northwestern blotting, and northern blotting, enzyme assay, ELISA, ligand-binding assay, immunoprecipitation, ChIP, ChIP-seq, ChIP-ChiP, radioimmunoassay, fluorescence polarization, FRET, surface plasmon resonance, filter-binding assay, affinity chromatography, immunocytochemistry, gene expression profiling, DNA profiling PCR, DNA microarray, serial analysis of gene expression, real-time polymerase chain reaction, differential display PCR, RNA-seq, mass spectrometry, DNA methylation detection, acoustic energy, lipidomic-based analysis, quantification of immune cells, detection of cancer-related markers, affinity purification of specific cell types, DNA sequencing, next-generation sequencing, detection of cancer-related fusion proteins, and detection of chemotherapy resistance-related markers.

[0014] As used herein, the terms "leading electrolyte" and "leading ion" generally refer to ions having a higher effective electrophoretic mobility compared to the sample ions and / or trailing electrolyte used for the purposes during ITP and / or epitacophoresis. In some embodiments, a leading electrolyte for use in anionic epitacophoresis can include, but is not limited to, chlorides, sulfates, and / or formates buffered to a desired pH with a suitable base such as histidine, TRIS, creatinine, etc. In some embodiments, a leading electrolyte for use in cationic epitacophoresis can include, but is not limited to, potassium, ammonium, and / or sodium together with acetate or formate. In some embodiments, an increase in the concentration of the leading electrolyte can result in a proportional increase in the sample region and a corresponding increase in the current (power) for a given applied voltage. Typical concentrations can generally range from 10 to 100 mM. However, higher or lower concentrations may be used.

[0015] As used herein, the terms "trailing electrolyte", "trailing ion", "terminating electrolyte", and "terminating ion" generally refer to ions having a lower effective electrophoretic mobility compared to the electrophoretic mobility of the sample ions and / or leading electrolyte used for the purposes during ITP and / or epitacophoresis. In some embodiments, a trailing electrolyte for use in cationic epitacophoresis can include, but is not limited to, MES, MOPS, acetate, glutamate, and other anions of weak acids and low mobility anions. In some embodiments, a trailing electrolyte for use in anionic epitacophoresis can include, but is not limited to, reactive hydroxonium ions at the moving boundary formed by any weak acid during epitacophoresis.

[0016] As used herein, the term “focused region” generally refers to the volume of solution containing a component that has been enriched (“focused”) as a result of performing epitacophoresis. The component may include a target analyte or any molecule having an ionic component affected by the voltage applied in the ETP. The focused region may be collected or removed from the apparatus and may contain an enriched and / or concentrated amount of the desired sample, e.g., a target analyte, e.g., a target nucleic acid. In the epitacophoresis methods described herein, the target analyte is generally focused to the center of the apparatus, e.g., a circular, spheroidal, or other polygonal apparatus.

[0017] As used herein, the terms “band” and “ETP band” generally refer to a region (e.g., a focusing region) of ions, analytes, or a sample that moves independently of other ions, analytes, or samples during electrophoresis (e.g., isophatic electrophoresis or epitaphoresis). A focusing region within an epitaphoresis instrument may alternatively be referred to as an “ETP band.” In some embodiments, an ETP band may comprise one or more types of ions, analytes, and / or samples. In some examples, an ETP band may comprise a single type of analyte whose separation from other substances present in the sample, e.g., separation of a target nucleic acid from cellular debris, is desired. In some examples, an ETP band may comprise more than one target analytes, e.g., polypeptides or nucleic acid sequences with very similar sequences, e.g., allele variants. In some examples, an ETP band may comprise analytes of similar size or with different electrophoretic mobilities. In such cases, one or more target analytes may be separated by further ETP runs, for example, under different conditions that facilitate the separation of the one or more analytes, and / or the one or more analytes may be separated by other techniques known in the art for the separation of analytes, such as those described herein. In some embodiments, the ETP bands may be collected and optionally subjected to further analysis after isolation / purification and collection of one or more ETP-based materials. In some embodiments, the ETP bands may include one or more target analytes that have undergone or are undergoing isolation / purification and optionally collected, for example, as part of an ETP run.

[0018] As used herein, the term “target nucleic acid” is intended to mean any nucleic acid that is the subject of detection, measurement, amplification, isolation, purification, and / or further assays and analyses. Target nucleic acids may include any single-stranded and / or double-stranded nucleic acids. Target nucleic acids may exist as isolated nucleic acid fragments or as part of a larger nucleic acid fragment. Target nucleic acids may be derived from or isolated from essentially any source, such as cultured microorganisms, uncultured microorganisms, complex biological mixtures, samples containing biological specimens, urine samples, tissues, serum, old or preserved tissues or specimens, and environmental isolates. Furthermore, target nucleic acids may include or be derived from cDNA, RNA, genomic DNA, cloned genomic DNA, genomic DNA libraries, enzymatically fragmented DNA or RNA, chemically fragmented DNA or RNA, physically fragmented DNA or RNA, etc. In some embodiments, target nucleic acids may include an entire genome. In some embodiments, target nucleic acids may include the total nucleic acid content of a specimen and / or biological specimen, such as a urine sample. Target nucleic acids may be in a variety of different forms or substantially purified forms, including, for example, simple or complex mixtures. For example, the target nucleic acid may be part of a sample containing other components, or it may be the sole or primary component of the sample. Furthermore, the target nucleic acid may have either a known or unknown sequence.

[0019] As used herein, the term “target microorganism” is intended to mean any single-celled or multicellular microorganism found in specimens such as blood, plasma, other bodily fluids, biological specimens, and / or tissues, for example, in relation to infectious symptoms or diseases. Examples include bacteria, archaea, eukaryotes, viruses, yeasts, fungi, protozoa, amoebas, and / or parasites. Furthermore, the term “microorganism” generally refers to a microorganism that can cause disease, whether a disease is mentioned or a disease-causing microorganism is mentioned.

[0020] As used herein, the terms “biomarker” or “biomarker of interest” refer to biological molecules found in tissues, blood, plasma, urine, and / or other bodily fluids that are signs of a normal or abnormal process, or of a symptom or disease (such as cancer). Biomarkers may be used to determine how well the body responds to treatment for a disease or symptom. In the context of cancer, a biomarker refers to a biological substance that indicates the presence of cancer in the body. A biomarker may be a molecule secreted by a tumor or a specific response of the body to the presence of cancer. Genetic biomarkers, epigenetic biomarkers, proteomics biomarkers, glycome biomarkers, and imaging biomarkers may be used for the diagnosis, prognosis, and epidemiology of cancer. Such biomarkers may be assayed in non-invasively collected bodily fluids such as blood, serum, and / or urine. Biomarkers may be useful as diagnostic agents (e.g., to identify early-stage cancer) and / or prognostic diagnostic agents (e.g., to predict how invasive cancer will be and / or how a subject will respond to a particular treatment and / or how likely cancer is to recur).

[0021] As used herein, the term “sample” includes specimens or cultures (e.g., microbial cultures) that contain or are presumed to contain one or more target analytes. The term “sample” also means biological, environmental, and chemical specimens, as well as any specimens of which analysis is desired. Samples may include specimens of synthetic origin. Samples may include one or more microorganisms from any source from which one or more microorganisms may originate. Examples of samples, but not limited to, include whole blood, skin, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, cerebrospinal fluid, lavage fluids (e.g., bronchoalveolar epithelium, stomach, peritoneum, tube, ear, arthroscopic), tissue specimens, biopsy specimens, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, semen, lymph, bile, organs, bone marrow, tears, sweat, breast milk, maternal fluids, germ cells, and fetal cells.

[0022] As used herein, the term “target analyte” is intended to mean any analyte that is subject to detection, measurement, separation, concentration, isolation, purification, and / or further assays and analyses. In some embodiments, the analyte may be, but is not limited to, any ion, molecule, nucleic acid, biomarker, protein, cell or cell population, such as a desired cell, and its detection, measurement, separation, concentration, and / or use in further assays is desirable. In some embodiments, the target analyte may be derived from any of the samples described herein, such as a urine sample.

[0023] The term “communication” is used herein to describe a structural, functional, mechanical, electrical, optical, thermal, or fluid relationship, or any combination thereof, between two or more components or elements. Therefore, the fact that one component is said to communicate with a second component is not intended to preclude the possibility that an additional component may exist between the first and second components, and / or may be operably associated with or engaged with the first and second components.

[0024] As used herein, “subject” refers to the mammalian subject being treated (e.g., human, rodent, non-human primate, dog, cattle, sheep, horse, cattle, etc.) and / or subject from which a sample is obtained.

[0025] "Detecting" a sample within the context of an epitaphoresis apparatus, system, or machine may include detecting its location in one, several, or many aspects of the entire apparatus. Detection may generally be performed by one or more means that do not interfere with the function of the desired apparatus, system, or machine, and the methods performed using the apparatus, system, or machine. In some embodiments, detection may include any means of electrical detection, such as the detection of conductivity, resistivity, voltage, or current. Furthermore, in some embodiments, detection may include one or more of the following: electrical detection, thermal detection, optical detection, spectroscopic detection, photochemical detection, biochemical detection, immunochemical detection, and / or chemical detection. In some embodiments, one or more target analytes may be detected during ETP-based isolation / purification and optionally during the collection of the one or more target analytes. Furthermore, sample detection within the context of ETP apparatus and ETP methods is further described in U.S. Patent Application No. 62 / 744,984; U.S. Patent Application Publication No. 2020 / 0282392; and PCT / EP2018 / 081049 and PCT / EP2019 / 077714, all of which are incorporated herein for all purposes.

[0026] In sample analyzers or systems, the term “sample collection volume” refers to the volume of sample intended to be collected, for example, by a robotic liquid handler, during or after analysis. In an apparatus for performing epitachophoresis, or a system including such an apparatus, the sample collection volume is the volume intended for collection, including the sample during or after epitachophoresis. In some embodiments, the sample collection volume may be located in the central well of the apparatus or system described herein. In some embodiments, the sample collection volume may be located anywhere that allows for the collection of the desired sample. In some embodiments, the sample collection volume may be anywhere between the sample loading area and the pre-electrolyte electrode / collection reservoir. The sample collection volume may consist of any suitable area, container, well, or space in the apparatus or system. In some embodiments, the sample collection volume may consist of a well, membrane, compartment, vial, pipette, etc.

[0027] As used herein, the term “ETP-based isolation / purification” generally refers to apparatus and methods including ETP, e.g., apparatus capable of performing ETP, e.g., methods including performing ETP, which focuses one or more target analytes into one or more focused regions (e.g., one or more ETP bands), thereby isolating / purifying one or more target analytes from other substances in the initial sample. Note that the terms “isolate” and “purify” are used interchangeably. Furthermore, ETP-based isolation / purification generally allows for the subsequent collection of the one or more focused regions (one or more ETP bands) containing the one or more target analytes. The degree of isolation / purification of one or more target analytes resulting from one or more ETP-based isolation / purification may be any degree or amount of one or more target analytes from other substances. In some embodiments, ETP-based isolation / purification of a target analyte from a sample may result in a purity of 1% or less, 1% or more, 5% or more, 10% or more, 60% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 85% or more, 90% or more, 95% or more, or 99% or more of the target analyte, as measured by analytical techniques for determining the composition of an ETP-isolated / purified sample containing one or more target analytes. In some embodiments, ETP-based isolation / purification of a target analyte from a sample may result in the recovery of less than 1%, more than 1%, more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 85%, more than 90%, more than 95%, or more than 99% of the target analyte from the original sample. In some embodiments, one or more ETP-based isolation / purifications may be performed to isolate / purify one or more target analytes, for example, one or more nucleic acids.For example, in some cases, an ETP-based isolation / purification can be performed on a sample containing one or more target analytes to focus one or more target analytes into a single focusing region (ETP band), thereby substantially separating one or more target analytes from other substances in the original sample. The sample may be collected after ETP isolation / purification, and the isolated / collected sample may undergo further ETP-based isolation / purification. Optionally, the second ETP-based isolation / purification may, in the case of more than one target analyte, be performed under conditions that isolate each of the one or more target analytes into a separate focusing region, each of which may be optionally collected individually, thereby separating the target analytes from each other, if desired.

[0028] As used herein, the term “mixed sample” generally refers to a sample containing substances from one or more sources.

[0029] As used herein, the term “ETP top marker” generally refers to a compound or molecule that is larger in size and / or longer in length compared to the target nucleic acid, thereby indicating a cutoff point at which the collection of the target analyte can be stopped during ETP-based isolation / purification and subsequent collection of the target analyte. For example, a fluorescently labeled or otherwise detectably labeled ETP top marker may be generated in a size larger than the target DNA to be isolated / purified and collected during ETP-based isolation / purification. By monitoring the marker throughout the entire ETP run, a user or automated machine can stop the run before the marker falls into the collection tube, thereby capturing DNA smaller than the marker, while larger contaminating DNA is excluded as it is positioned behind the top marker. Furthermore, since the ETP top marker itself is not collected and therefore does not interfere with downstream assays, e.g., one or more IVD assays, it can be used in large quantities with various detectable labels. In some examples, ETP top markers may be used in ETP-based isolation / purification methods to aid in the isolation / purification of one or more target analytes and the exclusion of genomic DNA from collected samples. [Modes for carrying out the invention]

[0030] Detailed explanation In the implementation of epitachophoresis (ETP), the concentration and / or separation of a sample proceeds in planar space. The components to be concentrated or separated move along one plane. Embodiments of the present invention include apparatus and methods for adding dimension to epitope separation. The first dimension is electromigration along one plane and is the first part of the migration of components. After ETP focusing, electromigration continues in a second dimension perpendicular or substantially perpendicular to the first dimension. This second migration proceeds in a space of at least 10 times the volume of the first dimension. The term "volume-bound" is used to describe the volume reduction of electromigration in ETP.

[0031] The advantages of ETP instruments and methods include focusing the sample into a much smaller volume and providing a higher concentration of the sample. Furthermore, the smaller volume of the second separation dimension can enable online connectivity to other analytical systems, including optical and electrochemical detection, liquid chromatography (LC), capillary electrophoresis (CE), NMR, and mass spectrometry.

[0032] I. Epitachorrhea Apparatus for epitacophoresis generally uses a concentric or polygonal disk architecture, as shown, for example, in Figures 1-4. Glass or ceramics may be used to manufacture the system (i.e., the material of the concentric or polygonal disks) because these materials provide beneficially improved heat transfer characteristics during apparatus operation. For example, flat channels in epitacophoresis apparatus have better heat transfer capabilities compared to narrow channels, thus generally preventing overheating (or boiling) of the focused material. Current / voltage programming is also suitable for adjusting the Joule heating of the apparatus. Plastic materials may also be used in the manufacture of the apparatus. Generally, the apparatus is manufactured to dimensions that accommodate the desired sample volume, for example, a sample volume on a milliliter scale, e.g., up to 15 mL.

[0033] Referring to Figures 1-3, two concentric disks are separated by a spacer, thereby forming a flat channel for epitaphoresis sample processing. Current is applied through multiple high-voltage (HV) connections, and a ground connection may be located in the center of the system (see, for example, Figures 1 and 3). In some examples, the sample is injected into the apparatus through an opening, e.g., the top or side (see, for example, Figure 3). The application of electricity focuses the target analyte of the sample as a concentric ring moving to the center of the disk, and the target analyte can then be collected through a syringe at the bottom of the apparatus (see, for example, Figure 3). As shown in Figures 2A (plan view) and 2B, an example of the apparatus configuration includes an outer circular electrode (1), a terminal electrolyte (2), and a leading electrolyte (3). Generally, the diameter of the outer circular electrode (1) is about 10-200 mm, and the diameter of the leading electrolyte is in the range of about 10 μm to about 20 mm in thickness (height). The leading electrolyte can be stabilized by a gel or viscous additive, or hydrodynamically separated from the terminal electrolyte by, for example, a membrane. Gel or hydrodynamic separation prevents mixing of the leading and terminal electrolytes during the operation of the device. Furthermore, in some devices, mixing is prevented by using a very thin (less than 100 μm) layer of electrolyte, as will be discussed later.

[0034] Referring to Figures 2A and 2B, at the center of the leading electrolyte is an electrolyte reservoir (4) with an electrode (5). The assembly of electrodes (1, 5) and electrolytes (2, 3) is placed on a flat, electrically insulating support (8). The electrolyte reservoir (4) is used to remove concentrated sample solution after a separation process, such as pipetting the sample from the reservoir. The electrolyte reservoir (4) is also a sample collection reservoir. An outer circular electrode (1) may be placed at the end of the circular channel where the leading electrolyte (3) and terminal electrolyte (2) are located.

[0035] In an alternative configuration (see Figure 4), the central electrode (5) is moved to a preceding electrolyte reservoir (10) connected to the concentrator by a tube (9). The tube (9) is either directly connected at one end or closed by a semipermeable membrane (not shown). This arrangement facilitates collection by stopping the movement of large molecules according to the properties of the membrane used. This arrangement simplifies sample collection and provides a means of connecting the concentrator online to other instruments such as capillary analyzers, chromatographs, PCR machines, and enzyme reactors. The tube (9) can also be used to supply countercurrent of the preceding electrolyte in a configuration that does not include a gel containing the preceding electrolyte.

[0036] Generally, the gel for pre-electrolyte stabilization is formed from any uncharged material, such as agarose, polyacrylamide, or pullulan. In some devices, the top is left open, depending on the nature of the separation performed, while in others the top is closed. If closed, the material used to cover the device is preferably a thermally conductive insulating material to prevent evaporation during the operation of the epitaphoresis device.

[0037] Generally, ring (circular) electrodes are preferably gold-plated or platinum-plated stainless steel rings to allow for maximum chemical resistance and electric field uniformity. Alternatively, stainless steel and graphite electrodes may be used in some devices, especially disposable devices. Also, ring (circular) electrodes can be replaced by other structures that provide similar functionality, such as arrays of wire electrodes. Furthermore, two-dimensional arrays of regularly spaced electrodes may be used additionally or alternatively in epitacophoresis devices. Arrays of regularly spaced electrodes in a circular orientation can also be used in epitacophoresis devices. Furthermore, other electrode configurations may also be used to produce different electric field shapes based on desired sample separation (e.g., to direct the focusing region). Such configurations are described as polygonal configurations of electrodes. When divided into electrically isolated segments, a switched electric field is generated with respect to the time-dependent shape of the driving electric field. Such configurations facilitate sample collection in some devices.

[0038] Epitaphoresis apparatuses, such as those shown in Figures 1-4, can operate with any of the following configurations, as shown in Figure 5: a two-electrolyte reservoir arrangement, a sample mixed with a leading electrolyte followed by a terminal electrolyte, or a sample mixed with a leading electrolyte followed by a terminal electrolyte. In such configurations, the sample can be mixed with any conductive solution. Alternatively, if the sample contains suitable terminal ions, the terminal electrolyte region can be excluded. Referring to Figures 2A-2B, when the region of the terminal electrolyte (2) is filled with a mixture of the sample and a suitable terminal electrolyte and the power supply (6) is turned on, the ions begin to move toward the central electrode (5) and form a region at the boundary between the leading electrolyte and the terminal electrolyte (7). The concentration of the sample region during electrophoresis is adjusted according to the general principle of isokinetic electrophoresis [Foret, F., Krivankova, L., Bocek, P., Capillary Zone Electrophoresis. Electrophoresis Library, (Editor Radola, BJ) VCH, Verlagsgessellschaft, Weinheim, 1993.], the entire details of which are incorporated herein by reference for all purposes. This concentrates low concentrations of sample ions and dilutes high concentrations of sample ions. In discontinuous electrolyte systems, the concentration of a region is adjusted by the concentration of the preceding region. Discontinuous electrolyte systems may include different gel structures (or the presence of gels), pH values ​​of buffers, ionic strengths of buffers, and / or ions. Thus, the concentration of a region with small amounts of sample components increases, but if there are high concentrations (higher than the preceding region), they are diluted. When the sample region enters the electrolyte reservoir (4), the separation process is stopped and the focused material is collected at the center of the apparatus. In practice, the final concentration in the electrophoretic region is comparable to the concentration of the preceding ion. Typically, any concentration factor from 2 to 1,000 or more can be achieved using epitaphoresis.

[0039] In a three-electrolyte reservoir configuration, the sample is applied between the leading and terminal electrolytes (see, for example, Figure 5), and such a configuration may result in slightly faster sample concentration and separation compared to a two-electrolyte reservoir configuration.

[0040] To avoid mixing, the leading and trailing electrolytes may be stabilized by a neutral (uncharged) viscous medium, such as an agarose gel (see Figures 2A-2B, (3) for example, the leading electrolyte optionally contained within the gel or hydrodynamically separated from the terminal electrolyte).

[0041] All common electrolytes used in isophatic electrophoresis can be used with the epitaphoresis apparatus of the present invention if the leading ion has a higher effective electrophoretic mobility than the effective electrophoretic mobility of the target sample ion. The opposite is true for selected terminal ions.

[0042] The apparatus can operate in either positive mode (separation / concentration of cation species) or negative mode (separation / concentration of anionic species). The most common leading electrolytes for anion separation using epitacophoresis include, for example, chlorides, sulfates, or formates buffered to the desired pH with a suitable base, such as histidine, TRIS, or creatinine. The concentration of the leading electrolyte for epitacophoresis for anion separation is in the range of 5 mM to 1 M relative to the major ion. In this case, the terminal ion often includes MES, MOPS, HEPES, TAPS, acetate, glutamate, and other anions, including weak acids and low-mobility anions. The concentration of the terminal electrolyte for epitacophoresis in positive mode is in the range of 5 mM to 10 M relative to the terminal ion.

[0043] For cation separation, common preceding ions for epitacophoresis include, for example, potassium, ammonium, or sodium, with acetate or formate being the most common buffer counterions. The reaction hydroxonium ion transfer boundary then functions as a universal terminal electrolyte formed by any weak acid.

[0044] In both positive and negative modes, increasing the concentration of the preceding ion results in a proportional increase in the sample region at the expense of increasing the current (power) for a given applied voltage. Typical concentrations are in the range of 10–100 mM; however, higher concentrations are also possible.

[0045] Furthermore, if regional electrophoretic separation alone is sufficient, the apparatus can be operated with only one background electrolyte.

[0046] Current and / or voltage programming is suitable for adjusting the sample's movement speed. Note that in this concentric configuration, the cross-sectional area changes during movement, and the speed of region movement is not constant over time. Therefore, this configuration does not strictly adhere to the principle of isokinetic electrophoresis, where the region moves at a constant speed. Depending on the operating mode of the power supply (6), three basic cases can be distinguished: 1. constant current isolation; 2. constant voltage isolation; 3. constant power isolation.

[0047] The variables in the following equation are as follows: d = displacement (d < 0; r > 0); E = electric field strength; H = electrolyte (gel) height; I = current; J = current density; κ = electrolyte conductivity; r = radius; S = cross-sectional area (area between the two electrolytes); u = electrophoretic mobility; v = velocity; X = length from the central electrode to the epitaphoresis boundary. Figure 6 shows the relationship between the variables d, r, and X in the apparatus.

[0048] In a common operating mode using a constant current supplied by a high-voltage power supply (HVPS), the moving region is accelerated as it approaches the center due to the increasing current density. With respect to separation at constant current, when using a device including a circular structure, for example, a device including one or more circular electrodes, the relative velocity at distance d depends only on the mobility (conductivity) of the preceding electrolyte, as demonstrated by the derivation of the epitachophoresis boundary velocity at v at distance d from the starting radius r, as follows. General formula:

number

[0049] ETP devices can also operate at constant voltage or constant power. The rate of electromigration is also accelerated during analysis performed at constant voltage and constant power.

[0050] II. Volume Bonding Systems Embodiments of the present invention may include systems that enable volume bonding for concentrating components from a sample. These volume bonding systems may involve adapting the apparatus shown in Figures 1 to 6 to include a second dimension for transport and a second volume for further concentration of the sample.

[0051] Figure 7 shows a system 700 for concentrating components in a sample using epitacophoresis. The system 700 includes a base 704. The base 704 may be an electrically insulating support 8 in Figures 2A, 2B, and 4. The base 704 may define a cylindrical portion in which a manifold 708 is placed on the base 704. A pipette tip 712 may be placed through the center of the manifold 708. The manifold 708 may hold the pipette tip 712 in place. The manifold 708 is optional. The system 700 may include electrical leads 716 and 720. One of the electrical leads 716 and 720 may be electrically connected to a first electrode (not shown in Figure 7). The first electrode may be a ring-shaped electrode on the outer edge of the cylindrical portion in the base 704. The first electrode may be any electrode described herein, including electrode 1 in Figures 2A, 2B, and 4. The other of the electrical leads 716 and 720 may be electrically connected to a second electrode (not shown in Figure 7). The second electrode may supply voltage to the lower end of the pipette tip 712. The second electrode may be similar to electrode 5 in Figures 2A, 2B, and 4, except that the electrode may be located further downstream from the sample collection point (e.g., where the pipette tip is placed).

[0052] The terminal electrolyte may be positioned between the manifold 708, toward the outer edge of the cylindrical portion within the base 704. The terminal electrolyte may be any terminal electrolyte described herein, including terminal electrolyte 2 in Figures 2A, 2B, and 4. The leading electrolyte is located in the center of the manifold 708 and may be in contact with the terminal electrolyte. The leading and terminal electrolytes may or may not be in contact with each other at the edges of the manifold 708. The outer edge of the leading electrolyte may be circular or rounded, the terminal electrolyte may be annular, or the edge of the terminal electrolyte may trace an annular portion. The leading electrolyte may be any leading electrolyte described herein, including leading electrolyte 3 in Figures 2A, 2B, and 4.

[0053] Figure 8 shows an exploded view of the system 700. The pipette tip 712 has a conical portion 724 that is not visible in Figure 7. The conical portion 724 fits into a cavity 728 within the base 704. The cavity 728 is also conical in shape. The cavity 728 may be slightly larger than the conical portion 724 of the pipette tip 712, resulting in an annular space between the base 704 and the pipette tip 712 when the pipette tip 712 is inside the cavity 728, as shown in Figure 7. The cavity 728 is different from the electrolyte reservoir 4 in Figures 2A, 2B, and 4. The cavity 728 is formed below the surface of the planar portion for the electrolyte within the base 704. Furthermore, unlike the electrolyte reservoir 4 in Figure 4, the cavity 728 is configured to receive the pipette tip 712 and generate an annular flow to the conical portion 724, rather than the entire electrolyte reservoir 4 which acts as a conduit to the sample collection container.

[0054] Figure 9 shows a cross-section of a pipette tip in system 900, similar to system 700. System 900 includes a base 904. A manifold 908 is positioned in the portion defined by the base 904. A pipette tip 912 is shown passing through the base 904. The pipette tip 912 is positioned in a cavity 928 defined by the base 904. The pipette tip 912 allows for the collection of components that are not possible with the manifold 908. An annular space 936 is formed between the cavity 928 and the pipette tip 924. A first electrode 916 is located in a first channel 932. The first channel 932 is defined by the base 904 and the manifold 908. A second electrode 920 is positioned in a reservoir 944, which is in fluid communication with the annular space 936. The liquid in the reservoir 944 can be separated from the liquid in the annular space 936 by a membrane 940. The membrane 940 may allow ions to pass through, but it may not allow analytes or other components in the sample to pass through. The membrane 940 may be any membrane described herein. The membrane 940 may have a diameter of 1 to 2 mm, 2 to 3 mm, 3 to 4 mm, 4 to 5 mm, 5 to 7 mm, 7 to 10 mm, or greater than 10 mm.

[0055] The first channel 932 may contain a first electrolyte and a sample that can form a first mixture. Components of the sample may pass through a manifold 908 that may contain a second electrolyte. The components may form a focusing band 948 as they pass through the manifold 908 and / or the second electrolyte. The focusing band 948 may move radially inward. The annular space 936 may contain a second electrolyte. A voltage difference applied to the first electrode 916 and the second electrode 920 may drive charged components upward through the membrane 940 from the first channel 932 to the annular space 936. Charged components may enter the pipette tip 924 by aspirating from the pipette via the pipette tip 924. The components may form a focusing band 952 within the annular space 936. The focusing band 952 may move away from the top of the system 900 and toward the orifice at the bottom of the membrane 940 and / or the pipette tip 924. The focusing band 952 can move radially inward.

[0056] In some embodiments, the system for concentrating components in a sample may include a base defining a first channel and a cavity. The base may be any base described herein, including base 704 and base 904. The base may be defined from a single material piece. The cavity may be any cavity described herein, including cavity 728 and cavity 928.

[0057] The first channel may include any first channel described herein, including the first channel 932. The first channel may be circular. The first channel is in fluid communication with the cavity. For example, components in the liquid may be allowed to move from the first channel to the cavity in the liquid. The outer diameter of the first channel may be greater than the first outer diameter of the top of the cavity. The outer diameter of the first channel may be the diameter of the cylindrical recess shown in the base 704 in Figures 7 and 8.

[0058] The cavity may include a conical shape having a first outer diameter greater than a second outer diameter at the bottom of the cavity. The narrower portion of the conical shape may be at the bottom of the system. As used herein, “bottom” and “top” refer to the orientation of the system during normal operation. The conical shape includes a cone or a substantially conical shape. The sides of the cavity may taper to a point (i.e., apex). Substantially similar shapes may include shapes in which the horizontal cross-section of the shape at any point has a maximum length (e.g., diameter) that is no more than 5%, 10%, or 15% of the dimensions of a cone defined by the same apex and cavity opening. The cone may be a right cone. In some embodiments, the conical shape may not include a apex. For example, the conical shape may be cut to include a base which may be circular. For example, the conical shape may have a cross-section similar to that of cavity 928 in Figure 9, where film 940 is one end of the conical shape. In some embodiments, the base surface may be conical. The cavity may have a depth of 5 to 10 mm, 10 to 15 mm, 15 to 20 mm, 20 to 30 mm, 30 to 40 mm, 40 to 50 mm, or greater than 50 mm.

[0059] In some embodiments, the cavity may have a cylindrical portion at the top of a conical portion. In this way, the cavity may have a shape similar to that of a container having conical and cylindrical portions (e.g., a pipette tip).

[0060] The system may further include a first electrode positioned in the first channel. The first electrode may be any electrode as described herein, including the first electrode 916. In some embodiments, the first electrode may be ring-shaped. The system may further include a second electrode. The second electrode may be any electrode as described herein, including the second electrode 920. The second electrode is configured to be in closer electrical communication with the cavity than the first channel, if the first channel and cavity contain an electrolyte. Closer electrical communication may mean lower resistance or higher current when the same voltage is applied. The cavity may be physically closer to the second electrode than the first channel is to the second electrode. If the second electrode is positioned in a liquid that contacts the cavity and the first channel, the amount of liquid between the second electrode and the cavity is less than the amount of liquid between the first channel and the second electrode. The second electrode may be ring-shaped, plate-shaped, or rod-shaped.

[0061] The first channel is characterized by a first volume. The first volume can be considered to be the volume of liquid that can be contained by the first channel. For example, in Figure 9, the first volume of the first channel 932 does not have to include the volume taken by the manifold 908. The first volume can be 0.25 ml or less, 0.25 to 0.50 ml, 0.50 to 0.75 ml, 0.75 to 1.0 ml, 1.0 to 2.5 ml, 2.5 to 5.0 ml, 5.0 to 7.5 ml, 7.5 to 10.0 ml, 10.0 to 12.5 ml, 12.5 to 15.0 ml, 15.0 to 30.0 ml, 30.0 ml to 50.0 ml, or greater than 50 ml.

[0062] The cavity is configured to receive the container and to form a second channel when the container is placed within the cavity. The container may be any container described herein, including pipette tips 112 and 912. In some embodiments, the container may be a trocar or syringe needle. The container may define a cannula from which liquid can enter from the cavity. The container may be configured to hold sample collection volumes from 1 μl to 500 μl (including 1 μl to 10 μl, 10 μl to 50 μl, 50 μl to 100 μl, 100 μl to 200 μl, 200 μl to 300 μl, 300 μl to 400 μl, 400 μl to 500 μl, or greater than 500 μl). The container may be held in place by a manifold. In some embodiments, the container may be held in place via a support attached to a base. In other embodiments, an external support on the base may hold the container in place. For example, the support structure may be the arm of a robotic handling system.

[0063] The second channel may be an annular space containing an annular space 936. The annular space may be defined by the surface of the base and the surface of the container when the container is placed in the cavity. The second channel is characterized by a second volume. The first volume is larger than the second volume. The first volume may be 5 to 10 times, 10 to 20 times, 20 to 50 times, 50 to 100 times, or more than 100 times the second volume. In some embodiments, the surface of the base and the surface of the container are concentric. The annular space may be the space between two concentric surfaces.

[0064] In some embodiments, the cavity is located below the bottom surface of the base defining a first channel. For example, as shown in Figures 8 and 9, the first channel may be a first recess in the base, and the cavity may be a second recess within the first recess. The bottom surface of the base defining the first channel may contact or intersect the surface of the base defining the second channel. The surfaces may intersect in a circular fashion.

[0065] In some embodiments, the system may include a membrane between the cavity and the second electrode. The membrane may be a semipermeable membrane. The membrane may be membrane 940 shown in Figure 9. The membrane may allow particles smaller than a certain size to pass through. Components in the biological sample intended to be analyzed do not need to pass through the membrane.

[0066] In some embodiments, the system may include a reservoir. A second electrode may be located within the reservoir. The reservoir may be any reservoir described herein, including reservoir 944. The reservoir may contain an electrolyte, including a preceding electrolyte. The reservoir may be separated from the cavity by a membrane. The same electrolyte may be present in both the reservoir and the cavity. An electric field from the second electrode located within the reservoir may propagate through the reservoir to the cavity.

[0067] In some embodiments, the system may include a power supply that is electrically connected to the first and second electrodes. In some embodiments, the system may include a computer configured to control the power supply. The power supply may provide a constant voltage, a constant current, or a constant power.

[0068] In some embodiments, the system may include an in vitro diagnostic assay apparatus comprising any in vitro diagnostic assay described herein. The assay apparatus may include a detector configured to detect components in a sample. The detector may include an optical detector, an electrochemical detector, or any detector described herein.

[0069] In some embodiments, the system may include a container. In some embodiments, the system may include a container placed within a cavity. In other embodiments, the system may include a container that is not placed within a cavity.

[0070] In some embodiments, the system may include a first electrolyte disposed in a first channel. The first electrolyte may be a terminal electrolyte. The system may include a second electrolyte, which may be a leading electrolyte. The second electrolyte may be disposed in a gel within the first channel or in a cavity. The system may include a gel. The electrolyte and gel may be any of those disclosed herein.

[0071] In some embodiments, the system may include a manifold. The manifold may define a plurality of channels having outlets to a second channel. The plurality of channels defined by the manifold may be between the first channel and the second channel.

[0072] In some embodiments, the base may define a plurality of structures extending within the cavity. The plurality of structures may include a first structure and a second structure located on the opposite side of the cavity as the first structure. The distance between the first and second structures may be the diameter of the vessel. The length of the structure may be the second thickness within the second channel. The structure may hold or support the vessel within the cavity.

[0073] Figure 10A shows a bottom view of a base 1000 having four supports, including support 1004. The supports may be multiple structures extending into the cavity. Support 1004 is a projection from the circular cavity. The four supports are arranged at equal intervals (90 degrees between adjacent supports). Each support may be identical to the other supports. The supports may be rectangular in shape. In some embodiments, the supports may be triangular, pyramidal, trapezoidal, or rounded in shape. The supports may have a length of 2% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 33%, or 33% to 40% of the diameter of the cavity. The distance between the supports on opposite sides of the cavity may be the outer diameter of the container (e.g., pipette tip). The number of supports at the top of the cavity may be 4 to 8, 8 to 10, 10 to 16, 16 to 20, 20 to 30, or 30 to 50. Additional supports may be present at different depths within the cavity.

[0074] Figures 10B, 10C, 10D, and 10E show plan views of different numbers of supports at the top of the cavity and supports at different depths within the cavity. Figures 10B, 10C, 10D, and 10E each show four supports at each depth below the top of the cavity. The number of supports at each depth may be any number provided on the supports at the top of the cavity, and this number may be the same as or different from the number of supports at the top of the cavity. Supports may be provided at different depth levels. For example, Figures 10B, 10C, 10D, and 10E show supports at three depth levels. Different depth levels including 2 to 5 and 5 to 10 are possible. Adjacent depth levels may be separated by the same or different vertical distances. Figure 10E shows a recess 1008 at the top of the base between the supports. The recess 1008 may ramp down into the cavity to allow more liquid flow than would be possible without the recess.

[0075] III. Exemplary Methods Figure 11 is a flowchart of an exemplary process 1100 related to volume binding in epitacophoresis. Process 1100 may concentrate components from a sample. Process 1100 may be for ETP-based isolation / purification. The sample may be any sample described herein. Components may include target nucleic acids, target microorganisms, biomarkers, or target analytes. The sample may contain multiple components. The sample may be derived from a subject. The sample may be a mixed sample containing materials from two or more sources. The sample may contain an ETP upper marker.

[0076] In some embodiments, one or more process blocks in Figure 11 may be executed by an epitacophoresis apparatus (e.g., system 900 in Figure 9). In some embodiments, one or more process blocks in Figure 11 may be executed by a separate apparatus or group of apparatus, either separate from or including the epitacophoresis apparatus. Additionally or alternatively, one or more process blocks in Figure 11 may be executed by one or more parts of system 1200, such as a logic system 1230, a processor 1250, memory 1235, external memory 1240, storage device 1245, and / or treatment apparatus 1260. Process 1100 may include additional embodiments, such as any single embodiment or any combination of embodiments relating to one or more other processes described below and / or elsewhere in this specification.

[0077] In block 1110, process 1100 may include applying a voltage difference between a first electrode and a second electrode. The first electrode may be placed in a first mixture containing a first electrolyte and a sample. In some examples, the first mixture may be in a first channel 932 in Figure 9. The second electrode may be placed in a second electrolyte. The second electrolyte may be placed in a gel in the first channel. The gel may be in or within the manifold 908. The first electrode may be different from the second electrode. The second electrolyte may be contained in a gel. The second electrolyte may be hydrodynamically separated from the first electrolyte. The first and second electrolytes may be separated by a membrane. The epitaphoresis apparatus may apply a voltage difference between the first electrode and the second electrode.

[0078] The first electrode may be circular. The first electrode may be a ring or may be positioned on the edge of a circular channel. For example, the first electrode may be any electrode described herein, including the first electrode 916 in Figure 9. The second electrode may be any electrode described herein, including the second electrode 920.

[0079] The voltage difference can be a constant voltage. In some embodiments, the voltage difference can be the result of a constant current being applied. In some embodiments, the applied voltage difference can be the result of a constant power being applied. The voltage can range from about 10V to about 10kV with power ranging from about 1mW to about 100W.

[0080] In block 1120, process 1100 may include using a voltage difference to flow components in a first channel in a first direction. The first channel may be any channel described herein, including the first channel 932. The first direction may be away from the first electrode and toward a second channel. Components may be focused into a band. A focused band may be a portion in which the target analyte is concentrated in the first or second electrolyte. The target analyte in a particular focused band may contain ions having the same or similar mobility in the applied electric field. The band may be ring-shaped and may be referred to as a focused region, such as the focused region in Figure 1. Focusing may result from the applied voltage and electrolyte. The first mixture is characterized by having a first thickness perpendicular to the first direction. The first thickness may be a height. For example, the first thickness may be the height of the first mixture in the first channel 932. The first thickness may be the vertical dimension of the focused band 948 shown in Figure 9.

[0081] In block 1130, process 1100 may include flowing the components focused in the band in a second channel in a second direction. The band may flow through the first channel, exit the first channel, and then enter the second channel. The second electrolyte may be in the second channel. The second direction may be from the first channel to the orifice of the vessel. The second channel may be an annular space. The second channel may include any second channel described herein, including the annular space 936. The second mixture containing the components and the second electrolyte in the second channel is characterized by having a second thickness perpendicular to the second direction. The first thickness may be greater than the second thickness. The thickness of the liquid may decrease as the liquid moves from the first channel to the second channel. The liquid may contain the components and either the first or second electrolyte. The second thickness may be height. The second thickness may be the thickness of the focusing band 952 shown in the annular space 936, and the thickness may be perpendicular to the wall defining the annular space 936. The amount of the component in the second mixture may be the same as the amount of the component in the first mixture.

[0082] In this embodiment, the annular space is defined by the outer surface of the container. The container may include a conical shape. The second channel may be defined by the first conical surface and the second conical surface. The first conical surface may be the outer surface of the container. The second conical surface may be the surface of the base.

[0083] Process 1100 may include, within the second channel, flowing the components focused within the band in a second direction, which reduces the radius of the band and flows the components in the direction of gravity. The second direction is not limited to the direction of gravity. The second direction may be angled downwards such that the second direction is the sum of a non-zero vector in the downward direction (direction of gravity) and one or more other vectors. The second direction may directly point to the vertex of a cone formed by the first or second conical surface. In some embodiments, the second direction may directly point to a cone having a surface in annular space.

[0084] In embodiments, the first channel and the second channel may each be defined by a base. The base may be any base described herein, including base 904. In some embodiments, process 1100 may include flowing components through a plurality of channels between the first channel and the second channel. The plurality of channels may be defined by a manifold, which may include manifold 908.

[0085] In the embodiment, the first direction does not have to be collinear with the second direction. The first direction may be horizontal and directed toward the center of the circle that forms the outside of the first channel. The second direction is directed toward the center of the circle but may be located below the bottom surface of the first channel.

[0086] A first channel may be characterized by a first volume, which may be defined as the maximum volume of liquid that can be placed within the first channel. The volume of the first channel may be determined based on the area of ​​the bottom surface of the first channel and the height of the side walls of the first channel. A second channel may be characterized by a second volume, which may be the volume of an annular space. The first volume may be at least 10 times larger than the second volume. In some embodiments, the first volume may be 5 to 10 times, 10 to 50 times, 50 to 100 times, 100 to 150 times, or more than 150 times the second volume.

[0087] In the embodiment, the second channel may have a length in the second direction of at least 2 mm. The length in the second direction may be 2 to 5 mm, 5 to 10 mm, 10 to 15 mm, or greater than 15 mm.

[0088] In block 1140, process 1100 may include collecting a second mixture in a container while applying a voltage difference. Collecting the mixture in the container may include flowing the second mixture into the container in a third direction against gravity. The concentration of the components in the second mixture in the container may be higher than the concentration of the components in the sample, including 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 500 times, 500 to 1,000 times, 1,000 to 2,000 times, or more than 1,000 times.

[0089] In embodiments, the sample may contain additional components from the sample, and these additional components may be focused into additional bands. For example, the component is a first component, and the sample may further contain a second component. The second component may be focused into a second band within the first channel. The method may further include using a voltage difference to flow the second component in the first channel. The method may also include flowing the second component, focused into a second band in a second direction, within the second channel. Process 1100 may include collecting the second component in a container.

[0090] Figure 11 shows an exemplary block of process 1100, but in some embodiments, process 1100 may include additional blocks, fewer blocks, different blocks, or blocks in different arrangements than those shown in Figure 11. Additionally or alternatively, two or more blocks of process 1100 may be executed in parallel.

[0091] IV. Exemplary Systems Figure 12 shows a measurement system 1200 according to an embodiment of the present disclosure. The shown system includes a sample 1205, such as a cell-free DNA molecule, in an assay device 1210, and an assay 1208 may be performed on the sample 1205. For example, the sample 1205 can be brought into contact with the reagents of assay 1208 to provide a signal of physical properties 1215. An example of an assay device may be a flow cell containing the assay probe and / or primers, or a tube through which a droplet moves (the droplet contains the assay). The assay device 1210 may include several modules, including any epitacophoresis (ETP) device described herein. The ETP device can concentrate or separate the sample 1205, and the concentrated sample may be sent to another module in the analyzer. Other modules may perform in vitro diagnostic assays.

[0092] Physical properties 1215 from the sample (e.g., fluorescence intensity, voltage, or current) are detected by the detector 1220. The detector 1220 can perform measurements at intervals (e.g., periodic intervals) to acquire data points that constitute a data signal. In one embodiment, an analog-to-digital converter converts the analog signal from the detector into digital format multiple times. The assay apparatus 1210 and the detector 1220 can form an assay system, for example, a sequencing system that performs sequencing according to the embodiments described herein. The data signal 1225 is transmitted from the detector 1220 to the logic system 1230. As an example, the data signal 1225 may be used to determine the sequence and / or position of a DNA molecule in a reference genome. The data signal 1225 may include various measurements performed simultaneously, for example, different colored fluorescent dyes, or different electrical signals to a sample 1205 of different molecules, and thus the data signal 1225 can correspond to multiple signals. The data signal 1225 may be stored in local memory 1235, external memory 1240, or storage device 1245.

[0093] The logic system 1230 may be a computer system, ASIC, microprocessor, graphics processing unit (GPU), etc., or may include them. It may also include a display (e.g., monitor, LED display, etc.) and a user input device (e.g., mouse, keyboard, buttons, etc.), or may be combined with them. The logic system 1230 and other components may be part of a standalone or networked computer system, or may be directly attached to or incorporated into an apparatus including the detector 1220 and / or assay apparatus 1210 (e.g., a sequencing apparatus). The logic system 1230 may also include software running within the processor 1250. The logic system 1230 may include a computer-readable medium that stores instructions for controlling the measurement system 1200 to perform any of the methods described herein. For example, the logic system 1230 can provide commands to a system including the assay apparatus 1210 so that sequencing or other physical operations are performed. Such physical operations may be performed in a specific order, for example, by adding and removing reagents in a specific order. Such physical operations may be performed by a robotic system, for example, including a robotic arm, so that the sample can be acquired and used to perform the assay. Furthermore, in some embodiments, the ETP apparatus may be used in conjunction with a liquid handling robot, which may be optionally used to perform downstream analysis of the sample that may have been focused and / or collected from the apparatus.

[0094] The measurement system 1200 may also include a treatment device 1260 that can provide a treatment to a subject. The treatment device 1260 can determine a treatment and / or can be used to perform a treatment. Examples of such treatments may include surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, hormone therapy, and stem cell transplantation. A logic system 1230 may be connected to the treatment device 1260, for example, to provide results of the methods described herein. The treatment device may receive inputs from other devices, such as an imaging device and user input (for controlling a treatment, such as control of a robotic system).

[0095] Any computer system referred to herein may utilize any appropriate number of subsystems. An example of such subsystems is shown in Figure 13 in computer system 1300. In some embodiments, the computer system comprises a single computer device, where subsystems are components of the computer device. In other embodiments, the computer system may include multiple computer devices, each having internal components, and each being a subsystem. The computer system may include desktop and laptop computers, tablets, mobile phones, and other mobile devices.

[0096] The subsystems shown in Figure 13 are interconnected via a system bus 75. Additional subsystems are shown, such as a printer 74, a keyboard 78, a storage device 79, and a monitor 76 (e.g., an LED display screen) coupled to a display adapter 82. External and input / output (I / O) devices coupled to an I / O control device 71 can be connected to the computer system by any number of means known in the art, such as input / output (I / O) ports 77 (e.g., USB, Lightning). For example, an I / O port 77 or an external interface 81 (e.g., Ethernet, Wi-Fi) can be used to connect the computer system 1300 to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via the system bus 75 allows the central processor 73 to communicate with each subsystem and control the execution of multiple instructions from the system memory 72 or storage device 79 (e.g., a fixed disk such as a hard drive, or an optical disk), as well as the exchange of information between subsystems. The system memory 72 and / or storage device 79 may embody computer-readable media. Another subsystem is a data acquisition device 85, such as a camera, microphone, accelerometer, and others. Any of the data described herein may be output from one component to another and to the user.

[0097] A computer system may include multiple identical components or subsystems connected together, for example, by an external interface 81, by an internal interface, or via removable storage devices that can be connected to and disconnected from one component to another. In some embodiments, computer systems, subsystems, or devices may communicate over a network. In such examples, one computer may be considered a client, another computer may be considered a server, and each of them may be part of the same computer system. Clients and servers may each include multiple systems, subsystems, or components.

[0098] Aspects of the embodiments may be implemented in the form of control logic using hardware circuits (e.g., application-specific integrated circuits or field-programmable gate arrays) and / or computer software with a modular or integrated generally programmable processor. As used herein, the processor may include a single-core processor, a multi-core processor on the same integrated chip, or a single circuit board or networked, as well as multiple processing units on dedicated hardware. Based on the disclosures and teachings provided herein, those skilled in the art will know and understand other forms and / or methods of implementing each embodiment of the invention using hardware, and combinations of hardware and software.

[0099] Any software component or function described in this application may be implemented as software code executed by a processor using any suitable computer language, such as Java, C, C++, C#, Objective-C, or Swift, or a scripting language, such as Perl or Python, using conventional or object-oriented techniques. The software code may be stored as a set of instructions or commands on a computer-readable medium for storage and / or transmission. Suitable non-temporary computer-readable media may include random access memory (RAM), read-only memory (ROM), magnetic media such as hard drives or floppy disks, or optical media such as compact discs (CDs), DVDs (Digital Versatile Discs), or Blu-ray discs, flash memory, etc. The computer-readable medium may be any combination of such storage devices or transmission devices.

[0100] Such programs may also be encoded and transmitted using carrier signals adapted for transmission over wired, optical, and / or wireless networks compliant with various protocols, including the Internet. Thus, computer-readable media may be created using data signals encoded with such programs. Computer-readable media encoded with program code may be packaged with compatible devices or provided separately from other devices (e.g., via internet download). Any such computer-readable media may reside on or within individual computer products (e.g., hard drives, CDs, or complete computer systems), or on or within different computer products within a system or network. Computer systems may include monitors, printers, or other suitable displays for providing users with any of the results described herein.

[0101] Any of the methods described herein may be performed entirely or partially using a computer system including one or more processors that can be configured to perform the steps. Thus, each embodiment may target a computer system configured to perform any of the steps of the methods described herein using different components that potentially perform each of the steps or each of the groups of steps. The steps of the methods herein are presented as numbered steps, but may be performed simultaneously or at different times, or in any logically possible order. Furthermore, some of these steps may be used in conjunction with some of other steps from other methods. Also, all or some of the steps may be optional. Furthermore, any of the steps of any of these methods may be performed using a module, unit, circuit or other means of a system for performing these steps.

[0102] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated or combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure.

[0103] The above description of exemplary embodiments of this disclosure is presented for illustrative and explanatory purposes and is written to provide a complete disclosure and description of how to create and use embodiments of this disclosure. It is not intended to be exhaustive or to limit this disclosure to the exact forms described, nor is it intended to represent all or only experiments that have been conducted. While this disclosure is described in some detail as examples and illustrations for clarity, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of this disclosure without departing from the spirit or scope of the appended claims.

[0104] Therefore, the above merely illustrates the principles of the present invention. Those skilled in the art will understand that various configurations embodying the principles of the present invention and falling within its spirit and scope can be devised, although not expressly described or shown herein. Furthermore, all examples and conditional language enumerated herein are primarily intended to assist the reader in understanding the principles of this disclosure, not limited to such specifically enumerated examples and conditions. Moreover, all descriptions herein enumerating the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and those to be developed in the future, i.e., any developed elements that perform the same function regardless of their structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims.

[0105] The use of “a,” “an,” or “the” is intended to mean “one or more” unless otherwise specifically indicated. The use of “or” is intended to mean “inclusive OR” rather than “exclusive OR” unless otherwise specifically indicated. A reference to a “first” component does not necessarily require a second component to be brought forth. Furthermore, a reference to a “first” or “second” component does not limit the referred component to a specific position unless explicitly stated. The term “based on” is intended to mean “based at least in part.”

[0106] Claims may be drafted to exclude any element that may be optional. Therefore, this reference is intended to serve as an antecedent for the use of exclusive terms such as “exclusively” or “only” in relation to the enumeration of elements in the claims or the use of “negative” limitations.

[0107] Where a range of values ​​is provided, each intervening value is understood to be up to one-tenth of the lower limit unit and between the upper and lower limits of the specifically disclosed range, unless the context explicitly indicates otherwise. Each smaller range between any stated or intervening value within the stated range and any other stated or intervening value within that stated range is included within embodiments of this disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and if a smaller range includes either the upper or lower limit, neither of them, or both, each range is also included in this disclosure and subject to any upper or lower limit specifically excluded within the stated range. If a stated range includes one or both limits, the range excluding either or both of the limits that they include is also included in this disclosure.

[0108] All patents, patent applications, publications, and descriptions referenced herein are incorporated herein by reference in whole for any purpose, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the relevant methods and / or materials from which the publications are cited. None of them are considered prior art.

Claims

1. A system for concentrating components in a sample, A base defining a first channel and cavity, wherein the cavity includes a conical shape in which a first outer diameter at the top of the cavity is greater than a second outer diameter at the bottom of the cavity, A first electrode disposed within the first channel, The second electrode and Equipped with, The first channel is in fluid communication with the cavity, The outer diameter of the first channel is larger than the first outer diameter at the upper part of the cavity. The second electrode is configured to be in closer electrical communication with the cavity than the first channel, when the first channel and the cavity contain an electrolyte. The first channel is characterized by a first volume, The cavity is configured to receive a container and to form a second channel when the container is placed within the cavity, The second channel is an annular space, The annular space is defined by the surface of the base and the surface of the container when the container is placed in the cavity. The second channel is characterized by a second volume, A system in which the first volume is greater than the second volume.

2. The system according to claim 1, wherein the surface of the base and the surface of the container are concentric.

3. The system according to claim 1, wherein the surface of the base is conical.

4. The system according to claim 1, wherein the cavity is located below the bottom surface of the base defining the first channel.

5. The system according to claim 1, further comprising a film between the cavity and the second electrode.

6. The system according to claim 5, further comprising a reservoir, wherein the second electrode is disposed within the reservoir.

7. The system according to claim 1, further comprising a power supply electrically connected to the first electrode and the second electrode.

8. The system according to claim 7, further comprising a computer configured to control the aforementioned power supply.

9. The system according to claim 1, further comprising a detector configured to detect components in the container.

10. The system according to claim 1, further comprising the container, wherein the container is a pipette tip.

11. A first electrolyte disposed within the first channel, A second electrolyte is disposed within the gel in the first channel and within the cavity, The gel and The system according to claim 1, further comprising:

12. The system according to claim 1, wherein the first electrode is ring-shaped.

13. The base defines a plurality of structures extending within the cavity, The plurality of structures include a first structure and a second structure located on the opposite side of the cavity from the first structure, The system according to claim 1, wherein the distance between the first structure and the second structure is the diameter of the container.

14. A method for concentrating components from a sample, The method is performed using the system described in claim 1, Applying a voltage difference between the first electrode and the second electrode, where, The first electrode is placed in a first mixture containing the first electrolyte and the sample. The second electrode is placed in the second electrolyte, The first electrolyte is different from the second electrolyte. Using the voltage difference, the component in the first channel is made to flow in the first direction, where, The first direction is away from the first electrode and toward the second channel, The aforementioned components are focused into a band, The first mixture is characterized in that it has a first thickness perpendicular to the first direction. In the second channel, the component focused within the band is flowed in a second direction, where, The second electrolyte is located within the second channel. The second direction is from the first channel toward the orifice of the container. The second channel is an annular space, The second mixture containing the component and the second electrolyte in the second channel is characterized in that it has a second thickness perpendicular to the second direction. The first thickness is greater than the second thickness. Furthermore, Collecting the second mixture in the container while applying the voltage difference, wherein the concentration of the component in the second mixture in the container is higher than the concentration of the component in the sample. Methods that include...

15. The component is a first component, The aforementioned sample contains a second component, The aforementioned band is the first band, The second component is focused into the second band in the first channel. The method described above is Using the aforementioned voltage difference, the second component in the first channel is made to flow, The method according to claim 14, further comprising flowing the second component, which is focused within the second band, in the second direction within the second channel.

16. The method according to claim 15, further comprising collecting the second component in the container.

17. The method according to claim 14, wherein the first thickness is the height.

18. The method according to claim 14, wherein the annular space is defined by the outer surface of the container.

19. The method according to claim 14, wherein the first channel and the second channel are each defined by a base.

20. The method according to claim 14, wherein the first direction is not collinear with the second direction.

21. The method according to claim 14, wherein the container includes a conical shape.

22. The method according to claim 14, wherein the second electrolyte is disposed within the gel in the first channel.

23. The method according to claim 14, wherein the band is ring-shaped.

24. The method according to claim 14, wherein the first electrode is circular.

25. The first channel is characterized by a first volume, The second channel is characterized by a second volume, The method according to claim 14, wherein the first volume is at least 10 times larger than the second volume.

26. The method according to claim 14, wherein the second channel has a length of at least 2 mm in the second direction.

27. ​​The method according to claim 14, wherein collecting the second mixture in the container involves flowing the second mixture into the container in a third direction against gravity.

28. The method according to claim 14, wherein in the second channel, the component focused within the band is flowed in a second direction, which reduces the radius of the band and causes the component to flow in the direction of gravity.

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