Flow cell and methods of making and using the same

Abstract

A flow cell comprising (a) a gasket interposed between a first substrate and a second substrate, wherein the gasket, the first substrate, and the second substrate are impermeable to an aqueous liquid and a liquid adhesive, wherein the gasket has a footprint on the first substrate that bounds a channel for containing the aqueous liquid; (b) a through-hole in the gasket, the through-hole comprising a solidified liquid adhesive that bonds the first substrate to the second substrate, wherein the solidified liquid adhesive in the through-hole is separated from the channel by the gasket; and (c) a channel port that connects the channel to an exterior of the flow cell, wherein the channel port is permeable to the aqueous liquid.

Description

[0001] This application is based on and claims the benefit of U.S. Provisional Application No. 62 / 969,845, filed on February 4, 2020, which is incorporated herein by reference. Background Technology

[0002] This disclosure relates in whole to analytical chemistry and biology, and has particular applicability to flow cells for analytical methods such as nucleic acid sequence analysis.

[0003] Flow cells are convenient containers for multi-step reactions in modern analytical chemistry or biology laboratories. A flow cell can contain a target analyte (e.g., cells, proteins, or nucleic acids) immobilized within a chamber, through which different liquid reagents can flow, and the chamber is configured to facilitate the detection of the target analyte. Allowing different reagents to flow through the flow cell in an ordered sequence to contact the immobilized target controls the nature of the biochemical reactions that may occur.

[0004] Some next-generation nucleic acid sequencing technologies have been automated to employ flow-through cells, where the flow of reagents through an array of surface-attached immobilized nucleic acids allows for convenient and efficient reagent exchange in a cyclic manner. For example, flow-through cells can be observed via luminescence microscopy to detect changes in the characteristics of the immobilized nucleic acids that indicate their sequence. Flow-through cells are typically disposable because they come into direct contact with the analyte being detected, and even small amounts of analyte residue from previous procedures can lead to erroneous results. For many procedures, nucleic acid sequencing is exemplary, and flow-through cells are constructed for high-precision detection. Even relatively small deviations in flow-through cell manufacturing or design can adversely affect the quality of analytical results.

[0005] Therefore, there is a need for a flow cell and a method for manufacturing it that are precise enough to support high-precision detection, yet efficient enough to limit costs while supporting single-use applications. This invention meets this need and also provides related advantages. Summary of the Invention

[0006] This disclosure provides a flow cell that optionally includes (a) a gasket inserted between a first substrate and a second substrate, wherein the gasket, the first substrate, and the second substrate are impermeable to aqueous liquids and liquid adhesives, wherein the gasket has a covering area on the first substrate defining a channel for containing an aqueous liquid; (b) a through-hole located in the gasket, the through-hole containing a cured liquid adhesive for bonding the first substrate to the second substrate, wherein the cured liquid adhesive in the through-hole is separated from the channel by the gasket; and (c) a channel port connecting the channel to the outside of the flow cell, wherein the channel port is permeable to aqueous liquids.

[0007] This disclosure also provides a method for manufacturing a flow cell. The method may include the steps of: (a) inserting a gasket between a first substrate and a second substrate to define a channel for containing an aqueous liquid, wherein the gasket, the first substrate, and the second substrate are impermeable to the aqueous liquid and the liquid adhesive; (b) delivering the liquid adhesive into a through-hole in the gasket; and (c) allowing the liquid adhesive to bond the first substrate to the second substrate, wherein the liquid adhesive is separated from the channel by the gasket.

[0008] Details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the following description. Further features and advantages of this subject matter will be apparent from the specification, drawings, and claims. Attached Figure Description

[0009] Figure 1A A perspective view of the assembled flow cell 100 is shown.

[0010] Figure 1B A top view of the assembled flow cell 100 is shown.

[0011] Figure 2A An exploded view of flow cell 200 is shown.

[0012] Figure 2B An exploded view of flow cell 300 is shown.

[0013] Figure 3A A top view of the assembled flow cell 500 is shown.

[0014] Figure 3B A perspective view of the assembled flow cell 500 is shown.

[0015] Figure 3C An exploded view of flow cell 500 is shown.

[0016] Figure 3D A cross-section of the assembled flow cell 500 is shown.

[0017] Figure 4A A perspective view of the flow cell 600 is shown.

[0018] Figure 4B An exploded view of the flow cell 600 is shown.

[0019] Figure 5A An adhesive rivet is shown that bonds an adhesive material to a non-adhesive material.

[0020] Figure 5B An adhesive rivet is shown that bonds two non-adhesive materials together.

[0021] Figure 6 A flow cell installed on an optical inspection system is shown.

[0022] Figure 7A A top perspective view of the fluid connector is shown.

[0023] Figure 7B A side view of the fluid connector is shown.

[0024] Figure 7C This shows a perspective view of the fluid connector before it engages with the container of the flow tank.

[0025] Figure 7D A cross-section of a fluid connector that engages with a container in a flow cell is shown.

[0026] Figure 7E A cross-section of the fluid connector and the pipe connected to the port of the flow tank is shown.

[0027] Figure 8A An adhesive rivet attached to a tube is shown.

[0028] Figure 8B An exploded view of the fluid connector, tube, and adhesive rivet is shown.

[0029] Figure 8C A perspective view of the assembled fluid connector is shown.

[0030] Figure 9A A top view of the assembled flow cell 700 is shown.

[0031] Figure 9B A photograph shows the liquid being displaced by foam in an assembled flow cell.

[0032] Figure 10 A top view of the flow cell with luminescent markers is shown.

[0033] Figure 11A A cross-section of the light-emitting doped medium in the flow cell is shown.

[0034] Figure 11B A cross-section of a flow cell dyed on a single layer by a light-emitting doped medium is shown.

[0035] Figure 11C A cross-section of a flow cell dyed with a light-emitting doped medium on multiple layers is shown.

[0036] Figure 11D A cross-section of a flow cell with a light-emitting doped gasket is shown.

[0037] Figure 12A A top view of a flow cell with a thermal expansion joint is shown.

[0038] Figure 12B A perspective view of the thermal expansion joint in the flow cell is shown.

[0039] Figure 13A A top perspective view of the two ports of the flow cell is shown.

[0040] Figure 13B A bottom perspective view of the two ports of the flow cell is shown.

[0041] Figure 14A and Figure 14B A perspective view of a pipette adapter configured to facilitate the introduction of nucleic acid library starting materials into a flow cell is shown.

[0042] Figure 15A and Figure 15B Another implementation of the pipette adapter is shown.

[0043] Figure 16 The bottom area of ​​a fluid connector with a pipe cover is shown. Detailed Implementation

[0044] This disclosure provides a flow cell that can be used in any of a variety of analytical or preparative methods. The flow cell provides high-accuracy detection and handling of analytes. High-accuracy detection is supported by the precise dimensions of the flow cell, such as by detecting the uniform flatness and thickness of the surface of the analyte within the flow cell. High-accuracy handling of analytes can be achieved due to robust mechanical properties, including, for example, the ability to withstand high internal fluid pressures and the ability to be conveniently and reliably connected to fluid components that deliver reagents to the flow cell.

[0045] This disclosure also provides methods for manufacturing flow cells. For example, a method is provided for bonding a gasket between two substrates using a liquid adhesive adhered to two substrates. The two substrates (e.g., optically transparent glass or plastic) are brought into contact with an inserted gasket layer (e.g., pressure-sensitive adhesive or rubber membrane). The gasket layer defines channels for analytes, and the gasket layer may also include one or more through-holes through the gasket. These through-holes are filled with liquid adhesive by injecting or directly injecting it into the gasket material through through-hole ports in one or both substrates. In some configurations, the gasket is bonded to the substrate due to a combination of the liquid adhesive in the through-holes and the adhesive at the interface between the gasket and the substrate.

[0046] Liquid adhesive bonding of flow cells provides a modular approach for bonding two substrates, particularly when forming microfluidic channels. This modular approach to bonding offers the advantage of separating the geometry of the flow cell design from its mechanical properties. Gasket layers can define the channel geometry (e.g., channel length, width, and height), and the liquid adhesive provides mechanical strength. This modularity allows for rapid prototyping and simplified manufacturing.

[0047] The liquid binder bonding of the flow cell also allows multiple layers to be bonded together, thus containing different materials (e.g., glass and plastic). Furthermore, reference points can be formed by constructing through-holes in the gasket to contain detectable liquid binders or other detectable materials. For example, one or more through-holes may contain luminescent binders or other luminescent materials. The shape of the through-holes and their position relative to the site of the analyte to be detected in the channel can be precisely controlled using efficient manufacturing techniques. Therefore, the through-holes provide reference points that can be relied upon during the analysis procedure to record the analyte site in the channel. The reference points of a flow cell with luminescent through-holes can be conveniently detected using the same detection hardware used to detect luminescent analytes in the detection channel of the flow cell. For example, the same excitation wavelength and the same emission wavelength can be used to achieve luminescence of the material in the through-hole and luminescence of the analyte in the detection channel. More specifically, the same excitation source can be used for both the reference point and the analyte. Alternatively or otherwise, the same emission detector can be used for both the reference point and the analyte.

[0048] Unless otherwise specified, the terms used herein should be understood to have their common meanings in the relevant fields. Several terms used herein and their meanings are described below.

[0049] As used herein, the term "array" refers to a group of molecules attached to one or more solid supports such that molecules at one site are distinguishable from molecules at other sites. An array may comprise different molecules at different addressable sites on a solid support. Alternatively, an array may comprise individual solid supports, each serving as a site for carrying different molecules, wherein different molecules can be identified based on the position of the solid support on the surface to which it is attached or based on the position of the solid support in a liquid such as a fluid flow. The molecules in an array may be, for example, proteins, nucleotides, nucleic acids, nucleic acid primers, nucleic acid templates, initiating template nucleic acids, or nucleases such as polymerases, ligases, exonucleases, or combinations thereof.

[0050] As used herein, the term "attachment" refers to a state in which two objects are joined, fastened, adhered, connected, bonded, or combined with each other. For example, nucleic acids can be attached to a solid carrier via covalent or non-covalent bonds. Similarly, two nucleic acids can be attached to each other via covalent (e.g., phosphodiester) bonds or non-covalent bonds (e.g., hydrogen bonds between the bases of the two nucleic acids). Covalent bonds are characterized by the sharing of electron pairs between atoms. Non-covalent bonds are chemical bonds that do not involve the sharing of electron pairs and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0051] As used herein, the term "closing portion," when used in relation to a nucleotide, refers to a nucleotide portion that inhibits or prevents the 3' oxygen of a nucleotide from forming a covalent bond with the next correct nucleotide during a nucleic acid polymerization reaction. An exemplary closing portion is a reversible terminator portion. The closing portion of a "reversible terminator" nucleotide can be removed from or otherwise modified from a nucleotide analog to allow the 3'-oxygen of the nucleotide to be covalently linked to the next correct nucleotide. This process is referred to as "declosing" the nucleotide analog. Such a closing portion is referred to herein as a "reversible terminator portion." Exemplary reversible terminator portions are described in U.S. Patent Nos. 7,427,673, 7,414,116, 7,057,026, 7,544,794, or 8,034,923, or PCT Publications WO 91 / 06678 or WO 07 / 123744, each of which is incorporated herein by reference. Nucleotides with a closing or reversible terminator portion can be located at the 3' end of a nucleic acid, such as primers, or can be monomers not covalently attached to the nucleic acid.

[0052] As used herein, the term "adhesion," when used in relation to an adhesive, means the process or result of attaching an adhesive to a substrate. Attachment can be performed mechanically or chemically. Exemplary mechanical attachment methods include, but are not limited to, the penetration of adhesive into pores in the substrate. Exemplary chemical methods include forming covalent bonds between the adhesive and the substrate, forming electrostatic forces between the adhesive and the substrate, forming van der Waals forces between the adhesive and the substrate, and forming hydrogen bonds between the adhesive and the substrate.

[0053] As used herein, the term "catalytic metal ion" refers to a metal ion that promotes the formation of a phosphodiester bond between the 3'-oxygen of a nucleic acid (e.g., a primer) and the phosphate ester of the introduced nucleotide by a polymerase. A "divalent catalytic metal cation" is a catalytic metal ion with a valence of two. The concentration of a catalytic metal ion present in a concentration that stabilizes the formation of a complex between the polymerase, the nucleotide, and the initiating template nucleic acid is referred to as the non-catalytic concentration of the metal ion, within a range that does not result in phosphodiester bond formation. The catalytic concentration of a metal ion is the amount of metal ion sufficient to enable the polymerase to catalyze the reaction between the 3'-oxygen of a nucleic acid (e.g., a primer) and the phosphate ester moiety of the introduced nucleotide. Exemplary catalytic metal ions include Mg. 2+ and Mn 2+ .

[0054] As used herein, the term "channel" refers to a passage in or on a substrate that guides the flow of fluid. A channel may take the form of a tube within the substrate. Alternatively, a channel may take the form of a groove or recess on the substrate.

[0055] The term "includes" is intended to be open-ended in this document, encompassing not only the listed elements but also any additional elements.

[0056] As used herein, the term "continuous" when used in relation to an adhesive that bonds two substrates together means that there is an uninterrupted adhesive path between the two substrates. An adhesive will be considered to form a continuous bond between the two substrates, provided that there is at least one uninterrupted adhesive path between the substrates.

[0057] As used herein, the term "each" is intended to identify a single item in a collection when used relative to a collection of items, but does not necessarily refer to every single item in the collection. Exceptions may occur if explicit public information or context clearly indicates otherwise.

[0058] As used herein, “equilibrium” refers to a state of equilibrium resulting from the equal action of opposing forces. For example, when the rate of formation of a ternary complex is in equilibrium with its rate of dissociation, the ternary complex formed between the initiating template nucleic acid, polymerase, and homologous nucleotide is in equilibrium with the unbound polymerase and unbound nucleotide. Under these conditions, the reversible binding reaction ceases to alter the net ratio of its product (e.g., the ternary complex) to reactants (e.g., polymerase, nucleotide, and nucleic acid). If the rate of the forward reaction (e.g., ternary complex formation) is in equilibrium with the rate of the reverse reaction (e.g., ternary complex dissociation), there is no net change in the product-to-reactant ratio.

[0059] As used herein, the term "exogenous" when used relative to a portion of a molecule means a chemical portion that is not present in the molecule's natural analogues. For example, an exogenous label on a nucleotide is a label that is not present on naturally occurring nucleotides. Similarly, an exogenous label present on a polymerase is not present on polymerases in their natural environment.

[0060] As used herein, the term "extension," when used in relation to nucleic acids, refers to the process of adding at least one nucleotide to the 3' end of a nucleic acid. The term "polymerase extension," when used in relation to nucleic acids, refers to a polymerase-catalyzed process of adding at least one nucleotide to the 3' end of a nucleic acid. Nucleotides or oligonucleotides added to nucleic acids via extension are allegedly incorporated into the nucleic acid. Therefore, the term "incorporation" can be used to refer to the process of attaching a nucleotide or oligonucleotide to the 3' end of a nucleic acid via the formation of a phosphodiester bond.

[0061] As used herein, the term "extensible" when used in relation to a nucleotide means that the nucleotide has an oxygen or hydroxyl moiety at the 3' position and is capable of forming a covalent bond with the next correct nucleotide if incorporated into a nucleic acid. An extensible nucleotide can be located at the 3' position of a primer, or it can be a monomeric nucleotide. An extensible nucleotide will lack a closing portion, such as a reversible terminator moiety.

[0062] As used herein, the terms “free” or “unbound” refer to components that are not in a bound state when used relative to components capable of forming a complex in a binding reaction. For example, an equilibrium binding reaction may include a product (e.g., a ternary complex) and reactants not bound to the product (e.g., free polymerase, free nucleic acid, or free nucleotide).

[0063] As used herein, a "flow cell" is a reaction chamber comprising one or more channels that direct fluid to a detection zone or reaction zone. The detection zone may be coupled to a detector, allowing observation of the reaction occurring within the reaction chamber. For example, a flow cell may contain a priming template nucleic acid molecule bound to a solid support, to which nucleotides and auxiliary reagents are repeatedly applied and washed away. The flow cell may contain a transparent material that allows imaging of the sample after the desired reaction has occurred. For example, a flow cell may comprise a glass or plastic slide containing small fluid channels through which polymerase, dNTPs, and buffer solutions can be pumped. The glass or plastic within the channels may be decorated with one or more priming template nucleic acid molecules to be sequenced.

[0064] As used herein, the term "fluid" refers to a liquid or gas capable of flowing and changing its shape to fill a container. In many cases, when a fluid is subjected to a force that tends to change its shape, the fluid will change its shape at a steady rate.

[0065] As used herein, the term "repressed metal ion" refers to a metal ion that, in the presence of a polymerase, inhibits the formation of the phosphodiester bond required for the chemical incorporation of nucleotides into primers. Repressed metal ions can interact with polymerases, for example, through competitive binding compared to catalytic metal ions. A "divalent repressed metal ion" is a repressed metal ion with a valence of two. Examples of divalent repressed metal ions include, but are not limited to, Ca. 2+ Zn 2+ Co 2+ Ni 2+ and Sr 2+ Trivalent Eu 3+ and Tb 3+ The ion is a suppressing metal ion with a valence of three.

[0066] As used herein, the term "label" refers to a molecule or portion thereof that provides a detectable characteristic. Detectable characteristics can be, for example, optical signals such as absorption of radiation, emission or fluorescence, emission or fluorescence lifetime, emission or fluorescence polarization, etc.; Rayleigh and / or Mie scattering; binding affinity to a ligand or acceptor; magnetic properties; electrical properties; charge; mass; radioactivity, etc. Exemplary labels include, but are not limited to, fluorophores, luminescent organisms, chromophores, nanoparticles (e.g., gold, silver, carbon nanotubes), heavy atoms, radioactive isotopes, mass labels, charge labels, spin labels, acceptors, ligands, etc.

[0067] As used herein, the term "liquid adhesive" refers to a substance that is bonded to a substrate in a fluid state when applied to the substrate. The substance does not need to remain in a fluid state after being activated to adhere to the substrate. Therefore, a liquid adhesive may be in a non-fluid state (e.g., "curing liquid adhesive" or "hardening liquid adhesive") after reacting to adhere to the substrate.

[0068] As used herein, the term "next correct nucleotide" refers to the type of nucleotide that will bind to and / or be incorporated into the 3' end of a primer to be complementary to a base in the template strand that hybridizes with the primer. The base in the template strand is referred to as the "next base" and immediately follows the 5' end of the base in the template that hybridizes with the 3' end of the primer. The next correct nucleotide can be referred to as a "homolog" of the next base, and vice versa. Homologous nucleotides that specifically interact with each other in a ternary complex or double-stranded nucleic acid are said to be "paired." Nucleotides with bases that are not complementary to the next template base are called "incorrect," "mismatched," or "non-homologous" nucleotides.

[0069] As used herein, the term "nucleotide" may refer to natural nucleotides or their analogues. Examples include, but are not limited to, nucleoside triphosphates (NTPs) such as ribonucleoside triphosphates (rNTPs), deoxyribonucleoside triphosphates (dNTPs), exogenously labeled nucleotides or their non-natural analogues such as dideoxyribonucleoside triphosphates (ddNTPs) or reversibly terminated nucleoside triphosphates (rtNTPs).

[0070] As used herein, the term "polymerase" can be used to refer to nucleic acid synthases, including but not limited to DNA polymerases, RNA polymerases, reverse transcriptases, primases, and transferases. Typically, a polymerase has one or more active sites capable of catalyzing nucleotide binding and / or nucleotide polymerization. A polymerase can catalyze the polymerization of nucleotides to the 3' end of the first strand of a double-stranded nucleic acid molecule. For example, a polymerase covalently incorporates a nucleotide into the first strand of a double-stranded nucleic acid molecule by catalyzing the addition of the next correct nucleotide to the 3' oxygen group of the first strand via a phosphodiester bond. Optionally, the polymerase does not need to be capable of nucleotide incorporation under one or more conditions used in the methods described herein. For example, a mutant polymerase can form a ternary complex but cannot catalyze nucleotide incorporation. The amount of polymerase in a fluid can be quantified in activity units. For example, a polymerase unit can be equal to the amount of enzyme that catalyzes the incorporation of 10 nmol dNTPs into DNA over 30 minutes at a specific temperature. For example, a thermostable polymerase can be measured at 75°C, while a thermostable polymerase can be measured at 37°C.

[0071] As used herein, the term "initiating template nucleic acid" refers to a nucleic acid hybrid having a double-stranded region such that one strand has a 3' end that can be extended by polymerase. The two strands can be part of a continuous nucleic acid molecule (e.g., a hairpin structure), or the two strands can be separable molecules that are not covalently linked to each other.

[0072] As used herein, the term "primer" refers to a nucleic acid having a sequence that binds to a nucleic acid sequence at or near a template sequence. Generally, primers bind in a conformation that allows template replication, for example, via primer polymerase extension. A primer can be a first part of a nucleic acid molecule that binds to a second part of the nucleic acid molecule, the first part being the primer sequence and the second part being the primer-binding sequence (e.g., a hairpin primer). Alternatively, a primer can be a first nucleic acid molecule that binds to a second nucleic acid molecule having a template sequence (e.g., a dissociable primer). Primers can consist of DNA, RNA, or analogues thereof. Primers can be closed at the 3' end, or they can be extendable.

[0073] As used herein, the term "site" in relation to an array refers to the location within the array where a particular molecule is present. A site may contain only a single molecule, or it may contain a group of molecules of the same kind (i.e., an ensemble of molecules). Alternatively, a site may contain a group of molecules of different kinds (e.g., a group of different template sequences). Sites in an array are typically discrete. Discrete sites may be continuous, or they may be spaced apart from each other.

[0074] As used herein, the term "substrate" refers to a solid support that is insoluble in aqueous liquids. The solid support may be non-porous or porous. The solid support may optionally be able to absorb liquids (e.g., due to its porosity), but will generally be rigid enough that the solid support does not substantially swell when absorbing liquids and does not substantially shrink when the liquid is removed by drying. Non-porous substrates are generally impermeable to liquids or gases. Exemplary substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutene, polyurethane, Teflon, etc.). TM Materials include cycloolefins, polyimides, nylon, ceramics, resins, Zeonor, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, fiber bundles, and polymers.

[0075] As used herein, the term "surface" refers to a portion of a solid carrier that comes into contact with a fluid or another solid carrier. The fluid may be a gas or a liquid. The surface may be substantially flat or planar. Alternatively, the surface may be circular or wavy. Exemplary profiles that may be included on the surface are recesses, depressions, pillars, ridges, channels, etc.

[0076] As used herein, the term "ternary complex" refers to the intermolecular association between a polymerase, a double-stranded nucleic acid, and a nucleotide. Typically, the polymerase promotes the interaction between the next correct nucleotide and the template strand of the initiating nucleic acid. The next correct nucleotide can interact with the template strand via Watson-Crick hydrogen bonds. The term "stable ternary complex" refers to a ternary complex with a promoted or elongated presence, or a ternary complex whose disruption is inhibited. Generally, stabilization of a ternary complex prevents the covalent incorporation of the nucleotide component of the ternary complex into the initiating nucleic acid component.

[0077] As used herein, the term "type" or "kind" is used to identify molecules that share the same chemical structure. For example, a mixture of nucleotides may contain several dCTP molecules. dCTP molecules should be understood as nucleotides of the same type (or kind) but different type (or kind) from dATP, dGTP, dTTP, etc. Similarly, individual DNA molecules with the same nucleotide sequence are DNA of the same type (or kind), while DNA molecules with different sequences are DNA of different types (or kinds). The term "type" or "kind" can also identify portions that share the same chemical structure. For example, cytosine bases in a template nucleic acid should be understood as bases of the same type (or kind) regardless of their position in the template sequence.

[0078] As used herein, the term "through-hole" when used in relation to a gasket or other substrate refers to a void in or through the substrate. A void through the substrate may have a circular, rectangular, elliptical, oval, triangular, square, rectangular, or polygonal cross-section (e.g., with more than 4 sides). The cross-section may be symmetrical (e.g., rotationally symmetrical, reflectively symmetrical, or translationally symmetrical), asymmetrical, or elongated (e.g., forming a channel). A through-hole can provide a continuous passage from the substrate on one side of the gasket to the substrate on the other side of the gasket (e.g., opposite side).

[0079] Based on the above definition, the embodiments described below and listed in the claims can be understood.

[0080] This disclosure provides flow cells that can be used for any of a variety of analytical or preparative applications. The features of the flow cells described herein can be modified to suit a particular application. Several features of the flow cells will be illustrated herein by example in the context of a particular application. However, those skilled in the art will understand that the exemplary flow cells, and more specifically, the exemplary features, can be used for purposes other than those explicitly illustrated.

[0081] Some useful features of flow pools are... Figure 1A Perspective and Figure 1BThe flow cell is shown in the top view. The flow cell may include detection channels where the analytical reaction of interest can be observed. Figure 1A and Figure 1B The flow cell shown includes two channels 101 and 102. The channels are formed by a gap in a spacer 105 sandwiched between two optically transparent substrates 110 and 111. Therefore, analytes in the channels can be optically detected through the substrates. Fluid can enter and exit channel 101 via channel ports 120 and 121, and fluid can enter and exit channel 102 via channel ports 123 and 124. Channel ports 120 and 124 are housed in container 125, and channel ports 121 and 123 are housed in container 126. Containers 125 and 126 are configured to connect with adapter 1000 (see...). Figure 7C In conjunction with this adapter, the flow cell channel is connected to a fluid delivery device. Containers 125 and 126 are further configured to mate with pipette adapter 1105, which is configured to facilitate the introduction of nucleic acid library starting materials into the flow cell, as described below. Figures 14A to 14B Further details are provided.

[0082] The flow cell also includes two wings 130 and 131 located in a z-plane different from channels 101 and 102. These wings respectively include apertures 132 and 133, which are configured to engage posts on the sliding frame of the optical detection device (see [link]). Figure 10 An image of the channel can be obtained by scanning along its length (i.e., along the x-axis). In some configurations, this is achieved by stepping the flow cell along the y-axis and then along... x Repeat the scan to obtain the next strip, thus imaging multiple strips of the channel.

[0083] The flow cell of this disclosure may have one or more openings for transferring fluid into a channel. In a particular configuration, a first opening may serve as an inlet for the fluid, and a second opening may serve as an outlet for the fluid. This is illustrated by channel ports 120 and 121, which may serve as either an inlet or outlet port for channel 101. Alternatively, the flow cell may have a single opening that serves as both an inlet and an outlet. The fluid may be a liquid, a gas, or a mixed-phase fluid (e.g., a foam, an emulsion, or a particulate slurry). Exemplary apparatuses and methods for generating and manipulating mixed-phase fluids are set forth in U.S. Patent Application Serial No. 16 / 700,422, which is incorporated herein by reference.

[0084] The analytical reaction can be carried out in a bulk solution within the flow cell of this disclosure. For example, two solutions can be mixed, and the products of the mixture can be observed in the detection channel. Alternatively, the analytical reaction can be carried out on a solid support within the detection channel. For example, a solid support to which the analyte of interest is attached can be present in channels 101 or 102, through which the reagent solution can flow, and the resulting reaction can be observed on the solid support. The flow cell allows for convenient fluid manipulation by allowing the solution to enter the detection channel through a channel port serving as an inlet and exit through a channel port serving as an outlet opening. The detection channel also has an observation area (e.g., observation area or observation volume), visible, for example, through an optically transparent window (e.g., optically transparent substrates 110 and 111). Particularly useful flow cells have windows that are transparent to the excitation and emission radiation for luminescence detection in the UV, VIS, or IR regions of the electromagnetic spectrum.

[0085] As in Figure 1A and 1B As illustrated in other figures herein, the flow cell may have one or more detection channels. One or more detection channels may be enclosed to the atmosphere (or other surrounding environment, such as the local environment directly surrounding the flow cell), for example, by forming tubes or tunnels within the flow cell structure. The shape of the flow cell channels may be derived from the shape of the substrates (e.g., substrates 110 and 111) and the shape of the void regions in the spacers used between the substrates (e.g., spacer 105). The spacers may be components of another substrate used to form the flow cell, such as... Figure 2A The flow cell 200 shown is an example. Alternatively, the spacer can be a gasket that can be separated from other substrates, such as... Figure 2B Examples of 300 circulation pools in the example.

[0086] Circulation pool 200 ( Figure 2A The system includes a top substrate 210 with a corrugated surface having cutouts for channels 201 and 202. The contours on the surface serve to define the channels. x and y Dimensional spacers. The top substrate 210 can be bonded to the bottom substrate 211 to form a channel with a closed profile. Bonding can be achieved using an adhesive between the materials of substrates 210 and 211, direct chemical bonding, melting one or both materials to fuse them together, etc. Channel 201 can be accessed through channel ports 220 and 221, and channel 202 can be accessed through channel ports 223 and 224. The ports are housed in containers 225 and 226. The flow cell 200 includes wings 230 and 231, which respectively include holes 232 and 233 for interacting with columns on the scanning gantry.

[0087] Flow pool 300 ( Figure 2BThe flow cell 300 includes channels 301 and 302 formed by mating a top substrate 310 and a bottom substrate 311 with a gasket 305. Channel 301 is accessible through channel ports 320 and 321, and channel 302 is accessible through channel ports 323 and 324. The ports are housed in containers 325 and 326. The flow cell 300 includes wings 330 and 331, each including holes 332 and 333 for interacting with a post on the scanning gantry.

[0088] like Figure 1A and 1B and Figure 2A and 2B As shown in the example, the depth of the channel (in) z The thickness of the spacer (in terms of dimension) is determined by the cutout in the spacer, whether the spacer is a feature of a substrate (e.g., substrate 210) or a separable gasket (e.g., gasket 305). Gaskets or other spacers can have any thickness required for a particular application, including, for example, at least 100 μm, 1 mm, 1 cm, 10 cm, or greater. Alternatively or otherwise, gaskets or other spacers can have a thickness of up to 10 cm, 1 cm, 1 mm, or 100 μm. A particular advantage of flow cells manufactured using liquid binders is their ability to withstand high positive pressures. High positive pressures can be used to allow fluid to flow through flow cells with relatively small channel thicknesses. Therefore, gaskets or other spacers can have a thickness of up to 100 μm, 80 μm, 50 μm, 30 μm, 10 μm, or less. Alternatively or otherwise, gaskets or other spacers can have a thickness of at least 10 μm, 30 μm, 50 μm, 80 μm, or 100 μm.

[0089] The detection channel in the circulation pool disclosed herein xy The cross-sectional area can be at least about 1 μm 2 10 μm 2 100 μm 2 1mm 2 10 mm 2 or 100 mm 2 Or larger. Alternatively, or otherwise, the detection channel... xy The cross-sectional area can be up to approximately 100 mm². 2 10 mm 2 1 mm 2 100 μm 2 10 μm 2 1 μm 2Or smaller. The volume of the detection channel in the flow cell can be at least about 1 nL, 10 nL, 100 nL, 1 μL, 10 μL, 100 μL, 1 mL or larger. Alternatively or otherwise, the volume of the detection channel in the flow cell can be at most about 1 mL, 100 μL, 10 μL, 1 μL, 100 nL, 10 nL, 1 nL or smaller. The length of the flow cell channel can be selected to provide the desired volume, surface area, etc. For example, the length of the flow cell channel can be at least 1 mm, 1 cm, 10 cm or longer. Alternatively or otherwise, the length of the flow cell channel can be at most 10 cm, 1 cm or 1 mm.

[0090] Due to the substrate forming the channel (in) y (above) and spacers (in) z (above) flat surface, Figure 1A and 1B and Figure 2A and 2B The detection channel in the example has a rectangle. yz Cross-sectional shape. Flow cell channels can have various shapes. yz Any of the following cross-sectional shapes, including, for example, circles, ellipses, triangles, squares, rectangles, polyhedra, or other closed shapes. Detection channel. yz Section in x The cross-section of the detection channel can be uniform in area (i.e., equal in area and shape). For example, a detection channel with a circular cross-section uniform in length will have a cylindrical shape, a detection channel with a rectangular cross-section uniform in length will have a cuboid shape, and a detection channel with a polyhedral cross-section uniform in length will have a prism shape. The cross-section of the detection channel does not need to be uniform in area. For example, a detection channel with a circular cross-sectional area that gradually increases or decreases in length will have a conical shape, and a detection channel with a square cross-sectional area that gradually increases or decreases in length will have a pyramidal shape.

[0091] The flow cell channel may also include a region in which analytes are detected. Fluid may flow into the flow cell via an inlet, then through this region, and finally out of the flow cell via an outlet. As an illustrative example, this region may be inspected or detected through a window in the flow cell. For example, an external optical detector may observe the internal region of the flow cell through an optically transparent window of the flow cell (such as a window formed by substrates 110, 111, 210, 211, 310, or 311). One or more regions of the flow cell channel may be inspected or observed using techniques other than optical techniques, including, for example, the detection techniques described herein. Thus, the flow cell may have a transmission surface that transmits signals from a region of the flow cell to a suitable detector device. It should be understood that the flow cell does not need to be constructed for detecting analytes. For example, the flow cell may provide a chamber for carrying out a reaction, and the reaction products may flow out of the flow cell for subsequent use or detection. Therefore, the flow cell does not need to have an optically transparent window or other surface constructed for transmitting analytical signals.

[0092] This disclosure provides a flow cell that optionally includes (a) a gasket inserted between a first substrate and a second substrate, wherein the gasket, the first substrate, and the second substrate are impermeable to aqueous liquids and liquid adhesives, wherein the gasket has a covering area on the first substrate defining a channel for containing an aqueous liquid; (b) a through-hole located in the gasket, the through-hole containing a cured liquid adhesive for bonding the first substrate to the second substrate, wherein the cured liquid adhesive in the through-hole is separated from the channel by the gasket; and (c) a channel port connecting the channel to the outside of the flow cell, wherein the channel port is permeable to aqueous liquids.

[0093] This disclosure also provides a method for manufacturing a flow cell. The method may include the steps of: (a) inserting a gasket between a first substrate and a second substrate to define a channel for containing an aqueous liquid, wherein the gasket, the first substrate, and the second substrate are impermeable to the aqueous liquid and the liquid adhesive; (b) delivering the liquid adhesive into a through-hole in the gasket; and (c) allowing the liquid adhesive to bond the first substrate to the second substrate, wherein the liquid adhesive is separated from the channel by the gasket.

[0094] An exemplary flow cell made using a liquid adhesive is shown. Figures 3A to 3D middle. Figure 3A A top view of the assembled flow cell 500 is shown, while Figure 3B A perspective view is shown. An exploded view of the flow cell 500 is shown in... Figure 3C In the middle, and the cross-section of the flow cell 500 is shown in Figure 3DThe flow cell 500 includes a body comprising a gasket 505 inserted between substrates 510 and 511. Gasket 505 includes two cutout areas defining channels 501 and 502. Gasket 505 may optionally have an adhesive on the surface contacting substrates 510 and 511. For example, gasket 505 may contain pressure-sensitive adhesive (PSA). Gasket 505, as well as substrates 510 and 511, are impermeable to aqueous liquids. Once assembled, channels 501 and 502 are fluid-tight except at inlet ports 512 to 515. One or both of substrates 510 and 511 may be optically transparent to provide optical inspection of channels 501 and 502. In this example of flow cell 500, the coverage area of ​​the gasket is identical on both substrates, resulting in windows of the same shape on both sides of the channel. It should be understood that the coverage areas on the two substrates may differ, resulting in windows of different shapes on both sides of the channel. 505 gaskets do not need to be optically transparent, but they can be optically transparent if required.

[0095] The assembled flow cell 500 includes a through-hole 550 containing a cured liquid adhesive for bonding substrate 510 to substrate 511. Figure 3D As shown in the view, the thickness of the cured liquid adhesive in through-hole 550 will be equal to the thickness of gasket 505. The liquid adhesive can be delivered into through-hole 550 through through-hole ports 556 or 557, which engage with bends 551 and 552 in through-hole 550, respectively. Through-hole 550 continuously surrounds channel 501 and also continuously surrounds channel 502. xy In the plane, the through-hole 550 has a figure-eight appearance forming grooves or channels surrounding each of channels 501 and 502. Thus, the curing liquid adhesive forms reinforcing beads around channels 501 and 502, enhancing their fluid impermeability. The through-hole 550 is spaced from channel 501 by a band 506 of gasket 505, and from channel 502 by a band 507 of gasket 505. Bands 506 and 507 prevent the liquid adhesive from entering channels 501 and 502, and conversely, prevent fluid in the channels from contacting the liquid adhesive. Thus, the liquid adhesive does not need to be inert to the fluid or fluid component that will be processed in channels 501 and 502.

[0096] Wings 530 and 531 are attached to the outer surface of substrate 510 (i.e., opposite to the inner surface of substrate 510). Wing 530 is attached to substrate 510 via a curing liquid adhesive in washer 570 and through-hole 571. Wing 531 is attached to substrate 510 via a curing liquid adhesive in washer 560 and through-hole 561. Wings 530 and 531 include holes 532 and 533, respectively, for attachment to a holder on a scanning device. Wing 530 includes a container 525 for receiving channel ports 520 and 524. Access to channel 501 is possible via a path including port 520, hole 572 in washer 570, and hole 515 in substrate 510, and also via a path including port 521, hole 562 in washer 560, and hole 512 in substrate 510. Access to channel 502 can be achieved via a path including port 524, hole 573 in gasket 570, and hole 514 in substrate 510, and also via a path including port 523, hole 563 in gasket 560, and hole 513 in substrate 510. Through-hole 571 forms a groove in gasket 570 surrounding holes 572 and 573 to create a fluid seal between wing 530 and substrate 510. Similarly, through-hole 561 forms a groove in gasket 560 surrounding holes 562 and 563 to create a fluid seal between wing 531 and substrate 510.

[0097] like Figure 4A and 4B As shown, the wing is not an essential feature of the flow cell. The flow cell 600 includes a gasket 605 inserted into a top substrate 610 and a bottom substrate 611. The gasket 605 may optionally be adhered to one or both of the top substrate 610 and the bottom substrate 611. Cutouts in the gasket 605 define channels 601 and 602. Channel 601 can be accessed through ports 612 and 615, while channel 602 can be accessed through ports 613 and 614. The assembled flow cell 600 includes a through-hole 650 containing a curing liquid adhesive for bonding substrate 610 to substrate 611. The thickness of the curing liquid adhesive in the through-hole 650 will be equal to the thickness of the gasket 605. The liquid adhesive can be delivered into the through-hole 650 through through-hole ports 656 or 657, which engage with bends 651 and 652 in the through-hole 650, respectively. The through-hole 650 continuously surrounds channel 601 and also continuously surrounds channel 602. The curing liquid adhesive forms reinforcing beads around channels 601 and 602, enhancing their fluid impermeability. Through-hole 650 is separated from channel 601 by band 606 of gasket 605, and through-hole 650 is separated from channel 602 by band 607 of gasket 605.

[0098] Any of a variety of substrates can be used in a flow cell. For example, a rigid material may be particularly useful when the flow cell may be subjected to mechanical stress. Optionally, the polymer material is shaped into a substantially rigid configuration. For example, the material may be shaped to have a thickness that resists deflection when used as a flat surface in the flow cell. Flat substrates are useful, as illustrated in the figures of this application. However, the substrate need not be flat, but may have other shapes completed by gaskets and / or one or more other substrates to form flow cell channels. In certain configurations, the substrate is made of a material that facilitates the transmission of optical signals, for example, in the UV, VIS, and / or IR regions of the spectrum. The material may also be selected based on its inertness to the fluid to be used in the flow cell. However, in some cases, it is desirable that at least a portion of the surface of the substrate be reactive for a particular chemical bonding process. Such procedures are illustrated herein with respect to attaching an analyte or an array of analytes to the substrate.

[0099] Exemplary materials that can be used as flow cell substrates include, but are not limited to, glass, borosilicate glass, silanized glass (e.g., aminosilane, glycidyl ether oxysilane, or mercaptosilane), silicon, silica, metal, carbon fiber, silicon nitride, fused silica, quartz, ceramic, acrylic, plastic, or polymer. Particularly useful plastics or polymers include, but are not limited to, polyimide, polybutene, polybutylene terephthalate (PBT); styrene-butadiene copolymer (SBC); polypropylene, polyethylene, cyclic olefin copolymer (COC); cyclic olefin polymer (COP); styrene-methyl methacrylate (SMMA); polystyrene (PS); polycarbonate (PC); copolyester; polysulfone; nylon; polyether ether ketone (PEEK); acrylonitrile-butadiene-styrene (ABS); Kapton and poly(methyl methacrylate) (PMMA).

[0100] For a flow cell using two substrates, the first and second substrates can be made of the same material. For example, both substrates can be glass, or both substrates can be the same plastic (e.g., both substrates can be COP). Alternatively, the materials of the two substrates can be different from each other. For example, one substrate can be glass, and the other substrate can be COP. Generally, it is preferred to select materials with similar coefficients of thermal expansion (CTE) to avoid warping when the bonded flow cell experiences significant temperature changes. However, materials with different CTEs can be combined using the following... Figure 12A and 12B The thermal expansion joint is described in further detail to accommodate this.

[0101] Gaskets or spacers used in flow cells can be made of any of a variety of materials. A useful material may be inert to the fluid to be used in the flow cell. The material used for gaskets may be transparent to optical signals, but for some applications of the flow cell, transparency is not required. For many applications, the material used for gaskets or spacers will be incompressible or inelastic, thus maintaining a fixed distance between two substrates when used as a spacer. The material used for gaskets may advantageously have a CTE similar to that of the material used for one or more other substrates in the flow cell. This can help prevent warping or breakage when the flow cell is subjected to significant temperature changes. For example, gaskets or spacers may include adhesive surfaces for adhesion to substrates used in the flow cell described herein. For example, gaskets may have pressure-sensitive adhesives on the surfaces where they engage with one or more substrates to which the gasket is bonded.

[0102] Exemplary materials for gaskets or spacers include, but are not limited to, the materials described above for substrates. Particularly useful materials include, for example, rubber, polytetrafluoroethylene (e.g., Teflon), polyethylene terephthalate (PET), polyimide, acrylic adhesives, silicone adhesives, polydimethylsiloxane (PDMS), glass, and latex. Elastomers can be used, for example, to form gaskets that create a tight seal with a relatively rigid material. Gaskets for a particular flow cell can be made of the same material as one or both substrates, or alternatively, the gasket can be made of a different material than the substrates.

[0103] Liquid adhesives can be selected based on their ability to bond with the material used as the substrate in the flow cell. In a flow cell configuration where the liquid adhesive is present in a through-hole connecting two substrates, the adhesive can bond to both substrates to assemble the flow cell. In this configuration, the adhesive does not need to bond to the gasket. However, if desired, the liquid adhesive can be selected based on its ability to bond to both the substrate and the gasket. Liquid adhesives can be selected based on the method of curing or solidifying the adhesive. For example, UV-curable adhesives can be useful when the material or contents of the flow cell are adversely affected by chemical-based or heat-based curing techniques used with other adhesives. Low or even no gas release can be a desirable characteristic of liquid adhesives, for example, when the adhesive is drawn into the through-hole using a vacuum, or when ventilation is inconvenient. Liquid adhesives can also be selected based on adhesive compatibility, or carrier solvent compatibility, and the structural or chemical integrity of the flow cell or its contents. In some cases, it is desirable to use liquid adhesives that do not expand or shrink after curing. For example, adhesives that experience less than 10% or even less than 1% expansion or shrinkage can be particularly useful. However, in other cases, expansion may be desirable, for example, to help the adhesive penetrate the contours of the surface. Shrinkage may be desirable to pull the substrates together after curing or to prevent warping of the substrates due to the volume of the liquid adhesive. In some configurations, low viscosity is desirable for the liquid adhesive because it will facilitate adhesive delivery by allowing the adhesive to flow into the through-holes or other physical features of the substrate to be adhered.

[0104] Useful liquid adhesives include, but are not limited to, epoxy resins, light-curing adhesives (e.g., UV-curing adhesives), and thermosetting adhesives. Optionally, liquid adhesives can be bonded by heat treatment (e.g., in an oven), exposure to UV light, or other treatments suitable for bonding adhesives.

[0105] like Figures 3A to 3D and Figure 4A and 4B As shown in the flow cell, the through-holes containing liquid adhesive can be configured as grooves surrounding a channel or other space containing fluid. In this way, the liquid adhesive forms continuous beads around the feature of interest within the flow cell. Alternatively, multiple through-holes filled with liquid adhesive can be positioned near the channel or other feature of the flow cell. In this way, the liquid adhesive can form a set of discrete adhesive fasteners, similar to adhesive rivets. For example, multiple through-holes filled with liquid adhesive can surround the flow cell channel, rather than... Figures 3A to 3D The continuous beads shown.

[0106] Liquid adhesive rivets do not need to adhere to all the materials that will be attached to each other. Instead, the liquid adhesive can be cured to form a solid material that holds two or more materials together using mechanical means. Figure 5A A cross-section of a liquid-bonded rivet 11 is shown, which adheres to a first substrate 15 but not necessarily to a second substrate 10. However, the liquid-bonded rivet 11 connects the substrate 15 to the substrate 10 through a combination of adhesion to the substrate 15 and providing a mechanical connection to the substrate 10. More specifically, the liquid-bonded rivet is cured to have a surface 17 adhering to the substrate 15, a relatively inelastic shaft 16, and a head 18 contacting a base on the substrate 10. Figure 5A (Seen as ridges 12 and 13 in the cross-sectional view). The rivet 11 holds the substrate together by the combined forces of the adhesion of the rivet 11 to the surface 17, the mechanical force of the head 18 of the rivet 11 on the base of the substrate 10, and the inelastic rigidity of the cured liquid adhesive 11 resisting tension or fracture.

[0107] Figure 5B A cross-section of a liquid-bonded rivet 21 is shown. This rivet is not necessarily adhered to substrate 20 or substrate 30, but the two substrates are still attached together by mechanical force. More specifically, the liquid-bonded rivet is cured to have a first head 28 (in contact with a base on substrate 20) Figure 5B The cross-sectional view shows ridges 22 and 23, a relatively inelastic shaft 26, and a second head 38 of the base on the contact plate 30 (in... Figure 5B (Seen as ridges 32 and 33 in the cross-sectional view). The rivet 21 holds the substrate together by a combination of the mechanical force of the head 28 of the rivet 21 on the base of the substrate 20, the inelastic rigidity of the cured liquid adhesive 21 against tension or fracture, and the mechanical force of the head 38 of the rivet 21 on the base of the substrate 30.

[0108] Another example of adhesive bonding provided by a mechanical device for bonding two components is... Figure 13A and Figure 13BThe dual-port component 980 shown is provided. The dual-port component 980 includes two ports 983 and 993 connected to each other via a base 987. Port 983 has a predominantly cylindrical body with an opening 982 at a narrow end of a tapered region 981. Port 993 has a similar shape, also a predominantly cylindrical body with an opening 992 at a narrow end of a tapered region 991. The cylindrical exterior of each port is shaped to complement the inner diameter of the flexible tube to form a tight fit between the port and the tube. The conical shape surrounding the opening of each port provides ease of insertion of the port into the tube. Ports 983 and 993 are held in a fixed orientation and distance from each other by the base 987. The lower side 990 of the base 987 and the lower sides 989 and 999 of ports 983 and 993 respectively have flat shapes that allow for a tight seal with the top of the flow cell. The flat lower side 989 of port 983 surrounds opening 988, and the flat lower side 999 of port 993 surrounds opening 998. Openings 988 and 998 can be aligned with holes in the flow cell, and the flat sides 989 and 999 can form a tight seal with the outer surface of the flow cell. Thus, fluid can enter through a tube into opening 982, pass through the internal channel of port 983, exit opening 988, and enter the flow cell channel through holes in the upper substrate of the flow cell. Similarly, fluid can enter through a tube into opening 992, pass through the internal channel of port 993, exit opening 998, and enter the flow cell channel through holes in the upper substrate of the flow cell.

[0109] The adhesion of the dual-port component 980 to the flow cell surface is facilitated by recesses 986 and 996 formed in the base 987. Recesses 986 and 996 surround ports 983 and 993, respectively. Recesses 986 and 996 can be filled with an adhesive that bonds to the flow cell surface. The adhesive can be bonded to the flow cell surface and the inner surfaces of recesses 986 and 996 to adhere the dual-port component to the flow cell surface. Figure 13A and Figure 13B As shown, recess 986 includes spokes 984 and 985 inserted into the recess, such that the spokes are encapsulated within the adhesive by filling the recess with adhesive. Recess 996 includes spokes 994 and 995 performing a similar function. As long as the adhesive adheres to the flow cell surface, the spokes and recess do not need to be chemically bonded to the adhesive. Instead, encapsulating the spokes in the adhesive provides a means of mechanical adhesion. Other features besides the spokes can be embedded in the liquid adhesive to provide these mechanical forces.

[0110] Any of a variety of analytes may be present in the flow cell described herein. Exemplary analytes include, but are not limited to, those exemplified herein or cited in the references cited herein. Particularly useful analytes are involved in the nucleic acid sequencing process. Therefore, the flow cell may contain one or more nucleic acids, polymerases, polymerase inhibitors, polymerase cofactors (e.g., those catalyzing metal ions), ternary complex stabilizers (e.g., those inhibiting metal ions), nucleotides, nucleic acid binding proteins, nucleotide deblocking agents, etc.

[0111] Other analytes that may be present in the flow cell include, for example, biological tissues and cells; organelles; protein-based enzymes; protein-based receptors such as antibodies, lectins, or streptavidin; peptides; RNA molecules; adaptors, etc. Exemplary protein-based enzymes that may be used include, but are not limited to, polymerases, transposases, ligases, recombinases, kinases, phosphatases, exonucleases, endonucleases, sulfatases, adenosine triphosphate diphosphatase, luciferases, green fluorescent protein (GFP), or phycobiliproteins (e.g., phycocyanin, allophycocyanin, or phycoerythrin).

[0112] In some configurations, the flow cell contains a ternary complex (e.g., a stable ternary complex) immobilized within the flow cell, wherein the ternary complex (e.g., a stable ternary complex) contains a polymerase, a template nucleic acid that initiates the process, and the next correct nucleotide of the template.

[0113] Flow cells may contain arrays of nucleic acids, proteins, or other analytes. In a particular configuration, ternary complexes (e.g., stable ternary complexes) are present at one or more sites on the array. The array provides the advantage of multiplexing analytes, allowing multiple different types of analytes to be manipulated or detected in parallel. Arrays may include at least 2, 10, 100, or 1 x 10n analytes. 3 1 x 10 4 1 x 10 5 1 x 10 6 1 x 10 9 One or more different analyte sites. Alternatively or otherwise, the array may include up to 1 x 10⁻⁶. 9 1 x 10 6 1 x 10 5 1 x 10 4 1 x 10 3 One, 100, 10, 2 or fewer different analyte sites.

[0114] The arrays used herein may have sites spaced, for example, less than 100 micrometers, 50 micrometers, 10 micrometers, 5 micrometers, 1 micrometer, or 0.5 micrometers apart. Alternatively or otherwise, the array may have sites spaced greater than 0.5 micrometers, 1 micrometer, 5 micrometers, 10 micrometers, 50 micrometers, or 100 micrometers apart. These sites may each have an area of ​​less than 1 square millimeter, 500 square micrometers, 100 square micrometers, 25 square micrometers, 1 square micrometer, or smaller. The site density in the array may be, for example, at least about 10 sites / cm². 2 100 sites / cm 2 1×10 3 loci / cm 2 1×10 4 loci / cm 2 1×10 5 loci / cm 2 1×10 6 loci / cm 2 Or higher.

[0115] The array can be attached to the inner surface of the flow cell wall or to a solid support inside the flow cell. The flow cell or solid support can be made of any of a variety of materials used for analytical biochemistry. Suitable materials may include glass, polymer materials, silicon, quartz (fused silica), Borofloat glass, silica, silica-based materials, carbon, metals, optical fibers or fiber bundles, sapphire, or plastic materials. Materials can be selected based on the properties required for a specific application. For example, materials that are transparent to radiation at a desired wavelength may be used in analytical techniques that utilize that wavelength. Conversely, it may be desirable to select materials that do not transmit radiation at a specific wavelength (e.g., opaque, absorptive, or reflective). Other properties of materials that can be utilized include inertness or reactivity to certain reagents used in downstream processes (such as those described herein), ease of manipulation, or low manufacturing cost.

[0116] Particularly useful solid carriers for flow-through cells or other containers are particles, such as beads or microspheres. Bead populations can be used to attach populations of analytes, such as stable ternary complexes or components capable of forming complexes (e.g., polymerases, templates, primers, or nucleotides). In some configurations, each bead contains a single type of stable ternary complex or a single type of component capable of forming a complex, or a single type of other analyte described herein or cited in the references cited herein. For example, a single bead may attach to a single type of ternary complex, a single type of template allele, a single type of template locus, a single type of primer, or a single type of nucleotide. Alternatively, different types of components do not need to be separated on a bead basis. Thus, a single bead can carry multiple different types of ternary complexes, template nucleic acids, primers, initiating template nucleic acids, and / or nucleotides. The composition of the beads can vary depending on, for example, the type to be used, the chemical properties, and / or the attachment. Exemplary bead compositions include, for example, plastics, ceramics, glass, polystyrene, melamine, methylstyrene, acrylic polymers, paramagnetic materials, thorium oxide sol, carbon graphite, titanium dioxide, controlled-porosity glass (CPG), latex, or cross-linked dextran such as Sepharose. TM Cellulose, nylon, cross-linked micelles and Teflon TM Other materials described in the “Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind., which are incorporated herein by reference.

[0117] The beads may have symmetrical shapes, such as spheres, polyhedra, cylinders, etc. Alternatively, the beads may have irregular or asymmetrical shapes. Exemplary sizes of the beads used herein may have an average diameter of at least about 10 nm, 100 nm, 1 μm, 5 μm, 10 μm, 100 μm, 1 mm, or greater. Alternatively or otherwise, the beads used herein may have an average diameter of at most about 1 mm, 100 μm, 10 μm, 5 μm, 1 μm, 100 nm, 10 nm, 1 nm, or smaller. Beads within these size ranges may be used as array features or as particles in fluid slurries.

[0118] Exemplary compositions and techniques that can be used to prepare bead arrays include, but are not limited to, those for BeadChip purchased from Illumina, Inc. (San Diego, CA). TMThose of the arrays, or those described in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570, or 7,622,294, or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. Beads may be located at discrete locations on a solid carrier, such as recesses, whereby each location accommodates a single bead. Alternatively, the discrete locations where the beads are located may each comprise multiple beads, as described, for example, in U.S. Patent Application Publication Nos. 2004 / 0263923 A1, 2004 / 0233485 A1, 2004 / 0132205 A1, or 2004 / 0125424 A1, each of which is incorporated herein by reference.

[0119] Other useful arrays include those used for nucleic acid sequencing applications. For example, arrays of amplicon arrays (often called clusters) for fixing genomic fragments can be particularly useful. Examples of nucleic acid sequencing arrays that can be used in this paper include those by Bentley et al. Nature The references to any of the following documents are incorporated herein by reference: 456:53-59 (2008), PCT Publication Nos. WO 91 / 06678, WO 04 / 018497 or WO 07 / 123744, U.S. Patent Nos. 7,057,026, 7,211,414, 7,315,019, 7,329,492 or 7,405,281, or U.S. Patent Application Publication No. 2008 / 0108082.

[0120] Nucleic acids or other analytes can be attached to the array in a manner that allows for detection at the single-molecule or ensemble level. For example, multiple different nucleic acids can be attached to the array in a manner that allows for the formation of a single stable ternary complex on a single nucleic acid molecule in the array, distinguishing it from all adjacent ternary complexes formed in the array. Alternatively, the arrays of this disclosure may include multiple ensembles, which are groups of analytes of the same type, such as groups of nucleic acids having a common template sequence. The array may have multiple ensembles, each formed using methods known in the art, such as bridging amplification, emulsion PCR, or other methods described herein.

[0121] The flow cells of this disclosure (e.g., flow cells bonded using a curing liquid adhesive) offer the advantage of withstanding high positive pressure. This can be advantageous when using positive pressure to drive fluid through the flow cell. Positive pressure is particularly useful for driving mixed-phase fluids (e.g., foams, emulsions, or particulate slurries) through the flow cell. Exemplary mixed-phase fluids and methods of employing such fluids in a flow cell are described in U.S. Patent Application Serial No. 16 / 700,422, which is incorporated herein by reference. The flow cells of this disclosure can be configured to withstand pressures greater than 5 PSI, 10 PSI, 50 PSI, 90 PSI, or higher.

[0122] The flow cell of this disclosure can be configured for use in any of a variety of analytical systems. The analytical system of this disclosure may optionally include an optical detection system configured to detect the interior of the flow cell, such as the interior of a detection channel within the flow cell. Particularly useful optical detection systems include those present in subsystems or components of nucleic acid sequencing systems. Several such detection devices are configured for optical detection, such as the detection of luminescence signals. Thus, the optical detection system may include an excitation system configured to irradiate the interior of the flow cell channel. The optical detection system may also include an emission system configured to detect luminescence from the interior of the flow cell channel. Detection of luminescence can be performed using methods known in the art for nucleic acid arrays or nucleic acid sequencing. The luminescent group can be detected based on any of a variety of luminescence characteristics, including, for example, emission wavelength, excitation wavelength, fluorescence resonance energy transfer (FRET) intensity, quenching, anisotropy, or lifetime.

[0123] Examples of nucleic acid sequencing systems and components thereof that can be used with the flow-through pool of this disclosure are described, for example, in U.S. Patent Application Publication No. 2010 / 0111768 A1 or U.S. Patent Nos. 7,329,860, 8,951,781, or 9,193,996, each of which is incorporated herein by reference. Other nucleic acid sequencing systems include those commercially available for nucleic acid sequencing, such as those developed by Illumina. TM , Inc. (e.g., HiSeq) TM MiSeq TM NextSeq TM or NovaSeq TM Systems), Life Technologies TM (For example, ION TORRENT) TM or SOLiD TM (systems), Pacific Biosciences (e.g., using SMRT) TM Technology systems, such as Sequel TMor RS II TM (system) or BGI (e.g., DNBSEQ) TM The system provides those. Other useful detectors that can be used with the flow cell of this disclosure are described in U.S. Patent Nos. 5,888,737, 6,175,002, 5,695,934, 6,140,489, or 5,863,722, or U.S. Patent Publication Nos. 2007 / 007991A1, 2009 / 0247414A1, or 2010 / 0111768 or WO2007 / 123744, each of which is incorporated herein by reference in its entirety.

[0124] Although the systems and methods of this disclosure are described in the context of optical detection in several exemplary embodiments herein, it should be understood that other forms of detection may be used as supplementary or alternative. For example, the detector may be an electronic detector for detecting protons or pyrophosphates (see, for example, U.S. Patent Application Publication Nos. 2009 / 0026082A1, 2009 / 0127589 A1, 2010 / 0137143 A1, or 2010 / 0282617 A1, each of which is incorporated herein by reference in its entirety, or an Ion Torrent commercially available from ThermoFisher, Waltham, MA). TM (system), or detectors such as those used in the detection of nanopores, such as those manufactured by Oxford Nanopore. TM Those commercialized by Oxford UK (e.g., MinION) TM or Promethion TM (System) or such as U.S. Patent No. 7,001,792, Soni & Meller, Clin. Chem. 53, 1996-2001 (2007), Healy, Nanomed. 2, 459-481 (2007) or Cockroft et al., J. Am. Chem. Soc.Those described in 130,818-820 (2008), each of which is incorporated herein by reference. FET detectors may be used, such as one or more of those described in U.S. Patent Application Serial No. 62 / 767,712, U.S. Patent Application Publication No. 2017 / 0240962 A1, 2018 / 0051316 A1, 2018 / 0112265 A1, 2018 / 0155773 A1 or 2018 / 0305727 A1, or U.S. Patent Nos. 9,164,053, 9,829,456, 10,036,064 or 10,125,391, each of which is incorporated herein by reference.

[0125] Figure 6 A flow cell 500 mounted on an optical inspection system 2000 is shown. Holes 532 and 533, passing through wings 530 and 531 respectively, engage posts on a bracket 2030. Hole 533 is circular and engages relatively tightly with the circular post on the bracket 2030, while hole 532 has an elongated shape, allowing for flexibility when the flow cell 500 is placed on the bracket 2030. When positioned on the bracket 2030, the flow cell 500 can be pushed against a reference surface on the back plate 2021 by the thrust of a preload 2010. The preload can be engaged and disengaged manually using lever 2011. The bracket 2030 allows the flow cell 500 to move along... x Dimensional sliding is used to scan the stripes of the flow cell channel, and the support 2030 is also configured to cause the flow cell 500 to slide along... y Directional stepping, so that other strips of the channel can move along x Dimensions are scanned. Focusing is achieved by moving the objective lens component within the optical device 2001 relative to the backplate 2021. Figure 6 The components and methods of the optical detection system type illustrated are described in U.S. Patent No. 10,501,796 and U.S. Patent Application Serial No. 16 / 700,422, each of which is incorporated herein by reference.

[0126] like Figure 6As illustrated in examples and systems described in U.S. Patent No. 10,501,796 and U.S. Patent Application Serial No. 16 / 700,422 (each of which is incorporated herein by reference), a flow cell can be a removable component of a fluid system. For example, the systems disclosed herein may include a platform configured for convenient placement and removal of the flow cell. Thus, the flow cell can be a consumable component dedicated to a first analytical test, which is then removed to replace a second flow cell for a second analytical test. The two flow cells can be constructed similarly to each other, for example, containing similar analytes, similar samples, or a small portion of a particular sample. Alternatively, the flow cell can be a fixed component of the fluid system, for example, requiring specialized tools and / or specialized training to remove.

[0127] Figures 7A to 7E An adapter or fluid connector 1000 is shown, configured to engage with containers (such as containers 125, 126, 225, 226, 325, 326, 525, 526, and 1526) on a flow-through pool. Engagement can be conveniently performed by hand by inserting end 1006 into the container while squeezing lever arms 1002 and 1003. Hooks 1005 and 1004 at the ends of the lever arms engage with complementary surfaces 1025 and 1024 in latches 1504 and 1505, as shown. Figure 7D As shown. Fluid line 1037 is inserted into hole 1007 of fluid connector 1000 and passes through channel 1017, such that fluid line 1037 engages with port 1521 in container 1526 of flow tank (see Figure 1000). Figure 7E Similarly, fluid line 1038 is inserted into hole 1008 of fluid connector 1000 and passes through channel 1018, such that fluid line 1038 engages with port 1523 in container 1526 of flow cell (see...). Figure 7E The other ends of fluid lines 1037 and 1038 engage with a fluid system, allowing reagents and other fluids to be delivered into the flow cell. Fluid connector 1000 can be easily removed from container 1526 by pulling fluid connector 1000 from latches 1505 and 1504 while squeezing lever arms 1002 and 1003 to disengage hooks 1005 and 1004. Ridged surfaces 1010 and 1011 provide friction for fingers or suitable tools when fluid connector 1000 is pulled from the container. Figure 7C The flow cell wing 1531 shown has two holes 1533 and 1534, which are configured to engage two posts on the sliding frame. These two holes prevent the user from placing the flow cell on the detector in the wrong orientation.

[0128] Figures 8A to 8CThe application of adhesive rivets for assembling fluid connectors is illustrated. Adhesive rivet 1031 is adhered to flexible tube 1037, as shown... Figure 8A As shown. Figure 8B An exploded view of six adhesive rivets 1031-1036 (i.e., 1031, 1032, 1033, 1034, 1035, and 1036) relative to the fluid connector 1000 is shown. The rivets pass through holes 1091-1096 (i.e., 1091, 1092, 1093, 1094, 1095, and 1096), such that the head of each rivet engages with a countersunk seat in the hole, and the tail of each rivet adheres to tube 1037 or tube 1038. Figure 8C An assembly diagram of the fluid connector 1000 is shown.

[0129] Figures 14A to 14B A pipette adapter 1105 is shown, which is configured to engage with containers such as containers 125, 126, 225, 226, 325, 326, 525, and 526 on a flow cell. Engagement can be easily performed by hand. Figure 14A A pipette adapter 1105 positioned within container 126 is shown, while Figure 14B An exploded view of a container adapter 1105 positioned above container 126 is shown. The size and shape of the pipette adapter 1105 are configured to fit within the bowl of container 126. In this respect, the pipette adapter 1105 may have a shape complementary to the shape of the bowl, such that the pipette adapter 1105 fits therein in a close-fitting manner.

[0130] The pipette adapter 1105 includes a pipette fluid inlet 1110 and a fluid outlet 1115. When the pipette adapter 1105 is located inside the container 126, the pipette fluid inlet 1110 is connected to port 121 of the container 126. Figure 1A The fluid outlet 1115 is aligned with port 123 of container 126. A pipette adapter 1105 is placed on ports 121 and 123 to facilitate the injection of the library into the flow cell. This step can be performed manually by the user. The size and shape of the pipette fluid inlet 1110 are configured to accommodate a pipette therein. As described above, the pipette adapter 1105 is configured to fit into any of containers 125, 126, 225, 226, 325, 326, 525, and 526.

[0131] Figure 15A and Figure 15B Another embodiment of the pipette adapter 1205 is shown, wherein the pipette adapter 1205 is made of a plurality of parts. Figure 14A and Figure 14BThe pipette adapter 1105 can optionally be a one-piece, monolithic structure. The pipette adapter 1205 is configured to engage with containers such as containers 125, 126, 225, 226, 325, 326, 525, and 526 on the flow cell. Engagement can be easily performed by hand. Figure 15A A pipette adapter 1205 positioned above container 126 is shown. As in a previous embodiment, the pipette adapter 1205 is sized and shaped to fit within the bowl of container 126. The pipette adapter 1105 may have a shape complementary to the shape of the bowl, such that the pipette adapter 1105 fits therein in a close-fitting manner.

[0132] Figure 15B It shows Figure 15A An exploded view of a pipette adapter 1205, which includes a lower base plate 1210 mechanically coupled to an upper retainer 1215. The upper retainer 1215 can mate with the lower base plate 1210, for example, by being mounted on the lower base plate. When mated, the retainer 1215 and the base plate 1210 define a space therebetween in which a core 1225, a diaphragm 1230, and at least one cover 1235 are positioned. The retainer 1215 defines a pipette fluid inlet 1240 and a fluid outlet 1245, which are aligned with the corresponding cover 1235, as described more fully below. When the pipette adapter 1205 is located within a container 126, the pipette fluid inlet 1240 aligns with port 121 of the container 126. Figure 1A The fluid outlet 1245 is aligned with the port 123 of the container 126.

[0133] When the retainer 1215 is attached to the substrate 1210, the retainer applies force to the diaphragm 1230 and the core 1225 to maintain a seal. The retainer may also have a snap-fit ​​engagement with the wing 131. The diaphragm 1230 serves as a seal for the pipette inserted into the pipette fluid inlet 1240 to suppress or prevent reagent leakage. The core 1225 is configured to soak or otherwise capture the reagent dispensed by the user.

[0134] The shroud 1235 also forms a seal with any tubing positioned therein. A first shroud is coaxially aligned with the pipette fluid inlet 1240, and a second shroud is coaxially aligned with the fluid outlet 1245. The shrouds facilitate proper alignment when the pipette is inserted into the pipette fluid inlet 1240 and also securely hold or fix the pipette in its position therein. The shrouds 1235 are tubular, each having an inner cavity whose dimensions and shape are configured to accommodate tubes or other structures, such as pipettes.

[0135] One or more coverings may also be referenced in the previous reference. Figures 7A to 7E The described adapter or fluid connector 1000 is used in conjunction with this. Figure 16 The lower region of the fluid connector 1000 is shown in the area of ​​port 1523. A tubing cover 1610 is positioned within port 1523. The tubing cover 1610 is configured for downstream chemical manipulation of the flow cell containing the introduced library. This chemical manipulation is automated and requires the tubing to be securely held in place as the flow cell is translated along the image capture surface. The cover 1610 has an enlarged lower locking region that holds the cover 1610 and the tubing in place as the flow cell is translated along the image capture surface. The tubing cover 1610 is configured to hold the tubing in place as the flow cell is mechanically translated back and forth through the image capture device during various sequencing cycles. The cover 1610 can also be used in conjunction with a high-pressure fluid flow through the tubing. The cover 1610 suppresses or prevents fluid leakage from the tubing and also suppresses or prevents disconnection of the interface between the tubing and the flow cell. In a non-limiting example, the high-pressure fluid flow is in the range of 40 psi to 80 psi.

[0136] The flow-through cell disclosed herein can be used for sequencing nucleic acids. Particularly useful sequencing methods are cyclic methods employing repeating cycles of reagent delivery and flow-through cell detection. Each cycle may include one or more steps. For example, each cycle may include all the steps required to detect a single nucleotide position in a template nucleic acid. Some sequencing methods employ cycle reversible terminator (CRT) chemistry, where each cycle includes the following steps: (i) adding a single reversibly terminating nucleotide to augment a nascent primer to the nucleotide position to be detected; (ii) detecting the nucleotide at the single nucleotide position; and (iii) deblocking the nascent primer to allow a return to step (i), thereby initiating subsequent cycles.

[0137] A concrete example of a useful CRT nucleic acid sequencing method is Sequencing By Binding. TM (SBB TM The reaction, for example, as described in commonly owned U.S. Patent Application Publication Nos. 2017 / 0022553 A1, 2018 / 0044727 A1, 2018 / 0187245 A1, or 2018 / 0208983 A1, each of which is incorporated herein by reference. Generally, SBBs used to determine the sequence of template nucleic acid molecules... TM The method can be based on the formation of a stable ternary complex (between polymerase, initiating nucleic acid, and homologous nucleotides) under specific conditions. The method may include an inspection phase followed by a nucleotide incorporation phase.

[0138] SBB TMThe method's testing phase can be performed in a flow cell containing at least one template nucleic acid molecule that is initiated by primers by delivering reagents to the flow cell to form a first reaction mixture. The reaction mixture may contain the initiating template nucleic acid, a polymerase, and at least one nucleotide type. The interaction between the polymerase and the nucleotide with one or more initiating template nucleic acid molecules can be observed under conditions where the nucleotide is not covalently added to one or more primers; and the observed interaction between the polymerase and the nucleotide with one or more initiating template nucleic acid molecules can be used to identify the next base in each template nucleic acid. The interaction between the initiating template, polymerase, and nucleotide can be detected in a variety of schemes. For example, the nucleotide may contain a detectable label. Each nucleotide may have a label that is distinguishable relative to other nucleotides. Alternatively, some or all of the different nucleotide types may have the same label, and the nucleotide types may be distinguished based on the individual delivery of the different nucleotide types to the flow cell. In some embodiments, the polymerase may be labeled. Polymerases associated with different nucleotide types may have unique labels that distinguish the types of nucleotides associated with them. Alternatively, polymerases may have similar labels, and different nucleotide types may be distinguished based on the individual delivery of the different nucleotide types to the flow cell. Detection can be performed by scanning the flow cell using the equipment or methods described herein or cited in the references cited herein.

[0139] At SBB TM The method's checking phase can facilitate the differentiation between correct and incorrect nucleotides through ternary complex stabilization. Various conditions and reagents can be useful. For example, primers may contain reversibly blocking portions that prevent covalent nucleotide linkage; and / or cofactors required for extension (such as divalent metal ions) may be absent; and / or inhibitory divalent cations that inhibit polymerase-based primer extension may be present; and / or the polymerase present in the checking phase may have chemical modifications and / or mutations that inhibit primer extension; and / or the nucleotides may have chemical modifications that inhibit incorporation, such as 5' modifications that remove or alter the native triphosphate moiety. The checking phase may include scanning the flow cell using the apparatus and methods described herein.

[0140] The extension phase can then be performed by creating conditions in a flow cell, where nucleotides can be added to primers on each template nucleic acid molecule. In some embodiments, this involves removing reagents used in the testing phase and replacing them with reagents that promote extension. For example, the testing reagents can be replaced with an extension-capable polymerase and one or more nucleotides. Alternatively, one or more reagents can be added to the testing phase reaction to create extension conditions. For example, a catalytic divalent cation can be added to a cation-deficient testing mixture, and / or a polymerase inhibitor can be removed or disabled, and / or an extension-capable nucleotide can be added, and / or a deblocking agent can be added to make one or more primers extension-capable, and / or an extension-capable polymerase can be added.

[0141] Another useful CRT sequencing method is sequencing-by-synthesis (SBS). SBS generally involves the enzymatic extension of nascent primers by repeatedly adding nucleotides to the template strand hybridized to the primer. In short, SBS is initiated by contacting a target nucleic acid (e.g., a site linked to a flow cell) with one or more labeled nucleotides, a DNA polymerase, etc. Those sites that use the target nucleic acid as a template to extend the primer will be incorporated with detectable labeled nucleotides. Detection may include scanning using the devices or methods described herein. Optionally, the labeled nucleotide may also include a reversible terminator that prevents further primer extension once the nucleotide has been added to the primer. For example, a nucleotide analog with a reversible terminator moiety may be added to the primer such that subsequent extension cannot be performed until a deblocking agent is delivered to remove that moiety. Thus, for embodiments using reversible termination, a deblocking agent may be delivered to a container (before or after detection). Washing may be performed between the various delivery steps. Cycling is possible. n This allows the primers to extend. n nucleotides, thus detecting lengths of nucleotides. n The sequence. Exemplary SBS procedures, reagents, and detection components that can be readily applied to the methods, systems, or apparatus of this disclosure, such as those described by Bentley et al. Nature The SBS method is described in U.S. Patent Nos. 456:53-59 (2008), WO 04 / 018497, WO 91 / 06678, WO 07 / 123744, U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019 or 7,405,281 and U.S. Patent Application Publication No. 2008 / 0108082 A1, each of which is incorporated herein by reference. Also useful is the SBS method, commercially available from Illumina, Inc. (San Diego, CA).

[0142] Some SBS implementations are cyclic but do not require the use of reversible terminator nucleotides. Particularly useful methods include detecting protons released when nucleotides are incorporated into the elongation product. For example, sequencing based on the detection of released protons can use reagents and electrodetectors commercially available from Thermo Fisher (Waltham, MA) or described in U.S. Patent Application Publication Nos. 2009 / 0026082 A1, 2009 / 0127589 A1, 2010 / 0137143 A1, or 2010 / 0282617 A1, each of which is incorporated herein by reference.

[0143] Other cyclic sequencing methods, such as pyrosequencing, can be used. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when nucleotides are incorporated into nascent primers that hybridize with the template nucleic acid strand (Ronaghi et al.). Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001); Ronaghi et al., Science 281 (5375), 363 (1998); U.S. Patents 6,210,891, 6,258,568, and 6,274,320, each of which is incorporated herein by reference. In pyrosequencing, the released PPi can be detected by conversion to adenosine triphosphate (ATP) by ATP sulfatase, and the resulting ATP can be detected by photons generated by luciferase.

[0144] Ligation sequencing reactions are also useful, including those by, for example, Shendure et al. Science 309:1728-1732 (2005); those described in U.S. Patent Nos. 5,599,675 or 5,750,341, each of which is incorporated herein by reference. Some embodiments may include, for example, Bains et al. Journal of Theoretical Biology 135 (3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science The hybridization sequencing methods described in WO 1989 / 10977, each of which is incorporated herein by reference. In ligation sequencing and hybridization sequencing methods, primers hybridizing with a nucleic acid template are repeatedly extended in cycles via oligonucleotide ligation. Typically, the oligonucleotides are luminescently labeled and can be detected to determine the sequence of the template, for example, using the systems or methods described herein.

[0145] The steps of the sequencing method described above can be repeated. For example, SBB can be repeated. TM The method includes checking and extension steps, such that in each cycle, a single next correct nucleotide (i.e., the next correct nucleotide is the nucleotide that correctly binds to a nucleotide in the template nucleic acid, which is located directly at the 5' of the base in the template that hybridizes to the 3' end of the hybridization primer) is checked, and the single next correct nucleotide is subsequently added to the primer. Any number of cycles of the sequencing method described herein can be performed, including, for example, at least 1, 2, 10, 100, 250, 500, or more cycles. Alternatively or otherwise, no more than 500, 250, 100, 10, 2, or 1 cycle may be performed.

[0146] Some implementations can utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interaction between the polymerase carrying a fluorophore and a γ-phosphate-labeled nucleotide, or using zero-mode waveguide (ZMW) waveguide. Techniques and reagents for sequencing via FRET and / or ZMW detection in the apparatus or methods described herein can be modified for use in, for example, Levene et al. Science 299, 682-686 (2003); Lundquist et al., Opt. Lett. 33, 1026-1028 (2008); Korlach et al., Proc. Natl. Acad. Sci. USA As described in U.S. Patent Nos. 105, 1176-1181 (2008); or U.S. Patent Nos. 7,315,019, 8,252,911, or 8,530,164, the disclosure of which is incorporated herein by reference.

[0147] Any of a variety of nucleic acid amplification techniques can be used to replicate nucleic acids in a flow-through cell. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), bridging amplification, random primer amplification (RPA), or other techniques known in the field of molecular biology. In a particular configuration, one or more primers used for amplification may be attached to the surface of the flow-through cell. In such embodiments, the extension of one or more primers attached to the surface along the template nucleic acid will result in a copy of the template being attached to the surface. This amplification method can be used for analytical purposes, such as real-time PCR or quantitative PCR. Alternatively, amplification can be used to prepare nucleic acids for downstream applications such as nucleic acid sequencing. Preparative amplification methods that generate one or more sites on a solid-phase support, wherein each site is attached to multiple copies of a particular nucleic acid template, are referred to as “clustering” methods.

[0148] In PCR technology, one or two primers used for amplification can be attached to a surface. The use of two ligating primers is often referred to as bridging amplification because the double-stranded amplicon forms a bridge-like structure between the two ligating primers located on the flanks of the replicated template sequence. Exemplary reagents and conditions that can be used for bridging amplification are described, for example, in U.S. Patent Nos. 5,641,658 or 7,115,400; U.S. Patent Publications 2002 / 0055100 A1, 2004 / 0096853 A1, 2004 / 0002090 A1, 2007 / 0128624 A1, or 2008 / 0009420 A1, each of which is incorporated herein by reference. PCR amplification can also be performed using either amplification primers attached to a surface or a second primer in solution. An exemplary form using a combination of a solid-phase attached primer and a liquid-phase primer is called primer walking, and can be performed as described in U.S. Patent No. 9,476,080, which is incorporated herein by reference. Another example is emulsion PCR, which can be performed, for example, by Dressman et al. Proc. Natl. Acad. Sci. USA The documents described herein are as follows: 100:8817-8822 (2003), WO 05 / 010145 or U.S. Patent Publication No. 2005 / 0130173 A1 or 2005 / 0064460 A1, each of which is incorporated herein by reference.

[0149] RCA technology can be used in the methods described herein. Exemplary reagents that can be used in RCA reactions and the principles of RCA amplicon generation are illustrated in, for example, by Lizardi et al. Nat. Genet. As described in 19:225-232 (1998) or U.S. Patent Application Publication No. 2007 / 0099208 A1, each of which is incorporated herein by reference. Primers used for RCA can be in solution or attached to the surface of the flow cell.

[0150] MDA techniques can also be used in the methods of this disclosure. Some reagents and useful conditions used in MDA are described, for example, in Dean et al. Proc Natl. Acad. Sci. USA 99:5261-66 (2002); Lage et al., Genome Research 13:294-307 (2003); Walker et al., Molecular Methods for Virus Detection , Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96 (1992); or as described in U.S. Patent Nos. 5,455,166, 5,130,238, or 6,214,587, each of which is incorporated herein by reference. Primers for MDA can be in solution or attached to the surface of a flow cell.

[0151] The nucleic acid template used in the methods or compositions described herein may be DNA, such as genomic DNA, synthetic DNA, amplified DNA, complementary DNA (cDNA), etc. RNA, such as mRNA, ribosomal RNA, tRNA, etc., may also be used. Nucleic acid analogs may also be used as templates in this paper. Primers used in this paper may be DNA, RNA, or analogs thereof.

[0152] The flow-through channel can extend directly from the inlet to the outlet, such as... Figure 1A and 1B to Figure 4A and 4B As shown. In some configurations, the channel can be constructed without any bends or curves in the bulk flow of the fluid within it. Alternatively, the channel may have curves or bends. For example, the channel may be wound around the surface of the flow cell, such as... Figure 9A The flow cell 700 is an example. Here, each channel includes three regions for detecting the analyte. More specifically, the channel connecting port 721 to port 720 includes detection regions 701, 705, and 709. The detection regions are relatively wide compared to the other regions in the channel to accommodate a relatively large surface area for the presentation array. Fluid flows directly from inlet port 721 into the relatively wide detection region 701, then through a bend 703 that narrows at 702 and ends at 704. o The channel narrows at the bend, then widens to the detection zone 705 at 704. After passing through the widened detection zone 705, the fluid passes through the bend 707, which narrows at 706 and ends at 708. o The channel remains narrow at the turn, widening into detection zone 709 at point 708. After passing through the widened detection zone 709, the fluid passes through the narrowing 90° channel that begins at point 710. o Turn and direct the fluid to outlet 720.180. o The shape of the curved section is used to maintain a relatively flat front section between two fluids during the exchange of fluids. This flat front section is achieved by configuring the inner wall of the curved section to have the same or very similar length as the outer wall of the curved section. This configuration creates a mushroom-shaped region 770 of washer on the inside of the bend. Figure 9A ) or 771 ( Figure 9B The channel connecting port 723 to port 724 has a similar shape and characteristics to the channel connecting port 720 to port 721. For example... Figure 9B As shown, the foam 791 that exchanges with the liquid 790 will remain flat at the front 799 when crossing the curve and on both sides of the curve, thus proving that the shape of the channel enables efficient and spatially uniform fluid exchange.

[0153] The circulation pool of this disclosure may include one or more reference points, which can be used by the circulation pool in... x and y 2D registration in one dimension. For example... Figure 10 As shown, reference points 881, 882, 883, and 884 can exist in a gasket 805 inserted between two transparent substrates. Gasket 805 also defines channels 801 and 802 in the flow cell 800. An effective method for generating reference point 881 is to use an IR or UV laser on a pressure-sensitive adhesive (e.g., white polyester ARcare from Adhesives Research, Glen Rock PA). ® A hole was cut out in 8939. The resulting hole has a luminescent edge 890, which produces a distinct ring 850 when observed with a photometer. Unwilling to be constrained by the mechanism, the luminescence is believed to be caused by the product of the interaction between the laser and the binder in the PSA.

[0154] The advantage of incorporating the reference point 881 into the gasket material is that conventional manufacturing processes can be used to accurately position the gasket relative to the channel and precisely place the reference point within the gasket, thereby creating a high-confidence spatial relationship between the reference point and the array present in the channel. Although Figure 10 The reference points in the model have a circular shape, but can form any of a variety of shapes, including, for example, shapes with symmetry that are advantageous to certain registration algorithms (e.g., squares, triangles, regular convex polygons, concentric circles, concentric polygons, etc.). Asymmetrical or irregularly shaped reference points can also be useful.

[0155] Various other reference points and techniques for generating reference points are shown in Figures 11A to 11D middle. Figure 11A A cross-section of a flow cell having a substrate 53 attached to a cap 51 via a PSA layer 52 is shown; an aperture 62 in the cap 51 extends through the PSA layer 52, wherein the aperture 62 is filled with a liquid binder 54 doped with a light-emitting element. When viewed from above the cap 51, a reference point will produce a filling shape defined by the shape of the aperture 62. For example, a cylindrical aperture will produce a circular reference point. The reference point may have a cross-sectional shape similar to that described herein in the context of through-holes for liquid binders.

[0156] Figure 11BThe process for staining a flow cell to generate reference points is illustrated. The flow cell is shown in cross-section, showing a substrate 53 attached to a cap 51 via a PSA layer 52. The cap 51 has a hole 62 extending through the PSA layer 52 into a surface 61 of the substrate 53. The hole 62 is filled with a liquid 54 doped with a light-emitting agent, and this liquid is removed, leaving a luminescent spot on the surface 61. In this configuration, the surface 61 is readily stained with the light-emitting agent, while the surfaces of the cap and PSA do not absorb the light-emitting agent. A configuration using a cap 71, PSA layer 72, and substrate 73 capable of absorbing the light-emitting agent is shown. Figure 11C In the middle, the surface of the cover 71 that contacts the light emitter will be stained (see surfaces 79 and 80 in the sectional view). Similarly, the surface of the PSA layer 72 that contacts the light emitter will be stained (see surfaces 75, 76, 77, and 78 in the sectional view). Surface 74 will also be stained. When viewed from above the cover, Figure 11B and Figure 11C The reference point in the hole will produce a filling shape defined by the shape of hole 62.

[0157] Figure 11D A cross-section of a flow cell with a substrate 87 attached to a cover 85 via a PSA layer 86 is shown, wherein the PSA layer contains a light-emitting material. In this configuration, the cover 85 is opaque to the excitation or emission of the light-emitting material. Because the aperture 82 in the cover 85 is wider than the aperture in the PSA layer 86, and because the apertures in both layers are circular, the reference point will appear as a light-emitting ring when viewed from above the cover 85. Portions 88 and 89 of the light-emitting ring are shown in... Figure 11D In the sectional view.

[0158] Exemplary luminescent agents that can be used as reference points or as markers for analytes include, but are not limited to, fluorescent nanocrystals; quantum dots; green fluorescent proteins and their color-shifting mutants; phycobiliproteins, such as phycocyanin and phycoerythrin; d-rhodamine acceptor dyes, including dichloro[R110], dichloro[R6G], dichloro[TAMRA], dichloro[ROX], etc.; fluorescein donor dyes, including fluorescein, 6-FAM, etc.; anthocyanin dyes, such as Cy3B; Alexa dyes, SETA dyes, Atto dyes, such as atto 647N which forms a FRET pair with Cy3B, etc. Others include, but are not limited to, MDCC (7-diethylamino-3-[([(2-maleimino)ethyl]amino)carbonyl]coumarin), TET, HEX, Cy3, TMR, ROX, Texas Red, Cy5, LC red 705, and LC red 640. Other luminescent agents known in the art include, Principles of Fluorescence Spectroscopy Joseph R. Lakowicz (ed.), Plenum Pub Corp, 2nd edition (July 1999) and Richard P. Hoagland Molecular Probes Handbook Those described in the 6th edition (each of which is incorporated herein by reference) may be useful. In certain embodiments, the luminescent or luminescent material used herein may have a fluorescence quantum yield of at least 0.2, 0.5, 0.8 or higher.

[0159] Figure 12A A top view of a flow cell cover 900 is shown, including expansion joints 970 and 971, which connect wings 931 and 930 to a window area 910, respectively. Expansion joints 970 and 971 are made of a low-hardness material (i.e., a material with low hardness). The expansion joints accommodate movement that occurs when the flow cell is subjected to temperature changes, such as during thermal cycling. The expansion joints prevent cracking or warping that may occur when the cover is made of a material with a different coefficient of thermal expansion (CTE) than the CTE of another substrate or gasket to which the cover is attached. Figure 12A The reference numerals 933, 932, 921, 920, 924, 923, 926 and 925 in 12B correspond to holes 133, 132, channel ports 121, 120, 124, 123 and containers 125, 126, respectively.

[0160] In one exemplary embodiment, a flow cell is disclosed, comprising: (a) a gasket inserted between a first substrate and a second substrate, wherein the gasket, the first substrate, and the second substrate are impermeable to an aqueous liquid and a liquid adhesive, wherein the gasket has a covering area on the first substrate defining a channel for containing the aqueous liquid; (b) a through-hole located in the gasket, the through-hole containing a cured liquid adhesive for bonding the first substrate to the second substrate, wherein the cured liquid adhesive in the through-hole is separated from the channel by the gasket; and (c) a first channel port connecting the channel to the outside of the flow cell, wherein the first channel port is permeable to the aqueous liquid. In an exemplary variation of any embodiment, a flow cell of any disclosed embodiment is disclosed, further comprising a through-hole port connecting the through-hole to the outside of the flow cell. In an exemplary variation of any embodiment, a flow cell of any disclosed embodiment is disclosed, further comprising a second through-hole port connecting the through-hole to the outside of the flow cell. In any exemplary variation of the embodiment, a flow cell of any disclosed embodiment is disclosed, wherein the gasket includes a plurality of through-holes, each through-hole containing a cured liquid adhesive for bonding a first substrate to a second substrate, wherein the cured liquid adhesive in the through-hole is separated from the channel by the gasket. In any exemplary variation of the embodiment, a flow cell of any disclosed embodiment is disclosed, wherein the channel includes an array of analytes. In any exemplary variation of the embodiment, the array of analytes is attached to a first substrate in the channel. In any exemplary variation of the embodiment, a second array of analytes is attached to a second substrate in the channel. In any exemplary variation of the embodiment, the analytes include nucleic acids, with different nucleic acids attached to corresponding sites on the array. In any exemplary variation of the embodiment, the array includes per mm 2At least 100 sites. In any exemplary variant of the embodiment, the gasket has a covering area on the second substrate defining a channel for containing an aqueous liquid. In any exemplary variant of the embodiment, the covering area on the first substrate has the same shape as the covering area on the second substrate. In any exemplary variant of the embodiment, the first substrate includes a flat surface forming a first side of the channel, and the second substrate includes a flat surface forming a second side of the channel. In any exemplary variant of the embodiment, a second channel port is disclosed connecting the channel to the outside of the flow cell, wherein the second channel port is permeable to the aqueous liquid. In any exemplary variant of the embodiment, the aqueous liquid is present in the channel. In any exemplary variant of the embodiment, the aqueous liquid applies a positive pressure of at least 10 PSI to the channel. In any exemplary variant of the embodiment, the gasket comprises a pressure-sensitive adhesive that contacts one or both of the first and second substrates. In any exemplary variant of the embodiment, the first and second substrates comprise the same material. In any exemplary variant of the embodiment, the material includes glass or plastic. In any exemplary variation of the embodiment, the glass comprises a silane or organosilane coating. In any exemplary variation of the embodiment, the first substrate comprises a material different from that of the second substrate. In any exemplary variation of the embodiment, the first substrate comprises glass, and the second substrate comprises plastic. In any exemplary variation of the embodiment, the first substrate comprises a transparent material and a thermal expansion joint. In any exemplary variation of the embodiment, a through-hole continuously surrounds a channel. In any exemplary variation of the embodiment, a through-hole continuously surrounds a respective channel among a plurality of channels in a flow cell. In any exemplary variation of the embodiment, a gasket defines a distance between the first and second substrates in the channel. In any exemplary variation of the embodiment, this distance is greater than 0.1 mm and less than 1 cm. In any exemplary variation of the embodiment, the volume of the channel is greater than 10 µl and less than 10 ml. In any exemplary variation of the embodiment, one or both of the first and second substrates are transparent to UV, VIS, or IR radiation. In any exemplary variation of the embodiment, the curing liquid adhesive comprises a luminescent material. In any exemplary variation of the embodiment, the gasket comprises a luminescent material. In an exemplary variation of any implementation, the passage includes a turn of at least ninety degrees, wherein the turn has an inner wall and an outer wall, the inner wall having substantially the same length as the outer wall.

[0161] In another exemplary embodiment, a method of manufacturing a flow cell is disclosed, the method comprising (a) inserting a gasket between a first substrate and a second substrate to define a channel for containing an aqueous liquid, wherein the gasket, the first substrate, and the second substrate are impermeable to the aqueous liquid and the liquid adhesive; (b) delivering the liquid adhesive into a through-hole in the gasket; and (c) allowing the liquid adhesive to bond the first substrate to the second substrate, wherein the liquid adhesive is separated from the channel by the gasket. In an exemplary variant of any embodiment, step (b) is performed prior to step (a). In an exemplary variant of any embodiment, the liquid adhesive is delivered through an inlet port of a through-hole in the first substrate, the second substrate, or the gasket. In an exemplary variant of any embodiment, the flow cell includes an outlet port of a through-hole through which air is discharged when the liquid adhesive is delivered through the inlet port. In an exemplary variant of any embodiment, step (c) includes applying heat or light to allow the liquid adhesive to bond the first substrate to the second substrate and the gasket. In an exemplary variant of any embodiment, forming an array of analytes in the channel is also disclosed. In any exemplary variant of the embodiment, the analyte includes nucleic acids, with different nucleic acids attached to corresponding sites on the array. In any exemplary variant of the embodiment, the array is formed by attaching the analyte to a first substrate of the channel. In any exemplary variant of the embodiment, a gasket is disclosed to be adhered to the first substrate using a pressure-sensitive adhesive. In any exemplary variant of the embodiment, a gasket is disclosed to be adhered to a second substrate using a pressure-sensitive adhesive. In any exemplary variant of the embodiment, the first and second substrates comprise the same material. In any exemplary variant of the embodiment, the material includes glass or plastic. In any exemplary variant of the embodiment, the glass comprises a silane or organosilane coating. In any exemplary variant of the embodiment, the first substrate comprises a material different from that of the second substrate. In any exemplary variant of the embodiment, the first substrate comprises glass and the second substrate comprises plastic. In any exemplary variant of the embodiment, a container is disclosed that houses at least a first channel port, wherein the container defines a basin. In any exemplary variant of the embodiment, the container is configured to mate with a pipette adapter, wherein the pipette adapter is configured to facilitate the introduction of nucleic acid library starting material into the flow cell. In an exemplary variant of any embodiment, a pipette adapter is disclosed, wherein the pipette adapter mates with a tray of a container. In an exemplary variant of any embodiment, the pipette adapter includes a pipette fluid inlet aligned with a first channel port, the pipette fluid inlet being configured to receive a pipette. In an exemplary variant of any embodiment, the pipette adapter includes a cover that aligns with the first channel port when the pipette adapter mates with the container. In an exemplary variant of any embodiment, the container is configured to mate with an adapter that connects a flow cell channel to a fluid delivery device.In any exemplary variant of the embodiment, the fluid delivery device includes at least one fluid line fluidly coupled to a fluid system, allowing reagents and other fluids to be delivered to a passage in a flow cell. In any exemplary variant of the embodiment, the adapter includes a covering that mates with the at least one fluid line. In any exemplary variant of the embodiment, the covering allows high-pressure fluid to flow through the at least one fluid line and prevents fluid leakage from the at least one fluid line. In any exemplary variant of the embodiment, the covering allows high-pressure fluid to flow through the at least one fluid line and prevents fluid leakage from the at least one fluid line at the interface between the at least one fluid line and the flow cell.

[0162] In another exemplary embodiment, an adapter device for facilitating the entry or exit of fluid relative to a flow cell is disclosed. The adapter includes: a housing configured to mate with the flow cell, the housing including a first port aligned with an inlet or outlet of the flow cell, the port configured to receive a tube for delivering or receiving fluid relative to the flow cell; and a first cladding positioned within the housing and aligned with the first port, the first cladding configured to hold the tube relative to the flow cell. In an exemplary variation of any embodiment, the tube is a pipette. In an exemplary variation of any embodiment, the tube is coupled to a fluid system such that reagents and other fluids can be delivered to a channel of the flow cell. In an exemplary variation of any embodiment, the first cladding is made of silicone. In an exemplary variation of any embodiment, the first port is aligned with the inlet of the flow cell. In an exemplary variation of any embodiment, the housing also includes a second port aligned with the outlet of the flow cell, and further includes a second cladding positioned within the housing and aligned with the second port. In an exemplary variation of any embodiment, the cladding allows high-pressure fluid to flow through the tube and inhibits fluid leakage from the tube.

[0163] Various publications, patents, and / or patent applications have been referenced throughout this application. The disclosures of these documents are incorporated herein by reference in their entirety. Several embodiments have been described. However, it should be understood that various modifications can be made. Therefore, other embodiments are also within the scope of the following claims.

[0164] While this specification contains numerous details, these should not be construed as limiting the scope of the claimed invention or the scope that may be claimed, but rather as descriptions of specific features of particular embodiments. Certain features described in the context of individual embodiments may also be implemented in a combined manner in a single embodiment. Conversely, various features described in the context of a single embodiment may also be implemented individually or in any suitable sub-combination in multiple embodiments. Furthermore, although features may be described above as functioning in certain combinations and even initially claimed in this way, one or more features from a claimed combination may be removed from that combination in some cases, and the claimed combination may be for sub-combinations or variations thereof. Similarly, although operations are depicted in a specific order in the drawings, this should not be construed as requiring these operations to be performed in the specific order or sequential sequence shown, or to perform all shown operations to achieve the desired result. Only a few examples and specific embodiments are disclosed. Variations, modifications, and enhancements may be made to the described examples and specific embodiments, as well as other specific embodiments, based on the disclosure.

Claims

1. A flow pool, comprising: (a) A gasket inserted between a first substrate and a second substrate, wherein the gasket, the first substrate and the second substrate are impermeable to aqueous liquids and liquid adhesives, wherein the gasket has a covering area on the first substrate defining a channel for receiving the aqueous liquid. (b) A through-hole located in the gasket, the through-hole containing a cured liquid adhesive for bonding the first substrate to the second substrate, wherein the cured liquid adhesive in the through-hole is separated from the channel by the gasket; as well as (c) A first channel port that connects the channel to the outside of the flow cell, wherein the first channel port is permeable to the aqueous liquid.

2. The flow cell of claim 1, further comprising a through-hole port connecting the through-hole to the outside of the flow cell.

3. The flow cell of claim 2, further comprising a second through-hole port connecting the through-hole to the outside of the flow cell.

4. The flow cell of claim 1, wherein the gasket includes a plurality of through holes, each through hole containing a cured liquid adhesive for bonding the first substrate to the second substrate, wherein the cured liquid adhesive in the through hole is separated from the channel by the gasket.

5. The flow cell of claim 1, wherein the channel comprises an analyte array.

6. The flow cell of claim 5, wherein the analyte array is attached to the first substrate in the channel.

7. The flow cell of claim 6, wherein the second analyte array is attached to the second substrate in the channel.

8. The flow cell of claim 5, wherein the analyte comprises nucleic acid, and different nucleic acids are attached to corresponding sites on the array.

9. The flow cell of claim 8, wherein the array comprises per mm 2 At least 100 sites.

10. The flow cell of claim 1, wherein the gasket has a covering area on the second substrate defining a channel for receiving the aqueous liquid.

11. The flow cell of claim 1, wherein the covering area on the first substrate has the same shape as the covering area on the second substrate.

12. The flow cell of claim 11, wherein the first substrate includes a flat surface forming a first side of the channel, and the second substrate includes a flat surface forming a second side of the channel.

13. The flow cell of claim 1, further comprising a second channel port connecting the channel to the outside of the flow cell, wherein the second channel port is permeable to the aqueous liquid.

14. The flow cell of claim 1, wherein the aqueous liquid is present in the channel.

15. The flow cell of claim 14, wherein the aqueous liquid exerts a positive pressure of at least 10 PSI on the channel.

16. The flow cell of claim 1, wherein the gasket comprises a pressure-sensitive adhesive that is in contact with one or both of the first substrate and the second substrate.

17. The flow cell of claim 1, wherein the first substrate and the second substrate comprise the same material.

18. The flow cell of claim 17, wherein the material comprises glass or plastic.

19. The flow cell of claim 18, wherein the glass comprises a silane coating.

20. The flow cell of claim 1, wherein the first substrate comprises a material different from that of the second substrate.

21. The flow cell of claim 20, wherein the first substrate comprises glass and the second substrate comprises plastic.

22. The flow cell of claim 20, wherein the first substrate comprises a transparent material and a thermal expansion joint.

23. The flow cell of claim 1, wherein the through-hole continuously surrounds the channel.

24. The flow cell of claim 1, wherein the through-hole continuously surrounds each of the plurality of channels in the flow cell.

25. The flow cell of claim 1, wherein the gasket defines the distance between the first substrate and the second substrate in the channel.

26. The flow cell of claim 25, wherein the distance is greater than 0.1 mm and less than 1 cm.

27. The flow cell of claim 1, wherein the volume of the channel is greater than 10 µl and less than 10 ml.

28. The flow cell of claim 1, wherein one or both of the first substrate and the second substrate are transparent to UV, VIS or IR radiation.

29. The flow cell of claim 1, wherein the cured liquid adhesive comprises a luminescent material.

30. The flow cell of claim 1, wherein the gasket comprises a luminescent material.

31. The flow-through pool of claim 1, wherein the channel includes at least a 90-degree bend, wherein the bend has an inner wall and an outer wall, the inner wall having substantially the same length as the outer wall.

32. The flow cell of claim 18, wherein the glass comprises an organosilane coating.

33. A nucleic acid sequencing system, comprising: (a) The flow cell as described in claim 1; and (b) an optical detection system configured to detect the interior of the flow cell.

34. A method for sequencing nucleic acids using the flow cell as described in claim 1.

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