Fabrication of flow cells using planar waveguides

Abstract

In one example, a method for manufacturing a flow cell is provided, the method comprising: forming a core layer, the core layer being disposed between a substrate and a nanopore layer, the nanopore layer having nanopores to receive a sample, the core layer having a higher refractive index than the substrate and the nanopore layer; and forming a grating to couple light to the core layer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to each of the following applications ("Related Patent Applications"): U.S. Provisional Patent Application No. 62 / 853,350, filed on May 28, 2019, and entitled "FABRICATION OF FLOW CELLS WITH PLANAR WAVEGUIDES," and Netherlands Patent Application No. N2023516, filed on July 17, 2019, and entitled "FABRICATION OF FLOW CELLS WITH PLANAR WAVEGUIDES." The contents of each Related Patent Application are incorporated herein by reference in their entirety. Background of the Invention

[0004] Samples of different materials can be analyzed using one or more of a variety of analytical methods. For example, sequencing such as high-throughput DNA sequencing can be the basis for genome analysis and other genetic studies. For example, modified deoxyribonucleotide triphosphates (dNTPs) are used by synthesis sequencing (SBS) technology, which includes terminators and fluorescent dyes with emission spectra. In this and other types of sequencing, the characteristics of the genetic material sample are determined by irradiating the sample and detecting the emitted light (e.g., fluorescence) generated in response to the irradiation. The quality of the illumination can determine the quality and efficiency of the emitted light detection. For example, if most of the illumination light does not reach the relevant sample material, it may result in a low level of efficiency in the system. As another example, if the illumination light inadvertently impinges on other parts of the sample, it may cause sample degradation. SUMMARY OF THE INVENTION

[0006] In a first aspect, a method for manufacturing a flow cell includes: forming a core layer, wherein the core layer is disposed between a substrate and a nanopore layer, the nanopore layer having nanopores to receive a sample, the core layer having a higher refractive index than the substrate and the nanopore layer; and forming a grating to couple light to the core layer.

[0007] Implementations may include any or all of the following features in any appropriate combination. Forming the grating includes photolithographic patterning, nanoimprinting, or both. The core layer is formed in the same process as forming the grating. The core layer is formed in a different process than forming the grating. The grating is formed by photolithographic patterning on a substrate, a core layer is formed on the grating, and a nanohole layer is formed on the core layer. The grating is formed by nanoimprinting on a substrate, a core layer is formed on the grating, and a nanohole layer is formed on the core layer. The grating is formed on a substrate, the core layer is formed on the grating, an additional layer is formed on the core layer, and a nanohole layer is formed on the additional layer. Prior to forming the nanohole, the additional layer and the nanohole layer are initially free of nanoholes, the method further comprising patterning the nanohole layer to form a nanohole while the additional layer remains intact, and then transferring the pattern of the nanohole layer to the additional layer to expose the core layer in the nanohole. The pattern is transferred by etching. The core layer is formed on the substrate, and wherein the grating and the nanohole layer are formed on the core layer. A core layer is formed on a substrate, a grating is formed on the core layer, and a nanopore layer is formed on the grating. A core layer is formed on a substrate, a first layer is formed on the core layer, a second layer is formed on the first layer, and a grating and a nanopore are formed on the first layer and the second layer, respectively. A core layer is formed on a substrate, a resin layer is formed on the core layer, and a grating and a nanopore are formed in the resin layer. The grating and the nanopore layer are formed in the same process. The grating and the nanopore layer are formed in the same layer of a flow cell. The grating and the nanopore layer are formed in different layers of the flow cell.

[0008] In a second aspect, a flow cell includes: a substrate; a nanopore layer having nanopores to receive a sample; a core layer disposed between the substrate and the nanopore layer, the core layer having a higher refractive index than the substrate and the nanopore layer; and a grating coupling light to the core layer.

[0009] Implementations may include any or all of the following features. A grating is disposed on a substrate, a core layer is disposed on the grating, and a nanohole layer is disposed on the core layer. A grating layer covers the substrate, the grating layer comprising a grating. A first resin layer comprising a grating is disposed on the substrate, and a second resin layer comprising a nanohole is disposed on the first resin layer. A grating is disposed on the substrate, a first polymer layer is disposed on the grating, and a second polymer layer is disposed on the first polymer layer, wherein nanoholes are disposed in the first and second polymer layers. A core layer is disposed on the substrate, a resin layer is disposed on the core layer, and a grating and a nanohole are disposed in the resin layer. A core layer is disposed on the substrate, a grating is disposed on the core layer, and a nanohole layer is disposed on the grating. The grating layer covers the core layer, the grating layer comprising a grating. A core layer is disposed on the substrate, a polymer layer is disposed on the core layer, and a resin layer is disposed on the polymer layer, wherein a grating is disposed in the polymer layer, and wherein a nanohole is disposed in the resin layer. A polymer layer is disposed on a substrate, and a resin layer is disposed on the polymer layer, wherein a grating is disposed in the polymer layer, and a nanohole is disposed in the resin layer.

[0010] It should be understood that all combinations of the foregoing concepts and the additional concepts discussed in more detail below (assuming such concepts are not mutually inconsistent) are considered part of the inventive subject matter disclosed herein and can achieve the benefits described herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are considered part of the inventive subject matter disclosed herein and can achieve the benefits described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 An example of fabricating a planar waveguide is shown.

[0013] Figure 2 Shows the manufacturing and Figure 1 A planar waveguide method related to the planar waveguide in FIG.

[0014] Figure 3 An example of fabricating a planar waveguide is shown.

[0015] Figure 4 Shows the manufacturing and Figure 3 A planar waveguide method related to the planar waveguide in FIG.

[0016] Figure 5 An example of fabricating a planar waveguide is shown.

[0017] Figure 6 Shows the manufacturing and Figure 5 A planar waveguide method related to the planar waveguide in FIG.

[0018] Figure 7 An example of fabricating a planar waveguide is shown.

[0019] Figure 8 Shows the manufacturing and Figure 7 A planar waveguide method related to the planar waveguide in FIG.

[0020] Fig. 9 An example of fabricating a planar waveguide is shown.

[0021] Fig.10 Shows the manufacturing and Fig. 9 A planar waveguide method related to the planar waveguide in FIG.

[0022] Fig.11 An example of fabricating a planar waveguide is shown.

[0023] Fig.12 Shows the manufacturing and Fig.11 A planar waveguide method related to the planar waveguide in FIG.

[0024] Fig.13 An example of fabricating a planar waveguide is shown.

[0025] Fig.14 Shows the manufacturing and Fig.13 A planar waveguide method related to the planar waveguide in FIG.

[0026] Fig.15 An example of fabricating a planar waveguide is shown.

[0027] Fig.16 Shows the manufacturing and Fig.15 A planar waveguide method related to the planar waveguide in FIG.

[0028] Fig.17 An example of a flow cell is shown.

[0029] Fig.18 is a diagram of a system including an instrument, a cartridge, and a flow cell.

[0030] Fig.19 is a diagram of an example lighting system. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present disclosure describes systems, techniques, articles, and / or compositions of matter that facilitate improved sample analysis. Sample analysis may include, but is not limited to, genetic sequencing (e.g., determining the structure of genetic material), genotyping (e.g., determining differences in individual genetic make-up), gene expression (e.g., using genetic information to synthesize gene products), proteomics (e.g., large-scale studies of proteins), or combinations thereof. As described herein, a substrate for holding a sample during analysis may be manufactured in a more efficient manner, and / or it may have improved properties. In some implementations, a flow cell may have an improved structure that facilitates efficient use of illumination light to excite active elements (e.g., fluorophores) in a sample. For example, the structure may include a core layer disposed between a substrate and a nanopore layer, and a grating that couples light (i.e., illumination or excitation light) to the core layer. The materials of the flow cell structure may be selected so that their respective refractive indices have a favorable ratio relative to each other. The core layer may facilitate efficient use of illumination light to excite the sample. For example, the structure may be based on the principle of total internal reflection (TIR) ​​and may be designed so that evanescent light reaches a sample in one or more dedicated regions (eg, a nanowell) in an efficient manner.

[0033] As scan speeds increase in sample analysis, and correspondingly data density also increases, systems can be designed with higher levels of illumination (e.g., laser) power. This can result in higher instrument costs. However, with this development, damage to optics and substrates (e.g., flow cells) can increase. Some implementations, such as those associated with planar waveguides, can be designed with an eye toward improving excitation efficiency and / or reducing background noise. For example, a planar waveguide flow cell can include a substrate (e.g., glass), one or more optical coupling gratings, a high refractive index core layer, and an aqueous buffer or a patterned polymer cladding layer. Improved fabrication techniques for such flow cells and / or structures thereof are described in the present disclosure.

[0034] Some embodiments described herein relate to sequencing of genetic material. Sequencing can be performed on a sample to determine which building blocks (called nucleotides) make up a particular genetic material in the sample. The genetic material can be purified first and then repeated multiple times to prepare a sample of the appropriate size before sequencing.

[0035] Imaging can be performed as part of the process of analyzing sample material. This can involve fluorescence imaging, such as when a sample of genetic material is irradiated with light (e.g., a laser beam), a fluorescent reaction is triggered by one or more markers on the genetic material. Some nucleotides of the genetic material can have fluorescent tags applied thereon, which allow the presence of nucleotides to be determined by shining light onto the sample and looking for characteristic reactions from the sample. Fluorescent reactions can be detected during the analysis process and used to establish a record of nucleotides in the sample.

[0036] Examples described herein relate to flow cells. A flow cell is a substrate that can be used to prepare and hold or carry one or more samples in at least one stage of an analytical process. The flow cell is made of a material that is compatible with genetic material, illumination, and the chemical reaction to be exposed. The substrate may have one or more channels in which the sample material can be deposited. A substance (e.g., a liquid) may flow through a channel in which the sample genetic material is present to trigger one or more chemical reactions and / or remove unwanted substances. The flow cell can be imaged by facilitating that the sample in the flow cell channel can be subjected to illumination light and any fluorescent reaction from the sample can be detected. Some embodiments of the system may be designed to be used with at least one flow cell, but may not include a flow cell during one or more stages (e.g., during transportation or when shipped to a customer). For example, the flow cell may be installed in an embodiment preset by the customer to perform analysis.

[0037] Examples herein relate to coupling light (e.g., a laser beam) into and / or out of a waveguide via one or more gratings. The gratings can couple the gratings to light impacts impinging on the gratings by diffracting at least a portion of the light, thereby causing a portion of the light to propagate in one or more other directions. In some embodiments, the coupling can involve one or more interactions, including but not limited to reflection, refraction, and / or transmission of a portion of the light. Implementations can be designed to meet one or more requirements, including but not limited to requirements related to mass production, cost control, and / or high light coupling efficiency.

[0038] Providing a planar waveguide in a substrate (such as a flow cell) can provide one or more advantages. Using TIR-based evanescent light excitation can provide higher illumination efficiency. In some previous methods, the entire laser beam is used to illuminate the substrate holding the sample, such as during scanning. This method may cause most of the light waves to propagate through the substrate without effectively irradiating the sample. As a result, only a small portion of the light applied by such a system can actually be used to excite the fluorophores in the sample. In contrast, the evanescent light can only penetrate the material (e.g., the cladding adjacent to the core layer) to a certain depth (e.g., in one example, between about 150nm to about 200nm, for example, between about 165nm to about 185nm. In some examples, the depth can be about 155nm, about 170nm, about 180nm, about 195nm, etc.). For example, the flow cell can be designed to configure one or more nanopores so that the evanescent field is largely limited to the hole area. As a result, evanescent light may be a very effective method for exciting fluorophores. For example, a system operating according to an earlier illumination method may require a laser with a certain power; in contrast, using evanescent light, a much lower laser power may be sufficient.

[0039] The examples described herein mention that one or more gratings can be provided for coupling of light. The gratings can be the same or similar to each other, or can be different types of gratings. The gratings can include one or more forms of periodic structures. In some embodiments, the gratings can be formed by removing or omitting material from a substrate (e.g., from a waveguide material included in a flow cell) or other material. For example, a flow cell can be provided with a set of slits and / or grooves therein to form a grating. In some embodiments, a grating can be formed by adding a substance to a flow cell (e.g., to a waveguide material included in a flow cell) or other material. For example, a flow cell can be provided with a set of ridges, bands, or other protruding longitudinal structures to form a grating. A combination of these methods can be used.

[0040] Examples herein relate to photolithography. The method based on photolithography may involve the use of photoresist, which is patterned by stepper or mask aligner, and the pattern present on reticle / photomask is transferred to photoresist by radiation exposure, and then developed to generate structured film (photoresist) on the top of substrate. Structured resist can be the final substrate that can be used for subsequent core layer coating. As another example, the pattern in resist can be transferred to substrate or other materials by additional treatment. Subsequent process (follow on process) operation can include reactive ion etching (plasma-based dry etching) or wet etching (chemical method-based) process. If the pattern is transferred to substrate / material, then the patterned photoresist is removed to produce patterned substrate (for example, for subsequent core layer coating). A sacrificial film of material, such as chromium or titanium or another metal, beneath the photoresist is first used to transfer the pattern in the photoresist to the metal film, which is then used as a hard mask through which the pattern is transferred to the substrate. After the pattern is transferred to the substrate, the film can be removed and is therefore considered sacrificial to the manufacturing process. One or more of a variety of materials can be applied in the photolithography process. In some embodiments, oxide materials are used. For example, SiO2 (silicon dioxide) can be applied. The lift-off process can be similar to the pattern photoresist process: instead of removing material by dry etching or wet etching, a material (such as SiO2) can be deposited and then lift-off, which involves removing the photoresist on top of it and the deposited material. A grating structure can also or alternatively be formed.

[0041] Examples herein relate to sputtering. Sputtering deposition may refer to a physical vapor deposition (PVD) method of depositing a thin film or coating. Such a process may involve ejecting a material from a source and depositing it onto a substrate. In some embodiments, sputtering deposition forms a thin layer of a waveguide on the surface of a substrate. One or more of a variety of materials may be used in sputtering. The waveguide materials used in sputtering deposition may include metals and metal oxides with high refractive index and low adsorption properties. For example, the waveguide material may include tantalum pentoxide (Ta2O5) or silicon nitride (e.g., one or more compounds represented by the general formula SiNx, including but not limited to Si3N4). When the lower surface is patterned, the waveguide layer / coating may adopt the pattern in the lower surface, thereby forming optical features on the surface of the substrate. Based on the design of the pattern, this may enable manipulation of light on the substrate surface in downstream imaging processes.

[0042] Examples herein relate to chemical vapor deposition. Chemical vapor deposition (CVD) can include all techniques, wherein a volatile material (sometimes referred to as a precursor) is caused to react and / or decompose on a substrate surface, forming a deposit on the substrate. CVD can be characterized by one or more aspects. For example, CVD can be characterized by the physical characteristics of steam (vapor) (for example, CVD is aerosol-assisted or involves direct liquid injection). For example, CVD can be characterized by the type of substrate heating (for example, the substrate is directly heated or indirectly heated, such as by a heating chamber). Examples of usable CVD types include, but are not limited to, atmospheric pressure CVD, low pressure CVD, ultra-low pressure CVD, ultra-high vacuum CVD, metal organic CVD, laser-assisted CVD, and plasma enhanced CVD.

[0043] Examples herein relate to atomic layer deposition. Atomic layer deposition can be considered a form of CVD and includes all techniques in which a thin film is grown on a substrate by exposure to a gas. For example, gaseous precursors can be introduced alternately into a chamber. Molecules of one of the precursors can react with the surface until a layer is formed and the reaction is terminated, and then the next gaseous precursor can be introduced to begin forming a new layer, and so on for one or more cycles.

[0044] Examples herein relate to spray coating. Spray coating may include any or all techniques by which a particular material is deposited onto a substrate. This may include, but is not limited to, thermal spraying, plasma spraying, cold spraying, warm spraying, and / or other procedures involving atomized or nebulized materials.

[0045] Examples herein relate to spin coating. Spin coating may include applying a quantity of coating to a substrate and distributing or spreading the coating on the substrate by centrifugal force due to rotation or spinning of the substrate.

[0046] Examples herein relate to nanoimprinting. In nanoimprint lithography, a prefabricated nanoscale template can mechanically displace a fluid resin to mold the desired nanostructure. The nanoscale template can then be used to cure the resin in place. After removing the nanoscale template, a molded solid resin attached to the desired substrate can be produced. In some embodiments, the nanoimprint process can begin with completely or partially covering a substrate or wafer with an imprinting resin (e.g., the resin of the following example). One or more nanostructures can be formed in the imprinting resin in a molding process using a nanoscale template. The imprinting resin can be cured for a substrate or wafer, and a resin removal process can be applied to remove residues from a wafer or substrate. For example, the resin removal can form a chamber channel adjacent to a nanostructure. The substrate or wafer so formed can have another substrate or gasket applied thereto to form a flow cell having the nanostructure and a flow cell chamber formed by closing a chamber channel. In some embodiments, the process of applying the imprinting resin can be configured to produce little or no resin residue, and in such an embodiment, the resin removal process can be omitted. In some applications, the cured resin may also be functionalized by chemical treatment or attachment of biomolecules, depending on the end use. In nanoimprint lithography, the imprinted photoresist may be a sacrificial material and similarly used as an intermediate tool to transfer the patterned resist into the substrate, or a variation of the resist may be used so that the imprinted resist is used as input for a subsequent coating process. An example of a resist that will remain after patterning is a material formed by a process that converts monomers into colloidal solutions as precursors to particles and / or polymer gels, sometimes referred to as sol-gel based materials.

[0047] Examples described herein mention that one or more resins can be used. Any suitable resin can be used for nanoimprinting in the method described herein. In some embodiments, organic resins can be used, including but not limited to acrylic resins, polyimide resins, melamine resins, polyester resins, polycarbonate resins, phenolic resins, epoxy resins, polyacetal resins, polyether resins, polyurethane resins, polyamide resins (and / or nylon), furan resins, diallylphthalate resins or combinations thereof. In some embodiments, resins can include inorganic siloxane polymers, which include Si-O-Si bonds between compounds (including silicon, oxygen and hydrogen), and are formed by using a material based on siloxane polymers represented by silicon glass as a starting material. The resin used may also be or alternatively be an organosiloxane polymer in which the hydrogen bonded to silicon is substituted by an organic group, such as a methyl or phenyl group, and represented by an alkylsiloxane polymer, an alkylsilsesquioxane polymer, a silsesquioxane hydride polymer, or an alkylsilsesquioxane hydride polymer. Non-limiting examples of siloxane polymers include polyhedral oligomeric silsesquioxane (POSS), polydimethylsiloxane (PDMS), tetraethyl ortho silicate (TEOS), poly(organo)siloxane (organosilicon), and perfluoropolyether (PFPE). An example of POSS may be described in Kehagias et al. Microelectronic Engineering 86 (2009), pp. 776-778, the entire contents of which are incorporated herein by reference. The resin may be doped with a metal oxide. In some embodiments, the resin may be a sol-gel material including, but not limited to, titanium oxide, hafnium oxide, zirconium oxide, tin oxide, zinc oxide, or germanium oxide, and a suitable solvent may be used. Any of a variety of other resins may be used appropriately according to the application.

[0048] Examples herein relate to substrates. A substrate can refer to any material that provides a substantially rigid structure, or a structure that maintains its shape without adopting the shape of a container in which it is placed in contact. The material can have a surface to which another material can be attached, including, for example, smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces), and textured and / or porous materials. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials, including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, fiber optic bundles, and a variety of other polymers. Typically, the substrate allows optical detection and does not fluoresce significantly itself.

[0049] Examples herein refer to polymers. The polymer layer may include a film of a polymer material. Exemplary film-forming polymers include, but are not limited to, acrylamide or copolymers with C1-C 12; aromatic and hydroxyl derivatives; acrylate copolymers; vinylpyrrolidine and vinylpyrrolidone copolymers; sugar-based polymers such as starch or polydextrin; or other polymers such as polyacrylic acid, polyethylene glycol, polylactic acid, silicone, siloxane, polyvinylamine, guar gum, carrageenan, alginate, lotus bean gum, methacrylate copolymers, polyimide, cycloolefin copolymers or combinations thereof. In some embodiments, the polymer layer includes at least one photocurable polymer. For example, photocurable polymers may include urethane, acrylate, silicone, epoxy, polyacrylic acid, polyacrylate, epoxysilicone, epoxy, polydimethylsiloxane (PDMS), silsesquioxane, acyloxysilane, maleate polyester, vinyl ether, monomer or copolymer with vinyl or ethynyl, or a combination thereof. In some embodiments, the layer may include a covalently linked polymer coating. For example, it may include a polymer coating that forms a chemical bond with the functionalized surface of the substrate compared to being attached to the surface in other ways such as adhesion or electrostatic interaction. In some embodiments, the polymer included in the functionalizable layer is poly (N-(5-azidoacetamidopentyl) acrylamide-co-acrylamide), sometimes referred to as PAZAM.

[0050] Figure 1 An example of making a planar waveguide 100 is shown. The planar waveguide 100 can be used in one or more examples described herein. For example, the planar waveguide 100 can hold one or more samples to facilitate illumination and excitation during sample analysis. Figure 2 Shows the manufacturing and Figure 1 The method 200 of a planar waveguide related to a planar waveguide in FIG. The method 200 may be combined with one or more other methods described herein. Unless otherwise indicated, more or fewer operations may be performed, and / or two or more operations may be performed in a different order.

[0051] At 210, photolithographic (PL) patterning of the grating can be performed. In some embodiments, the substrate 102 is provided with one or more gratings 104. For example, the grating 104 can be a laser coupled grating. In some embodiments, the photolithographic pattern may include a deposition or etching process. For example, an oxide (e.g., SiO2) can be patterned. The gratings 104 are shown longitudinally here so that the respective ridges 104' extend into the plane of the illustration. The grating layer 104 can include, for example, a plurality of ridges 104' organized in respective groups 106A and 106B. For example, the groups 106A-106B can be positioned so that the region 108 of the substrate 102 is substantially free of the gratings 104. The ridges 104' can have a pitch of about 200nm to about 300nm. For example, between about 220nm to about 280nm, to name just one example. In some embodiments, the pitch can be about 205nm, about 215nm, about 235nm, about 265nm, about 285nm, etc.

[0052] At 220, a core layer may be formed. In some embodiments, the core layer 110 is formed on the substrate 102. In some embodiments, the core layer 110 is formed on the grating 104. In some embodiments, the substrate 102 may act as a cladding for the core layer 110. The core layer 110 may have a higher refractive index than the substrate 102. For example, the substrate 102 may have a refractive index of about 1.5, and the core layer may have a refractive index of about 2.2, or in a range higher than about 1.5 to 2.2, such as about 1.6 to about 2.1. In some embodiments, the refractive index may be about 1.65, about 1.85, about 2.05, and the like. The core layer 110 may substantially cover the entire facing surface of the substrate 102. The core layer 110 may have a higher refractive index than the grating layer 104. In some embodiments, the core layer 110 includes Ta2O5 and / or SiN x For example, the core layer 110 may be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating, and / or spray coating.

[0053] At 230, nanopore layer patterning is performed. In some embodiments, the nanopore layer 112 is formed at the core layer 110. The nanopore layer 112 can facilitate a patterned flow cell. The nanopore layer 112 may include one or more nanopores 114 defined between two or more walls 116. In some embodiments, the nanopore 114 may have such a size that one or more of its size ranges are within the range of about one or more nanometers. For example, the nanopore 114 may be configured to receive and hold a sample in the form of a cluster during the analysis process. The end (e.g., bottom) of the nanopore 114 may have a thickness that accommodates the propagation of evanescent light. For example, the thickness may be about 0 to about 500 nm, such as between about 100 nm to about 400 nm. In some embodiments, the thickness may be about 10 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 450 nm, etc. The nanopore layer 112 may be formed by a nanoimprint process or a stripping process. In some embodiments, the nanopore layer 112 may include one or more resins. The nanoporous layer may substantially cover the entire facing surface of the core layer 110. For example, the resin may have a refractive index of about 1.5. In some embodiments, the nanoporous layer 112 may have an average pitch of at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more between the nanopores 114, and / or may have an average pitch of at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less. For example, the nanoporous layer 112 may have a pitch of about 600 to about 650 nm between the nanopores 114, for example, the pitch may be about 610 nm to about 640 nm. In some embodiments, the pitch may be about 605 nm, about 615 nm, about 635 nm, about 655 nm, etc. The depth of each nanopore 114 may be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth may be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or less.

[0054] Figure 3 An example of making a planar waveguide 300 is shown. The planar waveguide 300 can be used for one or more examples described herein. For example, the planar waveguide 300 can hold one or more samples to facilitate illumination and excitation during sample analysis. Figure 4 Shows the manufacturing and Figure 3The method 400 of the present invention relates to a planar waveguide in a planar waveguide. The method 400 can be combined with one or more other methods described herein. Unless otherwise indicated, more or fewer operations can be performed, and / or two or more operations can be performed in a different order.

[0055] At 410, nanoimprinting of the grating may be performed. In some embodiments, the substrate 302 is provided with one or more grating layers 304. For example, the grating layer 304 may include a laser coupled grating. For example, the resin may be nanoimprinted. The grating layer 304 is shown longitudinally here so that the respective ridges 304' extend into the plane of the illustration. The grating layer 304 may include, for example, a plurality of ridges 304' organized in respective groups 306A and 306B. For example, the groups 306A-306B may be separated by regions 308 of the grating layer 304 that also cover the surface of the substrate 302. The region 308 may form a thin residual layer on the surface of the substrate 302. The ridges 304 may have a pitch of about 200nm to about 300nm, for example, between about 220nm to about 280nm, to name just one example. In some embodiments, the pitch may be about 205nm, about 215nm, about 235nm, about 265nm, about 285nm, and the like. Applying the grating layer 304 by a nanoimprint process can provide one or more advantages. In some embodiments, nanoimprinting can be compatible with existing flow cell fabrication processes. For example, this can reduce the fabrication cost of the substrate.

[0056] At 420, a core layer may be formed. In some embodiments, the core layer 310 is formed on the substrate 302. In some embodiments, the core layer 310 is formed on the grating layer 304. In some embodiments, the grating layer 304 may function as a cladding for the core layer 310. The core layer 310 may have a higher refractive index than the substrate 302. For example, the substrate 302 may have a refractive index of about 1.5, and the core layer may have a refractive index of about 2.2, or in a range of greater than about 1.5 to about 2.2, such as about 1.6 to about 2.1. In some embodiments, the refractive index may be about 1.65, about 1.85, about 2.05, and the like. The core layer 310 may substantially cover the entire facing surface of the grating layer 304. The core layer 310 may have a higher refractive index than the grating layer 304. In some embodiments, the core layer 310 includes Ta2O5 and / or SiN x For example, the core layer 310 may be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating, and / or spray coating.

[0057] At 430, nanopore layer patterning is performed. In some embodiments, nanopore layer 312 is formed at core layer 310. Nanopore layer 312 can facilitate patterned flow pool. Nanopore layer 312 may include one or more nanopores 314 defined between two or more walls 316. In some embodiments, nanopore 314 may have such a size that one or more of its dimensions are within the range of about one or more nanometers. For example, nanopore 314 can be configured to receive and hold samples in the form of clusters during the sample analysis process. The end (e.g., bottom) of nanopore 314 may have a thickness that accommodates evanescent light propagation. For example, the thickness may be about 0 to about 500nm, such as between about 100nm to about 400nm. In some embodiments, the thickness may be about 10nm, about 50nm, about 100nm, about 200nm, about 300nm, about 450nm, etc. Nanopore layer 312 may be formed by a nanoimprint process or a stripping process. In some embodiments, nanopore layer 312 may include one or more resins. The nanoporous layer may substantially cover the entire facing surface of the core layer 310. For example, the resin may have a refractive index of about 1.5. In some embodiments, the nanoporous layer 312 may have an average pitch of at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more between the nanopores 314, and / or may have an average pitch of at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less. For example, the nanoporous layer 312 may have a pitch of about 600 to about 650 nm between the nanopores 314, for example, the pitch may be about 610 nm to about 640 nm. In some embodiments, the pitch may be about 605 nm, about 615 nm, about 635 nm, about 655 nm, etc. The depth of each nanopore 314 can be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth can be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or less.

[0058] Figure 5 An example of making a planar waveguide 500 is shown. The planar waveguide 500 can be used in one or more examples described herein. For example, the planar waveguide 500 can hold one or more samples to facilitate illumination and excitation during sample analysis. Figure 6 Shows the manufacturing and Figure 5 The method 600 of a planar waveguide related to a planar waveguide in FIG. The method 600 may be combined with one or more other methods described herein. Unless otherwise indicated, more or fewer operations may be performed, and / or two or more operations may be performed in a different order.

[0059] At 610, nanoimprinting of the grating can be performed in the core layer. In some embodiments, the substrate 502 is provided with one or more core layers 504. For example, the core layer 504 can include a laser coupled grating. For example, the resin can be nanoimprinted. The core layer 504 is shown longitudinally here so that the respective ridges 504' extend into the illustrated plane. The core layer 504 can include, for example, a plurality of ridges 504' organized in respective groups 506A and 506B. For example, the groups 506A-506B can be separated by regions 508 of the core layer 504 that also cover the surface of the substrate 502. The ridges 504' can have a pitch of about 200nm to about 300nm, for example, between about 220 and about 280nm, to name just one example. In some embodiments, the pitch can be about 205nm, about 215nm, about 235nm, about 265nm, about 285nm, etc. For example, substrate 502 may have a refractive index of about 1.5, and core layer 504 may have a refractive index higher than about 1.5. For example, core layer 504 may be made of a high refractive index polymer material. Core layer 504 may substantially cover the entire facing surface of substrate 502.

[0060] At 620, nanopore layer patterning is performed. In some embodiments, nanopore layer 510 is formed at core layer 504. Nanopore layer 510 can facilitate patterned flow pool. In some embodiments, nanopore layer 510 and substrate 502 can act as a cladding of core layer 504. Nanopore layer 510 may include one or more nanopores 512 defined between two or more walls 514. In some embodiments, nanopore 512 may have such a size that one or more dimensions thereof are within the range of about one or more nanometers. For example, nanopore 512 may be configured to receive and hold samples during the analysis process, such as in the form of clusters. The end (e.g., bottom) of nanopore 512 may have a thickness that accommodates evanescent light propagation. For example, the thickness may be from about 0 to about 500 nm, such as between about 100 nm and about 400 nm. In some embodiments, the thickness may be about 10 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 450 nm, etc. The nanoporous layer 510 may be formed by a nanoimprint process or a stripping process. In some embodiments, the nanoporous layer 510 may include one or more resins. For example, the resin may have a refractive index of about 1.5. In some embodiments, the nanoporous layer 510 may have an average pitch of at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm or more between the nanopores 512, and / or may have an average pitch of at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm or less. For example, the nanoporous layer may substantially cover the entire facing surface of the core layer 504. The nanoporous layer 510 may have a pitch of about 600 to about 650 nm between the nanopores 512, for example, the pitch may be about 610 nm to about 640 nm. In some embodiments, the pitch may be about 605 nm, about 615 nm, about 635 nm, about 655 nm, etc. The depth of each nanopore 512 may be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth may be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or less.

[0061] Figure 7 An example of making a planar waveguide 700 is shown. The planar waveguide 700 can be used in one or more examples described herein. For example, the planar waveguide 700 can hold one or more samples to facilitate illumination and excitation during sample analysis. Figure 8 Shows the manufacturing and Figure 7Method 800 for a planar waveguide related to a planar waveguide in FIG. Method 800 may be combined with one or more other methods described herein. Unless otherwise indicated, more or fewer operations may be performed, and / or two or more operations may be performed in a different order.

[0062] At 810, nanoimprinting of the grating may be performed. In some embodiments, the substrate 702 is provided with one or more grating layers 704. For example, the grating layer 704 may include a laser coupled grating. For example, the resin may be nanoimprinted. The grating layer 704 is shown longitudinally here so that the respective ridges 704' extend into the plane shown. The grating layer 704 may include, for example, a plurality of ridges 704' organized in respective groups 706A and 706B. For example, the groups 706A-706B may be separated by regions 708 of the grating layer 704 that also cover the surface of the substrate 702. The region 708 may form a thin residual layer on the surface of the substrate 702. The ridges 704' may have a pitch of about 200nm to about 300nm, for example, between about 220nm to about 280nm, to name just one example. In some embodiments, the pitch may be about 205nm, about 215nm, about 235nm, about 265nm, about 285nm, etc. Applying the grating layer 704 by a nanoimprint process can provide one or more advantages. In some embodiments, nanoimprinting can be compatible with existing flow cell manufacturing processes. For example, this can reduce the manufacturing cost of the substrate.

[0063] At 820, a core layer may be formed. In some embodiments, the core layer 710 is formed at the grating layer 704. In some embodiments, the grating layer 704 may act as a cladding for the core layer 710. The core layer 710 may have a higher refractive index than the substrate 702. For example, the substrate 702 may have a refractive index of about 1.5, and the core layer may have a refractive index of about 2.2, or in a range from greater than about 1.5 to about 2.2, such as from about 1.6 to about 2.1. In some embodiments, the refractive index may be about 1.65, about 1.85, about 2.05, and the like. The core layer 710 may substantially cover the entire facing surface of the grating layer 704. The core layer 710 may have a higher refractive index than the grating layer 704. In some embodiments, the core layer 710 includes Ta2O5 and / or SiN x For example, the core layer 710 may be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating, and / or spray coating.

[0064] At 830, additional layers may be formed. In some embodiments, layer 712 is formed at core layer 710. To name a few examples, layer 712 may be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating, and / or spray coating. Layer 712 has a lower refractive index than core layer 710. In some embodiments, layer 712 is formed by a polymer material having a refractive index similar to that of a water-based reagent (e.g., about 1.35). For example, layer 712 may include, but is not limited to, transparent, electrically insulating, water-repellent, oil-repellent, and / or chemically resistant fluoropolymers. Layer 712 may have a thickness of about 100 to about 200 nm, and to name just one example, for example, the thickness may be about 120 nm to about 180 nm. In some embodiments, the thickness may be about 105 nm, about 115 nm, about 135 nm, about 165 nm, about 195 nm, and the like.

[0065] At 840, nanopore layer patterning is performed. In some embodiments, nanopore layer 714 is formed at layer 712. Nanopore layer 714 can facilitate patterned flow pools. Nanopore layer 714 may include one or more nanopores 716 defined between two or more walls 718. In some embodiments, nanopore 716 may have such a size that one or more of its dimensions are within the range of about one or more nanometers. For example, nanopore 716 may be configured to receive and hold samples during the analysis process, such as in the form of clusters. The end (e.g., bottom) of nanopore 716 may have a thickness that accommodates evanescent light propagation. For example, the thickness may be about 0 to about 500nm, such as between about 100nm to about 400nm. In some embodiments, the thickness may be about 10nm, about 50nm, about 100nm, about 200nm, about 300nm, about 450nm, etc. Nanopore layer 714 may be formed by a nanoimprint process or a stripping process. In some embodiments, nanopore layer 714 may include one or more resins. The nanoporous layer may substantially cover the entire facing surface of layer 712. For example, the resin may have a refractive index of about 1.5. In some embodiments, the nanoporous layer 714 may have an average pitch of at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm or more between nanopores 716, and / or may have an average pitch of at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm or less. For example, the nanoporous layer 714 may have a pitch of about 600 to about 650 nm between nanopores 716, for example, the pitch may be about 610 nm to about 640 nm. In some embodiments, the pitch may be about 605 nm, about 615 nm, about 635 nm, about 655 nm, etc. Each nanopore 716 may have a depth of at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth may be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or less.

[0066] Having layer 712 between core layer 710 and nanopore layer 714 can provide one or more advantages. In some embodiments, during sample analysis, planar waveguide 700 is immersed in a water-based reagent, which can have a relatively low refractive index, such as about 1.35. Nanopore layer 714 can have a refractive index of about 1.5, which can be less than the ideal refractive index matched with the water-based reagent if layer 712 is not present. For example, scattering or other photonic crystal effects may occur in the case of refractive index mismatch. However, layer 712 can provide a relatively lower refractive index than other materials. The evanescent light can have only a specific penetration depth, such as about 150 to about 200 nm, for example, between about 165 nm to about 185 nm; in some embodiments, the depth can be about 155 nm, about 170 nm, about 180 nm, about 195 nm, etc. Therefore, the thickness of layer 712 can be controlled so that the evanescent light only encounters the water-based reagent in layer 712 and nanopore 716. That is, the evanescent light cannot (substantially) reach the nanoporous layer 714. This can avoid or reduce unwanted effects, such as scattering.

[0067] The pattern of nanoporous layer 714 can be transferred into layer 712. In some embodiments, layer 712 can be formed to substantially cover the entire surface of core layer 710. Nanoporous layer 714 can be formed at layer 712. Thereafter, nanoporous layer 714 can be imprinted with nanoporous layer 716. The nanoimprint process may not penetrate or perforate layer 712; instead, layer 712 can remain substantially intact after nanoimprinting. Thereafter, etching (e.g., reactive ion etching) can be performed to remove portions of layer 712 at the ends (e.g., bottom) of nanopores 716. Thus, the pattern of nanopores 716 can be transferred to layer 712.

[0068] Fig. 9 An example of making a planar waveguide 900 is shown. The planar waveguide 900 can be used in one or more examples described herein. For example, the planar waveguide 900 can hold one or more samples to facilitate illumination and excitation during sample analysis. Fig.10 Shows manufacturing and Fig. 9 The method 1000 of a planar waveguide related to a planar waveguide in FIG. The method 1000 may be combined with one or more other methods described herein. Unless otherwise indicated, more or fewer operations may be performed, and / or two or more operations may be performed in a different order.

[0069] At 1010, a core layer can be formed. In some embodiments, core layer 902 is formed at substrate 904. In some embodiments, substrate 904 can act as a cladding for core layer 902. Core layer 902 can have a higher refractive index than substrate 904. For example, substrate 904 can have a refractive index of about 1.5, while core layer can have a refractive index higher than about 1.5. Core layer 902 can substantially cover the entire facing surface of substrate 904. In some embodiments, core layer 902 includes Ta2O5 and / or SiN x or polymer material. For example, the core layer 902 can be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating and / or spray coating. In some embodiments, the core layer 902 includes a high refractive index polymer material, such as a resin. For example, a polymer can be spin coated to form the core layer 902.

[0070] At 1020, nanoimprinting of gratings and nanopores is performed. In some embodiments, layer 906 is formed at core layer 902. Layer 906 can substantially cover the entire facing surface of core layer 902. Layer 906 can facilitate patterned flow pools and laser coupling through gratings. For example, the resin can be nanoimprinted. Layer 906 is shown longitudinally here so that each ridge 908 extends into the illustrated plane. Layer 906 can include, for example, a plurality of ridges 908 organized in groups 910A and 910B. For example, groups 910A-910B can be input and output gratings for lasers. Ridge 908 can have a pitch of about 200 to about 300nm, for example, between about 220 to about 280nm, to name just one example. In some embodiments, the pitch can be about 205nm, about 215nm, about 235nm, about 265nm, about 285nm, etc.

[0071] Layer 906 may include one or more nanopores 912 defined between two or more walls 914. In some embodiments, nanopore 912 may be of such a size that one or more of its dimensions are within the range of about one or more nanometers. For example, nanopore 912 may be configured to receive and hold samples during the analysis process, such as in the form of clusters. The end (e.g., bottom) of nanopore 912 may have a thickness that accommodates the propagation of evanescent light. For example, the thickness may be about 0 to about 500 nm, such as between about 100 nm to about 400 nm. In some embodiments, the thickness may be about 10 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 450 nm, etc. In some embodiments, layer 906 can have an average pitch of at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more between nanopores 912, and / or can have an average pitch of at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less. For example, nanopores 912 can have a pitch of about 600 to about 650 nm, for example, the pitch can be about 610 nm to about 640 nm. In some embodiments, the pitch can be about 605 nm, about 615 nm, about 635 nm, about 655 nm, etc. The depth of each nanopore 912 can be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth can be at most about 1×10 3 um, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm, or less. For example, layer 906 may be formed of a resin having a refractive index of about 1.5.

[0072] Forming the ridges 908 and nanoholes 912 of the grating in layer 906 may involve nanoimprinting to more than one depth in layer 906. In some embodiments, a dual-depth stamp may be used in the nanoimprint process. For example, the ridges 908 may be formed in layer 906 at a relatively greater depth than the nanoholes 912.

[0073] Fig.11 An example of making a planar waveguide 1100 is shown. The planar waveguide 1100 can be used in one or more examples described herein. For example, the planar waveguide 1100 can hold one or more samples to facilitate illumination and excitation during sample analysis. Fig.12 Shows the manufacturing and Fig.11 The method 1200 of a planar waveguide related to a planar waveguide in FIG. The method 1200 may be combined with one or more other methods described herein. Unless otherwise indicated, more or fewer operations may be performed, and / or two or more operations may be performed in a different order.

[0074] At 1210, a core layer can be formed. In some embodiments, core layer 1102 is formed at substrate 1104. In some embodiments, substrate 1104 can act as a cladding layer for core layer 1102. Core layer 1102 can have a higher refractive index than substrate 1104. For example, substrate 1104 can have a refractive index of about 1.5, while core layer 1102 can have a refractive index higher than about 1.5. Core layer 1102 can substantially cover the entire facing surface of substrate 1104. In some embodiments, core layer 1102 includes Ta2O5, SiN x , polymer material or a combination thereof. For example, the core layer 1102 can be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating and / or spray coating. In some embodiments, the core layer 1102 includes a high refractive index polymer material, such as a resin. For example, a polymer can be spin coated to form the core layer 1102.

[0075] At 1220, nanoimprinting of the grating can be performed. In some embodiments, the core layer 1102 is provided with one or more grating layers 1106. For example, the grating layer 1106 can include a laser coupled grating. For example, the resin can be nanoimprinted. The grating layer 1106 is shown longitudinally here so that the respective ridges 1108 extend into the plane of the illustration. The grating layer 1106 can include a plurality of ridges 1108, for example, organized in respective groups 1110A and 1110B. For example, the groups 1110A-1110B can be separated by regions 1112 of the grating layer 1106 that also cover the surface of the substrate 1104. The regions 1112 can form a thin residual layer on the surface of the substrate 1104. The ridges 1108 can have a pitch of about 200 to about 300 nm, for example, between about 220 to about 280 nm, to name just one example. In some embodiments, the pitch can be about 205 nm, about 215 nm, about 235 nm, about 265 nm, about 285 nm, etc. Applying the grating layer 1106 by a nanoimprint process can provide one or more advantages. In some embodiments, nanoimprinting can be compatible with existing flow cell manufacturing processes. For example, this can reduce the manufacturing cost of the substrate.

[0076] At 1230, nanoimprinting of the nanopore layer is performed. In some embodiments, the nanopore layer 1114 is formed at the grating layer 1106. The nanopore layer 1114 can facilitate a patterned flow cell. The nanopore layer 1114 may include one or more nanopores 1116 defined between two or more walls 1118. In some embodiments, the nanopore 1116 may have such a size that one or more of its dimensions are within the range of about one or more nanometers. For example, the nanopore 1116 may be configured to receive and hold a sample, such as in the form of a cluster, during the analysis process. The end (e.g., bottom) of the nanopore 1116 may have a thickness that accommodates the propagation of evanescent light. For example, the thickness may be about 0 to about 500 nm, such as between about 100 nm to about 400 nm. In some embodiments, the thickness may be about 10 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 450 nm, etc. The nanopore layer 1114 may be formed by a nanoimprinting process or a stripping process. In some embodiments, the nanoporous layer 1114 may include one or more resins. The nanoporous layer may substantially cover the entire facing surface of the grating layer 1106. In some embodiments, the nanoporous layer 1114 may have an average pitch of at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more between the nanopores 1116, and / or may have an average pitch of at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less. For example, the nanoporous layer 1114 may have a pitch of about 600 to about 650 nm between the nanopores 1116, for example, the pitch may be about 610 nm to about 640 nm, to name just one example. In some embodiments, the pitch may be about 605 nm, about 615 nm, about 635 nm, about 655 nm, etc. Each nanopore 1116 may have a depth of at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth may be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or less.

[0077] Forming the grating layer 1106 at the core layer 1102 can provide one or more advantages. In some embodiments, the core layer 1102 can be pre-formed (e.g., by pre-sputtering) on ​​the substrate 1104 before delivery to a facility (e.g., a flow cell manufacturer's fabrication plant), and then the remainder of the planar waveguide 1100 can be formed (e.g., by nanoimprinting) without further transferring the planar waveguide 1100 from the facility.

[0078] The grating layer 1106 can have a higher refractive index than one or more other materials. In some embodiments, the grating layer 1106 has a higher refractive index than the core layer 1102. In some embodiments, the core layer 1102 has a higher refractive index than the nanohole layer 1114. For example, the refractive index of the grating layer 1106 can be higher than the refractive index of the nanohole layer 1114.

[0079] Fig.13 An example of making a planar waveguide 1300 is shown. The planar waveguide 1300 can be used in one or more examples described herein. For example, the planar waveguide 1300 can hold one or more samples to facilitate illumination and excitation during sample analysis. Fig.14 Shows the manufacturing and Fig.13 Method 1400 for a planar waveguide related to a planar waveguide in FIG. Method 1400 may be combined with one or more other methods described herein. Unless otherwise indicated, more or fewer operations may be performed, and / or two or more operations may be performed in a different order.

[0080] At 1410, a core layer can be formed. In some embodiments, core layer 1302 is formed at substrate 1304. In some embodiments, substrate 1304 can act as a cladding for core layer 1302. Core layer 1302 can have a higher refractive index than substrate 1304. For example, substrate 1304 can have a refractive index of about 1.5, while core layer 1302 can have a refractive index higher than about 1.5. Core layer 1302 can substantially cover the entire facing surface of substrate 1304. In some embodiments, core layer 1302 includes Ta2O5 and / or SiN x , or polymer material. For example, core layer 1302 can be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating and / or spray coating. In some embodiments, core layer 1302 includes a high refractive index polymer material, such as a resin. For example, a polymer can be spin coated to form core layer 1302.

[0081] At 1420, a polymer layer may be formed. In some embodiments, a polymer layer 1306 is formed at the core layer 1302. To name a few examples, the polymer layer 1306 may be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating, and / or spray coating. The polymer layer 1306 has a lower refractive index than the core layer 1302. The polymer layer 1306 may have a thickness of about 100 to about 200 nm, for example, the thickness may be about 120 nm to about 180 nm, to name just one example. In some embodiments, the thickness may be about 105 nm, about 115 nm, about 135 nm, about 165 nm, about 195 nm, and the like.

[0082] At 1430, a resin layer can be formed. In some embodiments, resin layer 1308 is formed at polymer layer 1306. Resin layer 1308 can substantially cover the entire facing surface of polymer layer 1306. Resin layer 1308 can be formed by spin coating and / or spray coating, to name a few examples. Resin layer 1308 has a lower refractive index than polymer layer 1306.

[0083] At 1440, nanoimprinting of the grating and nanopores is performed. The resin layer 1308 and the polymer layer 1306 can facilitate patterned flow cells and laser coupling through the grating. The polymer layer 1306 is shown longitudinally here so that the respective ridges 1310 extend into the illustrated plane. The polymer layer 1306 may include, for example, a plurality of ridges 1310 organized in respective groups 1312A and 1312B. For example, the groups 1312A-1312B may be the respective input and output gratings for the laser. The ridges 1310 may have a pitch of about 200 to about 300 nm, for example, between about 220 nm and about 280 nm, to name just one example. In some embodiments, the pitch may be about 205 nm, about 215 nm, about 235 nm, about 265 nm, about 285 nm, etc.

[0084] The resin layer 1308 may include one or more nanopores 1314 defined between two or more walls 1316. In some embodiments, the nanopore 1314 may have such a size that one or more of its dimensions are within the range of about one or more nanometers. For example, the nanopore 1314 may be configured to receive and hold samples during the analysis process, such as in the form of a cluster. The end (e.g., bottom) of the nanopore 1314 may have a thickness that adapts to the propagation of evanescent light. For example, the thickness may be about 0 to about 500nm, such as between about 100nm to about 400nm. In some embodiments, the thickness may be about 10nm, about 50nm, about 100nm, about 200nm, about 300nm, about 450nm, etc. In some embodiments, the resin layer 1308 can have an average pitch of at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more between the nanopores 1314, and / or can have an average pitch of at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less. For example, the nanopores 1314 can have a pitch of about 600 to about 650 nm, for example, the pitch can be about 610 nm to about 640 nm. In some embodiments, the pitch can be about 605 nm, about 615 nm, about 635 nm, about 655 nm, etc. The depth of each nanopore 1314 can be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth can be at most about 1×10 3μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or less.

[0085] Forming the ridges 1310 of the polymer layer 1306 and the nanopores 1314 of the resin layer 1308 may involve nanoimprinting to more than one depth. In some embodiments, a dual depth stamp may be used in the nanoimprint process. For example, the ridges 1310 may be formed at a relatively deeper depth than the nanopores 1314.

[0086] Fig.15 An example of making a planar waveguide 1500 is shown. The planar waveguide 1500 can be used in one or more embodiments described herein. For example, the planar waveguide 1500 can accommodate one or more samples to facilitate illumination and excitation during sample analysis. Fig.16 Shows the manufacturing and Fig.15 Method 1600 for a planar waveguide related to a planar waveguide in FIG. Method 1600 may be combined with one or more other methods described herein. Unless otherwise indicated, more or fewer operations may be performed, and / or two or more operations may be performed in a different order.

[0087] At 1610, a polymer layer can be formed. In some embodiments, polymer layer 1502 is formed at substrate 1504. Polymer layer 1502 may include a UV curable or thermally curable polymer material. Polymer layer 1502 may be formed by spin coating and / or spray coating, to name a few examples. Polymer layer 1502 may function as a core layer in planar waveguide 1500. Polymer layer 1502 has a higher refractive index than substrate 1504.

[0088] At 1620, a resin layer can be formed. In some embodiments, resin layer 1506 is formed at polymer layer 1502. Resin layer 1506 can substantially cover the entire facing surface of polymer layer 1502. Resin layer 1506 can be formed by spin coating and / or spray coating, to name a few examples. Resin layer 1506 has a lower refractive index than polymer layer 1502.

[0089] At 1630, nanoimprinting of the grating and nanopores is performed. The resin layer 1506 and the polymer layer 1502 can facilitate patterned flow pools and laser coupling through the grating. The polymer layer 1502 is shown longitudinally here so that the respective ridges 1508 extend into the illustrated plane. The polymer layer 1502 may include, for example, a plurality of ridges 1508 organized in respective groups 1510A and 1510B. For example, the groups 1510A-1510B may be the respective input and output gratings for the laser. The ridges 1508 may have a pitch of about 200 to about 300 nm, for example, between about 220 to about 280 nm, to name just one example. In some embodiments, the pitch may be about 205 nm, about 215 nm, about 235 nm, about 265 nm, about 285 nm, etc.

[0090] The resin layer 1506 may include one or more nanopores 1512 defined between two or more walls 1514. In some embodiments, the nanopore 1512 may have such a size that one or more of its dimensions are within the range of about one or more nanometers. For example, the nanopore 1512 may be configured to receive and hold samples during the analysis process, such as in the form of clusters. The end (e.g., bottom) of the nanopore 1512 may have a thickness that accommodates the propagation of evanescent light. For example, the thickness may be about 0 to about 500nm, for example, between about 100nm to about 400nm. In some embodiments, the thickness may be about 10nm, about 50nm, about 100nm, about 200nm, about 300nm, about 450nm, etc. In some embodiments, the resin layer 1506 can have an average pitch of at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more between the nanopores 1512, and / or can have an average pitch of at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less. For example, the nanopores 1512 can have a pitch of about 600 to about 650 nm, for example, the pitch can be about 610 nm to about 640 nm. In some embodiments, the pitch can be about 605 nm, about 615 nm, about 635 nm, about 655 nm, etc. The depth of each nanopore 1512 can be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth can be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or less.

[0091] Forming the ridges 1508 of the polymer layer 1502 and the nanopores 1512 of the resin layer 1506 may involve nanoimprinting to more than one depth. In some embodiments, a dual depth stamp may be used in the nanoimprint process. For example, the ridges 1508 may be formed at a relatively greater depth than the nanopores 1512.

[0092] The above examples illustrate a method of manufacturing a flow cell, which includes forming a core layer (e.g., core layer 110, 310, 504, 710, 902, 1102, or 1302). The core layer is disposed between a substrate (e.g., substrate 102, 302, 502, 702, 904, 1104, 1304, or 1504) and a nanoporous layer (e.g., nanoporous layer 112, 312, 510, 714, or 1114). The nanoporous layer has nanopores to receive a sample. The core layer has a higher refractive index than the substrate and the nanoporous layer.

[0093] The above embodiments describe methods of manufacturing a flow cell in which forming a grating includes photolithographic patterning (e.g., Figure 1 in) or nanoimprinting (e.g., in Figure 3 , 5 , 7, 9, 11, 13 or 15).

[0094] The above embodiments illustrate a method of manufacturing a flow cell in which the core layer is formed in the same process as the grating (e.g., in Figure 5 middle).

[0095] The above embodiments illustrate methods of manufacturing a flow cell in which the core layer is formed in a different process from forming the grating (e.g., in Figure 1 , 3 , 7, 9, 11, 13 or 15).

[0096] The above embodiments illustrate a method of manufacturing a flow cell in which a grating is formed by photolithographic patterning onto a substrate, a core layer is formed on the grating, and a nanopore layer is formed on the core layer (e.g., Figure 1 middle).

[0097] The above embodiments illustrate a method of manufacturing a flow cell in which a grating is formed by nanoimprinting onto a substrate, a core layer is formed on the grating, and a nanopore layer is formed on the core layer (e.g., Figure 3 middle).

[0098] The above embodiments illustrate a method of manufacturing a flow cell, wherein a grating is formed on a substrate, a core layer is formed on the grating, an additional layer is formed on the core layer, and a nanopore layer (e.g., Figure 7). The additional layer and the nanoporous layer may initially be free of nanopores, and the method may further comprise patterning the nanoporous layer while the additional layer remains intact, and then transferring the pattern of the nanoporous layer to the additional layer to expose the core layer in the nanopores. The pattern may be transferred by etching.

[0099] The above embodiments illustrate methods of manufacturing a flow cell, wherein a core layer is formed on a substrate, and wherein a grating and a nanopore layer (e.g., Figure 5 , 9 , 11, 13 or 15).

[0100] The above embodiments illustrate a method of manufacturing a flow cell, wherein a core layer is formed on a substrate, a grating is formed on the core layer, and a nanopore layer (e.g., Figure 5 , 11 , 13, or 15).

[0101] The above embodiments illustrate a method of manufacturing a flow cell, wherein a core layer is formed on a substrate, a first layer is formed on the core layer, a second layer is formed on the first layer, and wherein a grating and a nanopore (e.g., Fig.11 or 13).

[0102] The above embodiment illustrates a method of manufacturing a flow cell, in which a core layer is formed on a substrate, a resin layer is formed on the core layer, and a grating and a nanopore (e.g., Fig. 9 ).

[0103] The above embodiments illustrate methods of fabricating a flow cell in which the grating and nanopore layers (e.g., Fig. 9 , 13 or 15).

[0104] The above embodiments illustrate methods of fabricating a flow cell in which a grating and a nanopore layer are formed in the same layer of the flow cell (e.g., Fig. 9 ).

[0105] The above embodiments illustrate methods of fabricating a flow cell in which the grating and nanopore layers are formed in separate layers of the flow cell (e.g., Fig.13 or 15).

[0106] Fig.17An example of a flow cell 1700 is shown. The flow cell 1700 can be used in one or more other embodiments described elsewhere herein. For example, one or more of the above-described articles can be incorporated, and / or one or more of the above-described techniques can be used to make a flow cell. The flow cell 1700 can be produced according to one or more of the disclosed techniques. In the flow cell 1700, a set of sealed chambers may be created due to the negative space in the gasket layer. The chambers can be sealed at the top and bottom by a substrate layer.

[0107] Here, the flow cell 1700 comprises a base layer 1710 (e.g., of borosilicate glass), a channel layer 1720 (e.g., of etched silicon, etc.) overlying the base layer, and a cover or top layer 1730. When the layers are assembled together, a closed channel is formed with an inlet / outlet at either end through the cover. Some flow cells may include an opening to the channel at the bottom of the flow cell.

[0108] Fig.18 18 is a diagram of a system 1800 including an instrument 1812, a cartridge 1814, and a flow cell 1816. The system 1800 can be used for biological and / or chemical analysis. The system 1800 can be used with or in embodiments of one or more other examples described elsewhere herein.

[0109] Box 1814 can be used as a carrier for one or more samples, such as through flow cell 1816. Box 1814 can be configured to hold flow cell 1816 and transport flow cell 1816 to or from instrument 1812 for direct interaction. For example, instrument 1812 includes container 1818 (e.g., an opening in its housing) to receive and hold box 1814 at least during information collection from sample. Box 1814 can be made of any suitable material. In some embodiments, box 1814 includes molded plastic or other durable material. For example, box 1814 can form a frame for supporting or holding flow cell 1816.

[0110] The examples herein refer to samples being analyzed. Such samples may include genetic material. In some embodiments, the sample includes one or more template strands of genetic material. For example, using the techniques and / or systems described herein, SBS may be performed on one or more template DNA strands.

[0111] Flow cell 1816 can include one or more substrates configured to hold samples to be analyzed by instrument 1812. Any suitable material can be used for the substrate, including but not limited to glass, acrylic acid and / or other plastic materials. Flow cell 1816 can allow liquid or other fluids to flow selectively relative to the sample. In some embodiments, flow cell 1816 includes one or more flow structures that can hold the sample. In some embodiments, flow cell 1816 may include at least one flow channel. For example, the flow channel may include one or more fluid ports to facilitate the flow of fluid.

[0112] The instrument 1812 can be operated to obtain any information or data related to at least one biological and / or chemical substance. The operation can be controlled by a central unit or by one or more distributed controllers. Here, an instrument controller 1820 is shown. For example, the controller 1820 can be implemented using at least one processor, at least one storage medium (e.g., memory and / or drive) storing instructions for the operation of the instrument 1812, and one or more other components, as described below. In some embodiments, the instrument 1812 can perform optical operations, including but not limited to illumination and / or imaging of the sample. For example, the instrument 1812 can include one or more optical subsystems (e.g., illumination subsystem and / or imaging subsystem). In some embodiments, the instrument 1812 can perform thermal treatment, including but not limited to thermal treatment of the sample. For example, the instrument 1812 can include one or more thermal subsystems (e.g., heaters and / or coolers). In some embodiments, the instrument 1812 can perform fluid management, including but not limited to adding and / or removing fluids in contact with the sample. For example, the instrument 1812 can include one or more fluid subsystems (e.g., pumps and / or reservoirs).

[0113] Fig.19 1 is a diagram of an example illumination system 1900. Illumination system 1900 includes a light source assembly 1910, a mirror 1928, an objective lens 1934, a flow cell 1936, an emission dichroic filter 1938, a first optical detection subsystem 1956, and a second optical detection subsystem 1958. Illumination system 1900 is capable of imaging two color channels simultaneously. In some embodiments, another illumination system may be configured to enable imaging of more than two color channels (e.g., three color channels, four color channels, or more) simultaneously. It should be noted that there may be other optical configurations that can produce similar simultaneous imaging of multiple color channels.

[0114] Light source assembly 1910 produces excitation illumination that is incident on flow cell 1936. This excitation illumination will in turn produce emitted illumination or fluorescent illumination from one or more fluorescent dyes, which will be collected using lenses 1942 and 1948. Light source assembly 1910 includes a first excitation illumination source 1912 and a corresponding converging lens 1914, a second excitation illumination source 1916 and a corresponding converging lens 1918, and a dichroic filter 1920.

[0115] The first excitation illumination source 1912 and the second excitation illumination source 1916 illustrate an illumination system that can simultaneously provide respective excitation illumination light to the sample (e.g., corresponding to respective color channels). In some embodiments, each of the first excitation illumination source 1912 and the second excitation illumination source 1916 comprises a light emitting diode (LED). In some embodiments, at least one of the first excitation illumination source 1912 and the second excitation illumination source 1916 comprises a laser. Converging lenses 1914 and 1918 are each disposed at a distance from the respective excitation illumination sources 1912 and 1916 such that the illumination emerging from each converging lens 1914 / 1918 is focused on the field of view aperture 1922. The dichroic filter 1920 reflects the illumination from the first excitation illumination source 1912 and transmits the illumination from the second excitation illumination source 1916.

[0116] In some embodiments, the mixed excitation illumination output from the dichroic filter 1920 can be transmitted directly to the objective 1934. In other embodiments, the mixed excitation illumination can be further modified and / or controlled by additional intermediate optical components before being emitted from the objective 1934. The mixed excitation illumination can reach the filter 1924 through a focus in the field of view aperture 1922 and then pass through a color-corrected collimating lens 1926. The collimated excitation illumination from the lens 1926 is incident on the mirror 1928 where it is reflected and is incident on the excitation / emission dichroic filter 1930. The excitation / emission dichroic filter 1930 reflects the excitation illumination emitted from the light source assembly 1910 while allowing the emission illumination to pass through the excitation / emission dichroic filter 1930 to be received by one or more optical subsystems 1956, 1958, which will be further described below. The optical subsystems 1956 and 1958 illustrate a light collection system that can collect multiple fluorescent lights simultaneously. The excitation illumination reflected from the excitation / emission dichroic filter 1930 is then incident on a mirror 1932 , from which it is directed toward a flow cell 1936 and incident on an objective lens 1934 .

[0117] Objective lens 1934 focuses the collimated excitation light from mirror 1932 onto flow cell 1936. In some embodiments, objective lens 1934 is a microscope objective having a specified magnification, such as 1X, 2X, 4X, 5X, 6X, 8X, 10X, or higher. Objective lens 1934 focuses the excitation illumination incident from mirror 1932 onto flow cell 1936 at a cone of angle or numerical aperture determined by the magnification. In some embodiments, objective lens 1934 is movable on an axis ("z-axis") that is perpendicular (normal) to the flow cell. In some embodiments, illumination system 1900 independently adjusts the z position of tube lens 1948 and tube lens 1942.

[0118] The flow cell 1936 contains a sample to be analyzed, such as a nucleotide sequence or any other material. The flow cell 1936 may include one or more channels 1960 (shown schematically here in an enlarged cross-sectional view) configured to hold sample material and facilitate actions taken on the sample material, including but not limited to initiating a chemical reaction or adding or removing material. An object plane 1962 (shown schematically here using a dashed line) of the objective lens 1934 extends through the flow cell 1936. For example, the object plane 1962 may be defined to be adjacent to the channel 1960.

[0119] The objective lens 1934 can define a field of view. The field of view can define an area on the flow cell 1936 from which the image detector captures the emitted light using the objective lens 1934. One or more image detectors can be used, such as detectors 1946 and 1954. The illumination system 1900 can include separate image detectors 1946 and 1954 for each wavelength (or wavelength range) of the emitted light. At least one of the image detectors 1946 and 1954 can include a charge coupled device (CCD), such as a time-delay integrated CCD camera, or a sensor made based on complementary metal oxide semiconductor (CMOS) technology, such as a chemically sensitive field effect transistor (chemFET), an ion sensitive field effect transistor (ISFET) and / or a metal oxide semiconductor field effect transistor (MOSFET).

[0120] In some embodiments, the illumination system 1900 may include a structured illumination microscope (SIM). SIM imaging is based on spatially structured illumination light and reconstruction to obtain an image with higher resolution than an image produced using only the magnification of the objective lens 1934. For example, the structure may be composed of or include a pattern or grating that interrupts the illumination excitation light. In some embodiments, the structure may include a fringing pattern. Light fringes are generated by irradiating a light beam onto a diffraction grating, thereby causing reflection or transmission diffraction. The structured light can be projected onto the sample, and the sample is illuminated according to the respective fringes, which may occur in a certain period. In order to reconstruct an image using SIM, two or more patterned images are used when the patterns of the excitation illumination are at different phase angles to each other. For example, an image of a sample can be acquired at different stages of the fringes of the structured light (sometimes referred to as the individual pattern stages of the image). This can allow multiple locations on the sample to be exposed to multiple illumination intensities. The resulting collection of emission light images can be combined to reconstruct a higher resolution image.

[0121] The sample material in the flow cell 1936 is contacted with a fluorescent dye coupled to a corresponding nucleotide. The fluorescent dye emits fluorescent illumination after being illuminated by the corresponding excitation illumination incident on the flow cell 1936 from the objective lens 1934. The emitted illumination is identified by a wavelength band, each wavelength band being categorized as a respective color channel. The fluorescent dye is chemically bound to the respective nucleotide (e.g., containing the respective nucleobase). In this manner, when detected by the image detectors 1946, 1954, the dNTP labeled with the fluorescent dye can be identified based on the wavelength of light emitted within the respective wavelength band.

[0122] Objective lens 1934 captures the fluorescence emitted by the fluorescent dye molecules in flow cell 1936. After capturing the emitted light, objective lens 1934 collects and transmits collimated light. The emitted light then propagates back along the path in which the original excitation illumination came from light source assembly 1910. It should be noted that due to the lack of coherence between the emitted light and the excitation illumination, there is little or no expected interference between the emission and excitation illumination along this path. That is, the emitted light is the result of a separate light source, namely the result of the fluorescent dye contacting the sample material in flow cell 1936.

[0123] Upon reflection by mirror 1932, the emitted light is incident on excitation / emission dichroic filter 1930. Filter 1930 transmits the emitted light into dichroic filter 1938.

[0124] In some embodiments, the dichroic filter 1938 transmits illumination associated with the blue channel and reflects illumination associated with the green channel. In some embodiments, the dichroic filter 1938 is selected so that the dichroic filter 1938 reflects emitted illumination to the optical subsystem 1956 within a defined green wavelength band and transmits emitted illumination to the optical subsystem 1958 within a defined blue wavelength band, as discussed above. The optical subsystem 1956 includes a tube lens 1942, a filter 1944, and an image detector 1946. The optical subsystem 1958 includes a tube lens 1948, a filter 1950, and an image detector 1954.

[0125] In some embodiments, dichroic filter 1938 and dichroic filter 1920 operate similarly to each other (e.g., both can reflect light of one color and transmit light of another color). In other embodiments, dichroic filter 1938 and dichroic filter 1920 operate differently from each other (e.g., dichroic filter 1938 can transmit light having a color that is reflected by dichroic filter 1920, and vice versa).

[0126] In some embodiments, the emitted illumination encounters a mirror 1952 before the image detector 1954. In the illustrated embodiment, the optical path in the optical subsystem 1958 is angled so that the illumination system 1900 as a whole can meet space or volume requirements. In some embodiments, such subsystems 1956 and 1958 both have angled optical paths. In some embodiments, neither optical path in subsystems 1956 or 1958 is angled. Thus, one or more of the plurality of optical subsystems can have at least one angled optical path.

[0127] Each tube lens 1942 and 1948 focuses the emission illumination incident thereon onto a respective image detector 1946 and 1954. In some embodiments, each detector 1946 and 1954 comprises a charge coupled device (CCD) array. In some embodiments, each image detector 1946 and 1954 comprises a complementary metal oxide semiconductor (CMOS) sensor.

[0128] The lighting system 1900 does not need to be Fig.19 As shown. For example, each mirror 1928, 1932, 1940 can be replaced by a prism or some other optical device that changes the direction of the illumination. Each lens can be replaced by a diffraction grating, a diffractive optical device, a Fresnel lens, or some other optical device that produces collimated or focused illumination from the incident illumination.

[0129] The terms "substantially" and "about" used throughout the specification are used to describe and account for small fluctuations, such as fluctuations due to variations in processing. For example, they can mean less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. Also, when used herein, indefinite articles such as "a" or "an" mean "at least one".

[0130] It should be understood that all combinations of the aforementioned concepts and the additional concepts discussed in more detail below (assuming such concepts are not mutually contradictory) are considered to be part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are considered to be part of the inventive subject matter disclosed herein.

[0131] A number of implementations have been described, however, it will be appreciated that various modifications can be made without departing from the spirit and scope of the specification.

[0132] In addition, the logic flows depicted in the figures do not require the particular order shown or sequential order to achieve the desired results. In addition, other processes can be provided from the described flows, or processes can be eliminated from the described flows, and other components can be added to or removed from the described systems. Therefore, other implementations are within the scope of the appended claims.

[0133] Although certain features of the embodiments have been described as described herein, many modifications, substitutions, changes and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and changes that fall within the scope of the implementation. It should be understood that they are given only in an example and not in a limiting manner, and various changes in form and detail may be made. Except for mutually exclusive combinations, any part of the apparatus and / or method described herein may be combined in any combination. The embodiments described herein may include multiple combinations and / or sub-combinations of the functions, components and / or features of the different embodiments described.

Claims

1. A method for manufacturing a flow cell, the method comprising: forming a core layer disposed between a substrate and a nanoporous layer, the nanoporous layer having a plurality of nanopores for receiving a sample, wherein each nanopore of the plurality of nanopores comprises a side wall and a bottom wall, the core layer having a higher refractive index than the substrate and the nanoporous layer; and A grating is formed using nanoimprinting to couple light to the core layer, wherein the grating is disposed above the substrate and has a first set of ridges adjacent to a first side of the substrate and a second set of ridges adjacent to a second side of the substrate, wherein the first set of ridges is separated from the second set of ridges by a ridge-free region below a bottom wall of a nanohole, and wherein the core layer is formed in a process different from forming the grating. 2 . The method of claim 1 , wherein the grating is formed onto the substrate, the core layer is formed onto the grating, and the nanohole layer is formed onto the core layer by nanoimprinting.

3. The method of claim 1 , wherein the grating is formed onto the substrate, the core layer is formed onto the grating, an additional layer is formed onto the core layer, and the nanohole layer is formed onto the additional layer, Before forming the nanopore, the additional layer and the nanopore layer initially do not have the nanopore, and the method also includes patterning the nanopore layer to form the nanopore while the additional layer remains intact, and then transferring the pattern of the nanopore layer to the additional layer to expose the core layer in the nanopore. The method according to claim 3 , wherein the pattern is transferred by etching. 5 . The method of claim 1 , wherein the core layer is formed onto the substrate, the grating is formed onto the core layer, and the nanohole layer is formed onto the grating.

6. The method of claim 1, wherein the core layer is formed onto the substrate, the first layer is formed onto the core layer, the nanohole layer comprising the second layer is formed onto the first layer, and wherein the grating and the nanohole are formed into the first layer and the second layer, respectively. 7 . The method of claim 1 , wherein the core layer is formed on the substrate, a nanopore layer including a resin layer is formed on the core layer, and the grating and the nanopore are formed in the resin layer.

8. The method according to any one of claims 1 or 6, wherein the grating and the nanohole layer are formed in the same process.

9. The method of claim 8, wherein the grating and the nanopore layer are formed in the same layer of the flow cell.

10. The method of claim 8, wherein the grating and the nanopore layer are formed in separate layers of a flow cell.

11. A flow cell manufactured using the method of any preceding claim, comprising: substrate; a nanopore layer having a plurality of nanopores to receive a sample, wherein each nanopore of the plurality of nanopores comprises a side wall and a bottom wall; a core layer disposed between the substrate and the nanoporous layer, the core layer having a higher refractive index than the substrate and the nanoporous layer; and Light is coupled to a grating of the core layer, wherein the grating is disposed above the substrate and has a first set of ridges adjacent to a first side of the substrate and a second set of ridges adjacent to a second side of the substrate, wherein the first set of ridges is separated from the second set of ridges by a ridge-free region below a bottom wall of a nanopore.

12. The flow cell of claim 11, wherein the grating is disposed above the substrate, the core layer is disposed above the grating, and the nanopore layer is disposed above the core layer.

13. The flow cell of claim 12, wherein a grating layer covers the substrate, the grating layer comprising the grating. 14 . The flow cell according to claim 11 , wherein a first resin layer including the grating is disposed over the substrate, and the nanopore layer including a second resin layer including the nanopore is disposed over the first resin layer.

15. The flow cell of claim 11, wherein the grating is disposed above the substrate, the nanopore layer comprising a first polymer layer is disposed above the grating, and the nanopore layer comprising a second polymer layer is disposed above the first polymer layer, wherein the nanopores are disposed in the first and second polymer layers. 16 . The flow cell of claim 11 , wherein the core layer is disposed over the substrate, the nanopore layer comprising a resin layer is disposed over the core layer, and the grating and the nanopore are disposed in the resin layer.

17. The flow cell of claim 11, wherein the core layer is disposed above the substrate, the grating is disposed above the core layer, and the nanopore layer is disposed above the grating.

18. The flow cell of claim 17, wherein a grating layer overlies the core layer, the grating layer comprising the grating.

19. The flow cell of claim 11, wherein the core layer is disposed above the substrate, a polymer layer is disposed above the core layer, and the nanopore layer comprising a resin layer is disposed above the polymer layer, wherein the grating is disposed in the polymer layer, and wherein the nanopore is disposed in the resin layer.

20. The flow cell of claim 11, wherein a polymer layer is disposed over the substrate, and the nanopore layer comprising a resin layer is disposed over the polymer layer, wherein the grating is disposed in the polymer layer, and wherein the nanopore is disposed in the resin layer.

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