Devices and methods for isolation and utilization of extracellular chromosomal molecules

Abstract

Disclosed herein are devices and methods for isolation and analysis of exosomal molecules from cell DNA samples. Such devices and methods facilitate isolation of ecDNA for downstream applications, such as cellular analysis and design of probes or therapeutic agents, in order to detect or target ecDNA for degradation or cells carrying ecDNA. Similarly, disclosed herein are compositions enriched for ecDNA and their use in the development of therapeutic or diagnostic agents.

Description

1. Relevant Applications

[0001] This disclosure claims the benefit of priority to U.S. Provisional Application No. 63 / 482,587, filed February 1, 2023, which is incorporated herein by reference in its entirety. 2. Background

[0002] Extrachromosomal DNA (ecDNA) is a unit of circular DNA, ranging from hundreds of kilobases to over 5 megabase pairs, residing in the nucleus of a cell, and like... Figure 1 As shown, they are physically distinct from chromosomal DNA. These non-chromosomal circular particles can typically carry one or more full-length genes, sometimes oncogenes, as well as regulatory regions that drive gene expression. Often lacking any advanced compression, ecDNA has a relatively simpler chromatin conformation than regular chromosomes, and therefore their respective genes are more readily enzymatically reacted and thus can be transcribed efficiently, typically at a rate more frequent than their chromosomal counterparts. ecDNA is usually rare or absent in normal healthy tissues, but is present in 14% of primary cancers and >40% of metastatic cancers, and in nearly half of all cancer types in humans.

[0003] ecDNA accounts for over 50% of all high copy number amplifications in cancer, driving high copy number gene amplification and non-Mendelian genomic adaptation, enabling tumors to rapidly evolve and switch their oncogene dependence under therapeutic stress. However, little is known about the effects of non-chromosomal oncogene inheritance (through random ancestral identity), nor is the impact of ecDNA on somatic variation and selection, thus rendering current targeted and immunotherapies largely ineffective in patients with cancers exhibiting aggressive oncogene amplification. Another class of complex chromosomal aberrations arises from micronuclei. Micronuclei are tiny extranuclear bodies originating from acentric chromatids / chromosome fragments or intact chromatids / chromosomes that are delayed in late-phase division and are not contained within the host nucleus during telophase. They are small, DNA-containing nuclear structures spatially separated from the host nucleus. They are common in pathology, including cancer. Recent studies have shown that these nuclear structures not only serve as biomarkers of disease but also play an active role in tumor biology. Many consequences of micronucleus formation for tumor biology depend on the frequent and irreversible rupture of their nuclear envelope, leading to the exposure of their DNA contents to the cytoplasm. These consequences include extensive DNA rearrangements known as chromosome fragmentation and activation of the cGAS-STING innate immune signaling pathway, which can be a double-edged sword with both tumorigenesis and tumor prevention functions. Although micronuclei are small structures, their impact on cells and their microenvironment is considerable.

[0004] Due to their small size and highly variable copy number and size distribution between cells, traditional whole-genome analysis techniques, such as sequencing, mapping or quantification, isolation or probing of ecDNA or micronuclei or any other type of complex chromosomal damage and rearrangement, are challenging or inefficient. New methods and tools that can efficiently, accurately, and cost-effectively characterize ecDNA or micronuclei or any other type of complex chromosomal damage and rearrangement, their size, characteristics, abundance, and concentration, and better isolate or enrich them for further probing, such as imaging, mapping, sequencing, or genotyping, while maintaining single-molecule integrity and single-cell traceability, will be revolutionary and highly anticipated.

[0005] In particular, there is a strong demand for precise diagnostic methods and devices to identify cells, tissues, and patients with ecDNA, micronuclei, or complex lesion features associated with the risk, presence, or prognosis of disease (especially cancer). Such devices and methods will help determine the most effective therapeutic interventions for clinical treatment at the appropriate time and place.

[0006] In particular, there is a desire for methods and apparatuses that can identify ecDNA, micronuclei, or complex damage in patient samples / cells and that can isolate, enrich, and recover these specific types of ecDNA or micronuclei in sufficient quantities, integrity, and purity to deliver precise medical-grade information for therapeutic development and guidance, compatible with further downstream clinical analyses such as probe hybridization, optical mapping, and sequencing.

[0007] The genomic contents of ecDNA can be determined through sequencing (including common and low-cost massively parallel short-read sequencing). The presence of normal chromosomal DNA can interfere with sequencing, requiring partial or complete enrichment of the sample by physical methods prior to library construction. Existing methods such as CRISPR-Catch [Hung 2022], measured as the fraction of ecDNA into the total library contents, struggle to achieve purity better than 60%, yet this method requires prior knowledge of the ecDNA sequence to generate the guide RNA probes needed for the protocol. Other methods, such as hybridization of ecDNA-specific FISH probes, also require prior knowledge of the ecDNA sequence to generate the probes. These limitations collectively affect the ability of ecDNA reconstruction algorithms to identify heterogeneous ecDNA populations and limit the speed at which new ecDNA can be identified. 3. Overview of the Invention

[0008] This document discloses an apparatus and method for separating ecDNA molecules derived from one or more cells from cellular DNA, the separation being performed in a fluid apparatus. The separated ecDNA is then extracted from the fluid apparatus with a very high purity relative to the cellular DNA derived from the one or more cells. In some embodiments, the separated ecDNA undergoes an enzymatic reaction prior to extraction from the fluid apparatus. The purified ecDNA sample can then be used for applications. In some aspects, the applications include monitoring disease states. In some aspects, the applications include monitoring disease progression. In some aspects, this application includes generating hybridization probes. In some aspects, this application includes identifying the sequence location of at least a portion of the ecDNA within a long nucleic acid molecule, preferably within a long nucleic acid molecule derived from one or more ecDNA-source cells from a patient. In some aspects, the long nucleic acid molecule is a portion of a chromosome or an entire chromosome. In some aspects, the applications include treatment or therapy. In some aspects, a FISH probe library is generated from purified ecDNA, and in some aspects, a capture probe library can be generated from ecDNA. In some respects, FISH probes and / or capture probes can be used to analyze the ecDNA content of cells other than those used to generate the probes, and these cells can be derived from non-viable samples, such as FFPE or rapidly frozen tissue samples or preserved circulating tumor cells.

[0009] Some embodiments disclosed herein relate to methods for enriching, purifying, or isolating nonchromosomal DNA, such as ecDNA, from cells or cell populations, and fluid apparatuses for performing these methods.

[0010] Some of these methods involve the selective retention of cellular or nuclear chromosomes, allowing ecDNA to flow through barriers, such as filters or barriers, while preventing chromosomes from passing through. Some of these methods facilitate this by selecting filter pore sizes to allow ecDNA to pass through while preventing chromosomal DNA from passing through the filter. Exemplary sizes include less than 0.8 micrometers, 0.8 micrometers, 1 micrometer, 1.2 micrometers, 1.5 micrometers, 1 micrometer, 2.5 micrometers, 3 micrometers, or larger, or sizes that span or exceed the scope disclosed herein. Alternatively, porous materials such as gels or columns may be used to selectively allow ecDNA, but not chromosomal DNA, to pass through.

[0011] Alternatively, some such methods involve the selective retention of ecDNA or other circular nucleic acids, such as by flowing a sample in a first direction over a substrate having barriers or protrusions that capture or “block” the circular DNA while allowing the linear DNA to eventually slide off. If these protrusions are angled, then the circular nucleic acid, such as ecDNA, can be released from the surface by flowing the washing buffer in a second direction, such as antiparallel or “reverse.”

[0012] In the third set of embodiments, ecDNA and chromosomal DNA are differentially separated by flowing through a microfluidic device or other routes that include barriers that differentially orient chromosomal DNA and ecDNA to different output locations. Some such routes contain barriers, such as solid barriers, hydrophobic or hydrophilic barriers, or other structures that selectively redirect nucleic acids based on their size. Typically, these barriers are configured in an array having a parallel straight path through the array that deviates from the flow direction of the sample through the array. The offset can be, for example, about or exactly 15 degrees, such as 5, 10, 15, 20, 25, 30, 35, 45, or more than 45 degrees, or any value spanning or adjacent to the series disclosed herein. In these embodiments, ecDNA and other relatively small nucleic acids are able to follow the flow direction, while larger molecules such as chromosomes follow an offset parallel straight path through the array.

[0013] In some embodiments of each of these methods for ecDNA enrichment or purification, the sample is treated, such as by purification, prior to or as part of the methods disclosed above. Purification typically includes the removal of particulate or clogging cellular material, such as cell membranes or nuclei. Optionally or in combination, cells are treated before or after the preparation of cell extracts to enrich aggregated chromatin and, in some cases, dissolved nuclear membranes. Without being bound by theory, aggregated particulate chromosomes and ecDNA exhibit exemplary properties that facilitate their differential flow through the system and the practices of the methods described herein.

[0014] Consistent with the above disclosures, this document also discloses enriched cellular DNA compositions, such as enriched ecDNA compositions. The compositions are enriched by exhibiting, for example, an increased relative abundance relative to chromosomal DNA, which can be measured in moles, bases, or total molecular weight, and the enrichment can be at least about or more than: 2×, 3×, 4×, 5×, 6×, 10×, 20×, 30×, 40×, 50×, or more than 50×. In some cases, the enriched compositions lack chromosomes. In some cases, the relative increase in ecDNA may be higher than that measured by, for example, qPCR, because such methods may be sensitive to or unable to distinguish between intact chromosomal DNA and chromosomal fragments, such as those that may have been generated after sample preparation or purification. Some compositions also contain mitochondrial DNA, although alternative sample preparation methods include the removal of mitochondria before or after sample preparation.

[0015] Enriched ecDNA samples can be sequenced. Sequencing and alignment with genomic references or scaffolds allow for the identification of regions of chromosomal DNA they map to, and the identification of mutations in the ecDNA relative to their chromosomal origin. Such mutations can facilitate the identification of therapeutic targets, especially if the mutations suggest or are involved in specific pathways in the progression of cancer or other diseases. Similarly, sequencing can allow for the identification of ecDNA regions that cannot be used as ecDNA-specific probes because they are unlikely to identify genomic or chromosomal DNA.

[0016] This document also discloses compositions and methods that facilitate ecDNA enrichment or isolation. Some of these compositions and methods include ecDNA-specific probes, which can be used in many of the methods disclosed herein, such as quantification of ecDNA in cells isolated from an individual, such as isolating or independently identifying an individual from whom ecDNA is derived, such as information obtained by PCR amplification from ecDNA sequences isolated from the same or previous individuals. The probes can be used, for example, in in situ cellular assays such as FISH assays to visualize the presence, localization, or abundance of ecDNA in cells taken from an individual. Such assays can be used, for example, after treatment selection or monitoring of treatment efficacy. Similarly, ecDNA probes can be used to facilitate cell sorting, such as FACS cell sorting or emulsion-based cell sorting, to isolate a subpopulation of cells carrying ecDNA from a cell population in a sample taken from an individual. Exemplary samples include tumor samples, senescent or other affected samples, healthy samples, or circulating cell samples, etc. Further analysis can be performed on the sorted cell populations, such as one or more of genomic or transcriptomic sequencing, proteomic analysis, or cell culture.

[0017] Optionally or in combination, ecDNA can be labeled as aggregates and used to probe cells as described above. Such a method can convey the benefit of probing all ecDNA in a cell rather than a specific probe target. Native chromatin, if detected, is typically easily identified by its location in a cell image, such as visually or cytologically, and can be isolated from ecDNA analysis, or can be measured or evaluated to determine the chromosomal origin of the ecDNA.

[0018] Furthermore, in some cases, ecDNA-specific sequences can be used for probe design to facilitate CRISPR-mediated ecDNA isolation using an endonuclease-death complex, degradation using an active endonuclease, or cell death using a CRISPR-mediated RNase. Such compositions can be administered, for example, as a custom therapeutic to an individual with isolated ecDNA, or can be used as a general therapeutic, for example, to independently identify individuals carrying previously characterized ecDNA. ecDNAcrisper therapeutics or other sequence-specific therapeutics such as TALON, zinc fingers, or other programmable sequence-specific therapeutics can be delivered, for example, using ecDNA-expressing probes as carriers to guide oligonucleotides and CRISPR endonucleases or other enzymes, or by other methods known in the art or practice. 4. Brief description of the attached diagram

[0019] For all accompanying figures, Roman numerals are used to indicate the passage of time: i), ii), iii), iv), etc. Unless otherwise specified, the figures are not drawn to scale.

[0020] Figure 1 Fluorescent images of cell spreads containing chromosomes (101) and ecDNA (102) on an unpatterned glass slide surface are shown.

[0021] Figure 2 An implementation scheme is shown to generate a linear physical map along the length of a long nucleic acid molecule by cleaving the molecule at known recognition sites to produce an ordered length pattern.

[0022] Figure 3 An implementation scheme for generating a linear physical map by attaching markers at known identification sites to produce ordered segment patterns is demonstrated.

[0023] Figure 4 An implementation scheme for generating linear physical maps by attaching markers along the molecular length in a manner that correlates the density of the markers with the potential AT / CG ratio is demonstrated.

[0024] Figure 5 Different non-limiting implementations of confined and unconfined channel types within fluid devices are shown.

[0025] Figure 6 Different non-limiting embodiments of patterned surface properties on a substrate are shown.

[0026] Figure 7 The presents a workflow for an implementation of generating purified ecDNA using a fluid device and / or generating data about ecDNA by probing the ecDNA on or within the device.

[0027] Figure 8 The implementation workflow for generating probes specific to ecDNA rather than cellular DNA is demonstrated.

[0028] Figure 9 An implementation scheme for selecting ecDNA-specific probe target sequences is demonstrated.

[0029] Figure 10 A workflow for producing purified ecDNA from at least one cell by passing the lysed contents of at least one cell through a filter is demonstrated.

[0030] Figure 11 Fluorescent images of ecDNA and chromosome samples derived from cells that have passed through a 5-micron filter are shown.

[0031] Figure 12 Fluorescent images of ecDNA and chromosome samples derived from cells that have passed through a 1.2-micron filter are shown.

[0032] Figure 13 Various non-limiting embodiments of hook-shaped or piercing objects for capturing ecDNA within or on a fluid device are shown.

[0033] Figure 14 A fluid apparatus for separating ecDNA from cellular DNA by selectively puncturing ecDNA is demonstrated.

[0034] Figure 15 An implementation scheme for a method of manufacturing a puncture object or hook for capturing ecDNA within or on a fluid device is shown.

[0035] Figure 16 A fluid apparatus for separating ecDNA from cellular DNA by selectively puncturing ecDNA is demonstrated.

[0036] Figure 17 A fluid apparatus for separating ecDNA from cellular DNA by selectively puncturing ecDNA is demonstrated.

[0037] Figure 18 A fluidic apparatus for separating ecDNA from cellular DNA is illustrated. Fluid features on the surface of the apparatus include pillars that allow for the physical separation of chromosomes and ecDNA derived from lysed cells.

[0038] Figure 19An implementation method is shown for physically isolating ecDNA from chromosomes derived from cells lysed on the surface of an open fluid device, wherein the ecDNA is at least partially separated from the chromosome by positioning the ecDNA in a fluid feature that is physically large enough to locate the ecDNA but too small to locate the chromosome.

[0039] Figure 20 A fluid apparatus for separating ecDNA from cellular DNA is shown. Fluid features on the surface of the apparatus include columns that allow for the physical separation of chromosomes and ecDNA (circled in the figure) derived from lysed cells.

[0040] Figure 21 A fluid apparatus for separating ecDNA (2101) from cellular DNA (2102) is shown. Fluid features on the surface of the fluid apparatus include columns that allow for the physical separation of chromosomes and ecDNA (circled in the figure) derived from lysed cells.

[0041] Figure 22 A fluidic apparatus for separating ecDNA (2201) from cellular DNA (2202) is shown. Fluidic features on the surface of the apparatus include channels that allow for the physical separation of chromosomes and ecDNA (circled in the figure) derived from lysed cells.

[0042] Figure 23 Scanning electron microscope images show an embodiment of a fluid feature for a fluid device. In this particular embodiment, the substrate is silicon, and the feature comprises pillars approximately 1 micrometer long, 250 nm wide, and 1 micrometer high.

[0043] Figure 24 An implementation scheme for isolating single cells containing cellular DNA and ecDNA within a confined area of ​​a fluid device is demonstrated.

[0044] Figure 25 A workflow for separating ecDNA and cellular DNA is demonstrated by binding a magnet to two types of DNA and then separating the two types of DNA in a fluid device based on the differential force generated on the DNA types.

[0045] Figure 26 Fluorescent images of a fluid apparatus for separating ecDNA from cellular DNA by selectively allowing smaller ecDNA to be localized along the edges or corners of a fluid feature are shown.

[0046] Figure 27A fluid apparatus is shown as an embodiment for separating ecDNA from cellular DNA by selectively allowing smaller ecDNA to be localized along the edges or corners of a fluid feature.

[0047] Figure 28 A fluid apparatus is shown as an embodiment for separating ecDNA from cellular DNA by selectively allowing smaller ecDNA molecules to be positioned within a pocket along the edges or corners of a fluid feature.

[0048] Figure 29 A fluid apparatus is shown for separating ecDNA from cellular DNA by selectively allowing smaller ecDNA to be localized along the edges or corners of a fluid feature, and then removing the edges or corners at a later point in time.

[0049] Figure 30 Fluorescent images of ecDNA and chromosome blades coated on the surface of an open fluid device are shown, in which the ecDNA is localized at different locations on the surface due to the fluid characteristics of the device surface.

[0050] Figure 31 Fluorescent images of ecDNA and chromosome blades coated on the surface of an open fluid device are shown, in which the ecDNA is localized at different locations on the surface due to the fluid characteristics of the device surface.

[0051] Figure 32 A fluid apparatus is shown for separating ecDNA from cellular DNA by selectively allowing smaller ecDNA to flow in the flow direction while larger cellular DNA moves away from ecDNA through a DLD array.

[0052] Figure 33 A fluid apparatus is shown for separating ecDNA from cellular DNA by selectively allowing smaller ecDNA to flow through a filter structure while preventing cellular DNA from flowing through.

[0053] Figure 34 A fluid apparatus is shown as an embodiment for separating ecDNA from cellular DNA by selectively localizing ecDNA in an entropy-limited fluid region.

[0054] Figure 35 Fluorescent images of ecDNA (3501) and cellular DNA (3502) treated with EdU and stained with Alexafluor 647 are shown.

[0055] Figure 36Fluorescent images of ecDNA (3602) and cellular DNA (3601) treated with EdU and stained with Dapi are shown.

[0056] Figure 37 Fluorescent images of the DLD fluid apparatus used to separate ecDNA from chromosomes during operation are shown.

[0057] Figure 38 Fluorescent images of the DLD fluid apparatus used to separate ecDNA from chromosomes during operation are shown.

[0058] Figure 39 Fluorescent images of the DLD fluid apparatus used to separate ecDNA from chromosomes during operation are shown.

[0059] Figure 40 Fluorescent images of two cell slices with different populations of ecDNA lysed on the surface of an open fluid device are shown.

[0060] Figure 41 Fluorescent images of a fluid apparatus are shown for separating ecDNA from cellular DNA by selectively allowing smaller ecDNA to flow through a fluid feature including a filter while the filter prevents larger chromosomes from passing through.

[0061] Figure 42 The qPCR data are shown, indicating purification of ecDNA relative to chromosomes based on a filter followed by surface-adsorption-based purification. The Y-axis represents the cycle count.

[0062] Figure 43 The data presented indicate the purification of ecDNA relative to chromosomes using a DLD fluid apparatus. The Y-axis represents the cycle count.

[0063] Figure 44 Fluorescent images of purified ecDNA are shown.

[0064] Figure 45 Fluorescence images of purified ecDNA with two color channels are shown, one color (4501) comes from the nucleic acid staining agent (DAPI), and the second color (4502) is associated with the bound FISH probe.

[0065] Figure 46 Fluorescent images of ecDNA and chromosomes deposited on a glass surface are shown.

[0066] Figure 47Fluorescence images of a fluid apparatus used to separate ecDNA from cellular DNA by selectively capturing ecDNA (4704) of appropriate size to fit a single ecDNA in a depression are shown. In this figure, the depression is annotated with a white dashed box. 5. Definition

[0067] All publications, patents, patent applications, and information available on the Internet and mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, patent application, or information item is specifically and individually indicated to be incorporated by reference. Where any of the publications, patents, patent applications, and information items incorporated by reference contradict the disclosure contained in this specification, this specification is intended to substitute for and / or give precedence to any such contradictory material.

[0068] The terms used in this specification generally have their ordinary meaning in the art, in the context of this disclosure, and in the specific context in which each term is used. Some terms are discussed below or elsewhere in this specification to provide additional guidance to those skilled in the art in describing the apparatus and methods of this disclosure and how they are prepared and used. This should be understood in that manner. Therefore, alternative languages ​​and synonyms allow the same thing to be described in more than one language and synonym for any one or more terms discussed herein. Synonyms for certain terms are provided. However, the description of one or more synonyms does not preclude the use of other synonyms, and there is no particular significance in whether a term is set forth or discussed herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In case of conflict, the definitions included in this specification shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

[0069] This disclosure is also described by way of specific examples. However, such examples, including instances of any terms discussed herein, and their use anywhere in this specification, are illustrative only and do not in any way limit the scope and meaning of this disclosure or any exemplary terms. Similarly, this disclosure is not limited to any particular embodiment described herein. In fact, many modifications and variations to this disclosure will be apparent to those skilled in the art upon reading this specification and can be made without departing from the spirit and scope of this disclosure. Therefore, this disclosure is limited only by the terms of the appended claims and the full scope of their equivalents. As used herein, “about” or “approximately” in the context of numbers shall refer to a range spanning + / - 10% of that number, or in the context of ranges, to an extended range spanning 10% below the lower limit of the listed range to 10% above the upper limit of the listed range.

[0070] The term “or” as used in the claims is intended to mean “and / or” unless expressly indicated as referring only to substitutes or that the substitutes are mutually exclusive, although this disclosure supports the definitions of referring only to substitutes and “and / or”.

[0071] In the claims or description, when used with the word “comprising”, the words “a” and “an” mean one or more, unless otherwise specified.

[0072] Unless the context explicitly requires otherwise, throughout the specification and claims, the terms “comprise”, “comprising”, etc., shall be interpreted in an inclusive sense rather than an exclusive or exhaustive sense; that is, meaning “including but not limited to”. Terms using singular or plural numerals shall also include plural and singular numerals, respectively. Furthermore, when used in this application, the terms “this article,” “above,” “below,” and similar terms shall refer to the application as a whole, and not any specific part thereof.

[0073] The term "combination" is used to refer to selecting items from a set such that the order of selection is irrelevant, and that the selection of the empty set (nothing) is also a valid selection when explicitly stated. For example, unique combinations of selections that include the empty set of set {A, B} are: empty set, A, B, A, and B.

[0074] 5.1. Sample As used herein, the term "sample" generally refers to a biological sample of a subject that contains at least partially nucleic acids derived from said subject. A biological sample can contain any number of macromolecules, such as long cellular nucleic acid molecules. A sample can be a cell sample. A sample can be a cell line or cell culture sample. A sample can be a CTC (circulating tumor cells) or CFC (circulating fetal cells) sample. A sample can contain one or more cells. A sample can be one or more droplets containing biological material. A sample can contain one or more microorganisms. A biological sample can be a nucleic acid sample. A biological sample can be derived from another sample. A sample can be a tissue sample, such as a biopsy, core biopsy, needle aspiration, or fine needle aspiration. A sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. A sample can be a skin sample. A sample can be a buccal swab. A sample can be a plasma or serum sample. A sample can be a cell-free sample or a cell-free sample. Cell-free samples may include extracellular polynucleotides. Extracellular polynucleotides can be isolated from body samples, which can be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, and tears.

[0075] 5.2. Nucleic Acids The terms “nucleic acid,” “nucleic acid molecule,” “oligonucleotide,” “polynucleotide,” “nucleic acid polymer,” “nucleic acid fragment,” and “polymer” are used interchangeably and refer to a polymeric form of nucleotides of any length, specifically deoxyribonucleotides or ribonucleotides, or analogs thereof, including naturally occurring and synthetic ones. These terms cover, for example, DNA, RNA, and their modified forms. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, lncRNA (long non-coding RNA), lincRNA (long intergenic non-coding RNA), ribozymes, cDNA, ecDNA (exochromosomal DNA), artificial mini-chromosomes, cfDNA (circulating cell-free DNA), ctDNA (circulating tumor DNA), cffDNA (circulating fetal DNA), recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence and conformation, including circulating RNA, nucleic acid probes, and primers.

[0076] Unless otherwise specified, nucleic acid molecules can be single-stranded, double-stranded, or a mixture thereof. For example, hairpin bends or loops may be present. Unless otherwise specified, nucleic acid molecules may contain nicks.

[0077] 5.3. Long nucleic acid molecules Unless otherwise specified, a "long nucleic acid fragment" or "long nucleic acid molecule" is a double-stranded nucleic acid with a length of at least 100 bp, and is therefore a macromolecule that can span the entire chromosome. It can originate from any artificial or natural source, including single cells, cell populations, droplets, amplification processes, etc. It can include nucleic acids with additional structures (such as structural proteins histones), and thus include chromatin. It can include nucleic acids with additional bodies (e.g., markers, DNA-binding proteins, RNA) that bind to them.

[0078] 5.4. ecDNA “ecDNA” – extrachromosomal deoxyribonucleic acid (in the context of this document, excluding the background section but including all other sections) – refers to any long nucleic acid molecule having any conformation, including but not limited to circular form, derived from a cell that is not a regular chromosome, yet whose genetic contents are derived from at least one chromosome (though not necessarily from the chromosome of the source cell). For example, ecDNA may originate from the chromosome of a first cell and then be absorbed into the nucleus of a second cell. ecDNA may originate from the remnants of apoptotic cells. ecDNA may include long nucleic acid molecules at least 1,000 bp, or at least 5,000 bp, or at least 10,000 bp, or at least 50,000 bp, or at least 100,000 bp, or at least 500,000 bp, or at least 1,000,000 bp, or at least 5,000,000 bp. ecDNA may include higher-order nucleic acid structures. ecDNA may include small polydisperse circular DNA (eccDNA). ecDNA may include microDNA. ecDNA can include small, polydisperse circular DNA (spcDNA). ecDNA can include telomere loops. ecDNA can include double minutes (DM). ecDNA can include episomes. ecDNA can include micronuclei. The topological structure of ecDNA can be linear or circular. In some embodiments where the ecDNA molecule is circular DNA, the circular DNA can be positively supercoiled or negatively supercoiled. In some embodiments, due to the amount of strain in the circular molecule, the circular ecDNA itself may not present as a circular structure when locally probed. Circular ecDNA can take the form of plectonemes or toroids, or a combination of both.

[0079] 5.5. ecDNA purity ecDNA purity: A statistical measure of the fraction of all DNA in a sample derived from ecDNA. In some embodiments, purity can be assessed from sequencing reads using random primers and one or more accompanying contig maps describing the range of one or more ecDNAs being analyzed. In some embodiments, purity can be assessed by high-resolution visual or electron microscopy visualization of the contents, by screening for the circular shape of ecDNA absorbed onto a surface. In some embodiments, purity can be assessed by mass spectrometry, or gel, or fluid dynamics. In some embodiments, the fraction of all DNA refers to the proportion of total DNA mass in the sample, or the proportion of total DNA mass in the sample that does not include one or more specific mass ranges of DNA. In some embodiments, the fraction of all DNA refers to the proportion of total DNA bp in the sample, or the proportion of total DNA bp in the sample that does not include one or more specific bp length ranges of DNA. In some embodiments, the fraction of all DNA refers to the proportion of the amount of DNA in the sample, or the proportion of the amount of DNA in the sample that does not include one or more specific bp length ranges of DNA.

[0080] 5.6. Higher Nucleic Acid Structures "Higher-order nucleic acid structure" or "structure" or "higher-order structure" refers to any secondary, tertiary, or quaternary DNA structure, including any entity that binds to the nucleic acid molecule. Nucleic acid molecules can be linear or circular. Nucleic acids can have any of a variety of structural conformations, such as being single-stranded, double-stranded, triple-stranded, replication loops, or combinations thereof, and possess higher-order intramolecular or intermolecular secondary / tertiary / quaternary structures, such as chromosomal territories, chromosome boundaries, chromosome regions, compartments, topologically associated domains (TADs), chromatin loops binding to local direct regulatory factors, condensing protein-associated loops, adhesion protein-associated loops, guide nucleic acids, argonaut complexes, CRISPR-Cas9 complexes, nucleoprotein complexes, insulator complexes, enhancer-promoter complexes, ribonucleic acid (RNA), small interfering RNA (siRNA), microRNA (miRNA), and guide RNA. (gRNA), long non-coding RNA (lncRNA), repeat region binding proteins, telomere-modifying proteins, nucleic acid repair proteins, regulatory factor binding proteins, nucleic acid-binding proteins, proteins, histone deacetylases (HDACs), chromatin remodeling proteins, methyl-binding proteins, transcription factor transcription complexes, and bends in the genomic DNA polymer that bend cis or trans in a manner such as hairpins, replication loops, triple-stranded regions, etc. Nucleotides within nucleic acids can have any combination of epigenomic states, including but not limited to methylated or acetylated states. Nucleic acids can be derived from any source, artificial or natural, including single cells, cell populations, droplets, amplification processes, etc. In some embodiments, these structures include compounds and / or interactions between nucleic acids and proteins. In some embodiments, these structures include 2D and 3D conformations of nucleic acids outside of linear 1D polymer chains. These 2D and 3D conformations can be formed through interactions with proteins, other nucleic acid molecules, or external boundary conditions. Non-limiting examples of boundary conditions include microfluidic chambers or nanofluidic chambers, pores on or within a substrate or defined within a fluid device, droplets, and cores.Nucleic acids may include nucleic acids with additional structures, such as structural proteins, including but not limited to any regulatory binding site complexes, enhancer / transcription factor complexes and their interactions with nucleic acid molecules, SMC (chromosome structural maintenance) cohesin complexes, ATPase subunits (Smc1 and Smc3), non-SMC regulatory subunits (Rad21 / Scc1 / Mcd1 and SA1 / SA2 / Scc3), Sgo1, mitotic kinases (polo-like kinase 1 (Plk1) and aurora B), protein phosphatase 2A (PP2A), chromosome passenger complexes (CPC), topo II decatenation, condensates, CTCF proteins, PDS5 proteins, WAPL proteins, condensate I, condensate II, CAP-G, histones and their derivative complexes, and therefore chromatin. In some embodiments, higher-order structures may comprise exogenous nucleic acid genome integration complexes, particularly exogenous nucleic acid genome integration complexes comprising viral genome integration complexes or recombinant nucleic acid genome integration complexes. In some embodiments, higher-order structures may comprise extrachromosomal episome physical docking complexes, particularly wherein such complexes carry chromosomes via binding sites. In some implementations, the higher-order nucleic acid structure comprises extrachromosomal nucleic acids derived from the host chromosome. All of the above (without limitation) can be targets for labeling, physical or conformational biomarkers indicating the presence of certain states of genomic organization or transitions between states (which may be associated with pathogenic genomic outcomes).

[0081] Specifically, higher-level nucleic acid structures can refer to individual or aggregated genomic organizations at various levels within the cell nucleus [Jerkovic, 2021], [Kempfer, 2020], or subsets thereof. Such genomic organization begins with linear primary DNA coiled around histones to form nucleosomes, which are then organized into clusters, each containing ~1-2 kb of DNA. These nucleosome clusters form ~100 kb chromatin nanodomains (CNDs), where most enhancer-promoter (EP) contacts occur. At a ~1 Mb scale, CNDs and CCCTC-binding factor (CTCF)-cohesin-dependent chromatin loops form topologically associated domains (TADs) and loop domains. At even higher scales, up to hundreds of megabases, chromatin segregates into gene-active and gene-inactive compartments (A and B, respectively) and compartment-specific contact centers, forming sister chromatid axes. At the highest topological level, the cell nucleus is organized into chromosomal regions.

[0082] 5.7. Hybridization As used herein, the terms “hybridization,” “hybridizing,” “hybridize,” “annealing,” and “anneal” are used interchangeably when referring to the pairing of complementary or substantially complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of association between nucleic acids) are influenced by factors such as the degree of complementarity between nucleic acids, the stringency of the conditions involved, the Tm (melting temperature) of the resulting hybrid, and environmental conditions (e.g., temperature and pH). The “hybridization” method involves the annealing of one nucleic acid with another, a complementary nucleic acid, i.e., nucleic acids having complementary nucleotide sequences.

[0083] Pairing can be achieved through any process in which nucleic acid sequences join with substantially complementary or fully complementary sequences by base pairing to form a hybridization complex. For hybridization purposes, two nucleic acid sequences are considered "substantially complementary" if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary to each other.

[0084] In the context of this document, in the case of hybridization between nucleic acid strands and double-stranded nucleic acid molecules, it should be understood that such hybridization is carried out under conditions of partial or complete denaturation of the double-stranded nucleic acid molecules, unless otherwise specifically stated.

[0085] 5.8. (DNA) probe As used herein, a “probe” is a molecule or set of molecules capable of binding to a specific region of a target nucleic acid, including DNA, RNA, and higher-order compositions including chromatin, RNA-associated chromatin, and ribonucleic acid particles (RNPs). For clarity, this use of the term “probe” is separate from and distinct from descriptions of physical hardware (such as “contact probes” as defined elsewhere in this document). Non-limiting examples of probes include oligonucleotides, peptide nucleic acids, locked nucleic acids, proteins that recognize sequences (including methyltransferases and catalytically inactive restriction enzymes), mixtures of proteins that recognize sequences together, proteins and mixtures of proteins that recognize specific regions together with guiding nucleic acids (including CRISPR proteins), single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, polymers including pyrrole-imidazolium polyamides, single-stranded DNA or RNA with self-complementary sequences (such as those forming hairpins), probe mixtures, and probe mixtures that themselves avoid regions such that they do not have strict Watson-Crick complementarity or, more generally, purine / pyrimidine complementarity when aligned.

[0086] "FISH probes" (fluorescence in situ hybridization) are probes that include fluorescent moieties or affinity tags, reaction sites, docking strands, or other means of attaching fluorophores to the probe after deposition.

[0087] A “capture probe” is a probe that contains a sequence recognition portion and a reactive group or affinity tag portion capable of selectively isolating the capture probe from a solution or mixture. Non-limiting examples include libraries of 200-400 bp single-stranded DNA molecules having a biotin-16-dUTP nucleotide at the 5' end, derived from whole-genome amplification methods applied to purified chromosomes.

[0088] The term "hybridization" of a probe describes the process of bringing a probe into contact with one or more targets.

[0089] In some cases, the probe lacks any marker and acts as a competitive binder to alter the binding mode of another probe or in subsequent chemical or enzymatic reactions. In some cases, the probe contains a marker (as defined below) and a sequence recognizer. In some cases, the distinction between the marker and the sequence recognizer is imprecise, such as in the synthesis of DNA oligonucleotides by labeling with radioactive phosphorus or with fluorescent oligonucleotides such as Cy5-dUTP. In some cases, the probe has more than one marker, including interacting markers, such as hairpin probes having both a fluorophore and a quencher that interact differently when the probe binds to the target.

[0090] In some cases, probes have a well-defined and fully understood composition, such as DNA oligonucleotides that are designed and artificially synthesized using phosphoramide chemistry. In other cases, probes are naturally derived products with unknown or partially known compositions, including but not limited to DNA isolated from cells, DNA isolated from more than one cell, or DNA isolated from one or more cells and separated into containers based on physical properties or by random sorting and grading. In still other cases, probes are derived from copies or amplifications of such natural products, whether by perfect copies, error-prone copies, partial copies, or copies in the presence of additional nucleic acids.

[0091] In some cases, the probe contains a secondary sequence that can be detected itself. Non-limiting examples include probes that are amplified by branching signal amplification, FISH probes that contain a universal terminal sequence as a secondary sequence (which is used as a docking strand in DNA-PAINT super-resolution microscopy probing of FISH probes), and probes that contain a secondary sequence that can be amplified by in situ isothermal polymerization or PCR reaction specific to that secondary sequence.

[0092] Specificity is the ratio of the probe occupying the target region relative to other regions present in the target, and specificity varies with the probe's chemical composition and the chemical, biochemical, thermal, and environmental conditions that allow the probe to bind to the target. Epigenetic specificity can be modulated by pre-blocking the target, such as in the case where Cot1 DNA and / or DNA from heterologous organisms, such as salmon sperm DNA, are used to pre-block repetitive regions of the human genome before applying the actual probe. Target accessibility can be modulated by: washing, processing, enzymatic treatment, and extraction prior to probe application; non-limiting examples include washing with SSC buffer, formamide, hydration of over-dried samples, dehydration of over-wetted samples using an alcohol gradient, RNase digestion to improve probe accessibility to chromatin, protease digestion to remove unwanted proteins from the localized region of detection, treatment with a competitive binding agent to remove chromatin, competitive binding agents including PEI-g-PEG, heat treatment of the sample including baking at 90°C for 10 minutes, fixation including treatment with 10% neutral buffered formalin, and, if applicable, removal of the matrix from the mounting medium, such as FFPE-embedded samples. Samples can be cleaned to remove unwanted targets that may also bind to the probe. Non-limiting examples include RNaseic digestion of chromatin to remove RNA that itself may bind to the probe and generate a signal, and mild nuclease treatment to remove loose interphase DNA. Epigenetic specificity can also arise from competition between the probe and decoy probes that bind to similar sequences or regions with comparable or poor affinity. Background of probe binding can be minimized by using a blocking reagent during the assay. Non-limiting examples of such blocking reagents include nonionic detergents, IGEPA, proteins, BSA, serum, fetal bovine serum, and blocking reagents intended for immunostaining.

[0093] When the target is a double-stranded nucleic acid including dsDNA and the desired binding mode is a double-stranded hybrid of the probe and the target single strand, preferably when the target is one or more condensed portions of chromatin, the probe is enhanced by denaturing the target strand so that the probe encounters a larger portion of the single-stranded nucleic acid upon binding.

[0094] Denaturation can be performed before or simultaneously with probe application, and the basis includes conditioning of the temperature and chemical environment, preferably by immersion in a 70% formamide / 2× SSC solution at 72°C for about 2 minutes, followed immediately by a series of ice-cold alcohol dehydrations to preserve the denatured state, and then air drying. Rapid cooling enhances the degree of single-chain target availability of the probe. The specificity and total amount of the probe can be affected by the annealing rate experienced during the removal of chemical, thermal, and environmental denaturing conditions.

[0095] In some cases, the probe comprises a natural or synthetic product or a mixture of products that is determined by hybridization with one or more spatially defined target groups. A non-limiting example is the array comparative genomic hybridization (aCGH) method, in which a fluorescently labeled sample or a fluorescently labeled amplified product of a sample becomes a probe that hybridizes with an array of oligonucleotides printed on a surface in a predetermined pattern. In some cases, a mixture of probes can be used to generate a proportional readout of probe binding.

[0096] In some cases, the probe may contain at least one barcode, or at least one marker, or at least one primer, or at least a portion of a single-stranded nucleic acid, or at least a portion of a double-stranded nucleic acid, or at least one nucleic acid loop or vesicle, or at least one higher-order nucleic acid structure, or at least one cleavable adapter. In some cases, the probe may bind to an array, or beads, or dendritic polymer, or nanospheres, or quantum dots, or polymer chains, or DNA origami, or a fluid device.

[0097] 5.9. Marker As used herein, a “marker” is a physical entity or body that can bind to a nucleic acid molecule, directly or indirectly, and can be used to generate a signal that can be detected by a probe, unlike (or lacking) the detection signal produced by the nucleic acid without the entity. A marker can be a fluorescent intercalating dye, which, when bound to a nucleic acid, can be used in a fluorescence imaging system to identify the presence of the nucleic acid. In another example, a marker can be a compound that specifically binds to methylated nucleotides and provides a current-blocking signal when transported through a nanopore, thereby reporting a signal about the methylation state of the molecule. In another example, a fluorescent probe specifically hybridizes to a nucleic acid sequence, thereby providing confirmation of the presence of that sequence on the nucleic acid using a fluorescence imaging system. In yet another example, a fluorescent probe specifically binds to a specific protein (e.g., a DNA-binding protein) that binds to long nucleic acid molecules. In some cases, the absence of a marker is itself a signal. In some cases, the signal associated with a marker is a weakening, blocking, substitution, quenching, or modification of a signal from another marker. Non-limiting examples include: binding a dark label to a nucleic acid to displace an existing bound fluorophore; binding a dark label to the nucleic acid to block the binding of a fluorescent label; quenching a neighboring fluorescent label bound to the nucleic acid; and reacting directly or indirectly with the fluorescent label bonded to the nucleic acid to reduce its fluorescence. In some cases, the label is not physically attached to the nucleic acid molecule during probing. For example, the label can be attached to the nucleic acid molecule via a cleavable adapter. At a desired time, the adapter cleaves, releasing the labeled molecule, which is then detected by probing.

[0098] In some cases, the marker comprises a multi-step labeling system, preferably amplified in one or more steps. Non-limiting examples include immunohistochemical detection using a primary antibody targeting the site of interest and a secondary antibody targeting the primary antibody and attached to another marker.

[0099] In some cases, the marker is capable of chemical reactions, including but not limited to photosensitizers, preferably Rose Bengal, free radical generators, strong photoacids, chemical crosslinking agents, parts capable of click chemistry, strain-promoted alkyne-azide click chemistry (SPAAC) reactions, including markers having a dibenzocyclooctylene moiety, but preferably CuAAC reactions, initiator sites for free radical polymerization, most preferably initiator sites for controlled free radical polymerization, and most preferably 2-bromoisobutyrate groups for ATRP. In some cases, labeling includes two steps: a first step of depositing the label and optionally washing off excess label; and a second step of combining additional chemical reactants with the label to produce additional labels, including but not limited to covalent or non-covalent attachment of light-absorbing, light-emitting, or fluorescent molecules, generating light-absorbing, light-emitting, or fluorescent molecules from a precursor, growing a polymer matrix by growing a polymer from a directly attached label, final attachment of molecules with or without prior attachment to the surface of the fluid device, attaching a pre-synthesized polymer or matrix to a directly attached label, growth of the polymer matrix in the local vicinity of the label (wherein such growth is mediated by chemical diffusion in the local environment of the label), and continued growth of the polymer matrix in the local vicinity of the label (wherein such growth is mediated by chemical diffusion in the local environment of the label), and initial portions of the polymer matrix being attached to the fluid surface prior to deposition.

[0100] In some cases, the marker is an enzyme capable of performing a chemical reaction, including but not limited to alkaline phosphatase, horseradish peroxidase, β-galactosidase, an enzyme capable of producing a light reaction, including but not limited to bioluminescent protein luciferase, firefly luciferase, click beetle luciferase, kidney luciferase, NanoLuc, Gaussia luciferase, an enzyme capable of removing oxygen, preferably glucose oxidase, and polymerizable enzymes including oxidoreductases, peroxidases, peroxidases capable of polymerizing vinyl monomers, laccases, transferases, glycosyltransferases, phosphorylases, glycosyltransferases, acyltransferases, hydrolases, glycosidases, lipases, lipases capable of ring-opening polymerization of cyclic monomers, nucleases, endonucleases, exonucleases, RNases, and proteases.

[0101] 5.10. Exploration "Probing" is the process of assessing the state of nucleic acids, long nucleic acid molecules, higher-order nucleic acid structures, nucleic acid-protein complexes, or other biomolecules using a probing system. In some implementations, the state of a nucleic acid is assessed by probing the state of at least one marker on the nucleic acid, either directly or indirectly, from a marker. Probing can be a binary assessment, such as the presence or absence of a marker. Probing can be quantitative, such as how many markers are on the molecule. Probing can be signal density or intensity along a line, region, or volume. Probing can be a physical count along the length of the molecule or the distance between markers.

[0102] In some implementations, the digital or computer simulation (in-silico) representation used to generate the physical map is explored.

[0103] In some implementations, the physical state of the structure being probed is used to assess the higher-order structure of nucleic acids. The physical state of the structure being probed can include the molecular topology, such as the presence of loop structures, a set of hierarchical loop structures, the number of supercoils present in the loops, or the degree of entanglement of one or more loops from the same or different molecules. The physical state of the structure being probed can include the accessibility of nucleic acid regions to binding partner bodies or cis- or trans-acting factors. The physical state of the structure being probed can include the presence of markers of partially replicated nucleic acids that are still very close together (such as Okazaki fragments) or newly synthesized nucleic acids (such as results from BrdU pulses). The physical state of the structure being probed can include the level of cohesin remnants on metaphase chromosomes that have been experimentally manipulated or affected by genetic abnormalities (e.g., by consuming cohesin itself or Wapl), resulting in chromatids exhibiting substantially different lengths and shapes, becoming quantitatively measurable biomarkers indicating certain pathological conditions (Losada et al. 2005; Gandhi et al. 2006; Shintomi and Hirano 2009). The physical state of the structure being investigated can include the amount, ratio, and distribution of condensation proteins I and II in these chromatids. The physical state of the structure being investigated can also include dynamic changes in genome organization, such as the release of adhesion proteins and dynamic changes in sister chromatid resolution.

[0104] In some implementations, the signal being probed can be fluorescent, photoluminescent, electromagnetic, electrical, magnetic, physical, chemical, exhibiting plasmon resonance, or an enhanced Raman signal through surface-enhanced plasmon resonance.

[0105] The signal being probed can be analog or digital in nature. For example, the signal can be an analog density spectrum of markers along the length of the nucleic acid, where the measured signal originates from more than one marker. In some implementations, the state of the nucleic acid is probed directly without a marker, such as by directly probing long nucleic acid molecules in cells using phase microscopy, or by directly probing nucleic acids by blocking nanopores with an electric current. Non-exhaustive examples of different detection methods that can be used individually or in combination for the detection system include fluorescence imaging, bright-field imaging, dark-field imaging, contrast imaging, epi-florescent imaging, total internal reflection fluorescence imaging, near-field / eliminated-field imaging, waveguides, zero-mode waveguides, plasma signal transfer, confocal imaging, scattering, light sheets, structured illumination, stimulated emission loss, super-resolution, random activation super-resolution, random binding super-resolution, nanopore sensing of multiphoton, current, voltage, power, capacitance, inductance, or reactive signals (through Coulomb blockage of the pore or tunneling effect across the pore), chemical sensing (e.g., through reactions), physical sensing (e.g., interaction with the sensing probe), SEM, TEM, STM, SPM, and AFM. Furthermore, combinations of different markers and detection methods are also feasible. For example, fluorescence imaging of an embedded dye on a nucleic acid while simultaneously translocating the nucleic acid through a nanopore and measuring the pore current.

[0106] 5.11. Exploration System As used herein, a “probing system” is an automated, semi-automated, or manual system for probing samples. In some embodiments, the sample is probed while it is inside or on a fluid device, and the probing system is connected to and controls the operation of the fluid device. In some embodiments, the probing system comprises multiple separate systems that can be coordinated together by a controller or user. Examples include instruments for loading the sample into the fluid device, instruments for flowing the sample within the fluid device, instruments for imaging the sample in the fluid device, and a controller for operating software for analyzing the imaging data. In some embodiments, the probing system comprises an integration of all systems or subsets of systems.

[0107] In some embodiments where the sample is confined within or on a fluid device, the operation of the probe system on the device may include: manipulating the physical position and conformation of a package or long nucleic acid molecule by applying external forces to the entity; exposing the package or long nucleic acid molecule to environmental conditions or reagents for a period of time; optically probing the static or dynamic configuration of the package or long nucleic acid molecule to facilitate analysis of its composition, or as part of a feedback system to control the operation of the device; and retrieving the desired package or long nucleic acid molecule from the device. The fluid device and the probe system can be connected in a variety of ways. A non-exhaustive list includes: fluid ports (both open and sealed), electrical terminals, optical windows, mechanical pads, heating tubes or radiators, inductive coils, fluid dispensers, and surface scanning probes. The non-exhaustive list of potential functions that the probe system can perform on the device includes: temperature monitoring, applying heat, removing heat, applying pressure or vacuum to the port, measuring vacuum, measuring pressure, applying voltage, measuring voltage, applying current, measuring current, applying electrical power, measuring electrical power, exposing the device to focused and / or unfocused electromagnetic waves in far-field or near-field environments, collecting electromagnetic light generated or reflected from the device, generating and measuring temperature, electromagnetic force, surface energy, or chemical concentration difference or gradient, dispensing liquid into or onto the device orifice or port, or onto the device surface, and bringing the device surface or an entity on the device surface into contact with a contact probe (e.g., an AFM tip).

[0108] In some implementations, the confirmation of the presence of long nucleic acid molecules in a region of the fluid device and the control of their physical location within the device are controlled by a detection system using a feedback controller system. Detection of the long nucleic acid molecule is performed by detecting at least one probed signal. In a preferred embodiment, the signal is an electromagnetic signal derived from a marker bound to the long nucleic acid molecule. In one implementation, the control instrument feedback control system utilizes at least in part the identification of an intramolecular physical map of the long nucleic acid molecule or the absence of an intramolecular physical map as input information.

[0109] In some implementations, the exploration system includes a local computing processing module within the system, an adjacent computing processing module connected via direct communication, an external computing processing module connected via a network, or a combination thereof. Various examples of computing processing modules include: PCs, microcontrollers, application-specific integrated microchips (ASICs), field-programmable gate arrays (FPGAs), CPUs, GPUs, system-on-a-chip (SoCs), network servers, cloud computing services, or combinations thereof.

[0110] The probing system may include at least one fluid dispensing tip capable of dispensing fluid droplets at desired x, y, z coordinates on the surface of the device, and in some embodiments, extracting the fluid droplets at desired x, y, z coordinates on the surface of the fluid device. Fluid dispensing and extraction may be performed in volumes of microliters, nanoliters, picoliters, feliliters, or attoliters.

[0111] The detection system can illuminate more than one light source simultaneously or sequentially, and can image more than one color simultaneously or sequentially. If imaging of more than one color is performed simultaneously, this can be done on different cameras, on different regions of a single camera but a sensor array, or on the same sensor of the same camera. In some embodiments, the wavelength of the light irradiated by the control instrument is selected to interact in a certain way with the sample, sample label, or functionalized surface. Non-limiting examples include: photolysis of nucleic acids, photolysis of photolyzable linkers, manipulation of optical tweezers, activation of photoactivated reactions, deprotection of photoinstantaneous protecting groups, and IR heating.

[0112] 5.12. Sequence The term “sequence” or “nucleic acid sequence” or “oligonucleotide sequence” refers to a continuous string of nucleotide bases, and in a specific context, it also refers to the specific positions of the nucleotide bases relative to each other when they appear in an oligonucleotide, and the information conveyed therefrom.

[0113] Sequencing can be performed using a variety of currently available systems, including but not limited to those from Illumina, Pacific Biosciences, Oxford Nanopore Life Technologies (Ion Torrent), BGI, GenapSys, Element Biosciences, Singular Genomics Systems, and Ultima Genomics. Sequencing can also be performed using a variety of currently available technologies, including but not limited to: Sanger sequencing, nanopore sequencing, nanogap sequencing, ligation sequencing, combinatorial probe-anchored sequencing, synthetic sequencing, pyrosequencing, polopy sequencing, DNA nanosphere sequencing, tunneling current sequencing, hybridization sequencing, mass spectrometry sequencing, array-based sequencing, and RNA polymerase-based sequencing.

[0114] 5.13. Reference Object A “genomic reference” or “reference” is any genomic dataset that can be compared or aligned with another genomic dataset. It can be in any data format, including but not limited to sequence data, karyotype analysis data, methylation data, genomic functional element data such as cis-regulatory element (CRE) maps, primary structure variation maps, higher-order nucleic acid structure data, physical mapping data, genetic mapping data, optical mapping data, raw data, processed data, analog data, and signal spectra, including electron or fluorescence-generated signal spectra. A genomic reference may include more than one data format. A genomic reference may represent consistent content from more than one dataset, which may or may not originate from different data formats. A genomic reference may include all, a subset, or a representation of the genomic information of an organism or model. A reference may be representative of a portion of the genome. A reference may be representative of a portion of a chromosome. A reference may be representative of a gene or a portion thereof. A reference may be representative of a regulatory region or a portion thereof. A reference may be representative of a TAD, domain, region, or a portion thereof. A genomic reference may be an incomplete representation of the genomic information it represents.

[0115] Genome references can be derived from genomes indicating the absence of disease or disorder, or from genomes indicating disease or disorder. Furthermore, genome references (e.g., those longer than 100 bp, 1 kb, 100 kb, 10 Mb, or 1000 Mb) can be characterized in one or more aspects. Non-limiting examples include determining the presence (or absence) of specific features, haplotypes, genetic variations, structural variations, single nucleotide polymorphisms (SNPs), and combinations thereof, not only in the overall genome reference but also in specific regions of the genome reference, as defined by neighboring genomic contents. Additionally, any suitable type and number of features of the genome reference can be used to characterize sample nucleic acids as originating from (or not originating from) nucleic acids indicating disorder or disease based on whether the sample nucleic acids exhibit similar characteristics to the reference.

[0116] In some cases, the genomic reference is a physical map. This can be generated in any number of ways, including but not limited to: raw single-molecule data, processed single-molecule data, a digital or computer-simulated representation of a physical map generated from sequences or simulations, a digital or computer-simulated representation of a physical map generated by assembling and / or averaging more than one single-molecule physical map, or a combination thereof. For example, a simulated computer physical map based on a known or partially known sequence can be generated based on the method used to generate the physical map. In embodiments where the physical map includes markers at known sequences, a set of discrete, ordered segments of base pair length can be generated. In one embodiment, the physical map includes a continuous simulated signal along the sequence length based on simulated local hydrogen bond dissociation kinetics between double helices in terms of base pairs, based on nucleotide sequence and predicted functional element data maps in terms of chemical moiety modifications, regulatory factor associations, or structural folding patterns.

[0117] In some cases, genomic references are obtained from data derived from microarrays (e.g., DNA microarrays, MMChips, protein microarrays, peptide microarrays, tissue microarrays, etc.), karyotype analysis, or FISH analysis. In other cases, genomic references are obtained from proximity 3D mapping techniques or 3D physical mapping techniques.

[0118] In some cases, characterization by comparison or alignment with a genomic reference can be performed with the aid of a programmed computer processor. In some cases, such a programmed computer processor may be included in a computer control system.

[0119] 5.14. Physical Mapping The “physical mapping” or “mapping” of nucleic acids encompasses various methods for extracting genomic, epigenomic, functional, or structural information from physical fragments of long nucleic acid molecules, where the extracted information can be correlated with physical coordinates on the molecule. As a general rule, the resolution of the obtained information is lower than that of the actual potential sequence information, but these two types of information are spatially correlated (or anticorrelated) within the molecule, and therefore, the former typically provides a ‘map’ of the sequence content along the physical location of the nucleic acid. In some embodiments, the relationship between the map and the potential sequence is direct; for example, the map represents the density of AG content along the length of the molecule or the frequency of a particular recognized sequence. In some embodiments, the relationship between the map and the potential sequence is indirect; for example, the map represents the density of nucleic acids packaged together with proteins into a structure, which then varies at least partially with the potential sequence. In some embodiments, the physical map is a linear physical map, where the extracted information can be allocated along the length of an axis, for example, the AT / CG ratio along the main axis of a long nucleic acid molecule. In some embodiments, a “linear physical map” or “1D physical map” is generated by probing for markers bound to elongated portions along the main axis of a long nucleic acid molecule. To clarify, a string occupying 3D space in a coiled state can be represented as a straight line, and therefore the values ​​extracted along the 3D coil can be represented as binning values ​​along the 1D representation of the string, thus forming a linear physical map. In some embodiments, the physical map is a "2D physical map," where the extracted information can be distributed within a plane containing the molecule, e.g., karyotype analysis. In some embodiments, the physical map is a "3D physical map," where the extracted information can be distributed within a 3D volume occupied by the molecule. For example, super-resolution techniques are used to tag to identify the location of the tag within the chromosome in (x,y,z) space, as illustrated by OligoFISSEQ [Nguyen, 2020] or in situ genome sequencing [Payne, 2020].

[0120] In some embodiments, the physical map includes a physical pattern of higher-order nucleic acid structures within the long nucleic acid molecule. In some embodiments, the physical map includes the location of intramolecular TADs. In some embodiments, the physical map includes the location of intramolecular histones. In some embodiments, the physical map includes the location of intramolecular loops. In some embodiments, the physical map includes the location of intramolecular knots. In some embodiments, the physical map includes the location of intramolecular binding factors. In some embodiments, the physical map of the long nucleic acid molecule includes multiple physical map types merged into a single physical map. For example, a long nucleic acid molecule having a fluorescence physical map associated with local AT density along the molecular length and a second physical map indicating the location of loops along the molecular length. The first and most widely used form of physical mapping is karyotype analysis, in which metaphase chromosomes are treated with staining methods that preferentially bind to AT or CG regions, thereby producing “bands” associated with the potential sequence of the nucleic acid and the structure and epigenomic pattern of the nucleic acid [Moore, 2001]. However, due to the condensed nature of the imaged nucleic acids, such methods have relatively low resolution for nucleotide sequences, approximately 5–10 Mbp. More recent methods for linearly mapping elongated interphase genomic DNA involve imaging nucleic acids digested at known restriction sites [Schwartz, 1988, 6, 147, 198] (e.g., see...). Figure 2 Imaging of fluorescent probes attached to incision sites [Xiao, 2007] (e.g., see...) Figure 3 Imaging the fluorescence characteristics of methylation patterns in nucleic acid molecules [Sharim, 2019], imaging the fluorescence characteristics of histones in chromatin [Riehn, 2011], electrodetecting probes bound to nucleic acids using sensors [Rose, 2013, 2014 / 0272954], and electrodetecting methylation characteristics on nucleic acids using nanopore sensors [Rand, 2017].

[0121] Another linear physical mapping method is to measure the AT / CG relative density or local melting temperature along the length of the elongated nucleic acid molecule (e.g., see...). Figure 4Such signals can be used for comparison with other similar maps, or with maps generated from sequence data by a computer. There are many ways to generate such signals. For example, the signal can be inherently fluorescent or electrical. Nucleic acids can be uniformly stained with an intercalating dye and then partially unwound, resulting in a relative loss of dye in AT-rich regions [Tegenfeldt, 2009, 10, 434, 512]. Another approach is to expose the double-stranded nucleic acid to two different species that compete for binding. One species is non-fluorescent and preferentially binds to AT-rich regions, while the other is fluorescent and does not exhibit this bias [Nilsson, 2014]. Yet another approach is to use two different colored dyes that differentially label AT and CG regions.

[0122] Using such non-agglomerated interphase nucleic acid polymer chain mapping improves the resolution of primary sequence information; however, the maps remove any native structural folds or binding support protein information and are typically extracted from bulk solutions of samples with many potentially heterogeneous cells. Recently, 3D physical mapping has been demonstrated, in which tags attached to chromosomes at specific locations, directly or indirectly, are probed to determine their relative positions within the chromosome in 3D space (see [Jerkovic, 2021] for a review of various methods). These methods can include super-resolution microscopy methods such as SIM, SMLM, and STED, Oligopaint FISH, multiplexed oligopaint FISH, and OligoFISSEQ. Additionally, in situ sequencing methods such as OligoFISSEQ [Nguyen, 2020] are also included. Note that in this document, “3D physical mapping” differs from “proximity 3D mapping” as defined elsewhere in this document.

[0123] Figure 2 , Figure 3 and Figure 4 Several different implementation schemes for generating and probing linear physical maps of long nucleic acid molecules are demonstrated. Figure 2In this method, the physical map of a long nucleic acid molecule 104 is generated by cleaving the molecule at specific sequence sites (e.g., recognition sites for restriction endonucleases) to create gaps 205 at which cleavage events occur. Along the length of the molecule, a dye is nonspecifically attached (e.g., using an intercalating dye) so that daughter molecules from the source parent molecule can be probed to generate a signal 201 along the physical length (206) of the parent molecule. The signal can then be used to determine the length and order {203-x} of individual daughter molecules, thereby generating the physical map of the parent molecule. In most embodiments of this method, the parent molecule is combed onto a surface and then cleaved to maintain the physical proximity and relative order of the daughter molecules. However, such embodiments can also be implemented in an at least partially elongated state within an elongated channel of a confined fluid device, allowing the order of daughter molecules to be probed [Ramsey, 2015, 10, 106, 848]. In some embodiments, a mixture of different cleavage sites can be used simultaneously. Figure 3 In this process, the physical map of the long nucleic acid molecule 314 is generated by sparsely binding markers 115 along the molecule's length, where the binding sites are associated (or inversely associated) with a specific set of targets. In some methods, the markers bind directly to sequence motif targets. In some methods, the signal-generating markers bind indirectly, for example, by creating a sequence-specific nick and then incorporating nucleotides from the nick site, some of which may be capable of generating a signal. Probing the long nucleic acid molecule with the markers generates a signal 311 along the physical length of the molecule 316 from the markers 315. The set {313-x} of distances, lengths, and sequences between the signals then represents the molecule's physical map. In some embodiments, further information can also be generated by interpreting the relative amplitudes of the signals 312 from each marker site. When using fluorescence probing, different colored markers can be used to represent different specific sites.

[0124] exist Figure 4 In this method, the physical map of the long nucleic acid molecule 424 is generated by densely binding markers 425 along the molecule's length, such that the binding pattern is correlated (or inversely correlated) with the molecule's underlying physical sequence content. For example, relative AT / CG content, or relative melting temperature, or relative density of methylated CG. Due to the dense nature of the markers in this method, the physical map is not a collection of lengths and sequences, but rather a simulated signal 421 of intensity variation along the physical length of the molecule 426.

[0125] The detection method that generates physical spectra is usually fluorescence imaging; however, different implementations are also possible, including scanning probes along the length of combed molecules on a surface, or contraction devices that measure the Coulomb blocking current or tunneling current across the contraction as molecules shift through it.

[0126] Unless otherwise specifically stated, a physical spectrum refers to any of the previously mentioned methods, including combinations thereof. For example, a long nucleic acid molecule may have a physical spectrum generated from AT / TC density using fluorescent markers along the molecular length, and then also have a physical spectrum generated from methylation spectrum along the molecular length using the shrinkage device as the molecule is transported through the shrinkage device.

[0127] 5.15. Barcode As used herein, a “barcode” is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, or 35 nucleotides long) that encodes information. A barcode can be a continuous sequence or two or more non-contiguous subsequences. Barcodes can be used, for example, to identify molecules to which oligonucleotides are attached in partitions, beads, or bodies. In some embodiments, a bead-specific barcode is unique to a bead compared to barcodes in oligonucleotides attached to other beads. In another example, nucleic acids from each cell can be distinguished from those from other cells due to unique “cell barcodes.” Such partition-specific barcodes, cell barcodes, or bead barcodes can be generated using various methods. In some cases, partition-specific barcodes, cell barcodes, or particle barcodes are generated using separate and mixed (also known as separate and pooled) synthesis schemes, for example, as described in [Agresti, 2014, 2016 / 0060621]. In some embodiments, the oligonucleotides described herein may contain more than one type of barcode.

[0128] In some implementations, the information associated with the barcode may be the identification of a single microsome, a specific microsome, a class of microsomes, a subset of microsomes, specifically selected microsomes, randomly selected microsomes, or a group of microsomes, wherein the microsome can be a molecule, a higher-order nucleic acid structure, an organelle, a sample, or a subject. In some implementations, the information associated with the barcode may be a process, a time-stamp, a location, a relationship to another microsome and / or barcode, an experiment ID, a sample ID, or environmental conditions. In some implementations, more than one piece of information can be stored in the barcode using any encoding technology.

[0129] In some embodiments, the barcode is single-stranded. In some embodiments, the barcode is double-stranded. In some embodiments, the barcode has both single-stranded and double-stranded components. In some embodiments, the barcode at least partially comprises a 2D and / or 3D structure, such as a hairpin or DNA origami structure.

[0130] In some implementations, the information encoded in the barcode is handled using error checking and / or error correction techniques to ensure the accuracy of the stored information. For example, Hamming code is used. In cases where more than one piece of information is stored in the barcode, individual information segments are encoded separately using their corresponding nucleotides within the barcode. In other cases, nucleotides can be shared using an encoding scheme. In some cases, compression techniques can be used to reduce the number of nucleotides required.

[0131] In some implementations, the information encoded in the barcode includes unique identification of the molecule it is conjugated to. These types of barcodes are sometimes referred to as “unique molecular identifiers” or “UMIs.” In other instances, primers containing a “partition-specific barcode” unique to each partition and a “molecular barcode” unique to each molecule can be used. After barcoding, partitions can then be combined and optionally amplified while maintaining “virtual” partitions based on specific barcodes. Thus, for example, the presence or absence of target nucleic acids containing individual barcodes can be counted or tracked (e.g., by sequencing) without the need to maintain physical partitions.

[0132] The length of a barcode sequence determines how many unique barcodes can be distinguished. For example, a 1-nucleotide barcode can distinguish 4 or fewer different samples or molecules; a 4-nucleotide barcode can distinguish 256 or fewer samples; a 6-nucleotide barcode can distinguish 4096 or fewer different samples; and an 8-nucleotide barcode can index 65,536 or fewer different samples.

[0133] In some implementations, selection software is used to design or randomly generate barcode sequences. This selection software is used to select barcodes that: have no hairpins, or contain a uniform base composition (15%–30% A, T, G, and C), or have no homopolymers (the default allows for three identical nucleotides), or have no simple repetitive sequences, or have no low-complexity sequences, or are not identical to common vector or adaptor sequences. Furthermore, barcodes can be designed to be unique even with the presence of three mismatch sequencing errors. Barcodes are typically synthesized and / or aggregated (e.g., amplified) using inherently imprecise processes. Therefore, barcodes intended for consistency (e.g., cell barcodes, particle barcodes, or partition-specific barcodes common to all barcoded nucleic acids in a single partition, cell, or bead) may contain various N−1 deletions or other mutations from the prototype barcode sequence. Therefore, in some embodiments, a barcode referred to as an "identical" or "substantially identical" copy may include a barcode that differs due to one or more errors, such as errors in synthesis, polymerization, or purification, and thus may contain various N-1 deletions or other mutations from the prototype barcode sequence. However, such minor variations in a theoretically ideal barcode do not interfere with the methods, compositions, and kits described herein. Therefore, as used herein, the term "unique" in the context of particle barcodes, cell barcodes, partition-specific barcodes, or molecular barcodes covers various unintentional N-1 deletions and mutations from an ideal barcode sequence. In some cases, problems arising from the imprecise nature of barcode synthesis, polymerization, and / or amplification are overcome by oversampling the possible barcode sequences relative to the number of possible barcode sequences to be distinguished (e.g., at least about 2, 5, 10, or more possible barcode sequences) or by using error-correcting coding techniques. The use of barcode technology is well known in the art, see, for example, [Shiroguchi, 2012] and [Smith, 2010]. Other methods and compositions for using barcode technology include those described in [Agresti, 2014, 2016 / 0060621].

[0134] In some embodiments, at least a portion of the barcode may also serve as a primer binding site. In some embodiments, the primer binding site is used for PCR primers. In some embodiments, all barcodes forming a unique set of barcodes contain a globally identical primer binding site, such that a single primer sequence can be used to bind to all barcodes. In some embodiments, the primer will be a complementary sequence to the primer binding site. In other embodiments, the primer will be the same sequence as the primer binding site because the primer will bind to the previously amplified product of the original primer binding site. In some embodiments, combinations may be present.

[0135] In addition, in some implementations, at least a portion of the barcode can also be used as a primer.

[0136] 5.16. Combining As used herein, “binding,” “bound,” and “bind” generally refer to a covalent or non-covalent interaction between two entities (referred to herein as “binding partners,” such as substrate and enzyme or antibody and epitope). Any chemical binding between two or more entities is a bond, including but not limited to: covalent bonds, σ bonds, π bonds, ionic bonds, dipole bonds, metallic bonds, intermolecular bonds, hydrogen bonds, and van der Waals bonds. Since “binding” is a general term, the following are all examples of binding types: “hybridization,” hydrogen binding, minor groove binding, major groove binding, click binding, affinity binding, specific binding, and non-specific binding. Other examples include: transcription factor binding to nucleic acid and protein binding to nucleic acid.

[0137] 5.17. Specific binding As used herein, the terms “specific binding” and “non-specific binding” must be interpreted within the context in which these terms are used in the text. For example, a body may “specifically bind” to a nucleic acid molecule but does not have a significant preference or bias toward the potential sequence of said nucleic acid molecule at certain genome lengths and / or within certain genomic regions. Thus, in the context of molecular sequence, the body “non-specifically binds” to said nucleic acid molecule. When in the context of binding between physically dissimilar molecules, “specific binding” generally refers to the interaction between two binding couples that, under a specific set of conditions (e.g., physiological conditions), cause the couples to bind to each other but not at a significant or substantial level to other molecules that may be present in the environment (e.g., in a biological sample, in a tissue).

[0138] 5.18. Base As used herein, the term "substrate" is intended to refer to a solid or semi-solid support that can serve as the basis for defining a feature. Non-limiting examples of features include pores, fixed molecules, pillars, channels, and recesses. Features may be randomly positioned on the substrate or patterned. Substrates as provided herein can be modified to accommodate the attachment of biopolymers using a variety of methods well known to those skilled in the art. Exemplary types of substrate materials include glass, modified glass, functionalized glass, inorganic glass, silicon, silica, silicon nitride, quartz, fused silica, microspheres (including inert and / or magnetic particles), polysaccharides, nitrocellulose, hydrogels, films, membranes, plastics (including, for example, acrylic resins, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutene, polyurethane, Teflon™, cycloolefins, polyimide, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glass, fiber bundles, and polymers such as polystyrene, cycloolefin copolymers (COC), cycloolefin polymers (COP), polypropylene, polyethylene, polycarbonate, or combinations thereof.

[0139] Those skilled in the art will know or understand that the composition and geometry of the substrates provided herein can vary depending on the intended use and user preferences. Therefore, although planar substrates such as glass slides, chips, or wafers are frequently illustrated herein, those skilled in the art will understand, in light of the teachings and guidance provided herein, that a variety of other substrates illustrated herein or well known in the art can also be used in the methods and / or compositions described herein. Substrates may include more than one physically bonded substrate, for example, using any combination of bonding mechanisms, adhesives, thin films, or vacuum.

[0140] The substrate can include various combinations of coatings.

[0141] The substrate can have a patterned surface. Patterning can be added, subtracted, or a combination of both.

[0142] The substrate may include components of a microfluidic device or a flow cell.

[0143] The substrate can be a thin film, which can itself be in contact with another substrate.

[0144] The substrate can be any desired shape. For example, the substrate can typically be a thin, flat shape (e.g., square, rectangular, or elliptical). In some embodiments, the substrate structure has rounded corners (e.g., for increased safety or robustness). In some embodiments, the substrate structure has one or more cut-off corners (e.g., for use with slide clamps or cross stages). In some embodiments where the substrate structure is flat, the substrate structure can be any suitable type of support with a flat surface (e.g., a chip or slide, such as a microscope slide).

[0145] In some embodiments where the substrate is modified to include one or more features (including, but not limited to, pores, protrusions, ridges, features, or markings), the features may include physically altered sites. For example, a substrate modified with various features may include physical properties, including but not limited to physical configuration, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, surface energy altered sites, hydrophobic / hydrophilic altered sites, and / or electrostatic altered sites.

[0146] In some embodiments, the substrate includes one or more markings on its surface, for example, to provide guidance for relating spatial information to the characterization of interest. For example, the substrate may be marked with a line grid (e.g., to allow easy estimation of the size of an object observed at magnification and / or to provide a reference area for counting objects). In some embodiments, reference markings may be included on the substrate. Such markings can be fabricated using techniques including, but not limited to, printing, etching, sandblasting, and deposition on the surface.

[0147] In some implementations, reference marks may be present on the substrate to provide the orientation of the sample using features on the substrate or the substrate itself.

[0148] 5.19. Functionalization A "functionalized surface" is a surface of a substrate that has been modified or engineered by certain chemicals or macromolecules to elicit certain desired properties. For example, it may bind specifically or non-specifically to macromolecules, or provide reagents.

[0149] 5.20. Fixed As used herein, when referring to a molecule, macromolecule, or body directly or indirectly attached to a substrate via one or more covalent or non-covalent bonds, the term "fixed" means in a state of rest by physical confinement, local energy minimization, local entropy minimization, or held at rest by external force. It may be indirectly attached to the substrate via at least one additional intermediate molecule or body. In some embodiments, covalent attachment may be used, but only if the molecule, macromolecule, or body remains colocated to the substrate under the conditions of its intended use. Non-limiting examples include the entire molecule, macromolecule, or body remaining stationary relative to the substrate, or a portion of the molecule, macromolecule, or body remaining stationary relative to the substrate while the remaining portion of the molecule, macromolecule, or body has limited degrees of freedom of movement, or the molecule, macromolecule, or body being indirectly attached to the substrate via an intermediate and the entire molecule, macromolecule, or body having some limited degrees of freedom of movement. For example, fixing an oligonucleotide to a substrate can occur by hybridizing the oligonucleotide with a second oligonucleotide, the second oligonucleotide containing at least partially a sequence complementary to the first oligonucleotide, and the second oligonucleotide itself being fixed to the substrate.

[0150] In some implementations, molecules, macromolecules, or volumes can be fixed to a surface through processes including physical adsorption, hydrogen bonding, van der Waals forces, or static friction.

[0151] In some embodiments, the molecule or macromolecule may include biomolecules, nucleic acid molecules, proteins, peptides, nucleotides, or any combination thereof. In some embodiments, the body may include cells, droplets, dendritic polymers, packaging, nucleosomes, beads, nanospheres, nanoparticles, nanodots, or quantum dots.

[0152] Some implementations may utilize already functionalized substrates, for example by applying a layer or coating of an intermediate containing reactive groups that allow covalent attachment to biomolecules such as polynucleotides.

[0153] 5.21. Surface Energy The surface tension of a fluid is the energy parallel to the surface and opposite to the surface energy. Surface tension and surface energy are often used interchangeably. Here, surface energy is defined as the energy required to wet a surface. To achieve optimal wicking, wetting, and spreading, the surface tension of the fluid is reduced and is less than the surface energy of the surface to be wetted. The wicking motion of a fluid through a channel of a fluid device occurs through capillary flow. Capillary flow depends on the cohesive forces between liquid molecules and the adhesion between the liquid and the channel walls. The Young / Laplace equation states that the fluid will rise in the channel or column until the pressure difference between the fluid's gravity and the force propelling it through the channel equals. [Moore, 1962] Walter J. Moore, Physical Chemistry, 3rd ed., Prentice-Hall, 1962, p. 730.

[0154] Δp=(2γ cos θ) / γ Where Δp is the pressure difference across the surface, γ is the surface tension of the liquid, θ is the contact angle between the liquid and the channel wall, and r is the radius of the cylinder. If the capillary rises by h, and ρ is the density of the liquid, then the gravity of the liquid in the column is πγ²ghρ, or the force per unit area equal to the pressure difference is ghρ. Therefore: (2γ cos θ) / γ=ghρ For a capillary channel with maximum flow, the channel radius should be small, the contact angle θ should be small, and the fluid surface tension γ should be large. This phenomenon can be theoretically explained using the classical model known as the Young's equation: γSV = γSL + γLV cos θ This describes the relationship between the contact angle θ and the surface tensions γLV, γSV, and γSL at the liquid-gas, solid-gas, and solid-liquid interfaces. When the contact angle θ between a liquid and a solid is zero or close to zero, the liquid will diffuse on the solid. Contact angle measurement is an objective and simple method used to measure the relative surface tension of solids. Generally, when the contact angle in this test is less than 90°, the material is considered hydrophilic. If the contact angle is greater than 90°, the material is considered hydrophobic.

[0155] 5.22. Fluid apparatus As used herein, the terms "microfluidic device" or "fluid device" generally refer to a device configured for fluid transport and / or transport of an entity by fluid, and having fluid channels in which fluid can flow at least one minimum dimension not greater than about 100 micrometers. The minimum dimension can be any length, width, height, radius, or cross-sectional axis. A microfluidic device may also include more than one fluid channel. One or more dimensions of a given fluid channel in a microfluidic device can vary depending on, for example, the specific configuration of one and / or more channels and other features also included in the device.

[0156] Fluid devices can include any device that can contain a milliliter volume (or less) of fluid. The physical boundary containing the fluid does not necessarily have to be solid and may also include gaseous and / or secondary liquid boundaries. In some embodiments, a microfluidic device may be exposed to a large volume of fluid during at least some portions of its operation, thereby increasing the total volume beyond the milliliter scale. Non-limiting examples include immersing the device in a reservoir, immersing the device in a liquid nitrogen bath, allowing a large volume of liquid to flow through the device, or allowing a large volume of liquid to flow over the device.

[0157] It should be understood that some of the principles and design features described herein can be scaled to larger devices and systems, including devices and systems employing channels and features with cross-sectional areas reaching the millimeter or even centimeter scale. Therefore, when some devices and systems are described as “microfluidics,” it is intended in some embodiments that this description is equally applicable to some larger-scale devices. Additionally, it should be understood that some of the principles and design features described herein can be scaled to smaller devices and systems, including devices and systems employing channels and features with cross-sectional areas of hundreds of nanometers, or even tens of nanometers, or even single nanometers. Therefore, when some devices and systems are described as “microfluidics,” it is intended in some embodiments that this description is equally applicable to some smaller-scale devices. As an example, a device may have an inlet orifice to receive liquid loaded by a pipette with a diameter of several millimeters, fluidly connected to a channel several centimeters long, hundreds of micrometers wide, and hundreds of nm deep, the fluid channel being fluidly connected to a nanopore shrinkage device with a diameter of 0.1 nm–10 nm.

[0158] The microfluidic device described herein may also include any additional components, such as those that facilitate the regulation of fluid flow (e.g., fluid flow regulators, pumps, pressure sources, etc.), features that help prevent blockage of fluid channels (e.g., funnel features in the channels; reservoirs located between channels, reservoirs supplying fluid to the fluid channels, etc.), and / or features that remove debris from the fluid flow, such as filters, for example. Furthermore, the microfluidic device may be configured as a fluidic chip comprising one or more reservoirs supplying fluid to an arrangement of microfluidic channels, and also comprising one or more reservoirs receiving fluid passing through the microfluidic device. Additionally, the microfluidic device may be constructed of any suitable material, including polymeric substances and glass, or channels and cavities encapsulated by multiphase immiscible media. The microfluidic device may include numerous microchannels, valves, pumps, reactors, mixers, and other components for generating and manipulating (merging, sorting, splitting, etc.) droplets. The microfluidic device may include active and / or passive sensors, electronic and / or magnetic devices, and integrated optical or functionalized surfaces. The physical substrate defining the channels of a microfluidic device can be solid or flexible, permeable or impermeable, or a combination thereof, and the physical substrate can vary with location and / or time. The microfluidic device may include a material that is at least partially transparent to light of at least one wavelength and / or at least partially opaque to light of at least one wavelength.

[0159] Microfluidic devices are typically designed and operated to manipulate samples contained in solution to achieve desired results. Samples can be loaded into the device manually (e.g., using a pipette) or automatically (e.g., via an automated liquid handling system). Samples can also be introduced by exposing the microfluidic device to a fluid flow. Additionally, various other solutions containing buffers and reagents can be added simultaneously with the sample input, or individually at different input ports and / or at different time points. In some cases, microfluidic devices are fabricated to contain both liquids and reagents internally.

[0160] Microfluidic devices can be completely independent, possessing all the necessary functions to manipulate the desired sample contained within them. Operation can be entirely passive, such as using capillary pressure to manipulate the fluid flow [Juncker, 2002], or it can incorporate an internal power source, such as a battery. Alternatively, the fluid device can be operated with the assistance of an external device that can provide any combination of power, voltage, current, electrical waveforms, magnetic fields, pressure, vacuum, light, heat, cooling, sensing, imaging, digital communication, encapsulation, acceleration, movement of one part of the microfluidic device relative to another, movement of the fluid meniscus across at least a portion of the microfluidic device, acoustic waves or acoustic energy, environmental conditions, etc. The fluid device can also be operated by chemical energy provided by one or more fluids, solids, or gases applied to the device, such as energy from the decomposition, combustion, or chemical reaction of one or more fluids, solids, or gases; energy from the mixing, hydration, solvation, or desolvation of solids, fluids, and / or gases; wetting or meniscus forces; phase transition energy from one state to another, such as the energy of vaporization or fusion. External devices can be mobile devices, such as smartphones, microcontroller-based devices, embedded computing platforms, or larger instruments with or without direct human control.

[0161] In some embodiments, the fluid device includes an "electrowetting device" or "droplet microactuator," which is a class of microfluidic devices capable of controlling the operation of droplets within the fluid device by a specifically applied local electric field. Non-limiting examples of such devices include liquid droplets surrounded by air on an open surface, and liquid droplets surrounded by oil sandwiched between two surfaces. [Mugele, 2005] and [Zhao, 2013] provide detailed reviews of the various configurations used and the physics of droplet control, and these two documents are provided herein for reference.

[0162] Unless otherwise specified, a “fluid feature” is a feature within or on a fluid device that, on its own or in combination with other fluid features, at least partially directly or indirectly contains a liquid or volume, or that has the ability to interact with a volume. Examples of volumes include macromolecules, cells, nuclei, nucleosomes, microconidia, DNA, chromosomes, ecDNA, cyclic polynucleotides, proteins, enzymes, nucleotides, carbohydrates, RNA, beads, dendritic polymers, nanoparticles, lipids, nanospheres, quantum dots, Pickering emulsions, and droplets.

[0163] In some embodiments, the interaction between the fluid feature and the biological host may include: shear force, bonding, repulsion, attraction, puncture, compression, extrusion, expansion, electrochemical reaction, or charge exchange. In some embodiments, the fluid feature includes a physical barrier.

[0164] In some implementations, fluid features may include channels, walls, corners, edges, barriers, physical obstacles, pillars, depressions, cones, rods, spheres, partial spheres, ellipses, circles, polygons, cylinders, bulges, ramps, troughs, cubes, parallelograms, ellipsoids, triangles, trapezoids, trapezoidal buckets, crescents, cuboids, ellipsoids, triangular prisms, pyramids, tetrahedrons, octahedrons, lines, dashed lines, points, tori, prisms, and polygons.

[0165] In some embodiments, the fluid characteristics may include regions having desired surface energy properties, or desired surface hydrophobic properties, or desired surface hydrophilic properties, or desired antifouling properties, or desired surface roughness properties, or desired surface smoothness properties.

[0166] In some embodiments, the antifouling property or behavior includes preventing the adhesion of complete packaging, semi-complete packaging, macromolecules, or molecules to at least a portion of a surface having said property. In some embodiments, the antifouling property is also affected by the chemical composition of the delivery fluid or at least one environmental condition. The optimal antifouling surface treatment is not necessarily the same for various categories of complete packaging, semi-complete packaging, macromolecules, and molecules, and the effectiveness of a particular antifouling coating can be affected by the prior treatment of the packaging, macromolecules, or molecules.

[0167] In some embodiments, the surface roughness characteristics are related to preventing or promoting the adhesion of intact packaging, semi-incomplete packaging, macromolecules, or molecules to at least a portion of a surface having said characteristics. In some embodiments, the roughness exceeds 0.1 nm rms, or exceeds 0.2 nm rms, or exceeds 1 nm rms, or exceeds 2 nm rms, or exceeds 5 nm rms, or exceeds 10 nm rms, or exceeds 20 nm rms, or exceeds 50 nm rms, or exceeds 100 rms, or exceeds 200 nm rms, or exceeds 500 rms, or exceeds 1000 nm rms. In some embodiments, the surface roughness is selected to achieve a certain hydrophobic characteristic. In some embodiments, the surface roughness is selected to achieve a certain hydrophilic characteristic. In some embodiments, the surface roughness is selected to achieve a certain antifouling characteristic. In some embodiments, the surface roughness is adjusted to achieve a certain adhesion characteristic.

[0168] The restriction or flow of a fluid or body "within one or more fluid features of a fluid apparatus" can be achieved in any way in which at least a portion of the fluid or body can be maintained for a certain period of time within, in, along or on at least a portion of at least one fluid feature defined within or on the fluid apparatus.

[0169] In some implementations, at least a portion of the fluid is confined by the solid or semi-solid physical boundary of the channel wall. Figure 5 An embodiment is shown in which channel walls having cross-sections such as rectangular (502), triangular (503), elliptical (504), and mixed geometry (505) are all defined within a fluid device (501). In other embodiments, fluid confinement within the fluid device can be at least partially confined by a combination of solid physical characteristics and surface energy characteristics [Casavant, 2013] or by an immiscible fluid [Li, 2020]. Examples of fluids at least partially confined within physical boundaries include various channels, such as grooves (507, 508) and rectangles (509, 510), physically defined on the surface of the fluid device (506), all of which can confine at least a portion of the fluid by surface tension. In other embodiments, a channel (511) may be defined by a groove in a corner (512) of the fluid device, or a channel (514) may be defined by two physically separated boundaries (513 and 515) of the fluid device, or a channel (521) may be defined by a corner (520) of the fluid device. In other embodiments, the channel (517) is defined by a hydrophilic portion (518) on the surface of the fluid device (516), wherein the hydrophilic portion is defined by a hydrophobic portion (519) on the surface of the fluid device. In all cases, these embodiments are non-limiting examples, and further embodiments include combinations thereof. As used herein, an "open fluid device" is a fluid device comprising at least one fluid feature (e.g., a channel) in which a solution in said fluid feature is at least partially exposed to a gas phase interface. Examples include air, water vapor, solvent vapor, oxygen, nitrogen, or mixtures thereof. In particular, the selection of gas composition, pressure, and other environmental conditions can be controlled with regard to the operation of an open fluid device, and may be critical to the desired operation of the open fluid device. For example, specific temperatures, or humidity, or dew points, or solvent vapor partial pressures, or wavelength exposures may be desired for a particular time period.

[0170] It is possible to have more than one fluid feature having a region with the desired surface properties. Non-limiting examples of surface properties include degree of hydrophobicity, degree of hydrophilicity, or degree of antifouling, or degree of roughness, or degree of smoothness, or surface charge density, or surface functionalization, or surface energy. In some embodiments, the fluid feature may include a region having the desired surface properties. In some embodiments, the fluid feature may include at least one region having the desired surface properties. In some embodiments, the fluid feature may include more than one region, each region having a specific desired surface property.

[0171] Figure 6 Some embodiments of fluid features selectively patterning different desired surface properties of type "A" (602) and type "B" (603) on the surface of a fluid device (601) are shown. The fluid features defined on the fluid device can take on any shape. Furthermore, any surface region (including whole or part of a surface plane) on any said shape can include the desired surface properties. For example, a fluid feature including an open fluid rectangular cross-section channel (604, 605, 606) can have different regions having surface property "B" inside the channel and surface property "A" outside the channel. In another example, a fluid feature including an open fluid triangular cross-section channel (607, 608, 609) can have different regions having surface property "B" inside the channel and surface property "A" outside the channel.

[0172] There are many different methods for modifying the surface energy or antifouling properties of a surface. For example, by contact transfer stamping hydrophobic or antifouling molecules onto the surface of a glass fluid device, the naturally hydrophilic open and recessed channels on the surface of the fluid device are left unmodified. Other methods may include spin coating, dip coating, film growth, film deposition, film printing, film dispensing, film spraying, and self-assembling of monolayers. Surface modification can be selective for certain regions, where the modified regions are selected by the means of modification, their physical position relative to other regions, the use of masks, or the use of temporary sacrificial materials. Surface modification can also be selective for certain regions, where the modified regions are selected by chemical means such that only materials with certain surface chemical properties will participate in the modification process. Surface modification can also be selective for certain regions containing a seed or other materials containing surface properties that allow for selective modification.

[0173] Transfer contact printing (or microcontact printing) is a widely used method for transferring molecules from a donor substrate to a recipient substrate. The most common donor substrate is typically a flexible elastomer, which provides a degree of compliance with the recipient substrate. For reviews of different microcontact methods and materials, see [Smith, 2004], [Lamping, 2019], and [Qiu, 2021]. In some embodiments, the transferred molecules form a monolayer, bilayer, multilayer, multiplex, or thin film on the recipient substrate. In some embodiments, more than one contact printing step may be used.

[0174] In some embodiments, surface modifications modify the hydrophobicity of a portion of the fluid device and work in conjunction with topological features to guide fluid flow, particularly flow at the interface of separated phases, most preferably at the meniscus between the fluid and the gas phase. Highly hydrophobic surface modifications are not limited to molecular classes having one or more halogenated side chains, such as poly(heptadecyl acrylate), octadecyltrichlorosilane, or 1H,1H,2H,2H-perfluorodecylamine. Highly hydrophobic surface modifications are also not limited to molecular classes having aliphatic groups, such as lauryl methacrylate, octadecyltrimethoxysilane, or dodecylamine.

[0175] In some embodiments, the hydrophobicity of surface modifications applied to a portion of the device can be altered by mild reaction or environmental conditions. Adaptable hydrophobic surface modifications are not limited to polymers present in the form of homopolymers, copolymers, or block copolymers, said polymers being made of unstable or cleavable groups, including trimethylsilyl methacrylate, t-BOC-protected amines such as N-(3-BOC-aminopropyl)methacrylamide, 3-[(2-aminoethyl)dithio]propionic acid, enzymatically cleavable phenylalanine-rich peptides conjugated to polyacrylic acid, and polymers modified with terminal nitrobenzyl groups further conjugated to fluorinated alkanes. Adaptable hydrophobic surface modifications are not limited to the group of thermoresponsive polymers, which includes cellulose nanocrystals, poly(N-isopropylacrylamide), Pluronic F127, poly(organophosphazenes), and copolymers thereof, including poly(N-isopropylacrylamide) copolymerized with poly(ethylene glycol).

[0176] In some embodiments, surface modifications are added to chemical moieties that interact with a portion of the packaging or a portion of the macromolecule. Furthermore, surface modifications can be combined with one or more of the strategies described above for dynamic modification of the surface. Surface modifications are not limited to polymers and molecules containing multivalent moieties, including linear and branched polyamines including spermine and spermine moieties, peptides rich in charged amino acids (including lysine, glutamic acid, arginine, histidine, and aspartic acid), oligonucleotides, homopolymers of charged amino acids, copolymers of charged amino acids, graft polymers and coblock polymers, polyacrylic acid, poly(methacryloyl-1-lysine), poly(ethyleneimine), poly(2-aminoethyl methacrylate), poly(ethylene glycol), poly(N-[3-(N,N-dimethylamino)propyl]acrylamide), poly(2-(tert-butylamino)ethyl methacrylate), and poly(2-diisopropylaminoethyl methacrylate). Surface modifications include, but are not limited to, molecules, polymers, and macromolecules that react with packaging and macromolecules to form covalent bonds, such as homopolymers, copolymers, or coblock polymers, or self-reactive or reactive with chemical crosslinking agents such as formaldehyde, paraformaldehyde, and EDC / NHS combinations. This group includes poly(ethylene glycol) diamine, cinnamon methacrylate, poly(glycidyl methacrylate), poly(propargyl methacrylate), and poly(2-aminoethyl methacrylate).

[0177] Surface modifications include, but are not limited to, molecules, polymers, and macromolecules that specifically interact with membranes, lipid bilayers, or other components forming packaging membranes, or that interact with menisci separating more than one fluid or gas phase. These surface modifications, whether as homopolymers, copolymers, graft polymers, or coblock polymers, are not limited to side phospholipids and aromatic groups added to the polymer terminus, including 1-decyne added to the terminus of NaN3-substituted ATRP polymers via Cu-catalyzed cycloaddition.

[0178] In some embodiments, modification is a "grafting" reaction that adds small molecules, polymers, or macromolecules to at least a portion of a fluid device or fluid feature. Some embodiments utilize the reaction of silanes with glass, silicon, or oxide surfaces, said silanes including, but not limited to, aminopropyl (3-aminopropyl)triethoxysilane, mPEG5K-silane, and 3-(trimethoxysilyl)propyl acrylate. In some embodiments, non-covalent interactions are formed with surface-modifying chemicals including poly-1-lysine, most preferably with coblock polymers including the Pluoronic detergent family, Tween 20 and Tween 80, and other substances described herein.

[0179] According to certain aspects of this disclosure, various materials and methods can be used to form articles or components, such as those described herein, including channels such as microfluidic channels, chambers, etc. For example, various articles or components can be formed from solid materials, where channels can be formed through micromachining, thin-film deposition processes such as spin coating and chemical vapor deposition, laser manufacturing, photolithography, bonding, deposition, lamination, molding, imprinting, 3D printing, fused deposition modeling 3D printing, stereolithography 3D printing, selective laser sintering 3D printing, etching methods (including wet chemical or plasma processes), and encapsulation with multiphase immiscible media. For patterning, various methods can be employed, including but not limited to: photolithography, electron beam etching, nanoimprint etching, AFM etching, STM etching, laser engraving, focused ion beam etching, stamping, embossing, molding, and dip pen lithography. Various methods can be used for bonding, including but not limited to: thermal bonding, adhesive bonding, pressure bonding, vacuum bonding, surface-activated bonding, fusion bonding, anodic bonding, plasma-activated bonding, laser bonding, and acoustic bonding.

[0180] In one set of embodiments, various fluid features, structures, or components of the objects described herein may include polymers, such as elastomeric polymers, such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE” or Teflon®), etc. For example, according to one embodiment, microfluidic channels can be implemented by fabricating a fluid system separately using PDMS or other soft etching techniques [Xia, 1998, Whitesides, 2001].

[0181] Other examples of potentially suitable polymers include, but are not limited to, polyethylene terephthalate (PET), polyacrylate, polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, polypropylene, polyvinyl chloride, cyclic olefin copolymers (COC), cyclic olefin polymers (COP), polytetrafluoroethylene, fluorinated polymers, silicones such as polydimethylsiloxane, polyvinylidene chloride, bisbenzocyclobutene (“BCB”), polyimide, fluorinated derivatives of polyimide, nylon, etc. Compositions, copolymers, or blends including those described above are also contemplated.

[0182] In some embodiments, various structures or components of the object are made of polymeric and / or flexible and / or elastomeric materials and can be readily formed by phase change fluids, thereby facilitating manufacturing by molding (e.g., replication molding, injection molding, casting molding, etc.). Phase change fluids can essentially be any fluid that can be induced to solidify or spontaneously solidify into a solid capable of containing and / or delivering fluids intended for use in and with a fluid network. In one embodiment, the phase change fluid comprises a polymeric liquid or a liquid polymer precursor (i.e., a “prepolymer”). Suitable polymeric liquids may include, for example, thermoplastic polymers, thermosetting polymers, waxes, metals, or mixtures or composites thereof heated above their melting point. As another example, suitable polymeric liquids may comprise a solution of one or more polymers in a suitable solvent, which forms a solid polymeric material after the solvent is removed, for example, by evaporation. Such polymeric materials, which can solidify from, for example, a molten state or by solvent evaporation, are well known to those skilled in the art. For embodiments in which one or both of the mold master include elastomeric materials, a variety of polymeric materials (many of which are elastomeric) are suitable and also adapted to form the mold or mold master. A non-limiting list of examples of such polymers includes polymers of the general categories of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three-membered cyclic ether group, commonly referred to as an epoxy group, 1,2-epoxide, or ethylene oxide. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amines, triazines, and alicyclic backbones. Another example includes well-known phenolic varnish polymers. Non-limiting examples of silicone elastomers suitable for use according to this disclosure include those formed from precursors including chlorosilanes such as methylchlorosilane, ethylchlorosilane, phenylchlorosilane, dodecyltrichlorosilane, etc.

[0183] In some embodiments, silicone polymers, such as the silicone elastomer polydimethylsiloxane, are used. Non-limiting examples of PDMS polymers include those sold by Dow Chemical Co., Midland, Mich. under the trademark Sylgard, and particularly Sylgard 182, Sylgard 184, and Sylgard 186. Silicone polymers incorporating PDMS have several advantageous properties that simplify the manufacture of various structures of this disclosure. For example, such materials are low-cost, readily available, and can be cured from a prepolymer liquid by heat curing. For example, PDMS can typically be cured by exposing a prepolymer liquid to a temperature of about, for example, about 65°C to about 75°C for, for example, about 1 hour. Furthermore, silicone polymers (such as PDMS) can be elastomers and are therefore used to form very small features with relatively high aspect ratios, which is necessary in some embodiments of this disclosure. Flexible (e.g., elastomer) molds or master molds can be advantageous in this regard.

[0184] One advantage of forming structures (such as microfluidic structures or channels) from silicone polymers (such as PDMS) is the ability of such polymers to be oxidized, for example by exposure to oxygen-containing plasma (such as air plasma), such that the oxidized structure contains chemical groups on its surface that can crosslink with other oxidized silicone polymer surfaces or oxidized surfaces of various other polymeric and non-polymeric materials. Therefore, structures can be fabricated and then oxidized and substantially irreversibly sealed to other silicone polymer surfaces, or to the surface of other substrates reactive with the oxidized silicone polymer surface, without the need for separate adhesives or other sealing devices. In most cases, sealing can be accomplished simply by bringing the oxidized silicone surface into contact with another surface, without the need to apply auxiliary pressure to form a seal. That is, the pre-oxidized silicone surface acts as a contact adhesive against a suitable mating surface. In particular, in addition to being irreversibly sealed by itself, oxidized silicones such as oxidized PDMS can also be irreversibly sealed to a range of oxidized materials other than themselves, including, for example, glass, silicon, silica, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a manner similar to that of PDMS surfaces (e.g., by contact with oxygen-containing plasma). Oxidation and sealing methods and integral molding techniques useful in the context of this disclosure are described, for example, in [Duffy, 1998].

[0185] Devices may include glass, silicon, silicon nitride, silicon oxide, quartz, metal, fused silica, mica, ceramics, aluminum, BK7 glass, linbo3, borofloat 33 glass, SD2 glass, 7740 pyrex glass, flexinity® glass, D263 glass, AF32 glass, memmax glass, B270 glass, AS87 ECO glass, foturan II glass, flexible glass, SchottUTG glass, flat panel display glass, KG2 glass, KG3 glass, NG11 glass, BG63 glass, EAGLE XG® glass, Astra™ glass, Lotus™ NXT glass, gallium arsenide, germanium, graphene, ITO, litau3, nitrides, polycrystalline silicon, sapphire, silicon carbide, silicon-on-insulator, soda-lime glass. Glass), InP, polymers, plastics, polycarbonate (PC), PMMA, acrylic acid, polyethylene terephthalate, amorphous copolyester (PETG), polyvinyl chloride (PVC), liquid silicone rubber (LSR), cyclic olefin copolymer (COC), polyethylene (PE), ionomer resins, polypropylene (PP), fluorinated ethylene propylene (FEP), styrene-methyl methacrylate (SMMA), styrene-acrylonitrile resin (SAN), polystyrene, methyl methacrylate-acrylonitrile-butadiene styrene (MABS), acrylonitrile-butadiene styrene (ABS), polylactic acid (PLA), polyethylene terephthalate (PETG), nylon, thermoplastic polyurethane (TPU), polyvinyl alcohol (PVA), high-impact polystyrene (HIPS), resins, polyurethane resins, elastic resins, ESD resins, ceramic resins, nylon 12, nylon 11, TPU, titanium, stainless steel, nickel alloys, gels, hydrogels, dry gels, colloidal gels, organic gels, thermoplastic plastics, thermosetting plastics.

[0186] The apparatus may include filter paper, paraffin membrane, permanent fabricator ink or wax.

[0187] The apparatus may include patternable materials, particularly patternable polymers, patternable organic polymers, patternable inorganic polymers, or patternable gels. In some embodiments, the patternable material may include photo-patternable materials, or electronically patternable materials, or embossed materials, or imprintable materials, or 3D printable materials, or materials dispensed from a dispensing system, or materials patternable by ion beam etching, or materials patternable by soft etching, or materials patternable by colloidal etching, or materials patternable by self-assembly methods, or materials patternable by area-selective deposition (ASD), or materials patternable by microcontact printing, or materials patternable by casting, or materials patternable by lamination, or materials patternable by roll-to-roll processing, or... Materials that can be modeled by microthermoforming, or by micromolding in capillary (MIMIC), or by capillary force etching (CFL), or by scanning probe etching (SPL), or by micro-dicing, or by sonomechanics, or by laser ablation, or by plasma etching, or by biotemplate assembly, or by electrical discharge machining (EDM), or by microelectrochemical micromachining (ECM), or by X-ray LIGA. For a non-limiting review of several non-photolithographic methods for modeling materials, particularly polymers, see [Qiu, 2023], [Acikgoz, 2011], and [Scott 2021], and especially for 3D methods see [Yazdi, 2015].

[0188] In some embodiments where the patternable material is a patternable polymer, the patternable polymer may include any of the previously listed polymeric materials.

[0189] In some embodiments where the photomaturable material is a photomaturable material, the photomaturable material may include a photoresist. In some embodiments, the photoresist is a negative photoresist, or a positive photoresist, or a photocurable photoresist, or a UV-curable photoresist. Examples of different photomaturable photoresists include DNQ (diazonaphthoquinone) and phenolic varnish resin-based resists, epoxide-based resists, off-stoichiometry thiol-enes (OSTE) polymer resists, and hydrogen silsesquioxane (HSQ)-based resists. In some embodiments, the photomaturable polymer is based on FOX-16 or a derivative thereof, or based on poly(methyl methacrylate) (PMMA) or a derivative thereof, or based on poly(methylglutarimide) (PMGI) or a derivative thereof, or an OSTE polymer or a derivative thereof, or based on Ma-N photoresist, or Shipley photoresist, or SPR photoresist. In some implementations, the photoresist includes sensitivity to G lines, or I lines, or KrF, or ArF, or EUV, or electron beams. In some implementations, the photo-modetable polymer may include any DuPont SPR series photoresist, or any DuPont MCPR series photoresist, or any DuPont UV series photoresist, or any DuPont UVN series photoresist, or any DuPont LR series photoresist, or any DuPont EPIC series photoresist, or any Microchem AZ series photoresist, or any Microchem TI series photoresist, or any Microchem SU8 series photoresist, or any Microchem Ordyl series photoresist, or any Microresist MA-N series photoresist, or any Microresist MR-DWL series photoresist, or any Microresist EpoCore series photoresist, or any Microresist EpoClad series photoresist, or any Microresist MA-P series photoresist, or any Microresist MR-P series photoresist, or any Microresist InkEpo series resists, or any MicrochemInkOrmo series resists, or any MicrochemSUEX series resists, or any Microresists ADEX series resists, or any Microresist UV series resists, or any Microresist Microposit series resists, or any Microresist Ultra-I series resists, or any Microresist Microposit series resists.LOL series photoresist, or any Microresist Cyclotene series photoresist, or any Microresist Intervia series photoresist, or any Microresist MX series photoresist, or any Microresist WBR series photoresist, or any Microresist SU-8 series photoresist, or any Microresist KMPR series photoresist, or any Microresist Perminex series photoresist, or any Microresist KMSF series photoresist, or any Microresist LOR series photoresist, or any Futurrex NR series photoresist, or any Futurrex PR series photoresist, or any Sumitomo PFI series photoresist, or any Sumitomo PFM series photoresist, or any Sumitomo PXi series photoresist, or any Sumitomo NX series photoresist, or any Sumitomo PEK series photoresist, or any Sumitomo PAR series photoresist, or any Tok TSMR series photoresist, or any Tok THMR-iP series photoresist, or any Tok TDMR-AR series photoresist, or any Tok OFPR series photoresist, or any Tok OEBR-CAP series photoresist, or any Tok TFR-Di series photoresist, or any Fujifilm OiR series photoresist, or any Fujifilm GiR series photoresist, or any Fujifilm HiPR series photoresist, or any Fujifilm OiR series photoresist, or any Fujifilm SC series photoresist, or any Fujifilm IC series photoresist, or any Fujifilm HNR series photoresist, or any Fujifilm HR series photoresist, or any Fujifilm GKR series photoresist, or any Fujifilm GAR series photoresist, or any Fujifilm FAiRS series photoresist. In some implementations, the photo-patternable polymer may include polymer resins, photoactive compounds, photoactivator compounds, photoinitiator compounds, detection additives, resist solvents, crosslinking agents, additives that help the photoresist achieve the highest resolution, surfactants, quenchers, stabilizers, plasticizers, colorants, adhesive additives, surface leveling agents, or combinations thereof.

[0190] In some embodiments where the patternable material is an electron-patternable material, the electron-patternable material may include an electron beam resist. Examples of electron-patternable resists include any ZEP series resist, or any HSQ and its variants, or any PMMA and its variants, or any polycarbonate and its variants, or any resist with increased sensitivity by the addition of a halogenated acid-producing agent, or any CSAR series resist and its variants, or any AR series resist, or any SML series resist, or any microresist mr-EBL series resist, or any microresist XR series resist.

[0191] In some embodiments where the patternable material is an imprintable material, the imprintable material may include an imprinting (or nanoimprinting) resist. In some embodiments, the imprinting resist includes a UV-curable polymer. In some embodiments, the imprinting resist includes a thermosetting polymer. In some embodiments, the imprinting resist includes a thermoplastic polymer. In some embodiments, the imprinting process may include nanoimprinting, electrochemical imprinting, laser-assisted direct imprinting, UV imprinting, ultrafast nanoimprinting, and roll imprinting. Examples of imprint resists include any Microresist MR-NIL series resist, or any Microresist MR-I series resist, or any Microresist SIPOL series resist, or any Microresist OrmoComp series resist, or any Microresist OrmoStamp series resist, or any Microresist OrmoClear series resist, or any Microresist OrmoCore series resist, or any Microresist OrmoClad series resist, or any Microresist MR-XNIL series resist, or any Microresist KER series resist, or any Microresist MR-UVCur series resist, or any NanonexNRX series resist.

[0192] In some implementations, the patternable material can be applied to the fluid device by any method, including spin coating, lamination, roll coating, spraying, dip coating, dry film attachment, air knife coating, curtain coating, wire coating, wire-bar coating, gravure coating, extraction coating, doctor blade coating, dispensing from a dispensing system, inkjet printing, jetting, tape dispensing, or combinations thereof.

[0193] The device may include an organic thin film chemically bonded or grown on the surface of the device. In some embodiments, the organic thin film comprises a polymer. In some embodiments, the exact thickness of the organic thin film is controlled by the bonding or growth conditions of the film. In some embodiments, the growth of the organic thin film is self-limiting. In some embodiments, the growth of the organic thin film can be controlled by adjusting the reaction time during growth or by changing the number of growth cycles. In some embodiments, the organic thin film may include hydrophobic surface properties, or hydrophilic surface properties, or positive surface charge, or negative surface charge, or antifouling surface properties. In some embodiments, the organic thin film at one or more locations in the fluid device can be controlled by bonding or growing the organic thin film on a seed material, such that the organic thin film will only be bonded or grown on said seed material. In some embodiments, the seed material may include silicon oxide, or silicon nitride, or silicon, or glass, or quartz, or gold, or nickel, or platinum, or ITO, or silver, or a polymer, or a photomoldable polymer, or an imprintable polymer, or an embossed polymer, or a dispensable polymer. In some embodiments, the seed material is first chemically modified on its surface before bonding or growing the organic thin film. In some embodiments, one or more sites in the fluid device where the organic thin film is absent are at least partially controlled by the position of the sealing material. In some embodiments, the surface modification of the fluid device includes modification with a polymerizable patternable material that has been partially or fully polymerized, wherein the degree of polymerization is influenced by factors including: photoinitiator dose, etch mask, dose of maskless lithography equipment, wavelength-dependent absorption of light used to activate the photoinitiator, local environment resulting from fine features and proximity effects, pre-baking thermal spectrum, post-baking thermal spectrum, development, aging, etching, plasma etching, and quenching. In a preferred embodiment, the modification of the patternable material employs a chemical reaction that specifically modifies the patternable material and does not modify the underlying substrate. In some embodiments, the above effects produce a non-uniformly reactive patternable material for further chemical modification.

[0194] In some embodiments, surface modification reacts in a generally uniform and specific manner to modify substantially all of the underlying substrate or substantially the patternable material applied to the substrate. In some embodiments, surface modification relies on further photoactivation of the developed or partially developed patternable material to enhance the reactivity of the remaining patternable material in localized regions, thereby adding an additional level of spatial control to subsequent surface modification deposition. In some embodiments, selective application of plasma alters the reactivity of the substrate and / or the patternable material. In some embodiments, surface modification reacts non-uniformly with partially polymerized or partially degraded patternable material prepared using the effects described above. In some embodiments, the extent of surface modification is also influenced by the diffusion of oxygen within the three-dimensional structure during the surface modification process.

[0195] In some embodiments, the partially polymerizable patternable material undergoes surface modification. In some embodiments, the modification utilizes residual acid from a superacid photoactivator and / or free radicals left from radical polymerization. In some embodiments, free radical polymerization is carried out using a mixture of one or more acrylates and methacrylates, including but not limited to: poly(ethylene glycol), methyl ether acrylate, poly(2-hydroxyethyl methacrylate), isobornyl methacrylate, lauryl methacrylate, 2-(trimethylsilyloxy)ethyl methacrylate, N-isopropylacrylamide, N,N'-methylenebis(acrylamide), 2-hydroxyethyl methacrylate, propyne acrylate, acrylamide, N,N-dimethylacrylamide, 2-N-morpholinoethyl methacrylate, pentafluorophenyl methacrylate, dextran methacrylate, methacrylated gelatin, methacrylated hyaluronic acid, methacrylated collagen, methacrylated alginate, methyl methacrylate, 2,2,2-trifluoroethyl acrylate, butyl acrylate, propyl acrylate, N-hydroxyethylacrylamide, 2-hydroxy-3-phenoxy-propyl acrylate, tert-butyl methacrylate, tert-butyl acrylate, sodium methacrylate, and benzyl acrylate. In some embodiments, the radical polymerization (including radical polymerization with the above monomers) is carried out in controlled living polymerization using one of the reversible deactivating radical polymerization methods, including but not limited to atom transfer radical polymerization (ATRP), nitride-mediated polymerization (NMP), and reversible addition-fragmentation chain transfer (RAFT).

[0196] In some embodiments, the unreacted epoxy moiety from the patternable material can be opened and used for further reactions, including acylation reactions, including succinic anhydride and α-bromoisobutyryl bromide. Furthermore, the unreacted epoxy moiety reacts with primary amines under acid and base catalytic conditions, and direct surface modification of the epoxy-containing patternable material is not limited to the addition of alkylamines (including dodecylamine, octylamine, tert-octylamine, propylamine), cyclic aliphatic amines (including cyclohexylamine), fluorinated amines (including 2,2,3,3,4,4,4-heptafluorobutylamine), zwitterionic or charged amines (including glutamic acid), polar amines (e.g., 2-(2-aminoethoxy)ethanol), and secondary amines (such as dioctylamine).

[0197] In some embodiments, at least a portion of the surface of the fluid device or at least a portion of the fluid characteristics is modified by a combination of two different reactions, applied one at a time or simultaneously, wherein the first reaction is the substrate of the second reaction. In such reactions, chemical amplification can be achieved under certain reaction conditions. In some embodiments, the first reaction is a surface-initiated free radical polymerization of an ATRP initiator (2-(2-bromoisobutyryloxy)ethyl methacrylate) alone or as a mixture of different acrylates or methacrylates or bifunctional or polyfunctional crosslinked acrylates or methacrylates, and the second step is a surface-initiated atom transfer radical polymerization (siATRP) of an acrylate or methacrylate as described above, employing one of the established methods of copper-catalyzed ATRP, said ATRP comprising an activator generated by electron transfer (AGET), an activator regenerated by electron transfer (ARGET), an initiator for continuous activator regeneration (ICAR), and an initiator for the oxidative regeneration of Cu(I) from Cu(O) in the presence of ligands (including PMDETA, Me6TREN, TPMA, TMPA-NME2). In other embodiments, the first reaction is a thiol epoxy condensation of thiol-containing ATRP reagents such as bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide and bis[2-(2-bromoisobutyryloxy)undecyl]disulfide, followed by siATRP using acrylates or methacrylates as described above. In other embodiments, surface modification continues by free radical polymerization of one or more carboxyl-containing monomeric substances (including acrylic acid and methacrylic acid), and a second reaction utilizes N-ethyl-NO-(3-(dimethylamino)-propyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to graft biomolecules onto polymers comprising commonly prepared proteins known to have blocking capabilities or the ability to generate hydrogels or matrices (including all or part of bovine serum albumin, collagen, gelatin, terminal collagen, and proelastin). In other embodiments, the first reaction is the free radical polymerization of glycidyl methacrylate, and the second reaction is the addition of a primary amine present on a protein as described above to a glycidyl ether, or the addition of hyaluronic acid, dextran, or carboxymethyl cellulose to the glycidyl ether.

[0198] The apparatus may include combinations of different materials that can be mixed, joined, laminated, layered, grown, adhered, patterned, connected, coated, deposited, evaporated, sputtered, melted, merged, or combined thereof.

[0199] 5.23. Physical obstacles Unless otherwise specified, a "physical obstacle" is a physical feature within a fluid device in which long nucleic acid molecules physically interact with the physical obstacle under the presence of applied forces, such that the physical conformation or position of the molecules differs from when the physical obstacle is not present. Non-limiting examples include: pillars, corners, depressions, traps, barriers, walls, protrusions, contractions, and extensions. Physical obstacles do not need to be physically continuous with the fluid channel, but can also be added to the device, and non-limiting examples include: beads, gels, and particles.

[0200] 5.24. Filter Assembly Filter assembly: A type of fluid device comprising an inlet chamber and an outlet chamber in fluid communication with each other through at least one or more filter structures, wherein the filter structures include pores or fluid features such that, for a given applied force and a sample solution containing particles having certain properties (size, charge, etc.) in the inlet chamber, the filter assembly allows sample fluid to pass through the outlet chamber but restricts at least a portion of the particles from entering the inlet chamber without significant deformation, topological rearrangement, or, in the case where the particles contain macromolecular components, without changes in the stoichiometry or count of the portions bound to the macromolecules.

[0201] 5.25. Droplets The terms “droplet” and “microdroplet” are used interchangeably herein to refer to a small, circular structure (typically spherical in an unrestricted state) comprising at least a first fluid phase, such as an aqueous phase (e.g., water), defined by a second fluid phase (e.g., oil) that is immiscible with the first fluid phase, or defined by the surface tension formed by the interface of the first fluid phase, the surface, and air.

[0202] In some embodiments, the droplets according to this disclosure may comprise a first fluid phase, such as oil, defined by a second immiscible fluid phase, such as an aqueous phase fluid (e.g., water). In some embodiments, the second fluid phase will be an immiscible carrier fluid. Therefore, the droplets according to this disclosure can be provided as water-in-oil emulsions or oil-in-water emulsions. The droplets according to this disclosure can be formed as more than one emulsion, such as double or higher density emulsions, for example, producing water-in-oil-in-water droplets. In some embodiments, the subject droplet has a dimension (e.g., diameter) from 0.1 μm to 1000 μm (including endpoints). Furthermore, in some embodiments, the discrete entity as described herein has a volume range of about 1 aL to 1 μL (including endpoints). The droplets according to this disclosure can be used to encapsulate cells, nucleic acids (e.g., DNA), enzymes, reagents, and various other entities. The droplet may contain a single entity (i.e., a single cell or a single long nucleic acid fragment) or more than one entity. The droplet may contain a mixture of different types of entities. The term droplet can be used to refer to a droplet that is in, on, or generated by a microfluidic device and / or flows out of or is applied by a microfluidic device. Droplets can be generated externally and applied to the microfluidic device. Alternatively, droplets can be generated within the microfluidic device and then removed from the device.

[0203] Depending on the desired operation, a droplet can be partitioned into two or more droplets, or merged with at least one other droplet. The droplets to be merged can have the same or different contents.

[0204] The composition and properties of droplets can vary. For example, in some cases, surfactants can be used to stabilize the droplets. Thus, droplets can comprise surfactant-stabilized emulsions. Any convenient surfactant that allows the desired reaction to occur within the droplet can be used. In other cases, the droplets are not stabilized by surfactants or particles.

[0205] In some implementations, droplets can be formed at the interface of a liquid, a surface, and air, and thus include droplets defined by an electrowetting device. Examples of such droplets are described by [Zhao, 2013] and [Mugele, 2005].

[0206] 5.26. Entropy barrier and entropy trap A specific region of a fluid device will be defined as an “entropy barrier” if (a) the geometry of the nano- or micro-fluidic device contains inhomogeneous features on the order of or smaller than the size of the analyte of interest, and (b) the diffusion or flow of the analyte around or through the feature is significantly impeded or slowed in a manner dependent on the aggregate size, extended shape, or conformation of the analyte. Furthermore, an “entropy trap” will be defined as a region in a fluid device where all fluid connections are directly through the entropy barrier, such that if left stationary, the analyte of interest will remain in the trap because the object occupying the trap is in a local minimum energy state. The definition of an entropy trap will be limited to traps that are inherently passive, meaning they do not require a continuous energy supply to control the item or prevent it from passing through the device, but do require energy to release the item or allow it to pass through the barrier. The definition is also limited to traps created by fabricating features such as bags, contractions, restrictions, and physical barriers within the fluid device, and these can be defined partly or entirely by their geometry, and subsequently by the etched pattern and process parameters.

[0207] Entropy traps allow for the spatial preservation and positioning of packaging, long polymer chain molecules of interest, and even subregions of long polymer chain molecules. For brevity, all these objects will be referred to as deformable objects, meaning that their physical conformation can change when placed within elements of a confined fluid device, and that these objects share many similarities in controlling their general behavior relative to entropy traps and entropy barriers. However, when the similarities among these objects diverge, or when it is necessary to refer to a specific feature of interest associated with a particular object, that particular object will be mentioned in the text.

[0208] Deformable objects can remain in a trap or against a barrier when a buffer solution or surrounding fluid flows at a low velocity, allowing the chemical environment to change for reactions, etc. Furthermore, traps can be designed to influence changes in the physical conformation of the deformable object being trapped. While the geometries of various traps can appear convincingly similar, their operating principles can differ significantly based on the size and composition of the trap, as well as the chemical and local environment of the deformable object to be trapped and the fluid apparatus surrounding it. The methods of operation differ accordingly. The precise mechanisms of trap operation are a rich and ongoing area of ​​physics research, but in most cases, the benefits of traps defined by their geometry and methods of use can be utilized without a detailed understanding of the multi-scale physics underlying their use. Larger traps, such as those used to confine intact cells or droplets, rely on the elastic deformation of the object to be trapped; however, for consistency, they will still be referred to as entropy traps in this disclosure because such objects similarly possess a local minimum energy when occupying the trap, and there exists a minimum force greater than which the deformable object can pass through any particular entropy barrier. This is not entirely contrived; elasticity is an entropic force and other macroscopic manifestations. Similarly, in the context of oil / water droplet systems, entropy traps can be used to manipulate the motion and behavior of droplets and are understood as being driven by minimizing surface tension energy.

[0209] Entropy traps and entropy barriers form a large family of building blocks that can be arranged to create fluid devices for manipulating deformable objects. They complement other building blocks such as channels for moving deformable objects and various reagents, manifolds that combine or separate channels, and probing regions that facilitate observation of the deformable object. They also complement stationary phase materials as understood in chromatography, which utilize the chemical attraction between the deformable object and the surface of the fluid device or mechanically constrained attachments (such as chromatographic resins or beads) and act to slow the flow of the deformable object through the mobile phase of the device. Entropy traps and entropy barriers are typically found at the intersection of channels and / or probing regions and can be placed inside or near channels and probing regions, areas with defined surface chemistry, or other building blocks. Specific parts of a fluid device can possess the properties of an entropy trap or entropy barrier, as well as the properties of another type of building block.

[0210] The confinement energy of an entropy trap is generally understood as the difference in free energy exhibited by a specific instance of a long polymer (rather than intended confinement) as it takes on different physical conformations throughout the structure. A long polymer undergoing random thermal motion in the presence of an entropy trap will move into the portion of the trap with the lowest free energy. Free energy has two components: an enthalpy component that remains constant at temperature, such as chemical state (stretched or confined chemical bonds) energy, electrostatic attraction or repulsion, etc.; and an entropy component, which decreases the free energy in a manner proportional to the temperature and entropy of the long polymer in that portion of the trap, representing the number of ways the long polymer can conform in the trap. Analysis of entropy traps typically considers only the entropy component of the free energy, neglecting the enthalpy component. Comparing the two regions of an entropy trap, it is necessary to count the number of ways a randomly coiled polymer can occupy the trap. For example, a tightly packed cylindrical tube only slightly larger than the polymer's outer diameter will only allow for linear molecules to be accommodated in two ways: forward or backward. Conversely, a large open volume will allow for a greater number of combinations of random movement, twisting, and conformation. In the latter case, the geometry has a higher entropy due to the presence of more states, and the free energy of the long polymer in that region is correspondingly lower.

[0211] As deformable objects move within a fluid apparatus to minimize their free energy, they are said to fall into and occupy an entropy trap when they occupy a region within the apparatus that allows for a local minimum energy state. A deformable object completely confined within a portion of the apparatus with a uniform geometry, but not extending into an adjacent trap, will not spontaneously move into that trap, but will instead diffuse and move freely in response to external forces. However, when a portion of a deformable object diffuses or moves reversibly into a region within the fluid apparatus that constitutes an entropy barrier, such a fluid element is an entropy slope, because molecules will be attracted across the slope. In other words, without external forces, a deformable object in a specific physical conformation and position A within a fluid apparatus can reach a new physical conformation and position B by reducing its total energy through an entropy slope. However, this reversal is impossible without adding a minimum external force that allows the object to move from B to A through the entropy barrier.

[0212] A deformable object is released from its trap when the difference in free energy between the trapped and released states changes, causing the free state to now have lower energy. For long polymers, this is typically achieved by modulating the enthalpy portion of the free energy by subjecting the molecules to external forces (such as hydrodynamic drag from fluid flow) or by applying an electric field to molecules with a net charge (such as DNA).

[0213] The strength of a trap can be understood in a probabilistic sense, meaning that the probability of escaping from a trap decreases as the trap's energy increases. A well-balanced trap will hold an item until it is removed (displaced) by external force applied to the item or by manipulation or adjustment of the trap itself.

[0214] The behavior of smaller traps that capture long polymers is influenced by and can be modulated by the chemical characteristics of the long polymer, which in turn can be modulated by buffering conditions and local chemical environments. At shorter length scales, the extension direction of a segment of the polymer depends on the orientation of its preceding segments and is quantified by an intrinsic parameter called the duration of the long polymer. A conformation requiring the long polymer to bend sharply relative to its duration will result in spring energy. At longer length scales, self-avoidance is dominant because when the polymer forms a ring, it cannot overlap with previous segments. This loss of entropy is described by the excluded volume energy, which is proportional to the molecular diameter and net electrostatic charge.

[0215] In some implementations, deformable objects can overcome the entropy barrier, at least in part, due to changes in environmental conditions (e.g., temperature, pH, pressure) that reduce or completely remove it. For example, long nucleic acid molecules can change their radius of gyration by altering the ion concentration of the solution, thereby allowing manipulation of the entropy barrier to be highly effective [Dai, 2016].

[0216] Long polymer chains (such as nucleic acids) that remain stationary in solution will form a random coil configuration, the outer boundary of which can be approximately spherical, and its radius is controlled by the properties of the solution and the molecule itself. This is the lowest energy state of the polymer in solution, and if left undisturbed in solution, the polymer will naturally return to this state. However, when the polymer is subjected to physical features and / or external forces that limit its ability to take a random coil conformation, the polymer chain will be physically manipulated to a higher energy state. Conversely, when the physical boundary and / or external force are removed, the polymer chain will return to a spherical random coil configuration [Reisner, 2005][Han, 2007][Dai, 2016].

[0217] Previously, the interaction between long nucleic acid fragments and entropy traps and entropy barriers in fluid environments was demonstrated [Craighead, 1999, 6, 635, 163]. Here, the entropy barrier is an increase in physical confinement such that the overall energy state of the nucleic acid increases as the fragment is transported to a more confined region. The amount of energy state change depends on the physical feature size, solution composition, and physical properties of the polymer. The increase in energy provides a barrier so that long nucleic acid fragments will not move into higher energy states without a sufficiently large external force. However, by applying a sufficiently large external force, long nucleic acid molecules can occupy more confined regions [Craighead, 1999, 6, 635, 163].

[0218] Similarly, long nucleic acid molecules in an entropy trap will not escape unless a sufficiently large external force is applied. Furthermore, long nucleic acid fragments that are physically in contact with the trap (e.g., by external force or Brownian motion) will relax into the trap. Long nucleic acid molecules relax into the trap until their total energy state is minimized. Therefore, if the physical size of the trap is small enough, only a portion of the long nucleic acid fragment can occupy the trap. This has been previously shown where, in each trap, small “depressions” (trapholes) are used to trap subunits of long nucleic acid molecules in each trap, consisting of deformable objects containing random curls, which are connected to each other by elongated portions of the molecules, forming a “pearls on string” configuration [Reisner, 2009].

[0219] Besides long nucleic acid molecules (long polymers), droplets (and in some cases, cells) are also deformable objects that can be manipulated by entropy barriers, entropy slopes, and entropy traps. A droplet flowing in a channel will stop at a contraction (entropy barrier) and will not pass through unless a sufficiently large force (e.g., pressure) is applied to the droplet. Furthermore, a droplet can be trapped between two contraction points, and thus trapped in an entropy trap, again until a sufficiently large external force (e.g., pressure) is applied to release the droplet from the trap. [Tan, 2004] [Fraden, 2007, 8,592,221] [Baroud, 2010]. [Abbyad, 2011] shows another example of an entropy trap where the droplet is “pinned” (“captured”) along the track because the droplet has a locally lower energy state by relaxing into the track structure. For all implementations, the physical confinement sizes of the entropy barrier and entropy trap will vary with the deformable object, where the entropy barrier and entropy trap are designed to interact with the deformable object within them. For example, a 300 nm nanodepression is suitable for capturing a 10 kbp segment of a 500 kbp long nucleic acid molecule, while a 20 μm constriction is suitable as an entropy barrier for a 1 nL water-in-oil droplet.

[0220] 5.27. Gel A “gel” is defined as a substantially thin or porous system containing a cross-linked (“gelling”) “gelling agent”. Non-limiting examples of gels include agarose, polyacrylamide, hydrogels [Caló, 2015], and DNA gels [Gačanin, 2020]. In the context of this document, gels and semi-gels are equivalent, wherein a semi-gel is a gel having incomplete cross-linking and / or a low concentration of gelling agent. In some embodiments, the gel may contain photodegradable properties. In some embodiments, the cross-linking of the gelling agent may include photoactivation.

[0221] 5.28. Environmental Conditions "Environmental conditions" can include any physical, material, or chemical properties surrounding a biomolecule that can affect its physical, thermodynamic, chemical state, or reactivity to other reagents. The influence on the biomolecule can be caused by the presence or change of environmental conditions. Environmental conditions can include temperature, pressure, humidity levels, pH, ion concentration, flow rate, or direction. Environmental conditions can include light flux, polarization, and wavelength intensity. Environmental conditions can include solution composition, such as the concentration of a specific reagent in the solution, the proportion of certain reagents in the solution, or the salt composition used in a specific buffer solution. Environmental conditions can include external forces acting on the biomolecule, such as solution or air flow rates. Environmental conditions can include thermal conductivity, electrical conductivity, optical opacity, or transparency. Environmental conditions can include electric or magnetic fields. Environmental conditions can include sound of a specific frequency or intensity. Environmental conditions can include sound waves of a specific frequency or intensity. Environmental conditions can include gravity or effective gravity. Environmental conditions can include vapor pressure. Environmental conditions can include the composition of gases in the atmosphere. Environmental conditions can include the composition of vapors in the atmosphere. Environmental conditions can include the relative volatility of the atmosphere.

[0222] 5.29. Applied force "External force" or "force applied from the outside" or "applied force" is any force applied to a body such that it can disturb the body from a state of rest or can accelerate (or decelerate) the object. Additionally, an external force is any force applied to a body such that removing the force from the object can disturb the object from a state of rest or can accelerate (or decelerate) the object. Non-limiting examples include hydrodynamic forces exerted by fluid flow [Larson, 1999] (which can be simulated by pressure difference, gravity, capillary action, thermocapillary convection, electroosmosis, convection, surface tension, surface tension gradient, and the Maragoni effect), electric fields, electrodynamic forces, electrophoretic forces, pulsed electrophoretic forces, magnetic forces, dielectric forces, centrifugal acceleration, or combinations thereof. Furthermore, external forces can be applied indirectly, for example, if a bead is attached to a body and then the bead is subjected to an external force, such as a magnetic field or an optical teaser.

[0223] 5.30. Distribution System As used herein, a “dispensing system” or “dispenser” is an instrument or a component of an instrument capable of dispensing a volume of liquid from a dispensing tip, nozzle, or orifice (collectively referred to herein as a “tip”) at a desired location in (x, y, z) space. In some embodiments, the liquid is dispensed in a continuous flow. In some embodiments, the liquid is dispensed as a series of droplets. Droplet sizes can be 100 microliters or less, 10 microliters or less, 1 microliter or less, 100 picoliters or less, 10 picoliters or less, 1 picoliter or less, 100 feli or less, 10 feli or less, 1 feli or less, 100 atl or less, or 10 atl or less. In some embodiments, the tip includes a consumable pipette tip. In some embodiments, the dispenser tip is also capable of extracting a solution from a target solution in (x, y, z) space, and therefore the dispenser is also an “extractor.” In some embodiments, the dispensing tip and the extraction tip are different tips. In some embodiments, they are the same. In some embodiments, the tip is a microsyringe, or the end of a capillary, or a nozzle. In some implementations, the dispensing of the liquid is controlled by air displacement through pressurized air conduits or by a syringe pump moved by an electromechanical system such as a stepper motor.

[0224] In some implementations, an inkjet dispenser can be used. Inkjet printing includes continuous jet (CJ) and drop-on-demand jet (DODJ). CJ, based on a transducer, charging electrodes, and an electric field, can continuously generate droplets, and the position of the droplets on the substrate can be determined by the charge density of the droplets. Several actuators are available for DODJ devices, including piezoelectric actuators, thermal actuators, solenoid actuators, pneumatic actuators, magnetostrictive actuators, and acoustic actuators. In particular, there are two actuation modes for piezoelectric microjet devices: single actuation and hybrid actuation. Single actuation modes include shearing, squeezing, bending, pushing, and needle-impact modes, while hybrid actuation refers to electrohydrodynamic (EHD) assisted actuation. [Li, 2019] provides a detailed review of different inkjet technologies, which are incorporated herein by reference in their entirety.

[0225] In some embodiments, the dispenser comprises a contact probe capable of transporting and depositing solution droplets through contact wetting. In some embodiments, droplet extraction from a surface is accomplished by the contact probe contacting and wetting the droplet.

[0226] 5.31. Contact Probe The "contact probe" system used in this paper refers to an instrument or component within an instrument capable of positioning the point or tip of the contact probe relative to a surface at a desired location in (x, y, z) space with nanometer-level or better positioning accuracy and measuring a signal that varies with the xy or xyz position. In a preferred embodiment, the contact probe is capable of measuring the signal based on its interaction with a physical object. In a preferred embodiment, the contact probe is part of a contact probe probing system, which is itself a type of probing system. In a preferred embodiment, the contact probe is a surface scanning probe capable of generating a signal as the probe physically moves relative to the surface in xyz space through the instrument. Different types of contact probes include SPM (Scanning Probe Microscopy), AFM (Atomic Force Microscopy), HS-AFM (High-Speed ​​Atomic Force Microscopy), STM (Scanning Tunneling Microscopy), SPE (Scanning Probe Electrochemistry), CFM (Chemical Force Microscopy), and LFM. Lateral force microscopy, magnetic force microscopy (MFM), high-frequency MFM, magnetoresistive sensitivity mapping (MSM), electric field microscopy (EFM), scanning capacitance microscopy (SCM), scanning diffusion resistance microscopy (SSRM), tunneling AFM and conductive AFM, contact AFM, non-contact AFM, dynamic contact AFM, tapping AFM, Kelvin probe force microscopy (KPFM), piezoelectric response force microscopy (PFM), photothermal microspectroscopy, scanning gate microscopy (SGM), scanning quantum dot microscopy (SQDM), scanning voltage microscopy (SVM), force-modulated microscopy (FMM), ballistic electron emission microscopy (BEEM), electrochemical scanning tunneling microscopy (ECSTM), scanning Hall probe microscopy (SHPM), spin polarization scanning tunneling microscopy (SPSM), photon scanning tunneling microscopy (PSTM), scanning The various scanning probe microscopy systems include: Scanning Tunneling Potential (STP), Synchrotron Radiation X-ray Scanning Tunneling Microscopy (SXSTM), Scanning Probe Electrochemistry (SPE), Scanning Electrochemical Microscopy (SECM), Scanning Ion Conductivity Microscopy (SICM), Scanning Vibrating Electrode Technology (SVET), Scanning Kelvin Probe (SKP), Fluid Dynamics Microscopy (FluidFM), Characteristic Orientation Scanning Probe Microscopy (FOSPM), Magnetic Resonance Force Microscopy (MRFM), Near-Field Scanning Optical Microscopy (NSOM), Scanning Near-Field Optical Microscopy (SNOM), Scanning Squid Microscopy (SSM), Scanning Scattering Resistance Microscopy (SSRM), Scanning Thermal Microscopy (SThM), Scanning Single Electron Transistor Microscopy (SSET), Scanning Thermionic Microscopy (STIM), Charge Gradient Microscopy (CGM), and Scanning Resistance Probe Microscopy (SRPM). For a review of different scanning probe microscopy systems, see [Takahashi, 2017].For clarity, contact probes do not necessarily require close physical contact with the sample or any object to measure signals from the sample.

[0227] In some embodiments of contact probes, including AFM systems, the system can operate in a variety of different modes and thus measure a variety of different signals, depending on the probe type, its operating mode, and the choice of probe tip sharpness. Non-limiting examples of different AFM modes include non-contact mode, contact mode, tapping mode, dry mode, wet mode, high-frequency mode, ultra-high-frequency mode, force-modulated mode, conductive mode, magnetic mode, ultra-sharp tip mode, diamond tip mode, high aspect ratio mode, electron beam deposition tip mode, and carbon nanotube tip mode. In some embodiments, the contact probe can operate in a dry, humid, or liquid environment. In some embodiments, the point of the contact probe can be functionalized with chemical parts, biosomes, or affinity groups to achieve biochemical interactions with the physical object being probed. For a review of the various functionalizations exhibited on contact probes, see [Ebner, 2019]. In some embodiments, the point of the contact probe may include carbon nanotubes, nanorods, or nanospiks. In some implementations, the tip of the contact probe may include a hole or nanopore that allows fluid to connect to a fluid channel or fluid chamber within the contact probe.

[0228] In AFM microscopy, the probe is attached to a spring-loaded or flexible cantilever that contacts the surface to be analyzed. Contact occurs within the molecular force domain (i.e., within the range of van der Waals interactions). Different operating modes are possible in AFM, including contact mode, non-contact mode, and TappingMode™.

[0229] In contact mode, the atomic forces between the probe tip and the sample surface are measured by maintaining a constant tip-sample distance and measuring the deflection of the cantilever, typically by reflecting laser light from the cantilever onto a position-sensitive detector. The cantilever deflection causes a change in the position of the reflected laser beam. As in STM, the height of the probe tip can be computer-controlled using a piezoelectric element with feedback control. In some embodiments of this disclosure, a relatively constant degree of deflection is maintained by raising or lowering the probe tip. Because the probe tip may actually (van der Waals) contact with the sample, contact mode AFM tends to deform non-rigid samples. In non-contact mode, the tip is maintained between approximately 50 and 150 angstroms above the sample surface, and the tip is oscillating. The van der Waals interaction between the tip and the sample surface is reflected in changes in the phase, amplitude, or frequency of the tip oscillation. The resolution achieved in non-contact mode is relatively low.

[0230] In TappingMode™, the cantilever uses a piezoelectric element to oscillate at or near its resonant frequency. The AFM tip periodically contacts (taps) the sample surface at a frequency of approximately 50,000 to 500,000 cycles per second in air and at a lower frequency in liquids. As the tip begins to contact the sample surface, the amplitude of the oscillation decreases. The change in amplitude is used to determine the morphological properties of the sample. 6. Detailed Explanation

[0231] 6.1. Isolation of ecDNA in a fluidic apparatus This document describes apparatus and methods for separating or enriching one or more ecDNA molecules derived from at least one cell and cellular DNA derived from the same at least one cell. In some embodiments, the method for separating or sorting ecDNA from cellular DNA uses information including the size of the ecDNA. In some embodiments, the method for separating, enriching, or sorting ecDNA from cellular DNA uses information including the topological structure of the ecDNA. In some embodiments, the method for separating, enriching, or sorting ecDNA from cellular DNA uses information including the circular conformation of the ecDNA. In some embodiments, the method for separating, enriching, or sorting ecDNA from cellular DNA uses information including the genomic sequence within the ecDNA. In some embodiments, the method for separating, enriching, or sorting ecDNA from cellular DNA uses information including genomic sequences excluded from the ecDNA. In some embodiments, the method for separating, enriching, or sorting ecDNA from cellular DNA uses information including markers specifically bound to the ecDNA. In some embodiments, the method for separating, enriching, or sorting ecDNA from cellular DNA uses information including markers specifically bound to the cellular DNA. In some embodiments, the method for separating, enriching, or sorting ecDNA from cellular DNA uses information including the methylation pattern or density within the ecDNA. In some embodiments, methods for separating, enriching, or sorting ecDNA from cellular DNA use information including the presence, type, or density of higher nucleic acid structures associated with the ecDNA. In some embodiments, methods for separating, enriching, or sorting ecDNA from cellular DNA use information including the identification of the cellular DNA. In some embodiments, at least one ecDNA is probed within a fluid device using a probing system. In some embodiments, the operation and control of the fluid device are at least partially under the control of the probing system.

[0232] 6.1.1. Implementation plan for cellular DNA In some embodiments, the cellular DNA comprises all nucleic acid or ribonucleic acid molecules derived from the cell, excluding the specific ecDNA to be isolated. In some embodiments, the cellular DNA comprises the entire chromosome. In some embodiments, the cellular DNA comprises a portion of the chromosome. In some embodiments, the cellular DNA comprises fragments of the chromosome. In some embodiments, the cellular DNA comprises mitochondria. In some embodiments, the cellular DNA comprises plasmids. In some embodiments, a portion of the chromosome is obtained by cutting the chromosome. In some embodiments, cutting is performed by cleaving the chromosome, or by digesting the chromosome, or by photolysis, or by chemical cleavage, or by acoustic treatment of the chromosome. In a preferred embodiment, the ecDNA is unaffected by the cutting method. In some embodiments, the ecDNA is less sensitive to the cutting method. Examples include digesting the chromosome with a sequence-specific target whose sequence is not present in the ecDNA (such as a centromere-specific cleaver or a telomere-specific cleaver). In some embodiments, the ecDNA is unaffected or less sensitive to cutting methods that apply enzymes, chemicals, heat, reagents, light energy, fluid shear force, or acoustic treatment only to cellular DNA and not ecDNA. In some implementations, ecDNA is unaffected by or less sensitive to methods that allow selective cleavage of cellular DNA by means of thresholding enzymes, chemicals, reagents, heat, light, or acoustic treatments, which result in the relatively higher physical integrity of ecDNA relative to cellular DNA.

[0233] In some embodiments, the cellular DNA itself is a type of ecDNA; however, the cellular DNA has at least one property that distinguishes it from at least one ecDNA to which it is desired to be separated. In some embodiments, both the at least one ecDNA and the at least one cellular DNA are ecDNAs of different lengths. In some embodiments, the length difference exceeds 100 bp, or exceeds 1,000 bp, or exceeds 10,000 bp, or exceeds 100,000 bp, or exceeds 1,000,000 bp. In some embodiments, both the at least one ecDNA and the at least one cellular DNA are ecDNAs of different qualities. In some embodiments, both the at least one ecDNA and the at least one cellular DNA are ecDNAs of different topologies, particularly linear or circular topologies. In some embodiments, both the at least one ecDNA and the at least one cellular DNA are ecDNAs with different sequences in the whole or in parts of each molecule. In some embodiments, both the at least one ecDNA and the at least one cellular DNA are ecDNAs with different genes or ecDNAs with the same gene but one or two molecules whose genes have been mutated or modified so that the genes are no longer the same. In some embodiments, both the at least one ecDNA and the at least one cellular DNA are ecDNAs with similar sequences but whose respective sequences differ due to at least one mutation. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs having sequences that differ due to at least one structural variation (e.g., SNP, insertion, deletion, inversion, or translocation). In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs with different breakpoints. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs with different origins within a chromosome or originating entirely from different chromosomes. For example, at least one ecDNA may originate from chromosome 1, and at least one cellular DNA may originate from chromosome 10. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs with different oncogenes or completely lacking such oncogenes. For example, at least one ecDNA may contain an oncogene, while at least one cellular DNA may not contain an oncogene, or may contain different oncogenes. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs with different states relative to the amount of binders. For example, at least one ecDNA may have one or more binders, while at least one cellular DNA may have none. In another example, at least one ecDNA may have one binder, while at least one cellular DNA may have two binders.In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs in different states relative to the type of binder. For example, at least one ecDNA may bind to a type A binder, while at least one cellular DNA may bind to a type B binder. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs in different states relative to the position or orientation of one or more binders. For example, one or more binders may bind at different positions along the sequence. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs from cells of different origins. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs from tissues of different origins. For example, at least one ecDNA may be derived from liver tissue, and at least one cellular DNA may be derived from heart tissue. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs from disease states of different origins. For example, at least one ecDNA may be derived from a tissue sample diagnosed with cancer cells, while at least one cellular DNA may be derived from a tissue sample from which cancer cells have not been identified. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs from tumor states of different origins. For example, at least one ecDNA may be derived from a tumor sample, while at least one cellular DNA may be derived from a non-tumor sample. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs of different organisms. For example, at least one ecDNA may be derived from a human patient, and at least one cellular DNA may be derived from a virus, bacteria, or a different patient. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs with different amounts of higher nucleic acid structures. For example, at least one ecDNA may have 100 binding nucleosomes, while at least one cellular DNA may have none or fewer than 10. In another example, at least one ecDNA may have several cohesion protein loops, while at least one cellular DNA may not. In some embodiments, at least one ecDNA and at least one cellular DNA are ecDNAs having higher nucleic acid structures at different locations or in different orientations. In some embodiments, at least one ecDNA and at least one cellular DNA may have a relationship that is a combination of any of the previous embodiments.

[0234] 6.1.2. Application Implementation Plan Figure 7The present disclosure illustrates an implementation workflow comprising a process of lysing a sample of aggregated cellular DNA (703) and ecDNA (702) derived from at least one cell (701) in solution to produce substantially intact cellular DNA (706) and ecDNA (705) (the solution is illustrated herein as contained within a laboratory tube (704)), purifying the cellular DNA (706) and ecDNA (705) together and away from nuclear and cytoplasmic debris, and then introducing at least a portion of the solution containing the cellular DNA and ecDNA into a fluid device (707), wherein the ecDNA is separated to produce purified ecDNA (708) from the input ecDNA and / or information (709) about the input ecDNA. In a preferred embodiment, the information is generated at least in part by probing the ecDNA and / or cellular DNA on / within the fluid device using a probing system. In a preferred embodiment, the cells are derived from the sample. In a preferred embodiment, the sample is derived from a human. In some embodiments, the output of the fluid device is purified cellular DNA, wherein the ecDNA is at least partially removed.

[0235] In some embodiments, at least a portion of the process of lysing at least one cell and releasing cellular DNA and ecDNA from at least one cell is also performed within a fluid device. In a preferred embodiment, the fluid device for lysing at least one cell and the fluid device for isolating ecDNA are the same fluid device.

[0236] In all embodiments, ecDNA is separated from cellular DNA within a fluidic apparatus. In some embodiments, the separation is physical. In some embodiments, the separation is accomplished via computer simulation by probing a sample within the fluidic apparatus and analyzing the probing data to identify at least one ecDNA.

[0237] In a preferred embodiment, probing the ecDNA within the fluid device allows for the generation of an ecDNA profile of the input sample. In some embodiments, the profile includes confirmation of the presence or absence of ecDNA within the input sample. In some embodiments, the profile includes the amount or quantity of ecDNA or cellular DNA, or a proportion thereof, within at least one cell containing the input sample within the fluid device. In some embodiments, the profile includes the amount or quantity of ecDNA or cellular DNA, or a proportion thereof, within a sample volume of the cell or within a certain quantity or range of quantities.

[0238] In a preferred embodiment, an output sample is obtained from a fluid device, wherein the output sample contains purified ecDNA. In some embodiments, the output sample has an ecDNA to cellular DNA ratio of more than 1:1 by mass, or more than 2:1 by mass, or more than 3:1 by mass, or more than 4:1 by mass, or more than 5:1 by mass, or more than 7:1 by mass, or more than 10:1 by mass, or more than 15:1 by mass, or more than 20:1 by mass, or more than 50:1 by mass, or more than 100:1 by mass. In some embodiments, the ratio of ecDNA to cellular DNA in the output sample is more than 1:1 by mass, wherein the total mass includes only nucleic acid molecules greater than 5 bp, or greater than 10 bp, or greater than 20 bp, or greater than 50 bp, or greater than 100 bp, or greater than 500 bp, or greater than 1000 bp, or greater than 10,000 bp, or greater than 100,000 bp.

[0239] In some embodiments, the output sample has an effective ecDNA molecule count to effective chromosomal DNA molecule count ratio of more than 1:1, wherein the effective ecDNA molecule count is defined as the total ecDNA mass divided by the average mass of ecDNA, and the effective chromosomal DNA molecule count is defined as the total chromosome mass divided by the average chromosome mass. In some embodiments, the output sample has an effective ecDNA molecule count to effective chromosomal DNA molecule count ratio of at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 100:1, at least 200:1, at least 500:1, or at least 1000:1. In some cases, the output sample does not contain chromosomal DNA.

[0240] In some embodiments, the proportion of ecDNA in the output sample obtained from the fluid device relative to the total mass of nucleic acids in the output sample exceeds 10%, or exceeds 20%, or exceeds 30%, or exceeds 40%, or exceeds 50%, or exceeds 60%, or exceeds 70%, or exceeds 80%, or exceeds 90%, or exceeds 95%, or exceeds 98%, or exceeds 99%, or exceeds 99.5%, or exceeds 99.9%. In some embodiments, the proportion of ecDNA in the output sample obtained from the fluid device relative to the total mass of nucleic acids in the output sample exceeds 70%, wherein only the mass of nucleic acid molecules with a length greater than 5 bp, or greater than 10 bp, or greater than 20 bp, or greater than 50 bp, or greater than 1000 bp is considered.

[0241] In some embodiments, the output sample has a 2× or greater increase in ecDNA concentration compared to the input sample, wherein the ecDNA concentration is defined as the percentage of the total DNA mass in the sample from which the ecDNA is derived. In some embodiments, the output sample has a 3× or greater increase in ecDNA concentration, or a 4× or greater increase, or a 5× or greater increase, or an 8× or greater increase, or a 10× or greater increase, or a 15× or greater increase, or a 20× or greater increase, or a 50× or greater increase, or a 100× or greater increase, or a 500× or greater increase, or a 1000× or greater increase.

[0242] In some embodiments, the output sample is collected using an automated or manual pipette. In some embodiments, the output sample is directly connected to another fluid device via a fluid connector interface. In some embodiments, the output sample is collected from the fluid device using a contact probe. In some embodiments, the output sample from the fluid device is encapsulated in at least one droplet. In some embodiments, the output sample is a solution that is then encapsulated in at least one droplet.

[0243] In some embodiments, output samples containing enriched or purified ecDNA can be used as templates to generate hybridization probes via various labeling methods such as nick translation or end-conjugation, and hybridize with chromosomes, chromatin, and long or short genomic DNA fragments from source cells to generate specific signals. This allows direct visualization of the association between the genomic contents of the ecDNA and conventional cellular genomic DNA, extrapolating the ecDNA contents from or similar locations to infer so-called "chromosomal scar regions or sequences" by combining them with specific intrinsic genomic contents information such as genome mapping barcodes or sequencing information. In some embodiments, output samples containing enriched or purified ecDNA can be used as templates to generate hybridization probes via various labeling methods such as nick translation, and hybridize with chromosomes, chromatin, and long or short genomic DNA fragments from source cells to generate specific signals. This allows direct visualization of the association between the genomic contents of the ecDNA and conventional cellular chromosomes and / or specific cells containing such hybridization signals. Such labeled cellular or subcellular samples are then applied to flow cytometry sorting mechanisms to separate ecDNA sequence-matched populations from non-ecDNA genomic contents-matched populations, and then the above embodiments are performed for further genomic analysis.

[0244] In some embodiments, output samples containing enriched or purified cDNA can be mapped and sequenced to gain insights into the sequence information of the fusion region, where two previously broken ends fused together to form a circular loop, and the flanking genomic region sequence forming the chromosomal scar can be derived from such knowledge of the circular fusion sequence to guide the design of hybridization probes for location identification or guide RNA probes for subsequent gene editing / gene therapy applications. In some embodiments, mapping includes physical mapping. In some embodiments, mapping includes karyotype analysis. In some embodiments, mapping includes linear mapping.

[0245] In another embodiment, genomic mapping or sequence information from any region of a specifically isolated and purified circular ecDNA molecule can be used to design and generate synthetic hybridization probes, such as guide RNA or any other hybridization probe, to target specific ecDNA that is quantified, visualized, and tabulated by a prospective probing system (e.g., imaging device, optical microscope, fluorescence microscope, or flow cytometry) to obtain statistical information on the distribution of ecDNA in a patient cell population sample for diagnostic and prognostic applications. In another embodiment, genomic mapping or sequence information from any region of a specifically isolated and purified circular ecDNA molecule can be used to design and generate synthetic hybridization probes, such as antisense probes, to introduce therapeutic modalities that reduce or neutralize harmful ecDNA molecules.

[0246] In another implementation, genomic mapping or sequence information from any region of a particular isolated and purified circular ecDNA molecule can be used to design and generate synthetic hybridization probes such as guide RNA or any other hybridization probe having some functional entity attached, such as an enzyme, CRISPR, antibody, or conjugate, to target a specific ecDNA to be modified, neutralized, or digested.

[0247] In another implementation, genomic mapping or sequence information of any region derived from a specific isolated and purified circular ecDNA molecule can be used to obtain insights into genomic regions carrying specific genes and functional regulatory sequences that drive such genes in a cis or trans-activation manner, in order to elucidate potential pathways that cause disease, including but not limited to carcinogenic processes and other potential complex disease etiologies.

[0248] In another implementation, specific epigenomic factors, such as specific conformations, shapes, histones, or chromatin protein factors, bound from isolated and purified ecDNA can be used as target sites to immunofluorescence stain the conjugated antibody set to identify which factors are involved in which stage of the disease, in order to monitor carcinogenesis or other disease progression.

[0249] In another implementation, specific epigenomic factors, such as specific conformations, shapes, histones, or chromatin factors, bound from isolated and purified ecDNA can be used as target sites for generating specific neutralizing factors, such as antibodies, and for subsequent therapeutic purposes of reducing or eliminating carcinogenic or other pathogenic ecDNA substances, such as ADC (antibody-drug conjugate) programs.

[0250] In some implementations, samples containing purified ecDNA can be analyzed to assess the severity and outcome of a disease, particularly cancer. For example, if the initial cell concentration used to generate the purified ecDNA sample is known, the average amount of ecDNA present in each cell can be determined. The initial cell concentration is typically in the range of ≈10-1. 5 -10 7 Cells / mL provides a robust statistical representation of ecDNA density in both circulating blood and tumor tissue.

[0251] In another implementation scheme, the isolated ecDNA particles can be pulled from the surface using laser tweezers or biotinylated chromosomes with magnetic streptavidin beads. The pulled-out ecDNA particles can be amplified and sequenced. The results can then be analyzed to understand the ecDNA content profile of single cells in the sample.

[0252] In another implementation, one or more of at least one ecDNA particle can be used to diagnose genetic disorders based on single nucleotide polymorphisms (SNPs) and to develop diagnostic tests to detect the likelihood of passing on hereditary traits to offspring.

[0253] In one embodiment of this disclosure, a sample enriched with ecDNA particles can be applied to a substrate surface, which may be flat or patterned (having channels and depressions). The sample is then brought into contact with a blade-shaped substrate material. By moving the blade or substrate in a unidirectional direction, the ecDNA-containing sample is uniformly spread as a thin film on the substrate surface. During the spreading process, ecDNA particles are deposited on the substrate in the form of a thin film, resulting in the substrate surface being covered with non-overlapping, easily countable ecDNA particles.

[0254] 6.1.2.1. Generation of ecDNA-specific probes In some embodiments, it is desirable to generate a probe containing a target sequence comprising a sequence of nucleotides capable of selectively hybridizing with at least one complementary target sequence within ecDNA without hybridizing with a secondary sequence. In a preferred embodiment, the secondary sequence comprises all or a portion of the genome of an organism or human from which the ecDNA sample is derived. In some embodiments, the secondary sequence includes a reference genome sequence. Figure 8The document illustrates exemplary embodiments of a workflow for determining the target sequence of a probe. In some embodiments, a reference genome sequence is generated at least in part by computer simulation including sequence information obtained by sequencing at least a portion of a human genome, which is also the source of the ecDNA. In a preferred embodiment, the ecDNA sequence for selecting the target sequence is generated at least in part by sequencing a purified ecDNA sample produced from a fluid device. In some embodiments, the ecDNA sequence for selecting the target sequence is generated at least in part by sequencing chromosomal DNA from which ecDNA has been purified and comparing the chromosomal sequence data with sequence data generated by sequencing a genomic sample from the same person having unpurified ecDNA.

[0255] In some embodiments, the probe includes a hybridization probe, a guide RNA probe, a FISH probe, or an array probe. In some embodiments, the target sequence is at least 5 bp in length, or at least 6 bp, or at least 7 bp, or at least 8 bp, or at least 9 bp, or at least 10 bp, or at least 12 bp, or at least 14 bp, or at least 16 bp, or at least 18 bp, or at least 20 bp, or at least 22 bp, or at least 24 bp, or at least 26 bp, or at least 28 bp, or at least 30 bp. In some embodiments, the resulting probe contains a single-stranded portion. In some embodiments, the resulting probe contains a double-stranded portion. In some embodiments, the probe contains a loop.

[0256] In some embodiments, the probe comprises more than one nucleic acid that has been directly isolated from and enriched with ecDNA by any of the methods described herein. In some embodiments, the cells are grown in the presence of a metabolic marker (preferably a metabolic marker containing a reactive group, most preferably EdU), and the probe is labeled with a marker containing a complementary moiety (most preferably a sulfonylcy5 azide) capable of reacting with the reactive group.

[0257] In some embodiments, the probe comprises more than one nucleic acid amplified from material isolated from cells by any of the methods described herein, preferably from a sample enriched with ecDNA, and amplified using the Ampli 1 whole genome amplification kit. The probe may also contain a polymerase label applied using random primers, preferably using the Aglient SureTag kit.

[0258] In some embodiments, the probe comprises more than one nucleic acid that is isolated or derived from cells by any of the methods described herein, preferably from a sample enriched with ecDNA, and subsequently depletes the nonspecific binding region by hybridization with a DNA library containing repetitive sequences such as Cotl-DNA.

[0259] In some implementations, the probe contains more than one nucleic acid derived from a clonal portion of DNA present in a vector, including large fragments from BAC, PAC, HAC, and YAC. The clonal portion may be derived from previously isolated ecDNA or from healthy chromosomal DNA selected based on prior knowledge of the ecDNA sequence, whether from previous genomic testing or sequencing of the ecDNA or from ecDNA identification by any of the methods described herein.

[0260] In some implementations, the probe includes a marker, primer, barcode, or polymer.

[0261] In some embodiments, the probe is bound to an array, or beads, or dendritic polymer, or nanospheres, or quantum dots. In some embodiments, the probe is bound via a cleavable connector.

[0262] In a preferred embodiment, at least a portion of the target sequence is determined by comparing the ecDNA sequence, generated from purified ecDNA using any of the methods described herein or a combination thereof, with a reference, and selecting at least a portion of the ecDNA sequence as a complementary target sequence (wherein the selected complementary target sequence is not included in the reference). In some embodiments, a complement to the target sequence is selected. In some embodiments where the ecDNA sequence contains a sequence portion that is identical or similar to a sequence portion in the reference, the target sequence may contain a fused sequence region spanning said portion. For example, the ecDNA may contain portions of the human genome fused together at breakpoints, one or more of said breakpoints not present in the normal human genome. In some embodiments, the target sequence may include at least 1 bp, or at least 2 bp, or at least 3 bp, or at least 4 bp, or at least 5 bp from each flanking side of the breakpoint. In some embodiments, the breakpoint region may contain an addition of at least 1 bp not present in the human genome. In some embodiments, the breakpoint region may contain a removal of at least 1 bp present in the human genome. In some embodiments, the breakpoint region may contain a modification of at least 1 bp not so modified in the human genome.

[0263] Figure 9A specific example of ecDNA genome sequence 901 is shown, which includes two sequence portions 903 and 907, both of which can be present in the genome of the organism from which this ecDNA is derived. However, in this example, the two portions do not contact each other, or are not in contact with each other. Figure 9 The orientation fusion shown in the figure ensures that sequence 905, containing the first breakpoint 906, and sequence 904, containing the second breakpoint 902, are not included in the genome of the source organism. In this example, the probe target sequence may contain either 905 or 904. 915 illustrates a particular embodiment of such a probe, wherein the probe contains sequence 912 originating from one side of the breakpoint (916) and sequence 913 originating from the other side of the breakpoint. In some embodiments, probe 915 may also contain additional sequences or material at one end (911) or the other end (914) or both ends.

[0264] In some implementations, the target sequence may comprise a portion of a sequence present in ecDNA but not derived from the human genome. In some implementations, this portion of the sequence may originate from a mutation in ecDNA, an insertion into ecDNA, or a deletion from ecDNA.

[0265] 6.1.2.2. FISH probe generation FISH probes are a relatively old technique that allows researchers or physicians to map the location of corresponding sequences within interphase or condensed nuclei with microscopic precision. Furthermore, signal intensity and spatial distribution can be used alone or in conjunction with internal controls such as multicolor FISH (in which ecDNA probes and chromosome sequence probes are added together in a mixture) to measure the frequency of occurrence of a particular DNA sequence. In this way, it is possible to distinguish between cells containing ecDNA and those without ecDNA under a microscope.

[0266] In some embodiments, this disclosure takes a sample of live cells and possibly one or more additional samples of cells, tissues, or mixtures, including but not limited to: live cells, fixed cells, cells or tissues rapidly frozen on a cryostat or by other freezing means, or formalin-fixed paraffin-embedded (FFPE) tissue samples. Live samples are cultured and agglutinated, released, and purified or enriched with chromosomes and ecDNA, as discussed by any of the methods described in this disclosure. After further filtration or size-based separation to isolate or enrich fractions of ecDNA, the fractions may be used with or amplified to generate various probes, such as FISH probes, capture probes, or sequencing primers. In the following discussion, labeling methods can be applied not only to ecDNA-rich samples but also to ecDNA-depleted samples; however, for the sake of brevity, the discussion will be limited to ecDNA-rich samples.

[0267] Many possible methods exist for constructing probes, and the following examples are non-limiting. If cells are grown with metabolically labeled nucleosides such as BrdU that can be recognized by antibodies, or nucleosides with click chemistry capabilities such as EdU (5-ethynyl-2'-deoxyuridine), and other nucleotides besides those capable of participating in strain-promoted azide-alkyne cycloaddition (SPAAC) and the anti-electron-demanding Diels-Alder (iEDDA) reaction (Ganz 2020), FISH probes can be generated from purified ecDNA without amplification and subsequently labeled with complementary fluorophores. Metabolic labeling can also be performed using enzymatic methods (Liu 2022). Probes can be enzymatically or non-enzymatically cleaved before or after labeling.

[0268] Amplification-based methods can insert modified nucleotides, either by directly inserting fluorescent nucleotides via polymerase-based incorporation or by adding nucleotides with reactive handles capable of covalently linking to fluorophores such as aminoallyl-UTP. Amplification methods are not limited to nick translation, random primer amplification, rolling circle replication, multiple substitution amplification, and reverse transcription.

[0269] Ligation-based methods can be used to ligate affinity-tagged oligonucleotides or oligonucleotides with markers such as fluorophores to fragments of enriched ecDNA samples. Transposon-based methods, including those present in commercial DNA sequencing library preparations, can be used to attach primers to ecDNA fragments that can then be amplified using complementary primers, or the added transposon fragments may contain affinity molecules or physical markers.

[0270] Secondary labeling steps can also be used for detection, such as enzymatic incorporation of digoxigenin-containing nucleotides or various haptens used for immunoassay, or the addition of biotin or desiobiotin to metabolically labeled nucleotides based on click chemistry, followed by detection by labeled streptavidin.

[0271] 6.1.2.3. Capture Probe Capture probes can be prepared via metabolic labeling and subsequent chemical reaction with a complementary moiety. In some embodiments, EdU is metabolically incorporated during cell growth, followed by enrichment of ecDNA as discussed in this disclosure, and then CUAAC is used to add azide-Peg3-biotin and cleave to produce a native ecDNA molecule with an affinity tag for subsequent capture. Similar to the polymerase-based methods for amplifying ecDNA described above, affinity tags can be added to a population of purified or enriched ecDNA.

[0272] 6.1.2.4. FISH probes generated from ecDNA for detecting ecDNA in histological samples After FISH probes are generated, they can be used to probe samples other than the samples used to create the probes.

[0273] In one embodiment, the FISH probe is used to investigate a fixed tissue sample to detect the abundance of ecDNA in cells associated with the original cells used to generate the FISH library. The original sample may be derived from cultured cells, an animal model of cancer, or a biopsy taken from a human or animal patient with suspected cancer. In the latter case, the pathology laboratory and medical practitioner who collected the sample processes it to the extent that a standard biopsy or sample collection for karyotype analysis is being performed. Karyotype analysis is typically not performed for some cancer types that do not produce long chromosomes, and therefore the resolution of karyotype analysis is poor, but this limitation is not intended to limit the scope of this disclosure, where short chromosomes are intentionally prepared. In some embodiments, the second sample to be probed by the FISH probe is derived from the same sample, and in other embodiments, the sample to be probed is derived from a second sample collected under conditions where the cells subsequently do not survive (such as rapid freezing or formalin-fixed paraffin-embedded (FFPE) sample preparation).

[0274] In such an implementation, FISH is performed using standard methods for slide preparation, fixation, blocking, hybridization, washing, and detection. This disclosure provides the use of FISH probes derived from ecDNA samples.

[0275] 6.1.2.5. FISH Comparison The absence of ecDNA is valuable information, but a false negative result can occur if the FISH probe fails to reach the probed region due to poor application of the probe-containing fluid, poor sample preparation, or inappropriate hybridization and washing conditions. To control for this, some embodiments use a second set of FISH probes in pairs, intended to hybridize with all cells (whether healthy or abnormal and containing ecDNA). The second set of FISH probes should be as chemically similar as possible to the first set, despite having different DNA sequences and different markers, such as fluorophores occupying different portions of the electromagnetic spectrum and distinguishable from the fluorophore. In a preferred embodiment, a tricolor fluorescence scheme is used, where a sequence-nonspecific staining agent is used as the blue-most fluorophore, a control FISH probe is used with a green or orange fluorophore, and the ecDNA FISH probe is used with the red-most fluorophore, most preferably a set of DAPI, Alexa488, and Alexa568, respectively.

[0276] 6.1.2.6. FISH probe coverage area Existing techniques for determining the location of ecDNA typically begin by obtaining sequence information about the ecDNA, at which point humans identify genes within the ecDNA that correspond to commercially available FISH probes. This illuminates localized regions of the ecDNA but does not allow for probing its entire extent. In contrast, the ability to generate FISH probes across the entire ecDNA, as described in this disclosure, allows for probing of the entire ecDNA location, leading to a more comprehensive determination of its nuclear localization and the extent of cis-interactions and loops within the ecDNA itself.

[0277] In some implementations, fluorescently labeled ecDNA FISH library fragments are used for super-resolution microscopy via oligopaint, PALM, STORM, or other techniques, and can be used to examine both the extent of ecDNA in fixed cells and the extent of ecDNA prepared in vitro and deposited on a substrate in a circular or linear manner. In the case of ecDNA evolution or heterogeneous populations, ecDNA FISH probes from one type of ecDNA are used to probe linearized ecDNA to determine an optical map of sequence similarity, which, in the case of sequence insertion, is expected to show gaps or unlabeled regions.

[0278] 6.1.2.7. Distribution of ecDNA copy number within a cell population In some embodiments, cell samples from an organism are probed to individually determine the ecDNA population count for each cell, or to collectively determine the ecDNA population count for all cell samples. In some embodiments, the ecDNA population is assessed by fluorescence imaging of each cell on a cytogenetic patch on a substrate. In some embodiments, the substrate surface is patterned with fluid features that allow for the physical separation of chromosomes and ecDNA. In some embodiments, the ecDNA selectively hybridizes to a fluorescent probe to enable differentiation from chromosomes.

[0279] 6.1.2.8. Monitor ecDNA population statistics to assess the impact of disease treatment, patient recovery, or environmental factors. In some implementations, the effects of applying chemicals, chemotherapeutic agents, biomolecules, biotherapeutic agents, CRISPR-based ecDNA cleavage, immunoderived therapeutic agents, or external physical perturbations (such as ionizing radiation or sonic shock) are assessed using the methods described in this document to determine the ecDNA population in individual cells or the total ecDNA population within a cell sample before, during, and / or after application. In such applications, the population-level distribution of ecDNA molecules per cell or the cell sampling can evolve, particularly if the therapeutic target is overexpressed by ecDNA molecules.

[0280] In some implementations, monitoring of ecDNA populations is used to adjust the dosage of a specific therapeutic agent, or to adjust the target region of the therapeutic agent, or to adjust the frequency of application of the therapeutic agent.

[0281] In some implementations, monitoring of ecDNA population statistics is used to determine the patient's recovery progress after treatment at different time points. In some implementations, monitoring is used to determine if the patient's disease has relapsed and whether further treatment is needed. In some implementations, monitoring is used to determine if the patient's disease has been cured and whether treatment can be discontinued.

[0282] In some embodiments, ecDNA population statistics are monitored to determine the impact of environmental conditions on human health. In some embodiments, environmental conditions may include exposure to radiation, including alpha, beta, or gamma radiation, or exposure to light of certain wavelengths, or exposure to certain chemicals, or exposure to certain gases, or exposure to certain toxins, or exposure to certain viruses, or exposure to certain pathogens, or exposure to certain bacteria, or exposure to certain foods.

[0283] 6.1.2.9. Distribution of ecDNA nuclear morphology in cell populations In some embodiments, cell populations are probed using ecDNA-derived FISH probes and fluorescence microscopy (preferably high-content fluorescence microscopy) to measure the subnuclear localization of ecDNA. In other embodiments, measurements are obtained multiple times to compare a baseline state with one or more perturbation states that alter the distribution of chromatin in the nucleus due to the introduction of stimuli, said stimuli being limited to the application of chemicals, chemotherapeutic agents, biomolecules, biotherapeutic agents, compounds that reduce the nuclear localization of chromatin-containing ecDNA or cause ecDNA to less frequently associate with each other or with healthy chromosomes, CRISPR-based ecDNA cleavage, immune-derived therapeutic agents, or external physical perturbations such as ionizing radiation or sonic shock.

[0284] 6.1.2.10. Flow cytometry-based FISH for ecDNA In some implementations, flow cytometry is performed using FISH probes, such as the FISH-Flow method in a flow cytometer (Arrigucci 2017), or equivalently within a cell sorter or microfluidic cell sorter that distinguishes cells based on FISH signals that differentiate cells containing ecDNA from those without ecDNA. Using FISH probes, preferably in conjunction with sequence-nonspecific staining agents such as DAPI, cell populations are analyzed by flow cytometry to determine the amount of ecDNA present in each cell and to quantify the ecDNA content distribution of large cell populations.

[0285] In some implementations, more than one FISH library is generated from different ecDNAs and labeled with mutually distinguishable markers, preferably markers that can be distinguished from each other in flow cytometry and also markers that can be distinguished from DNA sequence-independent staining agents such as DAPI. Analysis of the mixture of FISH library signals can estimate the relative abundance of each ecDNA substance within the cell population and can further resolve the cell population's response to stimuli, preferably to potential therapeutic agents.

[0286] 6.1.2.11. Cell sorting to enrich cells expressing specific ecDNA or derived ecDNA In some embodiments, sample cells are screened and sorted using ecDNA FISH probes, resulting in an enriched cell population containing more ecDNA than the bulk population. In some embodiments, the ecDNA FISH probes are used to screen circulating tumor cells in patients who have FISH libraries generated from previous biopsies or other samples believed to contain overlapping ecDNA regions.

[0287] Optionally, in some cases, they can be used to stain cells before and after therapy to predict whether a particular chemotherapy or other therapeutic intervention (such as targeted cleavage via CRISPR or other methods) is altering the amount of ecDNA or its interphase or mitotic distribution.

[0288] 6.1.2.12. Collection and Pre-amplification of ecDNA In some embodiments, the isolated or enriched ecDNA is collected in small quantities. A single 10 Mb mitotic (2-C) ecDNA is equivalent to 20 fg DNA, and a single 100 kb mitotic ecDNA is equivalent to 0.2 fg DNA, wherein the additional mass of proteins, metals, electrolytes, and other components of chromatin does not account for the indicated mass. In some embodiments, the total mass of isolated DNA is 0.2 fg, 1 fg, 3 fg, 10 fg, 30 fg, 100 fg, 300 fg, 1 pg, 3 pg, 10 pg, 30 pg, 100 pg, 300 pg, 1 ng, 3 ng, 10 ng, 30 ng, 300 ng, 1 μg DNA, 3 μg DNA, or more.

[0289] In some implementations, whole-genome amplification is used to pre-amplify isolated or enriched ecDNA by approximately 1 million-fold. Various methods exist for pre-amplification, such as those reviewed in Kroneis 2015, which are incorporated herein by reference. Pre-amplification methods are not limited to: scattered repeat sequence (IRS) PCR, IRS PCR with Alu elements, degenerate oligonucleotide priming (DOP) PCR, DOP PCR with substitution polymerase, primer extension pre-amplification (PEP) PCR, PEP PCR with high-fidelity Pwo polymerase, tagged random primer PCR (T-PCR), ligation-mediated PCR (LM-PCR), LM-PCR using nondeterministic random shearing, transposon insertion, chemical cleavage or enzymatic treatment for DNA shearing, LM-PCR using deterministic restriction enzyme digestion of DNA, LM-PCR using Msel restriction enzyme for deterministic digestion of DNA, LM-PCR using Msel and adaptor ligation schemes described in US10273538B2, multiple substitution amplification (MDA), MDA using Phi29 polymerase, MDA using Bst polymerase, and generating MDA with more than 10 MDA of amplicon with an average kb size; two-stage amplification using MDA with insertion of a fixed sequence motif followed by PCR amplification; PicoPlex amplification kit and derivatives; multiple annealing and circularization-based amplification cycle (MALBAC); MALBAC kit sold by Yikon Genomics; PicoPLEX single-cell WGA kit v3 sold by Takara Bio; transposon-based fragmentation followed by linear amplification; transposon-based fragmentation followed by insertion into the T7 promoter and RNA transcription. Preferred embodiments include using the Ampli1 WGA Plus kit sold by Menari Silicon Biosystems SpA and linear amplification via transposon insertion (LIANTI) (Chen 2017).

[0290] 6.1.2.13. Condensed genome mapping on chromosomes In some implementations, when probing condensed chromosomes or their portions or fragments, or abnormal chromosomes that may not be ecDNA, using karyotype analysis or other linear physical mapping methods, the application of PISH probes is used to reveal sites where ecDNA is integrating into the genome or has already produced or is producing active damage. This method is sometimes used to reveal chromosomal regions that interact trans-with ecDNA and have strong chromatin-based associations capable of withstanding the condensation process. In preferred implementations, the application of FISH probes is combined with physical mapping schemes capable of identifying chromosomes, chromosomal portions, or chromosomal fragments and providing probeable maps that can be compared to references. For various methods of generating and probing physical maps of long nucleic acid molecules, see the definition of physical mapping.

[0291] 6.1.2.14. Targeted Sequencing Libraries In a preferred embodiment, ecDNA-derived capture probes are used to generate targeted sequencing libraries of genomic DNA or RNA, wherein the capture library is used to reduce the complexity of the input library through physical interactions. The captured DNA or RNA can be sequenced after release. By sequencing only the captured nucleic acids, the limited throughput of the sequencing run is more efficiently targeted at sequences of research interest. The fact that the sequence has been captured provides additional information, particularly informing practitioners of the origin of the nucleic acid fragment and whether it is derived from ecDNA. Molecular biology methods known to those skilled in the art exist that are capable of generating capture probe libraries from large artificial DNA fragments, such as bacterial artificial chromosomes (BAC) (Day, 2014), yeast artificial chromosomes (YAC), P1 phage artificial chromosomes (PAC), and human artificial chromosomes (HAC). These methods are suitable for use with ecDNA. In some embodiments of the invention, the source is not BAC or YAC, but a purified or enriched ecDNA sample. After generation, the capture library is capable of processing DNA or RNA samples from a single cell or from more than one cell, and substantially captures only DNA or RNA that matches the sequence initially isolated from the ecDNA. In this way, sequencing of cells other than those initially used to generate the capture library allows for comparison of ecDNA in more than one different cell, which, in the case of cancer, are expected to exhibit heterogeneity in sequence, arrangement, and internuclear distribution during interphase. It should be emphasized that this method is most informative when the cells used for library analysis are closely related to those used to generate the library. Combining single-cell sequencing methods, such as droplet-based barcoding, with subsequent capture via ecDNA capture probe libraries, it is possible to generate cellular atlases based on ecDNA sequence contents, where key components are derived from one or more similarity measures, such as the presence of specific ecDNA sequences, relative or absolute copy numbers, order of contiguous regions, and the presence of translocations, insertions, and deletions, as well as epigenetic markers, such as methylation status determined by methods such as bisulfite sequencing or chromatin characterization methods such as ChlP-seq or interphase nuclear chromatin localization methods such as Hi-C.

[0292] 6.1.2.15. Single-cell ecDNA sequencing In some embodiments, a capture library is generated and applied to a single-cell-derived and barcoded DNA library, such as typically using droplet-based single-cell methods. Cells can be in any state of the cell cycle. In this embodiment, it is possible to enrich the sample with ecDNA similar to the original ecDNA used to generate the capture library, and to obtain the ecDNA distribution per cell by counting copy numbers. Where genetic variations such as SNPs accumulate near capture probes derived from the ecDNA capture probe library on the ecDNA, the SNPs can be measured on a single-cell basis.

[0293] 6.1.2.16. CTC-based ecDNA detection In some implementations, cellular DNA is derived from circulating tumor cell enrichment methods, such as commercially available kits. The DNA can then be enriched using capture probes derived from tumors suspected of having metastasized, and suspected CTC cells can be screened against ecDNA to see if they match the primary tumor sample.

[0294] 6.1.2.17. Correlation between RNA-seq and ecDNA sequencing In some implementations that collect total RNA along with DNA, it is possible to correlate the per-cell expression level of various RNAs with ecDNA copy number and SNPs. Cells can be in any state of the cell cycle.

[0295] 6.1.2.18. Oncogene Detection and Exome Sequencing In some implementations, the capture library is used to screen for the presence of well-known oncogenes on ecDNA by performing a combination of capture-based enrichment, enriching the ecDNA once with a derived ecDNA capture probe library, and generating a kit (such as a commercially available one) with an established exome or oncogene-specific targeted sequencing library.

[0296] 6.1.2.19. Determination of the interphase nuclear chromatin structure of ecDNA-associated regions In some implementations, the high-throughput chromosome conformation capture method Hi-C is first performed according to an established protocol (Lafontaine 2021 and references therein), which may include steps such as cross-linking DNA in cells, nuclease trimming, biotinylation, proximity ligation, removal of unligated fragments, DNA extraction and shearing, biotin pull-down, and PCR library preparation. The resulting PCR fragments are biotin-free, and some fractions will contain contributions from ecDNA, whether cis-ecDNA interactions within the same molecule, trans-interactions between adjacent ecDNA molecules, trans-interactions between ecDNA and chromosomal DNA corresponding to the region replicated on the ecDNA, or trans-interactions between ecDNA and chromosomal regions outside the region replicated on the ecDNA. The library will contain similar contributions from chromosomal regions from which the ecDNA capture probes are derived, and in most cases, these fragments will have low copy numbers. After PCR product generation, the PCR product is hybridized with capture libraries derived from ecDNA produced from different cells of the same sample or from related samples. The captured products are sequenced, and sequences derived from the capture process are inferred to represent nuclear interactions of intranuclear ecDNA.

[0297] In some implementations, a Hi-C plus ecDNA capture library approach is used to assess the effects of stimulation on cells, including evaluating the efficacy of potential therapeutic interventions.

[0298] 6.1.2.20. Sequence Quality Improvement Compared to total genomic DNA, the isolation of ecDNA allows for improved sequence quality and computational inference. In some implementations, purified ecDNA is sequenced using short-read or long-read sequencing and used to construct contigs of ecDNA, co-ecDNA, and co-ecDNA using data from multiple repeated sequencing runs, and to identify populations of different sequences within the same sample.

[0299] 6.1.2.21. Optical Mapping In some implementations, purified or enriched ecDNA is used as input DNA for physical mapping, such as with commercially available kits and systems or as described in the definition of physical mapping in the literature (Neely 2011) and this document. In other implementations, the map is used in combination with short-read or long-read sequencing data (possibly with healthy reference genome information) from the same or related ecDNA samples and an ecDNA-specific contig generation algorithm to generate and validate ecDNA contigs.

[0300] 6.1.2.22. Test ecDNA contig allocation. In some implementations, a combination of site-specific cleavage and gel purification of the intact ecDNA is used, preferably with pulsed-field gel purification, as performed in the CRISPR-Catch method, and thus the length of the digested fragment is determined. Most preferably, the cleavage site is derived from a contig map that describes insertions, deletions, or translocations (collectively, insertions / deletions) that differ from a healthy DNA reference sequence corresponding to a healthy DNA region from which the suspected ecDNA is derived, and the length of the cleavage fragment can be used to verify or refute an established contig map or to support various connection maps or equivalent representations of contigs during the contig generation process.

[0301] 6.1.2.23. Barcoded Individual cDNA In some embodiments, intact or substantially intact ecDNA, obtained from purification or enrichment, is placed in individual droplets and then barcoded using unique primers. A DNA sequencing library is constructed, and the sequences are assembled relative to the barcoded information. In some embodiments, statistical methods, preferably clustering methods, are used to describe the population of ecDNA molecules in the sample. In a preferred embodiment, the formation of droplets containing ecDNA or chromosomes is controlled such that the probability of more than a single ecDNA cell occupying a droplet or more than a single chromosome occupying a droplet is less than 5%, less than 1%, less than 0.1%, or less than 0.01%. In some embodiments, each droplet and its contents are associated with a unique barcode.

[0302] 6.1.2.24. Further purification In some embodiments, as described in this invention, the isolated ecDNA can be further purified, including but not limited to gel purification, surface immobilization and subsequence adsorption, pulsed-field gel separation, pulsed-field gel separation after specific or non-specific lysis, most preferably according to the CRISPR-Catch method using CRISPR-CAS9 and guide RNA complementary to a single ecDNA, using site-specific endonucleases or CRISPR selectively lysing the ecDNA, followed by exonuclease digestion of the cleaved fragments.

[0303] 6.1.2.25. Proteomics Analysis of ecDNA Unlike existing methods for ecDNA preparation (such as CRISPR-Catch) that require the protein to first be stripped from the DNA, the filtering and size- and density-dependent methods described herein utilize intact or semi-intact ecDNA containing the protein. The degree of protein preservation is determined in part by chromosome fixation and stabilization as discussed elsewhere in this disclosure and is assumed to be a statistical mixture. In some embodiments, the protein is detected on the ecDNA by immunohistochemistry. In other embodiments, the protein is extracted from the ecDNA and identified by mass spectrometry, and in some embodiments, chromatin modifications such as acetylation and methylation are identified.

[0304] 6.1.2.26. RNA silencing from ecDNA sequences In some embodiments, ecDNA sequences derived from purified ecDNA as described herein, most preferably exhibiting mutations relative to healthy DNA or producing different exons relative to RNA transcribed from healthy DNA, are used to generate siRNAs that preferentially target cells expressing ecDNA.

[0305] 6.1.2.27. Identification of CRISPR-guided RNA from ecDNA Sequence In some embodiments, an ecDNA sequence derived from the purified ecDNA described herein, most preferably comprising at least a portion of a mutation, breakpoint, fusion, or structural variation relative to healthy DNA, is used to generate a guide RNA probe that specifically targets the ecDNA for cleavage. In some embodiments, the targeted cleavage of the ecDNA is part of a therapy for treating a disease.

[0306] 6.1.2.28. Sequence-specific DNA probes for purifying ecDNA In some implementations, such as those demonstrated in Vitharana 2006, sequence-specific probes can be used to hybridize with specific chromosomes or biological samples containing ecDNA. Subsequently, the markers can be separated from the surrounding sample using the binding affinity of magnetic beads to antibodies. This separation process can be performed in a liquid phase, such as by pre-dispensing onto a substrate, or directly on the substrate after the sample has been dispensed onto its surface.

[0307] 6.1.3. Implementation plan for sample preparation of fluid device The following embodiments of this disclosure describe the preparation of cellular DNA samples suitable for purification or enrichment of ecDNA. In some embodiments, chromosomes and ecDNA are reliably compacted to form discrete particles that can be separated as further described in this disclosure. For all described embodiments, all, a subset, or a portion of the sample preparation embodiments can be performed within a fluidic apparatus.

[0308] 6.1.3.1. Chromosomal condensation While other embodiments of this disclosure may utilize fixed cells or, more generally, non-dividing cells, this embodiment requires live cells capable of forming aggregated chromosomes and ecDNA bodies. This disclosure can be applied to any type of live cell routinely used for routine cytogenetic analysis, including primary cell samples, and preferably, but not exclusively, to cells capable of culturing at least one cell cycle. The most straightforward approach is to culture cells as for karyotype analysis and aggregate chromosomes by adding a cell cycle inhibitor (most preferably colchicine at a concentration of 100 ng / ml or higher). Various chromosome aggregation methods are described in the AGT Handbook (Arsham 2017), which is incorporated herein by reference. For samples that cannot undergo cell cycle analysis, less common chromosome aggregation methods are possible, and chemical additives such as calyculin A can be used to induce premature chromosome aggregation, as described in Gotoh 2019, which is also incorporated herein by reference.

[0309] It is well known that the fraction of cells with aggregated chromosomes (called the mitotic index) can vary depending on cell type and chromosome condensation method. A negative correlation is often observed between chromosome length and mitotic index, with samples remaining in an aggregated state for a longer period exhibiting greater axial compaction. While greater compaction is generally considered undesirable for routine cytogenetic analysis, it is the preferred mode in this disclosure. A higher mitotic index yields more usable sample and less interphase DNA contamination. More compact chromosomes are better resistant to cell lysis, particularly mechanical shearing. When chromosomes come into contact with each other in the precipitate from centrifugation, more compact chromosomes are less likely to entangle in solution during filtration or other interactions with microfluidic surfaces, the roughness of which can be difficult to control due to fabrication steps such as plasma oxidation or chemical etching. Synchronization in more than one step, such as thymidine blocking and release followed by the addition of colchicine, can produce a higher mitotic index than colchicine alone, and at the cost of longer hands-on time, resulting in better input samples.

[0310] In existing cytogenetic methods, the aspect ratio of chromosomes is generally considered to be constant at cell harvest, with chromosome width determined by the degree of compaction of the condensin I complex, and axial compaction determined by the degree of extrusion of the condensin II ring in accordance with the non-essential aid of topoisomerase II and histones. Chromosomes exposed to colchicine for extended periods (>2 hours) are known to be more compact, and in a preferred embodiment, cells are treated with 200 ng / ml colchicine for 16-18 hours prior to harvest.

[0311] 6.1.3.2. Cell lysis & initial enrichment Cell lysis can be accomplished using a variety of common methods, not limited to incubation with detergents such as digitalis saponins, saponins, NP-40, and Triton X-100, as well as mechanical forces such as vortexing, dounce homogenization, acoustic treatment, passing through a fine needle, and repeated freeze-thaw cycles. Cell lysis buffers may contain chemical additives to preserve chromosome morphology, including protease and nuclease inhibitors, crowding agents (not limited to sucrose), 2-methyl-1,3-propanediol (MPD), polyethylene glycol (PEG), and polyvinylpyrrolidone (PVP), and monovalent, divalent, and polyvalent cations such as potassium, magnesium, and spermidine. Sample pH can vary from highly acidic with citrate buffer to neutral with Tris buffer, preferably pH 2.0–7.5.

[0312] In some embodiments, hypotonic and lysis conditions are used to favor one of two mechanisms of chromosome disintegration: DNA strand breaks due to nucleases and chromatin dissociation and DNA unwinding due to disrupted packaging interactions, as described in chromosome isolation protocols based on flow cytometry (summarized in Mukhopadhyay 2023 and incorporated herein by reference). In some embodiments, free Mg and chelating agents are used to generate high molecular weight DNA in the chromosomes, and monovalent polyamines are used to prevent chromatin aggregation at neutral pH, most preferably 80 mM KCl, 0.3 mM spermine tetrahydrochloride, or 0.75 mM spermidine trihydrochloride. In some embodiments, propidium iodide at a concentration of 50 μg / ml is used to stabilize the chromosomes in the absence of Mg or chelating agents. In some embodiments, high concentrations of Mg are used at neutral pH to stabilize the chromosomes, most preferably 10 mM MgSO4 together with 50 mM KCl, 0.5 mM HEPES, pH 8.0. In some implementations, low pH and crowding agents are used, most preferably 100 mM each of citric acid and sucrose.

[0313] Low-speed centrifugation can be used to separate chromosomes and ecDNA from rapidly settling contaminants such as cell lysis debris, intact and broken nuclei, while high-speed centrifugation can separate chromosomes and ecDNA from slowly settling contaminants such as unaggregated interphase chromatin, mRNA, ribosomes, and other substantial contaminants. As described in Yusuf 2014, filtration through coarse filters (40 μm, 10 μm, 5 μm pore sizes) can remove a large amount of cell debris, such as nuclei and cell membranes. Such filters are preferably made of nylon woven mesh. When the mixture is passed through a filter with coarse pores, such as a 10 μm nylon mesh filter (1007), filtration is unable to separate ecDNA ( Figure 10 The filter separates from chromosomes (1006) and (1005). The filter does prevent intact cells (1001), some intact nuclei (1002), and cell debris or some nuclei (1003) from passing through. The filter allows chromosomes, ecDNA, and linear strands of DNA or unwound chromosomes (1004) to pass through.

[0314] Samples can be further enriched by applying forces (including fluid shear forces) strong enough to break individual strands of unaggregated DNA but not enough to cause significant damage to chromosomes. Such shear forces can be experienced in a variety of ways, not limited to vigorous and repetitive pipetting, through narrow orifices (such as 25 ga needles), between two closely spaced plates, rotating the sample in a tube, retaining the sample on a porous substrate with pores smaller than chromosome or ecDNA particles, or when the sample is retained within a microfluidic device via a column structure or fluid equivalent structure that allows fluid to flow through the device.

[0315] In some embodiments, cells are treated with solutions (including but not limited to KI, KNO3, CH3COOK, and NaCl) using chemicals that promote a more invasive hypotonic process. In other embodiments, certain solutions may be enhanced with cell membrane stiffeners such as cholesterol or phosphatidylethanolamine. In some embodiments, swollen cells may exert peripheral thrust, causing ecDNA and chromosomes to move outwards from the cell. In other embodiments, ecDNA and chromosomes may spatially separate as a result of changes in peripheral thrust after cell swelling and before cell rupture.

[0316] 6.1.3.3. Changing chromosome size to achieve reproducible interactions with fluidic devices At the physical level, chromatin exhibits an axial scaffold ionic hydrogel (Beel 2001). After being removed from the cell and freed from the interference of proteases and phosphatases, chromosomes can reversibly expand and contract with varying ionic strength, ionic polyvalentity, and water activity. Following harvest, chromosomes can be further isotropically contracted or expanded by adjusting the ionic strength and pH of the containing solution (Beel 2021). Both the composition and ionic strength of the salts in the filtration buffer affect chromosome morphology, size, and interaction with the physical substrate. A variety of ions can be used, including, but not limited to, acetates, ammonium, cesium, chlorides, cobalt, citrates, hydrides, hydroxyl groups, magnesium, manganate, phosphates, potassium, spermine, spermidine, triethylamine, and zinc, as described in the classic literature on chromosome preparations [Sone 2002]. The application of proteases is known to isotropically expand chromosomes and to sensitize chromosomes to staining procedures such as trypsinization and Giemsa banding, as early and late replication bands are proteolytically hydrolyzed at different rates in the early stages of the biochemical proteolytic reaction. Other methods for chromatin expansion and contraction are not limited to altering the composition and ionic strength of salts, which are highly sensitive to the concentrations of divalent and polyvalent cations; adding phosphatases, acetyltransferases, and methyltransferases to alter the composition of chromatin; adding agents that regulate DNA length; agents that disrupt protein-nucleic acid interactions; cosolvents or ionizing agents that disrupt nucleic acid and protein structures, such as formamide; fluid flow; electrophoretic forces; magnetic forces consistent with magnetic handles on chromosomes; iron nanoparticles, most preferably; and centrifugal forces.

[0317] 6.1.3.4. Chromosome Fixation In some embodiments, cells are treated with agents that modify chromosomes and chromatin before, during, or shortly after lysis, such as heating, cooling, acoustic destruction, microwave radiation, ultraviolet radiation, beading, ionic chemicals, fixative chemicals, and RNA and chromatin-modifying chemicals. For modifications prior to lysis, cell permeabilizing agents can be used, preferably digitalis saponins, saponins, and low concentrations of nonionic detergents. A variety of ions can be used as chemicals, such as those described in the classic literature on chromosome preparations [Sone 2002], including acetates, ammonium, cesium, chlorides, cobalt, citrates, hydrides, hydroxyl groups, magnesium, manganate, phosphates, potassium, spermine, spermidine, triethylamine, and zinc.A variety of fixatives can be used, including but not limited to formalin fixative, formaldehyde, paraformaldehyde, ammonium sulfate fixative, Bouin fixative, Bouin-Hollande fixative for IHC, Bouin's reagent (4% formaldehyde), Bouin Allen fixative, Carnoy fixative (chloroform), Carnoy fixative (methanol and acetic acid), Carnoy fixative (formaldehyde-alcohol-acetic acid), Carnoy fixative (chloroform & ferric chloride (III)), chromic acid-acetic acid fixative, chromic acid 2%-15%, Davidson solution, Delaunay fixative, diethyl ether fixative, Esposit fixative for urine cytology, ethanol-glacial acetic acid fixative, Freiburg fixative, fixatives after Thiel, fixatives according to Stevie, formaldehyde fixative for Bulbi, and formalin 2%-37%. (Whether buffered or unbuffered, acid-free, containing eosin), formalin acetone, fixative F13, zinc fixative, glutaraldehyde 0.65%-6.5% (with or without buffer), glutaraldehyde fixative according to KARNOVSKY, glutaraldehyde carboxylate, JORES fixative, KAISERLING fixative, thimerosal formalin solution, NAWASHIN fixative, bifunctional imides, bifunctional NHS-esters (including disuccinimidyl glutarate), O,O'-bis[2-(N-succinimidyl-succinamido)ethyl]polyethylene glycol and dithiobis[succinimidyl propionate], bifunctional maleimide, isobifunctional crosslinking agents (including imide NHS-ester crosslinking agents), paraformaldehyde (PFA) 4%-10% (whether unbuffered or buffered), in glutaraldehyde 0.5% & PBS pH 7.4 contains 4% paraformaldehyde (PFA), picric acid, Rossmann's fixative, Saccomanno's fixative, Schaffer's fixative, Schaudin's fixative, sodium formalin after Kossa, trichloroacetic acid-mercuric formaldehyde after Romeis, wintergreen oil after Spaltholz, Zamboni solution, Zenker's fixative, and zinc chloride-acetic acid-formaldehyde. The RNA and chromatin modifiers are not limited to RNases, but preferably RNase A, polyamines, EDTA, proteases, preferably pepsin, polyvinylpyrrolidone, coblock polymers, coblock polymers incorporating positively charged regions, most preferably PEI-g-PEG, small RNA, preferably yeast tRNA-phe, bulk protein, preferably BSA, organic acids, preferably acetic acid, and inorganic acids, preferably dilute HCl.

[0318] Cells can be treated before or during lysis, or the cell lysate can be treated shortly after lysis with agents that prevent chromosome and chromatin modifications (such as inhibitors of nucleases and proteases, preferably PMSF, and antioxidants, preferably dithiothreitol).

[0319] Stabilizing chromosomes and preventing one chromosome from becoming entangled with another is generally desirable. In some embodiments, chromosomes are coated with or encapsulated in one or more of the following layers: dextran, starch, hyaluronic acid, chitosan, protein aggregates (including protein aggregates formed from collagen, gelatin, and albumin), poly(lactide-co-glycolic acid), (3-hydroxybutyrate-co-3-hydroxyvalerate), poly(sebacic anhydride) and poly(s-caprolactone), poly(N-isopropylacrylamide), Eudragit L100 and S100, and high molecular weight polymers, including homopolymers, coblock polymers, and dendritic polymers, said polymers comprising various components, including but not limited to polyethylene oxide, polyethyleneimine, polyacrylamide, poly(N-isopropylacrylamide), and polyamides. Some polymers contain cross-linking agents and / or heterofunctional molecules having one portion that binds to chromatin and another portion that binds to or participates in binding with the polymer matrix via covalent bonds. In all cases, the cross-linking agent may optionally have an additional portion that allows for selective cleavage, preferably disulfide bonds. The particles can then be degraded by adding chemical portions that do not degrade the chromosome, including alginate degradation via chelation or pH changes, Eudragit degradation via pH changes, and polymer bead degradation via enzymatic digestion.

[0320] Centrifugation can be used to separate chromosomes from fast-settling contaminants such as cell lysis debris, intact and broken nuclei, as well as from slow-settling contaminants such as unaggregated interphase chromatin, mRNA, ribosomes, and other solid contaminants. Centrifugation can occur in buffer solutions with densities and viscosities similar to phosphate-buffered saline, or in solutions with higher densities or viscosities such as sucrose, percoll, ficoll, or cesium chloride.

[0321] 6.1.4. Implementation scheme of the fluid apparatus for isolating ecDNA This document describes numerous non-limiting embodiments of fluidic devices for use in the separation, purification, or sorting of ecDNA relative to cellular DNA. In addition to the embodiments disclosed herein, numerous previously disclosed fluidic devices have been shown to efficiently separate (or sort) various bodies (typically cells or beads) within a fluidic device by means of standards (typically size). Broadly speaking, separation or sorting by a fluidic device can be classified as “active” or “passive” sorting, where active sorting involves the application of an externally applied force field to classify bodies, while passive sorting relies primarily on the interaction between the bodies in solution and the fluid flow and fluid characteristics within the fluidic device to classify bodies, although the distinction between the two types is not always absolute. Examples of various embodiments of active and passive sorting fluidic devices are referenced to [Tang et al., nature, 2022] and [Zhang et al., Rev Adv Mater Sci, 2021], which are incorporated herein by reference in their entirety.

[0322] In some embodiments using fluid devices to separate or sort bodies, the bodies are classified by the fluid device at least in part by the inherent properties of their respective bodies. Examples of such inherent properties may include shape, size, length, density, mass, total charge, charge density, surface properties, magnetism, dipole charge, dielectric electrophoretic response, elasticity, volume, and entropy energy. In some embodiments, the bodies are classified at least in part by the properties of a binding body to the body. In some embodiments, the binding body is a marker body. In some embodiments, the bodies are classified by the fluid device at least in part by the inherent properties of a corresponding secondary binding body, wherein the secondary binding body binds to the first body. In some embodiments, the binding body specifically binds to a target body.

[0323] For the purposes of this disclosure, the difference between a "smart" fluid separation or sorting device and a "non-smart" fluid separation or sorting device lies in the fact that, with a smart device, the fluid being sorted is probed in a certain way within the fluid device, such that the result of the probe provides data or information for generating a decision regarding whether to separate or sort the fluid within the device. In some embodiments, the decision is at least partially controlled manually. In some embodiments, the decision is at least partially automated, for example, by using a program running on a computer, or smartphone, or microprocessor, or microcontroller, or ASIC, or FPGA.

[0324] Non-limiting embodiments of fluid separation or sorting devices include electrophoretic sorting, acoustic sorting, magnetic sorting, optical sorting, inertial sorting, deterministic lateral displacement sorting, microfiltration sorting, entrainment sorting, and viscoelastic sorting.

[0325] In some implementations, the fluid device can separate or sort at least one ecDNA from at least one cellular DNA using a combination of at least one non-smart method and at least one smart method.

[0326] In one set of embodiments, the separation mechanism includes introducing at least one ecDNA and at least one cellular DNA into a separation region within a fluid device, wherein at least one force is applied to or removed from the molecules, the application or removal of the at least one force resulting in an increased physical separation distance between the molecules compared to a situation where no force is applied or removed. In a preferred embodiment, the magnitude and direction of the force applied to the at least one ecDNA vary at least in part with a property associated with the at least one ecDNA or the body bound to the at least one ecDNA. In some embodiments, the property includes the size of the at least one ecDNA. In some embodiments, the property includes the mass of the at least one ecDNA. For example, the size of the ecDNA, in moles or Daltons. In some embodiments, the property includes rigidity. In some embodiments, the property includes the number of single-strand nicks. For example, ecDNA with zero, one, or several nicks. In some embodiments, the property includes the rigidity of the at least one ecDNA. In some embodiments, the property includes the stiffness of the at least one ecDNA. In some embodiments, the property includes the radius of gyration of the at least one ecDNA. For example, the radius of gyration of at least one ecDNA can vary significantly depending on a variety of factors, including but not limited to: the number of nicks, the proportion of its double-stranded molecules, the density and type of conjugates, and the density and type of higher nucleic acid structures. The radius of gyration then affects the interaction of the molecule with confined spaces and fluid barriers within a fluid device; smaller gyratory molecules are more capable of sharp turns and close proximity interactions with smaller fluid features compared to molecules of similar size with higher gyrations, thus allowing for greater drag and more favorable confinement within confined spaces. In some embodiments, the characteristic includes the proportion of at least one ecDNA that is single-stranded. In some embodiments, the characteristic includes the amount of at least one ecDNA that is single-stranded. In some embodiments, the characteristic includes the proportion of at least one ecDNA that is denatured. In some embodiments, the characteristic includes the amount of at least one ecDNA that is denatured. In some embodiments, the characteristic includes the mass density of at least one ecDNA. In some embodiments, the characteristic includes the charge of at least one ecDNA. In some embodiments, the characteristic includes the charge density of at least one ecDNA. In some embodiments, the characteristic includes the surface charge of at least one ecDNA. In some embodiments, the characteristic includes the surface charge density of at least one ecDNA. In some embodiments, the characteristic includes the length of at least one ecDNA. For example, the length of ecDNA is measured in base pairs, or in nm, or in micrometers. In some embodiments, the property includes the magnetic moment of at least one ecDNA. In some embodiments, the property includes the elasticity of at least one ecDNA.In some embodiments, the characteristic includes the volume of space occupied by the 3D envelope containing at least one ecDNA. In some embodiments, the characteristic includes the length of the longest axis of the 3D envelope containing at least one ecDNA. In some embodiments, the characteristic includes the length of the shortest axis of the 3D envelope containing at least one ecDNA. In some embodiments, the characteristic includes the entropy energy of at least one ecDNA. In some embodiments, the characteristic includes the enthalpy energy of at least one ecDNA. In some embodiments, the characteristic includes the free energy of at least one ecDNA. In some embodiments, the characteristic includes the temperature of at least one ecDNA. In some embodiments, the characteristic includes the topology of at least one ecDNA. In some embodiments, the characteristic includes the mobility of at least one ecDNA. In some embodiments, the characteristic includes the average trajectory vector of at least one ecDNA. In some embodiments, the characteristic includes the relaxation time of at least one ecDNA. In some embodiments, the characteristic includes the inertia of at least one ecDNA. In some embodiments, the characteristic includes the drag coefficient of at least one ecDNA. In some embodiments, the characteristic includes the skin friction of at least one ecDNA. In some embodiments, the characteristic includes the form drag of at least one ecDNA.

[0327] In some embodiments, the property includes the binding affinity of at least one ecDNA to some other entity. In some embodiments, the property includes the binding specificity of at least one ecDNA to another entity. In some embodiments, the property includes the binding strength of at least one ecDNA to another entity. In some embodiments, the other entity is directly or indirectly linked to: a fluid device, or a bead, or a gel, or a dendritic macromolecule, or a long nucleic acid molecule, or a marker.

[0328] In some embodiments, the characteristics include the number, presence, absence, or density of binders that bind to at least one ecDNA. In some embodiments, the binder is a marker.

[0329] In some implementations, the feature includes a signal from a marker that binds to at least one ecDNA.

[0330] In some embodiments, the feature includes at least a portion of a sequence within at least one ecDNA. In some embodiments, the feature includes at least one methylation pattern within at least one ecDNA. In some embodiments, the feature includes at least one histone pattern within at least one ecDNA. In some embodiments, the feature includes at least one binding marker pattern within at least one ecDNA. In some embodiments, the pattern forms a physical map.

[0331] In some embodiments, the separation mechanism within the fluid device includes immobilizing at least one ecDNA and at least one cellular DNA to different locations within the fluid device. In some embodiments, the separation mechanism within the fluid device includes selectively immobilizing only ecDNA to regions of the device. In some embodiments, the separation mechanism within the fluid device includes selectively immobilizing only cellular DNA to regions of the device.

[0332] In some embodiments, the separation mechanism within the fluid device includes a filtration effect. In some embodiments, the separation mechanism within the fluid device includes entropy trapping. In some embodiments, the separation mechanism includes deterministic lateral displacement. In some embodiments, the separation mechanism includes entrainment-based fractional separation. In some embodiments, the separation mechanism includes a viscoelastic effect. In some embodiments, the separation mechanism includes an inertial effect. In some embodiments, the separation mechanism includes a hydrodynamic filtration effect. In some embodiments, the separation mechanism includes a microfiltration effect. In some embodiments, the filtration mechanism includes piercing at least one ecDNA with a column or hook through a topological pore of the DNA, wherein the DNA is circular DNA.

[0333] In some embodiments, at least one force applied to or removed from at least one ecDNA includes a fluid flow shear force. In some embodiments, the mechanism generating fluid flow includes capillary forces. In some embodiments, the mechanism generating fluid flow includes evaporation. In some embodiments, the mechanism generating fluid flow includes convection. In some embodiments, the mechanism generating fluid flow includes a temperature gradient. In some embodiments, the mechanism generating fluid flow includes thermocapillary convection. In some embodiments, the mechanism generating fluid flow includes electrodynamic forces. In some embodiments, the mechanism generating fluid flow includes surface tension. In some embodiments, the mechanism generating fluid flow includes a surface tension gradient. In some embodiments, the mechanism generating fluid flow includes wetting forces. In some embodiments, the mechanism generating fluid flow includes the Marangoni effect.

[0334] In some embodiments, at least one force applied to or removed from at least one ecDNA includes entropic forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes hydrophobic forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes van der Waals forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes pressure. In some embodiments, at least one force applied to or removed from at least one ecDNA includes drag forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes inertial forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes electrophoretic forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes dielectrophoretic forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes sonophoretic forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes magnetic forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes electromagnetic forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes electrostatic forces. In some embodiments, at least one force applied to or removed from at least one ecDNA includes an optical force.

[0335] In some embodiments, the separation region of the fluid device includes a surface comprising a groove, recess, channel, module, trench, railing, fence, functionalized surface, or textured surface to increase the drag force on the body when the body is pushed against the surface by an applied force. In some embodiments, the drag force is sufficient to collect the body at the surface.

[0336] In some embodiments, the separation region of the fluid device includes a filter. In some embodiments, the separation region of the fluid device includes a gel. In some embodiments, the separation region of the fluid device includes an artificial gel. In some embodiments, the separation region of the fluid device includes a deterministic gel. In some embodiments, the separation region of the fluid device includes an entropy trap. In some embodiments, the separation region of the fluid device includes an entropy barrier. In some embodiments, the separation region of the fluid device includes a functionalized surface. In some embodiments, the separation region of the fluid device includes a deterministic lateral displacement (DLD) device. In some embodiments, the separation region of the fluid device includes a pin-flow fractionation (PFF) device. In some embodiments, the separation region of the fluid device includes a viscoelastic effect microfluidic (VEM) device. In some embodiments, the separation region of the fluid device includes an inertial effect microfluidic (IMF) device. In some embodiments, the separation region of the fluid device includes a cross-flow filtration (CFF) device. In some embodiments, the separation region of the fluid device includes a hydrodynamic filter. In some embodiments, the separation region of the fluid device includes a microfiltration filter. In some embodiments, the separation region of the fluid device includes an electrophoretic sorting device. In some embodiments, the separation region of the fluid device includes a dielectric electrophoretic sorting device. In some embodiments, the separation region of the fluid device includes an acousto-electrophoretic sorting device. In some embodiments, the separation region of the fluid device includes a magnetic sorting device. In some embodiments, the separation region of the fluid device includes an electrostatic sorting device. In some embodiments, the separation region of the fluid device includes an array of protrusions. In some embodiments, the separation region of the fluid device includes an array of ratches. In some embodiments, the separation region of the fluid device includes a physical barrier. In some embodiments, the separation region of the fluid device includes a column. In some embodiments, the separation region of the fluid device includes a recess. In some embodiments, the separation region of the fluid device includes a channel. In some embodiments, the separation region of the fluid device includes a groove. In some embodiments, the separation region of the fluid device includes an orifice.

[0337] In some embodiments, the separation region of the fluid device includes fluid characteristics. In some embodiments, the fluid characteristics include the following lateral dimensions: less than 100 micrometers, or less than 90 micrometers, or less than 80 micrometers, or less than 70 micrometers, or less than 60 micrometers, or less than 50 micrometers, or less than 40 micrometers, or less than 30 micrometers, or less than 20 micrometers, or less than 10 micrometers, or less than 8 micrometers, or less than 6 micrometers, or less than 5 micrometers, or less than 4 micrometers, or less than 3 micrometers, or less than 2 micrometers, or less than 1.5 micrometers, or less than 1 micrometer, or less than 0.8 micrometers, or less than 0.6 micrometers, or less than 0.4 micrometers, or less than 0.2 micrometers. In some implementations, the fluid characteristics include the following vertical dimensions: less than 100 micrometers, or less than 90 micrometers, or less than 80 micrometers, or less than 70 micrometers, or less than 60 micrometers, or less than 50 micrometers, or less than 40 micrometers, or less than 30 micrometers, or less than 20 micrometers, or less than 10 micrometers, or less than 8 micrometers, or less than 6 micrometers, or less than 5 micrometers, or less than 4 micrometers, or less than 3 micrometers, or less than 2 micrometers, or less than 1.5 micrometers, or less than 1 micrometer, or less than 0.8 micrometers, or less than 0.6 micrometers, or less than 0.4 micrometers, or less than 0.2 micrometers.

[0338] In some embodiments, at least one fluid feature has a surface energy different from that of at least a second fluid feature within the fluid device. In some embodiments, at least a portion of the surface of the fluid feature has a surface energy different from that of at least a second portion of the surface of the fluid feature. In some embodiments, the first and second portions may include the top, sides, and bottom of the fluid feature. In some embodiments, methods for obtaining different surface energies include using different underlying materials for portions of the surface. In some embodiments, methods for obtaining different surface energies include applying different surface modifications to portions of the surface. In some embodiments, modifications may include plasma modification, chemical modification, thin film application, molecular monolayers, and molecular multilayers.

[0339] 6.1.4.1. Filter Selection and Treatment Not all filters with a given pore size filter chromosomes and ecDNA in the same way. The composition of the filter and how it processes data are related. The topology of the filter substrate influences the paths taken by chromosomes and ecDNA, and features prominent at the filter inlet or along the topology of the filter pores can hook chromatin loops in a Velcro-like manner. Filters with pores that include a straight-channel topology, such as orbital-etched membrane filters, provide a more controlled and consistent pathway for chromosome and ecDNA interactions. Figure 11 The output is shown using a nylon filter with a 5-micron pore size to capture the nucleus while allowing both chromosomes (1102) and ecDNA particles (1101) to pass through. Conversely,Figure 12 This demonstrates the use of a nylon filter with a pore size of 1.2 micrometers, allowing chromosomes to be trapped within the filter, with only ecDNA particles (1201) able to pass through. When comparing the nominal pore size with the measured chromosome size, chromosomes become stuck in filters nominally large enough to pass through, and flow is blocked unless the number of pores exceeds the number of chromosomes. Extrachromosomal DNA can pass through the filter pores, but can also be trapped by the rough edges of the pores.

[0340] In some embodiments, the filter or fluid device is pretreated or treated during sample application to alter the chemical interaction between the ecDNA-containing sample and the filter or fluid device. A combination of pretreatment during sample application and simultaneous treatment can be used, preferably depending on the nature of the treatment. The treatment is accomplished by introducing chemical additives, which can fall into one of three categories: additives that compact the chromatin of the ecDNA-containing sample and reduce the likelihood of chromatin protrusions being hooked; additives that alter the chemical properties of the filter or fluid device surface; or additives that alter the local roughness of the device to make it smooth.

[0341] Chemical additives for compacting chromatin are primarily described in the section describing chromosome stabilization through buffer components (such as ionic strength and composition, crosslinking, pH, and crowding agents that can affect water activity), and are not limited to additives (such as sucrose, alcohols, PEG solutions, and polyols).

[0342] Chemical additives that alter the chemical properties of filters or fluid devices include a variety of agents known for passivating surfaces, such as those sold in commercially available blocking kits for immunohistochemistry, nucleic acid hybridization and detection, and fluorescence in situ hybridization. In certain embodiments, mixtures of additives may be used, including detergents, preferably nonionic detergents, most preferably a mixture of Tween 20 and Tween 80. In other embodiments, passivating agents known in the field of electrophoresis, such as polyvinylpyrrolidone, may be used. In yet another embodiment, proteins such as bovine serum albumin may be added to coat the fluid device or filter, and similarly, uninteresting nucleic acids such as synthetic homopolymers, preferably multidC, may be added to block the filter or fluid device.

[0343] Chemical additives that alter surface roughness to prevent hooking include long polymers, preferably high molecular weight solutions of PEG. The polymer must be water-soluble and preferably have minimal interaction with DNA and proteins, such as dextran and Ficol. Polymers with strong positive charges will perform worse than polymers with negative charges, such as poly(sodium acrylate) or poly(ethylene oxide)-block-poly(sodium 4-vinylbenzenesulfonate).

[0344] In some embodiments, the filter substrate includes, but is not limited to, polycarbonate, cellulose, methylcellulose, nylon, polyvinylidene fluoride (PVDF), PTFE, polyethersulfone, mixed cellulose esters, polypropylene, regenerated cellulose, glass, silicon, silicon nitride, glass fiber, cellulose acetate, borosilicate glass, glass microfiber, glass fiber reinforced polypropylene, borosilicate glass fiber, nitrocellulose, paper, PVC, cellulose nitrate, polysulfone, alumina, cellulose fibers, composite regenerated cellulose, polyester, polyolefin, polystyrene, MF, PET, TF, and silver. In other embodiments, the filter comprises polymers, including but not limited to agarose, polyacrylamide, gelatin methacrylate, POEGMA, NIPAM, polyacrylic acid, Ficoll, polyvinyl alcohol, and cross-linked PEG. In some embodiments, the filter comprises a slurry of microscopic particles, including but not limited to glass, zirconium, coated particles, silica, porous silica, gel filter media, and Percoll. In some embodiments, the filter also includes a solid or glass frit structure to physically support a more fragile membrane material. In some embodiments, the filter is coated with a hydrophilic coating to accelerate wetting, including but not limited to polyvinylpyrrolidone (PVP), and in other embodiments, the filter is pre-wetted with an amphiphilic solvent, not limited to methanol, ethanol, butanol, and short-chain alcohols. In some embodiments, the filter comprises a combination of materials listed herein. In some embodiments, the filter is manufactured using techniques not limited to woven mesh, track etching, phase separation, non-solvent-induced phase separation (NIPS), thermally induced phase separation (TIPS), and fiber covering. In some embodiments, the pore size is 10 μm, 8 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1.2 μm, 1.0 μm, 0.8 μm, 0.5 μm, 0.3 μm, 0.2 μm, or 0.1 μm. In some embodiments, more than one filter is used in series, preferably such that the filter pore size decreases in the flow direction. In some embodiments, the filtrate contains fractions enriched with ecDNA, while in other embodiments, a small-pore filter is used to collect ecDNA from the solution and allow non-aggregated DNA to pass through, preferably a filter with a pore size of 0.3 μm.

[0345] 6.1.4.2. Multiple Implementation Schemes for Separation Mechanisms in Fluid Devices In some embodiments where the separation or sorting mechanism includes electrophoretic sorting, applying or removing the applied force includes applying a constant, time-varying, or space-varying electric field to directly or indirectly regulate the position or velocity of a body within the fluid device. In some embodiments, the body can be classified by the amount or density of its bulk charge or surface charge. In some embodiments, the body can be classified by the degree of polarization that produces a positive or negative dielectric electrophoretic response in the presence of the field. In some embodiments, the dielectric electrophoretic effect can be used to create virtual fluid flow channels within the fluid device, which can be used to guide fluid flow in a desired direction controlled by the modulating field. In some embodiments, the dielectric electrophoretic response can be varied by the location, number, size, and shape of the electrodes used to generate the electric field.

[0346] In some embodiments where the separation or sorting mechanism includes acoustic separation, applying or removing the applied force includes applying a constant, time-varying, or space-varying sound pressure to directly or indirectly adjust the position or velocity of a body within the fluid device. This can include standing acoustic wave devices acting as tweezers to manipulate the body, or highly focused surface acoustic waves to apply force to the desired body.

[0347] In some embodiments where the separation or sorting mechanism includes magnetic sorting, applying or removing the applied force includes applying a constant, time-varying, or space-varying magnetic field. In some embodiments, the body to be sorted is bonded to magnetic beads (such as iron nanoparticles) such that the application or removal of the magnetic field generates a force on the beads, and thus a force on the body bonded to the beads. In some embodiments, the separation region of the fluid device comprises a ferrofluidic material such that the flow direction and therefore the shear force from such flow can be manipulated by applying or removing a magnetic field. In some embodiments, the body to be separated is located directly within the ferrofluidic material. In some embodiments, the body to be separated is located in a droplet, and the droplet itself is located within the ferrofluidic material.

[0348] In some embodiments where the separation or sorting mechanism includes optical sorting, applying or removing the applied force involves applying constant, time-varying, or space-varying electromagnetic radiation. In a preferred embodiment, the source of the electromagnetic radiation is a laser. In some embodiments, the force applied to the body originates from light scattering and gradient forces generated between the bodies using optical tweezers.

[0349] In some embodiments where the separation or sorting mechanism includes inertial or viscoelastic sorting, the Reynolds number of the fluid device is high enough that fluid inertia cannot be ignored and / or non-Newtonian flow occurs. In some embodiments, inertial migration applies a force perpendicular to the fluid flow. In some embodiments, the force applied to the volume is a shear-induced Saffinan force. In some embodiments, the force applied to the volume is a number-induced lifting force, or rotational lift, or wall lift. In some embodiments, differences in migration distance can be used to separate volumes. In some embodiments, asymmetry in the velocity in the cross-sectional plane of a curved channel results in Dean flow and Dean drag. In some embodiments, the channel cross-section can be modified to optimize Dean flow and Dean vortices for a specific volume of interest. In some embodiments, vortices can be generated by adding features to the channel. Features can include side vortex channels, side chambers, topology-optimized structures, spirals, and serpentine channels. In some embodiments, the ratios of inertial forces, viscous forces, and elastic forces are adjusted to optimize the separation of the desired volume.

[0350] In some embodiments where the separation or sorting mechanism includes deterministic lateral displacement, this mechanism is used to separate volumes by size as a sample flows through a fabricated array of columns, wherein the column array is intentionally designed to separate volumes with different physical properties in laminar flow. Typically, when the fluid flow encounters a column, the flow bifurcates, and a certain number of streamlines closest to the column deflect. As a result, smaller volumes are able to deflect in a zigzag pattern and travel in a zigzag pattern, while larger volumes with diameters exceeding a critical diameter are unable to deflect in a zigzag pattern and travel in parallel, thus becoming physically separated from the smaller volumes. In some embodiments, the parameters of the array are modified to optimize performance relative to the desired volumes to be sorted. These parameters may include pitch, spacing, asymmetric velocity spectrum, column size, column shape, column protrusions and curvature affecting volume flow and trapping, column rotation, column material, column surface properties, column spacing, groups of columns of more than one type in the array, array orientation, array angle, array lattice vector, and topology optimization. In some embodiments, the column array may include more than one subarray with different geometric parameters to enable different sorting capabilities in each subarray. In some implementations, subarrays can be fluidly connected in a cascade. In some implementations, the spatially averaged laminar streamline vector of the fluid can be modified. In some implementations, the array can consist of an array of subarrays.

[0351] In some embodiments where the separation or sorting mechanism includes entrained flow classification, the mechanism separates volumes by size-based exposure of the volumetric confinement body to the secondary flow, ensuring that sufficiently small volumes do not fully interact with the secondary flow. In some embodiments, reduced flow resistance on the secondary flow side contributes to inclined streamlines and improved separation efficiency. In some embodiments, reduced flow resistance includes increased outlet channel diameter and / or reduced outlet channel width. In some embodiments, an asymmetric squeeze flow classification method is employed.

[0352] In some embodiments, the separation or sorting mechanism includes fluorescence-activated sorting. In some embodiments where ecDNA is contained within cells, the sorting mechanism includes fluorescence-activated cell sorting (FACS). In some embodiments where ecDNA is contained within cells, the sorting mechanism includes fluorescence-activated droplet sorting (FADS).

[0353] In some implementations, at least one body to be classified is contained within a droplet, and the fluid device classifies the at least one body within the droplet. For a review of various droplet-based classification systems, see [Tan, Lab on a Chip, 2017], all examples of which are incorporated herein by reference in their entirety. Various non-limiting implementations for classifying droplets include magnetic control, pneumatic control, thermal control, electrical control, and acoustic control.

[0354] In some embodiments, at least one ecDNA is identified as ecDNA in the fluid device. In a preferred embodiment, the ecDNA is identified as distinct from at least one cellular DNA.

[0355] In some embodiments, at least one ecDNA is identified by its location within a fluidic device, wherein the location is designed to separate DNA of a specific standard range (e.g., size, mass, topology, charge, magnetic moment, etc.), where the standard range includes at least one ecDNA but excludes cellular DNA. For example, the location could be an outlet port of the fluidic device, which includes a clamping and grading separation method to separate input DNA molecules of a specific standard range to the outlet port, while larger chromosomes are directed to different outlet ports. In another example, the location could be an outlet port of the fluidic device, which includes a deterministic lateral displacement method to separate input DNA molecules of a specific standard range to the outlet port, while larger chromosomes are directed to different outlet ports. In another set of embodiments, the location could be a set of fluidic features within the fluidic device designed to capture only DNA molecules of a specific standard range through the interaction of the dimensions of the fluidic features and a set of forces acting on the DNA molecules. For example, the location could be a depression designed to have a sufficiently large width and depth to allow ecDNA of a specific size range to enter, but not large enough to allow chromosomes. In another example, the location could be a fluid channel in an open fluid device, where the channel width is wide enough to allow entry of ecDNA within a specific size range, but not large enough to allow chromosomes. In another set of embodiments, the location could be a slit channel, where the vertical dimension is sufficiently constrained such that only molecules within a specific size range can enter the slit channel by a specific applied force, while larger chromosomes cannot enter. In yet another set of embodiments, the location could be on the opposite side of a filter, where the filter pore size is large enough to allow at least one type of ecDNA to pass through the filter, while cellular DNA cannot.

[0356] In some embodiments, at least one ecDNA is identified by probing. In some embodiments, the identification of at least one ecDNA includes identifying ecDNA-specific markers that bind to DNA. In some embodiments, the identification of at least one ecDNA includes identifying DNA having a circular topology. In some embodiments, the identification of at least one ecDNA includes identifying DNA of a specific size or length range. In some embodiments, the identification of at least one ecDNA includes identifying DNA of a specific mass range. In some embodiments, the identification of at least one ecDNA includes identifying the presence, absence, type, or amount of DNA having higher-order nucleic acid structures.

[0357] In some embodiments, the detection method includes microscopy, particularly fluorescence microscopy. In some embodiments, at least one ecDNA and at least one cellular DNA are stained to improve the detection sensitivity of the microscopy system. In some embodiments, the staining agent is a fluorescent staining agent, preferably an embedded dye. In some embodiments where the fluid device is a closed fluid device, the fluid device includes an observation window through which at least one ecDNA can be optically detected.

[0358] In some embodiments where the fluid device is an open fluid device, at least one ecDNA can be detected using a contact probe system. In a preferred embodiment, the contact probe system is an atomic force microscopy probe system.

[0359] In some implementations, the detection method may include more than one detection method, preferably in a coordinated manner. For example, at least one ecDNA may be detected by fluorescence microscopy and atomic force microscopy.

[0360] In some implementations, at least one ecDNA can be detected when contained within a droplet, which is contained within a fluid device.

[0361] In some embodiments, at least one ecDNA is probed within a fluidic device such that the at least one ecDNA can be identified as ecDNA, and the data output of the probe is used at least in part directly or indirectly to generate an ecDNA profile of the input sample of the fluidic device. In some embodiments, the ecDNA profile includes information confirming the presence of the ecDNA in the input sample. In some embodiments, the ecDNA profile includes information confirming the absence of ecDNA in the input sample. In some embodiments, the ecDNA profile includes information about the amount of ecDNA in the input sample. In some embodiments, the ecDNA profile includes information about the relative fraction of ecDNA in the input sample. In some embodiments, the ecDNA profile includes information about the size distribution of the ecDNA in the input sample. In some embodiments, the ecDNA profile includes information about the topological distribution of the ecDNA in the input sample. In some embodiments, the ecDNA profile includes information about the distribution of the higher nucleic acid structures of the ecDNA in the input sample. In some embodiments, the ecDNA profile includes information about the methylation distribution of the ecDNA in the input sample.

[0362] In some embodiments, the ecDNA profile includes information confirming the presence of the ecDNA within at least one cell of the input sample. In some embodiments, the ecDNA profile includes information confirming the absence of ecDNA in at least one cell of the input sample. In some embodiments, the ecDNA profile includes information regarding the amount of ecDNA in at least one cell of the input sample. In some embodiments, the ecDNA profile includes information regarding the relative fraction of ecDNA in at least one cell of the input sample. In some embodiments, the ecDNA profile includes information regarding the size distribution of ecDNA in at least one cell of the input sample. In some embodiments, the ecDNA profile includes information regarding the topological distribution of ecDNA in at least one cell of the input sample. In some embodiments, the ecDNA profile includes information regarding the distribution of higher nucleic acid structures of ecDNA in at least one cell of the input sample. In some embodiments, the ecDNA profile includes information regarding the methylation distribution of ecDNA in at least one cell of the input sample.

[0363] In some embodiments, ecDNA profiling is used to diagnose diseases. In some embodiments, ecDNA profiling is used to monitor the status of a disease. For example, ecDNA profiling is used to determine whether the disease's mortality rate is stable, in remission, or its mortality rate is increasing. In some embodiments, ecDNA profiling is used to select the type of treatment for the disease. For example, selecting a drug compound to be administered to a patient, or selecting the amount of dosage to be administered to a patient, or selecting the tissue targeted when administered to a patient, or selecting the duration or period of administration to a patient. In some embodiments, ecDNA profiling is used to modify the type of treatment for the disease. In some embodiments, ecDNA profiling is used to personalize drug administration to a patient. In a preferred embodiment, the disease is cancer.

[0364] 6.1.4.3. Solid Velcro Method and Apparatus for Separating Circular DNA In one set of embodiments, a closed loop of at least one circular ecDNA is punctured within a channel of a fluid device using a puncturing object. In a preferred embodiment, a non-zero external force is present that can be applied to the punctured circular ecDNA such that, when co-located and exposed to the same external force, the punctured circular ecDNA migrates less in solution than the linear long nucleic acid molecule, thereby allowing the circular ecDNA and the linear molecule to physically separate from each other. In a preferred embodiment, a non-zero external force is present such that, when averaged over time, the migration rate of the circular ecDNA is practically zero.

[0365] In some implementations, the object being pierced is a pillar, a tilted pillar, a hook, a "T" or a spike. Figure 13 The text shows various examples of objects being pierced.

[0366] In some embodiments, the migration rate of the circular ecDNA after puncture varies with the direction of the externally applied force. In a preferred embodiment, when an external force is applied in one direction, the migration rate of the punctured circular ecDNA is practically zero when averaged over time, and when an external force is applied in a second direction, the migration rate of the punctured circular ecDNA is similar to that of co-located non-punctured circular ecDNA, or in some cases, is greater than zero when averaged over time.

[0367] In some embodiments, a sample of a long nucleic acid molecule in a solution containing at least one ecDNA molecule and at least one cellular ecDNA is introduced onto the surface of an open fluid device containing more than one puncture object. In a preferred embodiment, the cellular ecDNA is a chromosome or part of a chromosome. In some embodiments, at least one circular ecDNA and at least one cellular DNA are brought to the surface while still contained within their source cell or nucleus, and said cell or nucleus lyses or breaks down upon collision with said surface or at a later time point after contact with said surface. In some embodiments, the sample is brought to more than one puncture object by an applied external force. In some embodiments, at least one perforated circular ecDNA and at least one cellular DNA are then separated by an applied external force. In some embodiments, an external force is applied to at least one circular ecDNA and at least one cellular DNA, and the separation of the two molecules is caused by the difference in migration rates between the two molecules due to the puncture of the circular ecDNA.

[0368] Figure 14 One embodiment is shown in which circular ecDNA (1413) and linear cellular DNA (1412) flow (1415) through a fluid channel (1411) of a fluid device under the application of an external force (here, fluid flow), in which more than one piercing object (1414) originating from the channel wall and occupying the channel is present. In this particular embodiment, the piercing object is oriented to retain the circular DNA on the piercing object after it has been pierced and is oriented (1415) in the presence of the original external force used to transport the circular DNA to the piercing object. After the circular DNA is hooked (1421), the linear cellular DNA (1422) can then be separated from the circular DNA by maintaining the external force such that it is used to expel the linear molecule, while the circular molecule remains on the piercing object in the channel. In some embodiments, the linear molecule is then collected for further analysis. In some implementations, after the linear molecules have been flushed out, an external force of different orientation (1433) is applied to the circular DNA (1431) to release them from the punctured object, after which they can be collected for further analysis.

[0369] In some embodiments, when the circular long nucleic acid molecule interacts with more than one piercing object, the external force applied to the circular long nucleic acid molecule is not constant over time, but varies in amplitude or direction over time. In a preferred embodiment, the external force is removed or its duration is substantially reduced in amplitude for at least a certain period of time, allowing the circular ecDNA to relax in solution. The relaxation of the circular ecDNA in the liquid allows the molecule to naturally avoid opening the loop and provides a larger circular surface area for piercing. Additionally, the random Brownian motion of the molecule within the fluid channel occupied by the pierced object allows for a greater chance of piercing.

[0370] In a preferred embodiment, the density, shape, size, height, and orientation of the puncture objects are selected to optimize the capture probability of a specific circular ecDNA size, topology, form, or shape. In some embodiments, the density, shape, size, height, and orientation of the puncture objects are all identical. In a preferred embodiment, at least one puncture object has a different shape, size, height, or orientation than at least one other puncture object. In some embodiments, a subset of the puncture objects is placed in a regular array. In some embodiments, a subset of the puncture objects is placed randomly. In some embodiments, at least two puncture objects may originate from different channel walls or surfaces.

[0371] In some implementations, the channel may contain two or more piercing objects, or 10 or more piercing objects, or 100 or more piercing objects, or 1,000 or more piercing objects, or 10,000 or more piercing objects, or 100,000 or more piercing objects.

[0372] In some implementations, the narrowest protruding feature that pierces the object may be less than 50 micrometers, or less than 10 micrometers, or less than 5 micrometers, or less than 2 micrometers, or less than 1 micrometer, or less than 0.5 micrometers, or less than 0.2 micrometers.

[0373] In some embodiments, the longest protruding feature of the pierced object (including any corners or bends from the channel surface) is at least 100 micrometers, or at least 25 micrometers, or at least 10 micrometers, or at least 5 micrometers, or at least 1 micrometer, or at least 0.5 micrometers, or at least 0.2 micrometers, or at least 0.1 micrometers.

[0374] In some embodiments, the surface of the punctured object can be modified or selected to promote nucleic acid adhesion. In other embodiments, the surface of the punctured object can be modified or selected to reduce the probability of nucleic acid adhesion.

[0375] In some implementations, surface-patterned fluid devices are micro / nano-fabricated with topological or chemical / energy contrast features, comprising arrays or more than one purposefully designed pattern and structure, such as a large number of parallel linear channels, linear or nonlinear grooves, column arrays with vertical or oriented tilt angles, asymmetric oriented hook structures in suspensions, micropores, and platforms whose geometry has variable width, depth, or contour dimensions to facilitate the capture of circular or loop-associated circular DNA or chromosome particles of certain sizes and shapes to enrich or exclude certain members of a sample population for hierarchical separation and isolation.

[0376] There are many different methods for creating one or more such piercing objects. Figure 15 In one embodiment shown, the hook is fabricated using a method well understood by those skilled in the art of microelectromechanical systems (MEMS) through patterning and etching. First, the tip of the hook is patterned in a photoresist (1511) on a substrate having a nitride film on its surface. Then, anisotropic plasma etching using the photoresist (1521) as a mask is used to etch through the nitride (1522) and across the nitride into the underlying glass substrate (1523). The photoresist is then removed, and isotropic timed chemical etching using diluted hydrofluoric acid is used to etch below the nitride to expose the hook tip, but the etching stops before all the glass is consumed, leaving the glass tower portion (1533) of the hook attached to the glass substrate (1534). This method is highly efficient for mass-producing thousands and millions of such hooks parallel to the surface of a glass substrate, offering considerable design freedom in choosing the location, density, and lateral (xy) feature dimensions. Hooks of varying heights can be fabricated using more than one patterning and etching step.

[0377] Another implementation involves fabricating the pierced object, at least partially, in a photocurable material (e.g., SU8) or in another negative resist that crosslinks upon exposure to light of a suitable wavelength. Such photocurable materials are highly advantageous for complex 3D configurations with micron and submicron features because they are constructed by adding the material to a substrate rather than etching it away. In some implementations, the photocurable material can be patterned through more than one step: spin-coating to the desired thickness, aligning a photomask, exposing the material to light through the mask, and rinsing away any unexposed areas of the photocurable material. In another implementation, a direct-write laser can be used to focus the desired photocurable material to be crosslinked in 3D space.

[0378] Other methods for manufacturing one or more puncture objects may include 3D printing puncture objects, self-assembling pre-formed puncture objects on the surface of a fluid device, stamping pre-formed puncture objects on the surface of a fluid device, embossing or imprinting puncture objects on the surface of a fluid device, and combinations of all the previous embodiments.

[0379] In another set of embodiments that selectively puncture one or more circular ecDNAs and exclude linear cellular DNA (particularly chromosomal DNA), cells containing both circular ecDNA and regular chromosomes are prevented from entering the metaphase by chemicals that disrupt spindle fibers, and then the cells are subjected to hypotonic treatment, swelling, and capsule weakening, so that cell rupture can be induced by a variety of methods, including dripping onto the surface of an open fluid device. Figure 16 In one particular embodiment shown, the surface of the device (1611) includes an assembly of piercing objects (1612), preferably rods, cones, or spikes, which act to facilitate both cell rupture and piercing of circular ecDNA. In this embodiment, a solution (1614) of cells (1615) containing linear cellular DNA (1617) (drawn as a chromosome) or a portion thereof and circular ecDNA (1616) is dispensed (1613) onto the assembly of piercing objects (1612). In a preferred embodiment, impact of the cells with the piercing objects promotes physical lysis or rupture of the cells, allowing intracellular contents to disperse into the assembly of piercing objects (1612), piercing circular long nucleic acid molecules (1621) but not linear long nucleic acid molecules (1622). In some embodiments, the sample is then optically probed to identify and quantify the circular ecDNA derived from the cells. In a preferred embodiment, the difference in migration rate between the pierced circular ecDNA and cellular DNA under applied force physically separates the pierced circular ecDNA from the cellular DNA. In some embodiments, the applied force may include fluid flow shear force, capillary flow shear force, convective flow shared force, or surface tension. The physical separation facilitates the optical detection of circular molecules by removing or reducing the confusion effects of overlapping or adjacent molecules during optical detection. In a preferred embodiment, the molecules undergo at least one washing or reaction step with an enzyme, dye, or reagent prior to optical detection. In some embodiments, the migration rate of cellular DNA in solution to the applied force is further increased by selectively cleaving the molecules into smaller molecules (1632) rather than circular ecDNA (1631). In some embodiments, linear cellular DNA (1643) can be selectively washed away from circular ecDNA (1642).

[0380] 6.1.4.4. Flexible Velcro method and apparatus for isolating circular DNA.

[0381] In another set of embodiments, the circular long nucleic acid molecule can be pierced by the head of a flexible polymer chain attached to a solid object, wherein after piercing, the head binds to a binding partner attached to the same or a different solid object, thereby "passing through" the circular DNA. In a preferred embodiment, the head cannot bind to the binding partner until a preliminary chemical, photochemical, enzymatic, additive, or substrate enabling process occurs first. In some embodiments, the enabling process is the addition of a catalyst to enable binding. In some embodiments, the enabling process is the addition or removal of light to enable binding. In some embodiments, the enabling process is the addition or removal of a reagent to enable binding. In some embodiments, the enabling process is the un-caging of the head or the binding partner, or both, to enable binding. In some embodiments, the binding partner itself is attached to the solid object via a flexible polymer chain.

[0382] Figure 17An embodiment of piercing circular ecDNA with a flexible polymer chain is illustrated, wherein the flexible polymer chain (1712) and its binding head (1715) are attached to a wall of a channel (1711) of a fluid device, while the binding mating body (1714) is attached to a second wall within the channel. At time point (i), the flexible polymer chain is unable to bind to its corresponding binding mating body because the binding mating body is caged or blocked in activity by a photolytically cleavable binding inhibitor (1713). At a later time point (ii), the binding inhibitor is removed (1723) (or inactivated) from the binding mating body (1714) by exposure to light of an appropriate wavelength (1727). An applied external force (1726) holds the flexible polymer chain in an elongated manner (1722) against the wall of the channel (1721) to which it is attached, and...

Claims

1. A method for separating at least one ecDNA from at least one cellular DNA, wherein the at least one ecDNA and the at least one cellular DNA are derived from at least one cell, and the separation is performed in a fluid apparatus.

2. The method according to claim 1, wherein the at least one cellular DNA is not ecDNA.

3. The method according to claim 2, wherein the at least one cellular DNA comprises a chromosome or a portion thereof.

4. The method according to claim 2, wherein the at least one cellular DNA comprises chromatin.

5. The method according to claim 2, wherein the at least one cellular DNA comprises mitochondrial DNA.

6. The method according to claim 1, wherein the cell DNA itself is a type of ecDNA having properties different from those of the at least one ecDNA.

7. The method of claim 6, wherein the characteristic is length.

8. The method of claim 6, wherein the characteristic is quality.

9. The method of claim 6, wherein the characteristic is a topological structure.

10. The method of claim 6, wherein the characteristic is a sequence.

11. The method of claim 6, wherein the characteristic is a gene.

12. The method of claim 6, wherein the characteristic is a mutation.

13. The method of claim 6, wherein the characteristic is structural variation.

14. The method of claim 6, wherein the characteristic is a single nucleotide polymorphism.

15. The method of claim 6, wherein the characteristic is a breakpoint.

16. The method of claim 6, wherein the characteristic is the location of origin within the chromosome.

17. The method of claim 6, wherein the characteristic is the source chromosome number (or type).

18. The method of claim 6, wherein the characteristic is an oncogene.

19. The method of claim 6, wherein the characteristic is the amount of the bound body.

20. The method of claim 6, wherein the characteristic is the type of the bonded body.

21. The method of claim 6, wherein the characteristic is the amount of bound markers.

22. The method of claim 6, wherein the characteristic is the position or orientation of the bonded body.

23. The method of claim 6, wherein the characteristic is derived from cells.

24. The method of claim 6, wherein the characteristic is a source organization.

25. The method of claim 6, wherein the characteristic is a source of disease state.

26. The method of claim 6, wherein the characteristic is derived from a tumor state.

27. The method of claim 6, wherein the characteristic is derived from a patient.

28. The method of claim 6, wherein the characteristic is a type of higher nucleic acid structure.

29. The method of claim 6, wherein the characteristic is the amount of higher nucleic acid structure.

30. The method of claim 6, wherein the characteristic is the position or orientation of an higher-order nucleic acid structure.

31. The method of claim 1, wherein an output sample is then extracted from the fluid device, the output sample comprising at least 70% ecDNA based on the total nucleic acid mass in the output sample.

32. The method of claim 31, wherein the total nucleic acid mass in the output sample comprises only nucleic acid molecules longer than 100 bp.

33. The method of claim 31, wherein the total nucleic acid mass in the output sample comprises only nucleic acid molecules longer than 1000 bp.

34. The method of claim 1, wherein an output sample is then extracted from the fluid device, the output sample comprising no more than 30% cellular DNA based on the total nucleic acid mass in the output sample.

35. The method of claim 34, wherein the total nucleic acid mass in the output sample comprises only nucleic acid molecules longer than 100 bp.

36. The method of claim 34, wherein the total nucleic acid mass in the output sample comprises only nucleic acid molecules longer than 1000 bp.

37. The method of claim 1, wherein an output sample is then extracted from the fluid device, the output sample comprising at least 90% ecDNA based on the total nucleic acid mass in the output sample.

38. The method of claim 37, wherein the total nucleic acid mass in the output sample comprises only nucleic acid molecules longer than 100 bp.

39. The method of claim 37, wherein the total nucleic acid mass in the output sample comprises only nucleic acid molecules longer than 1000 bp.

40. The method of claim 1, wherein an output sample is then extracted from the fluid device, the output sample comprising no more than 10% cellular DNA based on the total nucleic acid mass in the output sample.

41. The method of claim 40, wherein the total nucleic acid mass in the output sample comprises only nucleic acid molecules longer than 100 bp.

42. The method of claim 40, wherein the total nucleic acid mass in the output sample comprises only nucleic acid molecules longer than 1000 bp.

43. The method of claim 1, wherein a hybridization probe is generated comprising at least a portion of the at least one isolated ecDNA (or an amplified copy thereof, or synthesized from its sequence).

44. The method of claim 43, wherein the length of said at least a portion is at least 5 base pairs.

45. The method of claim 43, wherein the length of said at least a portion is at least 10 base pairs.

46. ​​The method of claim 43, wherein the length of said at least a portion is at least 50 base pairs.

47. The method of claim 43, wherein the hybridization probe comprises a marker.

48. The method of claim 43, wherein the hybridization probe comprises a hybridization capture probe.

49. The method of claim 43, wherein the hybridization probe then binds to at least a portion of a long nucleic acid molecule.

50. The method of claim 49, wherein the long nucleic acid molecule and the isolated at least one ecDNA originate from the same patient.

51. The method of claim 49, wherein the long nucleic acid molecule is a chromosome.

52. The method of claim 49, wherein the long nucleic acid molecule is bound to at least two markers, the markers comprising a physical map of the long nucleic acid molecule.

53. The method of claim 1, wherein a guide RNA is generated, the guide RNA comprising at least a portion of the at least one isolated ecDNA (or an amplified copy thereof, or synthesized from its sequence).

54. The method of claim 53, wherein the length of said at least a portion is at least 5 base pairs.

55. The method of claim 53, wherein the length of said at least a portion is at least 10 base pairs.

56. The method of claim 53, wherein the length of said at least a portion is at least 50 base pairs.

57. The method of claim 1, wherein the treatment for the disease is generated, the treatment comprising at least a portion of the at least one isolated ecDNA (or an amplified copy thereof, or synthesized from its sequence).

58. The method of claim 57, wherein the length of said at least a portion is at least 5 base pairs.

59. The method of claim 57, wherein the length of said at least a portion is at least 10 base pairs.

60. The method of claim 57, wherein the length of said at least a portion is at least 50 base pairs.

61. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA is sequenced.

62. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA is amplified.

63. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA is replicated.

64. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA is genotyped.

65. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA is hybridized.

66. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA hybridizes with the probe.

67. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA hybridizes with the array.

68. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA is bound to the body.

69. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA is bound to a marker.

70. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA participates in the chemical reaction.

71. The method according to claim 1, wherein at least a portion of the at least one isolated ecDNA participates in the enzymatic reaction.

72. The method of claim 1, wherein at least one isolated ecDNA is lysed.

73. The method of claim 1, wherein at least one isolated ecDNA is cleaved.

74. The method of claim 1, wherein at least one isolated ecDNA is used for DNA fragmentation.

75. The method of claim 1, wherein at least one isolated ecDNA is used to ligate the adaptor to the fragmented product.

76. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA (or its amplified product or sequence) is used to generate an array.

77. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA (or its amplified product or sequence) is used to generate primers.

78. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA (or its amplified product or sequence) is used to generate a marker.

79. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA (or its amplified product or sequence) is used to generate a barcode.

80. The method of claim 1, wherein at least a portion of the at least one isolated ecDNA (or its amplified product or sequence) is used to generate a unique molecular identifier.

81. The method according to claims 1-80, wherein at least a portion of the method occurs within the fluid device.

82. The method according to claims 1-80, wherein at least a portion of the method occurs outside the fluid device.

83. The method of claim 1, wherein the at least one cell comprises circulating tumor cells.

84. The method of claim 1, wherein the fluid device comprises an open fluid device.

85. The method of claim 84, wherein the isolated at least one ecDNA is extracted from the fluid device by rinsing.

86. The method of claim 84, wherein the isolated at least one ecDNA is extracted from the fluid device by dispensing a volume of solution onto the surface of the fluid device to absorb the at least one ecDNA.

87. The method of claim 84, wherein the isolated at least one ecDNA is extracted from the fluid device by bringing a contact probe to the vicinity of the ecDNA and forming a bond directly (or indirectly) between the ecDNA and the contact probe.

88. The method of claim 1, wherein the fluid device comprises a closed fluid device.

89. The method of claim 1, wherein at least one cell is lysed within the fluid device.

90. The method of claim 1, wherein at least one cell is ruptured outside the fluid device.

91. The method of claim 1, wherein the nucleic acid contents of the at least one cell are contained within a single droplet.

92. The method of claim 1, wherein the fluid device includes an inlet port and at least two outlet fluid ports, the inlet port and the at least two outlet fluid ports being fluidly connected to each other through a separation region.

93. The method of claim 92, wherein the at least one ecDNA and at least one cellular DNA are introduced into the inlet port, and the at least one ecDNA is collected at a specific outlet port, and the at least one cellular DNA is collected at a cell outlet port.

94. The method of claim 92, wherein at least one output port is the same as the input port.

95. The method of claim 1, wherein the separation mechanism comprises introducing the at least one ecDNA and the at least one cellular DNA into a separation region within the fluid device, wherein at least one force is applied to or removed from the molecules, and the application or removal of the at least one force results in an increased physical separation distance between the molecules compared to the case where no force is applied or removed.

96. The method of claim 95, wherein the magnitude and direction of the force applied to the at least one ecDNA vary at least in part with the properties associated with the at least one ecDNA or the organism bound to the at least one ecDNA.

97. The method of claim 96, wherein the characteristic includes size.

98. The method of claim 96, wherein the characteristic includes quality.

99. The method of claim 96, wherein the characteristic includes rigidity.

100. The method of claim 96, wherein the characteristic includes stiffness.

101. The method of claim 96, wherein the characteristic includes the radius of gyration.

102. The method of claim 96, wherein the characteristic includes the number of single-chain cuts.

103. The method of claim 96, wherein the characteristic includes the proportion of single-stranded ecDNA.

104. The method of claim 96, wherein the characteristic includes the proportion of denatured ecDNA.

105. The method of claim 96, wherein the characteristic includes mass density.

106. The method of claim 96, wherein the characteristic includes charge.

107. The method of claim 96, wherein the characteristic includes charge density.

108. The method of claim 96, wherein the property includes surface charge.

109. The method of claim 96, wherein the characteristic includes surface charge density.

110. The method of claim 96, wherein the characteristic includes length.

111. The method of claim 96, wherein the characteristic includes magnetic moment.

112. The method of claim 96, wherein the characteristic includes elasticity.

113. The method of claim 96, wherein the characteristic includes the volume of the space occupied by the 3D coating containing the molecule.

114. The method of claim 96, wherein the characteristic includes the surface area of ​​the space occupied by the 3D coating containing the molecule.

115. The method of claim 96, wherein the characteristic includes the length of the longest axis of the 3D coating containing the molecule.

116. The method of claim 96, wherein the characteristic includes the length of the shortest axis of the 3D coating comprising the molecule.

117. The method of claim 96, wherein the characteristic includes entropy energy.

118. The method of claim 96, wherein the characteristic includes enthalpy.

119. The method of claim 96, wherein the property includes free energy.

120. The method of claim 96, wherein the characteristic includes temperature.

121. The method of claim 96, wherein the characteristic includes topology.

122. The method of claim 96, wherein the characteristic includes mobility.

123. The method of claim 96, wherein the characteristic includes an average trajectory vector.

124. The method of claim 96, wherein the characteristic includes relaxation time.

125. The method of claim 96, wherein the characteristic includes inertia.

126. The method of claim 96, wherein the characteristic includes a drag coefficient.

127. The method of claim 96, wherein the characteristic includes surface friction.

128. The method of claim 96, wherein the characteristic includes shape resistance.

129. The method of claim 96, wherein the property includes bonding affinity.

130. The method of claim 96, wherein the characteristic includes bonding specificity.

131. The method of claim 96, wherein the property includes bond strength.

132. The method of claim 96, wherein the characteristic includes a signal from the marker.

133. The method of claim 96, wherein the characteristic includes the higher nucleic acid structure.

134. The method of claim 96, wherein the characteristic includes a sequence.

135. The method of claim 96, wherein the characteristic includes a physical spectrum.

136. The method of claim 96, wherein the characteristic includes the density of the body bound to the molecule.

137. The method of claim 96, wherein the characteristic includes the number of bodies bound to the molecule.

138. The method of claim 96, wherein the characteristic includes the presence of a body that binds to the molecule.

139. The method of claim 96, wherein the characteristic includes the absence of an organism bound to the molecule.

140. The method of claim 95, wherein the separation mechanism includes a filtering effect.

141. The method of claim 95, wherein the separation mechanism includes entropy capture.

142. The method of claim 95, wherein the separation mechanism comprises deterministic lateral displacement.

143. The method of claim 95, wherein the separation mechanism comprises entrained flow staged separation.

144. The method of claim 95, wherein the separation mechanism includes a viscoelastic effect.

145. The method of claim 95, wherein the separation mechanism includes an inertial effect.

146. The method of claim 95, wherein the separation mechanism includes a cross-flow filtering effect.

147. The method of claim 95, wherein the separation mechanism includes a hydrodynamic filtration effect.

148. The method of claim 95, wherein the separation mechanism includes a microfiltration effect.

149. The method of claim 95, wherein the separation mechanism comprises piercing the at least one ecDNA with a column or hook through a topological pore of the DNA, wherein the DNA is circular DNA.

150. The method of claim 95, wherein the applied force comprises fluid flow shear force.

151. The method of claim 95, wherein the at least one applied force comprises surface tension.

152. The method of claim 95, wherein the at least one applied force comprises a surface tension gradient.

153. The method of claim 95, wherein the at least one applied force comprises convection.

154. The method of claim 95, wherein the at least one applied force comprises thermocapillary convection.

155. The method of claim 95, wherein the at least one applied force comprises an entropic force.

156. The method of claim 95, wherein the at least one applied force comprises a hydrophobic force.

157. The method of claim 95, wherein the at least one applied force comprises a capillary force.

158. The method of claim 95, wherein the at least one applied force comprises a van der Waals force.

159. The method of claim 95, wherein the at least one applied force comprises pressure.

160. The method of claim 95, wherein the at least one applied force comprises the Marangoni effect.

161. The method of claim 95, wherein the at least one applied force comprises an electrophoretic force.

162. The method of claim 95, wherein the at least one applied force comprises a dielectric electrophoretic force.

163. The method of claim 95, wherein the at least one applied force comprises an acoustic force.

164. The method of claim 95, wherein the at least one applied force comprises a magnetic force.

165. The method of claim 95, wherein the at least one applied force comprises an electromagnetic force.

166. The method of claim 95, wherein the at least one applied force comprises an electrostatic force.

167. The method of claim 95, wherein the at least one applied force comprises an optical force.

168. The method of claim 95, wherein the separation region comprises a filter.

169. The method of claim 95, wherein the separation region comprises a gel.

170. The method of claim 95, wherein the separation region comprises an artificial gel.

171. The method of claim 95, wherein the separation region comprises a deterministic gel.

172. The method of claim 95, wherein the separation region comprises an entropy trap.

173. The method of claim 95, wherein the separation region includes an entropy barrier.

174. The method of claim 95, wherein the separation region comprises a functionalized surface.

175. The method of claim 174, wherein the functionalized surface specifically binds to ecDNA (directly or indirectly).

176. The method of claim 174, wherein the functionalized surface specifically binds to cellular DNA (directly or indirectly).

177. The method of claim 95, wherein the separation region includes a deterministic lateral displacement (DLD) device.

178. The method of claim 95, wherein the separation region comprises a fractional flow separation (PFF) device.

179. The method of claim 95, wherein the separation region comprises a viscoelastic effect microfluidic (VEM) device.

180. The method of claim 95, wherein the separation region comprises an inertial effect microfluidic (IMF) device.

181. The method of claim 95, wherein the separation region comprises a cross-flow filter (CFF) device.

182. The method of claim 95, wherein the separation region comprises a fluid dynamics filter.

183. The method of claim 95, wherein the separation region comprises a microfiltration filter.

184. The method of claim 95, wherein the separation region comprises an electrophoretic sorting device.

185. The method of claim 95, wherein the separation region comprises a dielectric electrophoretic sorting device.

186. The method of claim 95, wherein the separation region comprises an acoustic separation device.

187. The method of claim 95, wherein the separation region comprises a magnetic sorting device.

188. The method of claim 95, wherein the separation region comprises an electromagnetic sorting device.

189. The method of claim 95, wherein the separation region comprises an electrostatic sorting device.

190. The method of claim 95, wherein the separation region comprises an optical sorting device.

191. The method of claim 95, wherein the separation region comprises a bump array.

192. The method of claim 95, wherein the separation region comprises a ratchet array.

193. The method of claim 95, wherein the separation region comprises a column.

194. The method of claim 95, wherein the separation region includes a depression.

195. The method of claim 95, wherein the separation region comprises a channel.

196. The method of claim 95, wherein the separation region comprises a groove.

197. The method of claim 95, wherein the separation region includes a hole.

198. The method of claim 197, wherein the pore has a size of 0.1-0.2 micrometers.

199. The method of claim 197, wherein the pore has a size of 0.2-0.4 micrometers.

200. The method of claim 197, wherein the pore has a size of 0.4-0.6 micrometers.

201. The method of claim 197, wherein the pore has a size of 0.6-0.8 micrometers.

202. The method of claim 197, wherein the pore has a size of 0.8-1.0 micrometers.

203. The method of claim 197, wherein the pore has a size of 1.0-1.2 micrometers.

204. The method of claim 197, wherein the pore has a size of 1.2-1.4 micrometers.

205. The method of claim 197, wherein the aperture has a size of 1.4-1.6 micrometers.

206. The method of claim 197, wherein the aperture has a size of 1.6-1.8 micrometers.

207. The method of claim 197, wherein the pore has a size of 1.8-2.0 micrometers.

208. The method of claim 197, wherein the pore has a size of 2.0-2.2 micrometers.

209. The method of claim 197, wherein the pore has a size of 2.2-2.4 micrometers.

210. The method of claim 95, wherein the separation region includes fluid characteristics.

211. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 100 micrometers.

212. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 80 micrometers.

213. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 70 micrometers.

214. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 60 micrometers.

215. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 50 micrometers.

216. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 40 micrometers.

217. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 30 micrometers.

218. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 20 micrometers.

219. The method of claim 210, wherein the fluid feature comprises an object having dimensions of less than 10 micrometers.

220. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 8 micrometers.

221. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 5 micrometers.

222. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 3 micrometers.

223. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 2 micrometers.

224. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 1 micrometer.

225. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 0.8 micrometers.

226. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 0.5 micrometers.

227. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 0.3 micrometers.

228. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 0.2 micrometers.

229. The method of claim 210, wherein the fluid feature comprises an object having a dimension of less than 0.1 micrometers.

230. The method of claim 1, wherein the separation mechanism comprises introducing the at least one ecDNA and the at least one cellular DNA into a confined region of the fluid device, whereby the probed region physically confines the molecules within a fluid channel having a certain confined dimension, such that the probability of the molecules overlapping along any axis parallel to the confined dimension is reduced relative to a similar channel having a larger confined dimension.

231. The method of claim 230, wherein the constraint dimension is less than 10 micrometers.

232. The method of claim 230, wherein the constraint dimension is less than 8 micrometers.

233. The method of claim 230, wherein the constraint dimension is less than 6 micrometers.

234. The method of claim 230, wherein the constraint dimension is less than 5 micrometers.

235. The method of claim 230, wherein the constraint dimension is less than 4 micrometers.

236. The method of claim 230, wherein the constraint dimension is less than 3 micrometers.

237. The method of claim 230, wherein the constraint dimension is less than 2 micrometers.

238. The method of claim 230, wherein the constraint dimension is less than 1 micrometer.

239. The method of claim 230, wherein the constraint dimension is less than 0.8 micrometers.

240. The method of claim 230, wherein the constraint dimension is less than 0.6 micrometers.

241. The method of claim 230, wherein the constraint dimension is less than 0.5 micrometers.

242. The method of claim 230, wherein the constraint dimension is less than 0.4 micrometers.

243. The method of claim 230, wherein the constraint dimension is less than 0.3 micrometers.

244. The method of claim 230, wherein the constraint dimension is less than 0.2 micrometers.

245. The method of claim 230, wherein the at least one ecDNA and at least one cellular DNA are probed within the confined region.

246. The method of claim 245, wherein the probing includes optical microscopy.

247. The method of claim 245, wherein the exploration includes fluorescence microscopy.

248. The method of claim 245, wherein the exploration includes bright-field microscopy.

249. The method of claim 245, wherein the exploration includes scanning electron microscopy.

250. The method of claim 245, wherein the probing includes contact probe probing.

251. The method of claim 250, wherein the contact probe comprises an atomic force microscope.

252. The method of claim 245, wherein at least a portion of the probe output is used as at least one input to a logic function, the output of which includes selectively applying (or removing) at least one applied force to the at least one ecDNA (or the at least one cellular DNA) such that the physical location of the at least one ecDNA (or the at least one cellular DNA) differs from the location it would occupy in the fluid device if the at least one force were not applied.

253. The method of claim 1, wherein the separation mechanism comprises introducing the at least one ecDNA and the at least one cellular DNA into a region of the fluid device, thereby enabling the selective application (or removal) of at least one force to the at least one ecDNA (or the at least one cellular DNA) such that the physical location of the at least one ecDNA (or the at least one cellular DNA) differs from the location it would occupy in the fluid device if the at least one force were not applied (or removed); wherein the decision to selectively apply the at least one force is based at least in part on the results of the probe.

254. The method of claim 253, wherein the probing includes quantifying the physical mass of the at least one ecDNA or at least one cellular DNA.

255. The method of claim 253, wherein the probing includes quantifying the physical topology of the at least one ecDNA or at least one cellular DNA.

256. The method of claim 253, wherein the probing includes quantifying the physical length of the at least one ecDNA or at least one cellular DNA.

257. The method of claim 253, wherein the probing includes quantifying the higher nucleic acid structure of the at least one ecDNA or at least one cellular DNA.

258. The method of claim 253, wherein the probing includes quantifying the physical mass of the at least one ecDNA or at least one cellular DNA.

259. The method of claim 253, wherein the probing includes identifying a conjugate that binds to the at least one ecDNA or at least one cellular DNA.

260. The method of claim 253, wherein the probing includes identifying individuals that specifically bind directly or indirectly to ecDNA.

261. The method of claim 253, wherein the probing includes identifying a body that specifically binds directly or indirectly to cellular DNA.

262. The method of claim 1, wherein the at least one isolated ecDNA is identified as ecDNA.

263. The method of claim 262, wherein identification includes determining the physical location of the ecDNA within the fluid device.

264. The method of claim 263, wherein the physical location is the output port of the fluid device.

265. The method of claim 263, wherein the physical location is a fluid characteristic of the fluid device.

266. The method of claim 265, wherein the fluid feature is a hook.

267. The method of claim 265, wherein the fluid feature is a depression.

268. The method of claim 265, wherein the fluid feature is a column.

269. The method of claim 265, wherein the fluid feature is a channel.

270. The method of claim 262, wherein identification comprises probing for markers that specifically bind directly or indirectly to ecDNA.

271. The method of claim 262, wherein identification comprises probing for markers that specifically bind directly or indirectly to cellular DNA.

272. The method of claim 262, wherein identification includes probing the physical size of the molecule.

273. The method of claim 262, wherein identification includes probing the topological structure of the molecule.

274. The method of claim 262, wherein identification includes probing the higher-order structure of the molecule.

275. The method of claim 262, wherein identification includes probing the length of the molecule.

276. The method according to claims 270-275, wherein the probing includes optical imaging.

277. The method according to claims 270-275, wherein the probing includes a contact probe.

278. The method according to claims 270-275, wherein the probing includes a shrinking device.

279. The method according to claims 270-275, wherein the probing includes the at least one ecDNA contained within the droplet.

280. The method according to claims 270-275, wherein the probing includes at least one type of cellular DNA contained within the droplet.

281. The method of claim 262, wherein the ecDNA profile is generated by the identification of the at least one isolated ecDNA.

282. The method of claim 281, wherein the ecDNA profile includes the presence of ecDNA in the at least one cell.

283. The method of claim 281, wherein the ecDNA profile includes the absence of ecDNA in the at least one cell.

284. The method of claim 281, wherein the ecDNA profile comprises the amount of ecDNA in the at least one cell.

285. The method of claim 281, wherein the ecDNA profile comprises an approximate number of ecDNAs in the at least one cell.

286. The method of claim 281, wherein the ecDNA profile includes the number of ecDNA in the at least one cell.

287. The method of claim 281, wherein the ecDNA profile includes the physical size of the at least one ecDNA in the at least one cell.

288. The method of claim 281, wherein the ecDNA profile includes the physical mass of the at least one ecDNA in the at least one cell.

289. The method of claim 281, wherein the ecDNA profile includes the physical topology of the at least one ecDNA in the at least one cell.

290. The method of claim 281, wherein the ecDNA profile includes higher nucleic acid structures associated with the at least one ecDNA in the at least one cell.

291. The method of claim 281, wherein the ecDNA profile includes a topological structure associated with the at least one ecDNA in the at least one cell.

292. The method of claim 281, wherein the ecDNA profile comprises higher nucleic acid structures associated with the at least one ecDNA in the at least one cell.

293. The method of claim 281, wherein the ecDNA profile includes sequences associated with the at least one ecDNA in the at least one cell.

294. The method of claim 281, wherein the ecDNA profile includes genotypes associated with the at least one ecDNA in the at least one cell.

295. The method of claim 281, wherein the ecDNA profile includes methylation patterns associated with the at least one ecDNA in the at least one cell.

296. The method of claim 281, wherein the ecDNA profile comprises a sequence associated with the at least one ecDNA in the at least one cell.

297. The method of claim 281, wherein the ecDNA profile includes genes associated with the at least one ecDNA in the at least one cell.

298. The method of claim 297, wherein the gene comprises an oncogene.

299. The method of claim 281, wherein the spectrum is used for diagnosing diseases.

300. The method of claim 281, wherein the spectrum is used to monitor the state of the disease.

301. The method of claim 281, wherein the spectrum is used to select the treatment type for the disease.

302. The method of claim 281, wherein the spectrum is used to change the treatment type of the disease.

303. The method of claim 281, wherein the spectrum is used to individualize the treatment type for the disease.

304. The method according to claims 299-303, wherein the disease includes cancer.

305. The method of claim 1, wherein the ecDNA comprises circular ecDNA.

306. The method of claim 1, wherein the ecDNA comprises linear ecDNA.

307. The method of claim 1, wherein the ecDNA comprises a fragment of ecDNA.

308. The method of claim 1, wherein the cellular DNA comprises chromosomes.

309. The method of claim 1, wherein the cellular DNA comprises segments of chromosomes.

310. The method of claim 1, wherein the at least one cell comprises a single cell.

311. The method according to claim 1, wherein the at least one ecDNA and the at least one cellular DNA originate from the same cell.

312. A fluid device capable of separating at least one ecDNA from at least one cellular DNA, wherein the at least one ecDNA and the at least one cellular DNA are derived from at least one cell, and the separation is performed within the fluid device.

313. A method for culturing cells in the presence of a first metabolic marker integrated into DNA.

314. A method for allowing early replication of DNA in the absence of a second marker.

315. A method for adding a second metabolic marker integrated into DNA.

316. A method for preparing a mixture of fixed or stable chromosomes from cells.

317. A method for sorting chromosomes that do not exist based on a second marker.

318. A method for amplifying DNA sorted by a method inhibited by a first label using nucleotides or primers containing an affinity label.

319. A method for generating an ecDNA FISH probe library, the method comprising: (a) Culture cells in the presence of a chromosome condenser, (b) Lyse the cells and preserve the mixture of ecDNA and chromosome, (c) Purify the ecDNA from the chromosome using a fluidic apparatus, (d) Generate labeled FISH probes from the purified ecDNA sample, (e) Hybridize the FISH probes with biological material, and (f) Probe the FISH probes.

320. The method of claim 319, wherein the fluid device comprises a syringe filter.

321. The method of claim 319, wherein the FISH probe label comprises more than one fluorophore.

322. The method of claim 319, wherein the lysis step is followed by pre-filtration to separate cell debris from DNA-containing material.

323. The method of claim 319, wherein the lysis step is followed by a precipitation step to separate DNA that is not a dense chromatin portion.

324. A method for generating an ecDNA capture probe library, the method comprising: (a) Culture cells in the presence of a chromosome condenser, (b) Lyse cells and preserve a mixture of ecDNA and chromosomes, (c) Purify ecDNA from chromosomes using a fluidic apparatus, (d) Generate affinity-tagged probes from the purified ecDNA sample, (e) Purify biological material with the affinity-tagged probes, and (f) Analyze the purified biological material.

325. The method of claim 324, wherein the fluid device comprises a syringe filter.

326. The method of claim 324, wherein the affinity probe label comprises a biotinylated random primer.

327. The method of claim 324, wherein the lysis step is followed by pre-filtration to separate cell debris from DNA-containing material.

328. The method of claim 324, wherein the lysis step is followed by a precipitation step to separate DNA that is not a dense chromatin portion.

329. The method of claim 324, wherein the biomaterial is a neighbor-linked oligonucleotide derived from a Hi-C experiment.

330. A method comprising flowing a sample containing aggregated nucleic acids through a fluid device in one direction, the fluid device including an array of barriers having a proximal end and a distal end, the array of barriers being configured to exhibit a series of parallel straight paths passing through at least a portion of the array of barriers, the series of parallel straight paths not parallel to the direction of sample flow; and collecting a first sample fraction at a first region at a distal end of the array of barriers remote from the barriers and collecting a second sample fraction at a second region at a distal end of the array of barriers.

331. The method of claim 330, wherein the obstacle comprises a solid obstacle.

332. The method of claim 330, wherein the obstacle comprises an entropy barrier.

333. The method of claim 330, wherein the first region spans the endpoints of a line drawn parallel to the direction in which the sample flows from the deposition point of the sample at the proximal end to the distal end.

334. The method of claim 330, wherein the second region spans a region including the endpoints of a path in the parallel series of straight paths, the path having a starting point in the proximal half of the obstacle array.

335. The method of claim 333, wherein the second region spans the region forming a first side of a triangle, the triangle having a second and a third side, the second and third sides comprising a path in the parallel series of straight paths and a line drawn parallel to the direction causing the sample to flow.

336. The method of claim 330, wherein the second region does not overlap with the first region.

337. The method of claim 330, wherein the obstacle array is a regular array.

338. The method of claim 330, wherein the obstacle array includes a regular array portion.

339. The method according to any one of claims 330 to 338, wherein the sample comprises chromosomes, and wherein the chromosomes flow through a straight path in the parallel series of straight paths, the straight path being not parallel to the direction of sample flow.

340. The method of any one of claims 330 to 338, wherein the sample comprises chromosomes, and wherein the chromosomes flow through the array to a point traversed by the second region.

341. The method of claim 330, comprising collecting a first sample fraction at the first region and passing the first sample fraction through a second obstacle array.

342. The method of claim 341, wherein the second obstacle array exhibits an inter-object distance smaller than the inter-object distance of the array.

343. The method of claim 341, wherein the second obstacle array exhibits an inter-object distance greater than the inter-object distance of the array.

344. The method of claim 341, wherein the second obstacle array exhibits an inter-object distance equal to the inter-object distance of the array.

345. The method of claim 330, further comprising collecting a second sample fraction at the second region and passing the second sample fraction through a second barrier array.

346. The method of claim 345, wherein the second obstacle array exhibits an inter-object distance smaller than the inter-object distance of the array.

347. The method of claim 345, wherein the second obstacle array exhibits an inter-object distance greater than the inter-object distance of the array.

348. The method of claim 345, wherein the second obstacle array exhibits an inter-object distance equal to the inter-object distance of the array.

349. The method of claim 330, further comprising, prior to flowing the sample, treating the sample precursor such that the deoxyribonucleic acid of the sample is condensed by chromosomes.

350. The method of claim 349, wherein treating the sample precursor such that the deoxyribonucleic acid of the sample is condensed by chromosomes comprises treating the cellular precursor of the sample with a cell cycle process inhibitor.

351. The method of claim 350, wherein the cell cycle process inhibitor stops the cell cycle process at the point where the DNA of the sample is condensed by chromosomes.

352. The method of claim 330, further comprising treating a sample precursor to remove lipids from the sample prior to flowing the sample.

353. The method of claim 330, further comprising treating a sample precursor to remove nuclei from the sample prior to flowing the sample.

354. The method of claim 330, wherein the method does not include ecDNA sequence-specific binding, exonuclease treatment, or incubation for more than 24 hours, more than 12 hours, or more than 4 hours.

355. The method of claim 330, further comprising treating a sample precursor to remove ribonucleic acid from the sample prior to flowing the sample.

356. The method of claim 330, comprising collecting a first sample fraction at the first region.

357. The method of claim 356, wherein the first sample fraction comprises ecDNA.

358. The method of claim 356, wherein the first sample fraction comprises mtDNA.

359. The method of claim 356, comprising sequencing the nucleic acids of the first sample fraction.

360. The method of claim 356, further comprising designing probes that specifically anneal to the nucleic acids of the first fraction.

361. The method of claim 360, wherein the probe is not annealed with human chromosomes.

362. The method of claim 360, wherein the probe is not annealed with a second nucleic acid sample collected at the second region.

363. The method of claim 357, comprising designing probe-specific annealing with the ecDNA of the first fraction.

364. The method of claim 363, wherein the probe is not annealed with human chromosomes.

365. The method of claim 363, wherein the probe is not annealed with a second nucleic acid sample collected at the second region.

366. The method according to any one of claims 360 to 365, wherein the probe is a CRISPR-guided nucleic acid.

367. The method according to any one of claims 360 to 365, wherein the probe is a hybridization probe.

368. The method according to any one of claims 360 to 365, wherein the probe is a FISH probe.

369. The method according to any one of claims 360 to 365, wherein the probe is an array probe.

370. The method of claim 360, wherein the probe is a CRISPR endonuclease component.

371. The method of claim 360, comprising administering the probe to cells of an individual from which the sample is derived.

372. The method of claim 370, comprising administering the probe to cells of an individual from which the sample is derived.

373. The method of claim 39, wherein the administration comprises transfection using a vector system expressing the CRISPR enzyme and the guiding oligonucleotide.

374. The method of claim 373, comprising monitoring the individual.

375. The method of claim 374, wherein monitoring the individual comprises separating a sample from the subject after the administration.

376. A method for treating an ecDNA-related disorder in an individual, comprising obtaining cells associated with the disorder, obtaining ecDNA from the cells, generating at least one probe that cleaves the ecDNA without cleaving the chromosome of the cells, and administering the probe to the individual such that the probe contacts the ecDNA in the individual's cells.

377. The method of claim 376, wherein the disorder is cancer.

378. The method of claim 376, wherein the disorder is aging.

379. The method of claim 376, wherein the cells are obtained from the individual.

380. The method of claim 376, wherein the cells are not obtained from the individual.

381. The method of claim 376, wherein obtaining ecDNA from the cells comprises treating the cells to enrich aggregated nucleic acids.

382. The method of claim 376, wherein obtaining ecDNA from the cells comprises treating the cells to enrich them with active cells.

383. The method of claim 376, wherein obtaining ecDNA from the cells comprises treating the cells to cause cell cycle arrest.

384. The method of claim 376, wherein obtaining ecDNA from the cells comprises treating the cells to arrest the cell cycle at mid-phase.

385. The method of claim 376, wherein obtaining ecDNA from the cell comprises lysing the cell and discarding the cell nucleus.

386. The method of claim 376, wherein obtaining ecDNA from the cells comprises lysing the cells and treating the cell lysates with an RNase.

387. The method of claim 376, wherein obtaining ecDNA from the cell comprises lysing the cell and discarding the cell membrane.

388. The method of claim 376, wherein obtaining ecDNA from the cells comprises filtering lysates from the cells.

389. The method of claim 388, wherein filtering the lysate comprises removing lipids from the lysate.

390. The method of claim 388, wherein filtering the pyrolyte comprises removing nuclei from the pyrolyte.

391. The method of claim 388, wherein filtering the lysate comprises removing cell membranes from the lysate.

392. The method of claim 388, wherein filtering the lysate comprises size-selecting the deoxyribonucleic acid of the lysate to obtain a filtrate.

393. The method of claim 388, wherein filtering the lysate includes size selection of the deoxyribonucleic acid of the lysate, excluding ecDNA sequence-specific binding.

394. The method of claim 392, wherein size selection includes making the pyrolysis stream through a porous membrane.

395. The method of claim 394, wherein the porous membrane is treated with magnesium ions.

396. The method of claim 394, wherein the porous membrane comprises pores having a diameter of about 1 micrometer.

397. The method of claim 394, wherein the porous membrane comprises pores having a diameter of about 1.2 micrometers.

398. The method of claim 394, wherein the porous membrane comprises pores having a diameter of about 0.8 micrometers.

399. The method of claim 394, wherein the porous membrane comprises pores having a diameter of about 0.5 micrometers.

400. The method of claim 394, wherein the porous membrane comprises pores having a diameter of about 2 micrometers.

401. The method of claim 392, wherein size selection includes causing the pyrolyzed stream to flow through an array of obstacles.

402. The method of claim 401, wherein the obstacle array is configured to form parallel straight paths through the obstacles.

403. The method of claim 402, wherein the parallel straight path through the array of obstacles is not parallel to the flow direction of the pyrolyte through the array of obstacles.

404. The method of claim 403, comprising collecting a first fraction and a second fraction of the pyrolyte.

405. The method according to claim 404, wherein, The first segment is collected at the first path endpoint, which includes a greater number of obstacle-mediated directional changes, compared to the second segment collected at the second endpoint.

406. The method of claim 404, wherein the first segment is collected at a first path endpoint, the first path endpoint comprising the longest path segment in the parallel straight path through the obstacle that is shorter than the second segment collected at the second path endpoint.

407. The method of claim 392, further comprising sequencing the nucleic acids of the filtrate to generate nucleic acid reads.

408. The method of claim 407, further comprising assigning at least some of the reads to ecDNA.

409. The method of claim 408, wherein allocating at least some of the reads to ecDNA comprises aligning the nucleic acid reads to a genomic sequence, and allocating the nucleic acid reads to ecDNA when the nucleic acid reads are mapped to a region of the genomic sequence having at least 3× coverage of overlapping reads.

410. The method of claim 408, wherein allocating at least some of the reads to ecDNA comprises aligning the nucleic acid reads to a genomic sequence, and allocating the nucleic acid reads to ecDNA when the nucleic acid reads are mapped to a region of the genomic sequence having at least 5× coverage of overlapping reads.

411. The method of claim 408, wherein allocating at least some of the reads to ecDNA comprises aligning the nucleic acid reads to a genomic sequence, and allocating the nucleic acid reads to ecDNA when the nucleic acid reads are mapped to a region of the genomic sequence having at least 10× coverage of overlapping reads.

412. The method of claim 408, wherein generating at least one probe that cleaves the ecDNA comprises identifying an ecDNA sequence segment different from the genomic sequence, and using the sequence segment as a probe target.

413. The method of claim 412, wherein the probe is a CRISPR guide.

414. The method of claim 412, wherein the probe is a CRISPR-guided nucleic acid.

415. The method of claim 412, wherein the probe is a hybridization probe.

416. The method of claim 412, wherein the probe is a FISH probe.

417. The method of claim 412, wherein the probe is an array probe.

418. The method of claim 412, wherein administering the probe to the individual comprises transfecting ecDNA into the cells of the individual using an expression system.

419. The method of claim 418, wherein the expression system encodes a CRISPR enzyme directed by the probe.

420. The method of claim 418, wherein the cells comprise cancer cells.

421. The method of claim 412, further comprising monitoring disease progression in the individual.

422. The method of claim 412, further comprising monitoring ecDNA levels in cells associated with the disorder.

423. The method of claim 412, further comprising monitoring ecDNA levels in cells unrelated to the disorder.

424. A method of treating cancer in an individual, comprising obtaining an ecDNA sequence from the individual and administering to the individual a CRISPR-guided endonuclease that specifically targets the ecDNA sequence from the individual.

425. The method of claim 424, wherein the ecDNA sequence comprises a sequence having a point mutation relative to a genomic reference.

426. The method of claim 424, wherein the ecDNA sequence comprises a sequence having an insertion mutation relative to a genomic reference.

427. The method of claim 424, wherein the ecDNA sequence comprises a sequence having a deletion mutation relative to a genomic reference.

428. The method of claim 424, wherein the ecDNA sequence comprises a sequence having a translocation mutation relative to a genomic reference.

429. The method of claim 424, wherein the ecDNA sequence comprises a sequence at the junction of separate segments spanning a genomic reference.

430. The method of claim 424, wherein obtaining the ecDNA sequence from the individual comprises filtering cell extracts from the individual.

431. The method of claim 424, wherein obtaining the ecDNA sequence from the individual comprises flowing a cell extract from the individual through an array of barriers.

432. The method of claim 424, wherein obtaining the ecDNA sequence from the individual does not include ecDNA sequence-specific binding.

433. The method of claim 424, wherein obtaining the ecDNA sequence from the individual comprises lysing cells from the individual and discarding any cell nuclei.

434. The method of claim 424, wherein obtaining the ecDNA sequence from the individual comprises processing cells from the individual to enrich aggregated chromosomes.

435. The method of claim 424, wherein obtaining the ecDNA sequence from the individual comprises arresting cells from the individual in the intermediate stage.

436. An enrichment composition comprising ecDNA, wherein the composition is derived from a cell extract, and wherein the concentration of the ecDNA in the enrichment composition is at least 2 × that of the cell extract.

437. The enrichment composition according to claim 436, wherein the concentration of the ecDNA in the enrichment composition is at least 4 times the concentration of the cell extract.

438. The enrichment composition according to claim 436, wherein the concentration of the ecDNA in the enrichment composition is at least 10 × the concentration of the cell extract.

439. The enrichment composition according to claim 436, wherein the concentration of the ecDNA in the enrichment composition is at least 20 times the concentration of the cell extract.

440. The enrichment composition according to claim 436, wherein the concentration of the ecDNA in the enrichment composition is at least 30 times the concentration of the cell extract.

441. The enriched composition according to claim 436, wherein the enriched composition does not contain chromosomes.

442. The enriched composition according to claim 436, wherein the enriched composition is obtained from the individual by a method comprising filtering a cell extract from the individual in a fluid device.

443. The enriched composition of claim 436, wherein the enriched composition is obtained from the individual by a method comprising flowing a cell extract from the individual through an array of barriers.

444. The enriched composition according to claim 436, wherein the enriched composition is obtained from the individual by a method that does not include ecDNA sequence-specific binding and / or does not include exonuclease treatment and / or does not include incubation for more than 24 hours.

445. The enrichment composition of claim 436, wherein the enrichment composition comprises mtDNA.

446. A CRISPR complex comprising a guide nucleic acid that directs the cleavage of ecDNA without cleaving chromosomes.

447. The complex according to claim 446 is delivered to cancer cells.

448. The complex of claim 447, wherein the complex is delivered by transfection with an expression vector encoding the complex.

449. The complex of claim 446, wherein the guiding nucleic acid targets an ecDNA target identified in a cell extract.

450. The complex of claim 449, wherein the cell extract is produced by filtration.

451. The complex of claim 449, wherein the cell extract is produced by flowing through an array of barriers.

452. The complex of claim 446, wherein the guiding nucleic acid of the complex is annealed with a portion of an ecDNA target region comprising two distinct segments of a genomic reference.

453. The complex of claim 446, wherein the guiding nucleic acid of the complex is annealed with an ecDNA target region containing a point mutation relative to a genomic reference.

454. The complex of claim 446, wherein the guiding nucleic acid of the complex is annealed with an ecDNA target region comprising an insertion relative to a genomic reference.

455. The complex of claim 446, wherein the guiding nucleic acid of the complex is annealed with an ecDNA target region containing a deletion relative to a genomic reference.

456. The complex of claim 446, wherein the guiding nucleic acid of the complex is annealed with an ecDNA target region comprising a translocation relative to a genomic reference.

457. The complex of claim 446, wherein the guiding nucleic acid of the complex is annealed to an ecDNA target region that is substantially different from that of a genomic reference, such that the CRISPR does not cleave the chromosome of the individual.

458. The complex of claim 446, wherein the genomic reference comprises a sequence of the chromosome.

459. A method for isolating circular DNA from a subject, comprising flowing lysate of at least one cell of the subject through a surface in a fluid device in a first flow direction, and then flowing a washing buffer through the surface in a second flow direction, the surface including protrusions configured to form an acute angle with the surface in the first flow direction and to form an obtuse angle with the surface in the second flow direction.

460. The method of claim 459, wherein the lysate is treated to remove the cell nucleus.

461. The method of claim 459, wherein the lysate is treated to remove the cell membrane.

462. The method of claim 459, wherein the lysate is treated to remove RNA.

463. The method of claim 459, wherein the lysate comprises ecDNA.

464. The method of claim 459, wherein the lysate comprises mtDNA.

465. The method of claim 459, wherein the first direction and the second direction are antiparallel.

466. The method according to claim 459, wherein, The first direction and the second direction form an obtuse angle with each other.

467. The method of claim 459, comprising flowing the lysate in the first flow direction until substantially all unbound lysate leaves the surface, and then flowing the wash buffer in the second direction.

468. The method of claim 459, comprising flowing the wash buffer in the first direction after the lysate has flowed in the first direction but before the wash buffer has flowed in the second direction.

469. The method of claim 459, further comprising sequencing nucleic acids captured in the effluent in the second flow direction to generate sequence reads.

470. The method of claim 469, further comprising identifying sequence reads mapped to mitochondrial genome sequences.

471. The method of claim 469, comprising identifying sequence reads at a given locus that are mapped to a nuclear genome sequence with low coverage.

472. The method of claim 471, wherein the low coverage does not exceed 4×.

473. The method of claim 471, wherein the low coverage does not exceed 3×.

474. The method of claim 471, wherein the low coverage does not exceed 2×.

475. The method of claim 471, wherein the low coverage does not exceed 1×.

476. The method of claim 471, further comprising designating sequence reads mapped to the nuclear genome sequence with low coverage as artifacts.

477. The method of claim 469, further comprising identifying sequence reads mapped to a nuclear genome sequence with high coverage at a given locus.

478. The method of claim 477, wherein the high coverage is at least 5×.

479. The method of claim 477, wherein the high coverage is at least 8×.

480. The method of claim 477, wherein the high coverage is at least 10×.

481. The method of claim 477, further comprising designating a sequence read mapped to a nuclear genome sequence with high coverage as ecDNA.

482. The method of claim 477, comprising generating a CRISPR enzyme having guide nucleotides from a high-coverage sequence different from the nuclear genome sequence reference, such that the CRISPR enzyme does not target the chromosomes of the subject's cells.

483. The method of claim 482, comprising delivering the CRISPR enzyme to the subject.

484. The method of claim 483, wherein the delivery is to cancer cells of the subject.

485. The method of claim 483, wherein the delivery comprises transfection using a vector system expressing the CRISPR enzyme and the guiding oligonucleotide.

486. The method of claim 483, further comprising monitoring the subject.

487. The method of claim 486, wherein monitoring the subject includes separating a second set of circular DNA from the subject after the delivery.

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