Systems and methods for patterning flow cell substrates

Abstract

1. A method for patterning a flow cell substrate using photoinitiated chemical reactions, the method comprising: fabricating a planar waveguide flow cell by forming a layer of optical coupling grating on a glass substrate layer; depositing a core layer on the layer of optical coupling grating; depositing a cladding layer on the core layer; and forming nanowells in the cladding layer; silanizing the cladding layer; coating the silanized cladding layer and the nanowells with a first group of reactants; introducing a second group of reactants into the nanowells, the second group of reactants including a target reactant and a photosensitive photoinitiator system; and coupling a light source to the optical coupling grating and directing light inward within the planar waveguide flow cell to photoinitiate chemical reactions between the first group of reactants and the second group of reactants, wherein the photoinitiated chemical reactions covalently bond the target reactants only to the bottom of each nanowell.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 884,753, filed August 9, 2019, and Dutch Patent Application No. 2023679, filed August 21, 2019, the disclosures of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Fabrication of nanowell substrates for use in industrial processes such as sequencing-by-synthesis can involve complex processes, including, for example, surface functionalization of microfluidic devices, such as flow cells, that house or contain the nanowell substrates. In some embodiments, a particularly challenging aspect of nanowell substrate fabrication involves polishing the interstitial spaces of the nanowells to remove pre-existing surface chemistry found in the interstitial areas between the nanowells. Challenges associated with this type of surface preparation include over-polishing, under-polishing, and scratching or damaging the patterned surface. Thus, a need exists for a method of fabricating nanowell substrates that does not suffer from the aforementioned complications and difficulties. Summary of the Invention

[0003] The following provides a summary of certain embodiments. This summary is not an extensive overview and is not intended to identify key or critical aspects or elements of the invention or to delineate the scope of the invention.

[0004] Embodiments of the described systems and methods enable spatially resolved chemical patterning of nanoscale features within microfluidic devices, such as flow cells, and advantageously improve the fabrication of nanowell substrates by eliminating polishing steps entirely. The described methods utilize planar waveguides or similar devices to spatially control the chemical functionalization of specific, predefined areas of each nanowell substrate fabricated within a flow cell. The nanowell substrates are patterned into microfluidic flow cell devices using known flow cell fabrication processes. The planar waveguides direct excitation light only to the underside of each nanowell substrate, where the light photoinitiates a chemical reaction that covalently bonds target reactants to the lower regions of the nanowell substrate. Unreacted molecules are removed during a washing step following photoinitiation without any adverse effect on the patterned surface of the flow cell. In some embodiments, the disclosed systems and methods utilize the same light source used during sequencing-by-synthesis processes and a specific blue-light (e.g., 470 nm wavelength)-sensitive photoinitiator system.

[0005] According to one embodiment, a first method for patterning a flow cell substrate is provided, the method comprising preparing a flow cell for photoinitiated chemical reactions, the flow cell comprising: a substrate having an optical coupling grating formed thereon; a first material layer disposed on the substrate; a second material layer disposed on the first material layer; and nanowells formed in the second material layer, each nanowell comprising an upper portion and a lower portion, the preparing the flow cell comprising: silanizing the second material layer; and coating the silanized second material layer and the nanowells with a first set of reactants. introducing a second group of reactants into the nanowells, the second group of reactants comprising at least one target reactant, a copper chelating ligand, and a light-sensitive photoinitiator system; and directing light inwardly within the flow cell through the light coupling grating to only a lower portion of each nanowell to photoinitiate a chemical reaction between the first group of reactants and the second group of reactants, the photoinitiated chemical reaction covalently binding the target reactant to only the lower portion of each nanowell. In certain embodiments, the method further includes washing unreacted materials from the nanowells; using a polymer and an azide moiety bound to the polymer as the first group of reactants; using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer; using a camphorquinone-amine photosensitizer system using a light wavelength of about 470 nm as the photosensitive photoinitiator system; using an alkyne-conjugated primer as the target reactant; using an alkyne-conjugated fluorophore as the target reactant; using a laser as the light source; and using a material having a refractive index in the range of 1.0 to 1.3 for the substrate and a material having a refractive index in the range of 2.0 to 2.15 for the first material layer, although other values ​​are possible.

[0006] According to another embodiment, a second method for patterning a flow cell substrate is provided, which includes fabricating a planar waveguide flow cell by forming a layer of an optical coupling grating on a glass substrate layer, depositing a core layer on the layer of optical coupling grating, depositing a cladding layer on the core layer, and forming nanowell substrates in the cladding layer, each nanowell substrate including an upper and lower portion and defining a gap region therebetween; silanizing the cladding layer; and coating the silanized cladding layer and nanowell substrates with a first group of reactants. introducing a second group of reactants into the nanowell substrate, the second group of reactants comprising at least one target reactant, a copper chelating ligand, and a light-sensitive photoinitiator system; and directing light inwardly within the planar waveguide flow cell through the optical coupling grating to only a lower portion of each nanowell substrate to photoinitiate a chemical reaction between the first group of reactants and the second group of reactants, the photoinitiated chemical reaction covalently binding the target reactant to only the lower portion of each nanowell substrate. In certain embodiments, the method further includes washing unreacted materials from the nanowells; using a polymer and an azide moiety bound to the polymer as the first group of reactants; using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer; using a camphorquinone-amine photosensitizer system using a light wavelength of about 470 nm as the photosensitive photoinitiator system; using an alkyne-linked primer as the target reactant; using an alkyne-linked fluorophore as the target reactant; using a laser as the light source; and using materials having refractive indices in the range of 1.0 to 1.3 for the layers of the optical coupling grating and materials having refractive indices in the range of 2.0 to 2.15 for the core layer, although other values ​​are possible.

[0007] In yet another embodiment, a third method of spatially patterning a flow cell substrate is provided, the method comprising: fabricating a planar waveguide flow cell by forming a layer of optical coupling grating on a glass substrate layer, depositing a core layer on the layer of optical coupling grating, depositing a cladding layer on the core layer, and forming nanowell substrates in the cladding layer, each nanowell substrate including an upper and lower portion and defining a gap region therebetween; silanizing the cladding layer; and coating the cladding layer and nanowell substrates with a first group of reactants, the first group of reactants further comprising a polymer, an azide moiety bound to the polymer, a copper ligand, and a photosensitive photoinitiator system; and silanizing each nanowell substrate to photoinitiate a chemical reaction between reactants in the first group of reactants. directing light of a predetermined wavelength inwardly within the planar waveguide flow cell only toward the lower portion of each nanowell substrate, wherein the photoinitiated chemical reaction covalently bonds a polymer only to the lower portion of each nanowell substrate; introducing a second group of reactants into the nanowell substrate, wherein the second group of reactants comprises at least one target reactant, a copper ligand, and a photosensitive photoinitiator system; and directing light of a predetermined wavelength inwardly within the planar waveguide flow cell only toward the lower portion of each nanowell substrate to photoinitiate a chemical reaction between the covalently bonded polymer and the second group of reactants, wherein the photoinitiated chemical reaction covalently bonds the target reactant only to the lower portion of each nanowell substrate.In certain embodiments, the method further includes washing unreacted materials from the nanowell substrate after each photoinitiated chemical reaction; using 3-azidopropyltrimethoxysilane to silanize the cladding layer; using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer; using a camphorquinone-amine photosensitizer system using a light wavelength of about 470 nm as the photosensitive photoinitiator system; using an alkyne-linked primer as the target reactant; using an alkyne-linked fluorophore as the target reactant; using a laser as the light source; and using materials having refractive indices in the range of 1.0 to 1.3 for the layers of the optical coupling grating and materials having refractive indices in the range of 2.0 to 2.15 for the core layer, although other values ​​are possible.

[0008] In another embodiment, the methods herein include silanization by vapor deposition of norbornene silane. In one example, poly(N-(5-azidoacetamidylpentyl)acrylamide) is spin-coated onto the silanized layer by the following procedure: step 1—600 rpm, 5 seconds, acceleration 1500 rpm / sec; step 2—1500 rpm, 30 seconds, acceleration 5000 rpm / sec; step 3—4000 rpm, 5 seconds, acceleration 5000 rpm / sec; step 4—600 rpm, 5 seconds, acceleration 5000 rpm / sec, followed by heating, preferably at 65-75°C for 1 hour. In another example, silanization is performed by vapor deposition of 3-azidopropyltrimethoxysilane, followed by poly(N-(5-azidoacetamidylpentyl)acrylamide). Poly(N-(5-azidoacetamidylpentyl)acrylamide) is preferably crosslinked to the azide groups using a photoinitiated reaction using a bifunctional crosslinker such as NH-bis(PEG-2 propargyl), a photoinitiator (e.g., camphorquinone at 470 nm), and copper sulfate bearing a ligand, e.g., PMDTA (Pentamethyldiethylenetriamine). In yet another embodiment, poly(N-(5-azidoacetamidylpentyl)acrylamide) is covalently attached to the surface at the underside of the nanowell using light, preferably laser light.

[0009] According to another embodiment, the method herein includes directing light through an optical coupling grating only to the bottom of each nanowell to photo-initiate a chemical reaction between a first group of reactants and a second group of reactants. In one example, the copper is then removed using a dilute solution of EDTA (0.1 M). In another example, the method does not include a polishing step to remove any existing surface chemistry found in the interstitial areas between the nanowells.

[0010] In yet another embodiment, a flow cell is provided. In one example, the flow cell includes a substrate; an optical coupling grating layer disposed on the substrate, the optical coupling grating layer having a refractive index; a core layer disposed on the optical coupling grating layer, the core layer having a refractive index greater than the refractive index of the optical coupling grating layer; and a silanized layer disposed on the core layer, the nanowells of the silanized layer being separated by gap areas. In one example, the substrate is glass. In another example, the optical coupling grating layer and the core layer are formed from a resin. In yet another example, the optical coupling grating layer and / or the core layer are formed from tantalum pentoxide. In yet another embodiment, the flow cell comprises poly(N-(5-azidoacetamidylpentyl)acrylamide) covalently bound to a surface at the underside of the nanowell. In another embodiment, the flow cell comprises a second group of reactants disposed on the first group of reactants, the second group comprising at least one target reactant, a copper chelating ligand, and a photosensitive photoinitiator system. In yet another embodiment, the flow cell is a planar waveguide flow cell. This flow cell can be used for nucleic acid sequencing, including high-throughput sequencing-by-synthesis, and can be fabricated according to any of the methods disclosed herein for fabricating flow cells and spatially patterning flow cell substrates.

[0011] It should be understood that any corresponding features / embodiments of each of the aspects of the present disclosure as described herein may be implemented together in any combination to achieve the benefits and results as described herein, and that any feature / embodiment from any one or more of these aspects may be implemented with any of the features of the other aspect(s) as described herein in any combination to obtain the benefits as described herein.

[0012] This disclosure also includes the following clauses: 1. A method for patterning a flow cell substrate, comprising: 1. Preparing a flow cell for photoinitiated chemical reactions, the flow cell comprising: a substrate having an optical coupling grating formed thereon; a first layer of material disposed over a substrate; and preparing the flow cell, the flow cell including: a second material layer disposed on the first material layer; and nanowells formed in the second material layer, each nanowell including an upper portion and a lower portion. silanizing the second layer of material; coating the silanized second material layer and the nanowells with a first group of reactants; introducing a second group of reactants into the nanowell, the second group of reactants including at least one target reactant, a copper chelating ligand, and a light-sensitive photoinitiator system; directing light inwardly within the flow cell through the optical coupling grating only to a lower portion of each nanowell to photo-initite a chemical reaction between a first group of reactants and a second group of reactants; The photoinitiated chemical reaction comprises covalently attaching a target reactant only to the bottom of each nanowell. 2. The method of clause 1, further comprising washing unreacted materials from the nanowells. 3. The method of clause 1 or 2, further comprising using a polymer and an azide moiety attached to the polymer as the first group of reactants. 4. The method of claim 3, further comprising using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer. 5. The method of any one of clauses 1 to 4, further comprising using a camphorquinone-amine photosensitizer system using a light wavelength of 470 nm as the photosensitive photoinitiator system. 6. The method of any one of clauses 1 to 5, further comprising using an alkyne-conjugated primer as the target reactant. 7. The method of any one of clauses 1-6, further comprising using an alkyne-linked fluorophore as the target reactant. 8. The method of any one of clauses 1 to 7, further comprising using a laser as the light source. 9. The method of any one of clauses 1 to 8, further comprising using a material having a refractive index in the range of 1.0 to 1.3 for the substrate and a material having a refractive index in the range of 2.0 to 2.15 for the first material layer. 10. A method for patterning a flow cell substrate, comprising: Fabricating a planar waveguide flow cell, the fabricating the planar waveguide flow cell comprising: forming a layer of an optical coupling grating on a glass substrate layer; depositing a core layer on the layer of the optical coupling grating; depositing a cladding layer on the core layer; forming nanowell substrates within the cladding layer, each nanowell substrate including an upper portion and a lower portion, the nanowell substrates defining a gap region therebetween; silanizing the cladding layer; coating the silanized cladding layer and nanowell substrate with a first group of reactants; introducing a second group of reactants into the nanowell substrate, the second group of reactants including at least one target reactant, a copper chelating ligand, and a light-sensitive photoinitiator system; directing light inwardly within the planar waveguide flow cell through an optical coupling grating only to a lower portion of each nanowell substrate to photo-initite a chemical reaction between a first group of reactants and a second group of reactants; the photoinitiated chemical reaction covalently attaches the target reactant only to the bottom of each nanowell substrate. 11. The method of clause 10, further comprising washing unreacted materials from the nanowell substrate. 12. The method of clause 10 or 11, further comprising using a polymer as the first group of reactants and an azide moiety attached to the polymer. 13. The method of claim 12, further comprising using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer. 14. The method of any one of clauses 10-13, further comprising using a camphorquinone-amine photosensitizer system using a light wavelength of 470 nm as the photosensitive photoinitiator system. 15. The method of any one of clauses 10-14, further comprising using an alkyne-conjugated primer as the target reactant. 16. The method of any one of clauses 10-15, further comprising using an alkyne-linked fluorophore as the target reactant. 17. The method of any one of clauses 10 to 16, further comprising using a laser as the light source. 18. The method of any one of clauses 10 to 17, further comprising using a material having a refractive index in the range of 1.0 to 1.3 for the layers of the optical coupling grating and a material having a refractive index in the range of 2.0 to 2.15 for the core layer. 19. A method for patterning a flow cell substrate, comprising: Fabricating a planar waveguide flow cell, the fabricating the planar waveguide flow cell comprising: forming a layer of an optical coupling grating on a glass substrate layer; depositing a core layer on the layer of the optical coupling grating; depositing a cladding layer on the core layer; forming nanowell substrates within the cladding layer, each nanowell substrate including an upper portion and a lower portion, the nanowell substrates defining a gap region therebetween; silanizing the cladding layer; coating the cladding layer and the nanowell substrate with a first group of reactants, the first group of reactants further comprising a polymer, an azide moiety bound to the polymer, a copper ligand, and a light-sensitive photoinitiator system; directing light of a predetermined wavelength inwardly within the planar waveguide flow cell only to a lower portion of each nanowell substrate to photoinitiate a chemical reaction between reactants in the first group of reactants, wherein the photoinitiated chemical reaction covalently bonds a polymer to only the lower portion of each nanowell substrate; introducing a second group of reactants into the nanowell substrate, the second group of reactants including at least one target reactant, a copper ligand, and a light-sensitive photoinitiator system; directing light of a predetermined wavelength inwardly within the planar waveguide flow cell only to a lower portion of each nanowell substrate to photoinitiate a chemical reaction between the covalently attached polymer and a second group of reactants, wherein the photoinitiated chemical reaction covalently attaches a target reactant only to the lower portion of each nanowell substrate. 20. The method of clause 19, further comprising washing unreacted materials from the nanowell substrate after each photoinitiated chemical reaction. 21. The method of clause 19 or 20, further comprising using 3-azidopropyltrimethoxysilane to silanize the cladding layer. 22. The method of any one of clauses 19 to 21, further comprising using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer. 23. The method of any one of clauses 19-22, further comprising using a camphorquinone-amine photosensitizer system using a light wavelength of 470 nm as the photosensitive photoinitiator system. 24. The method of any one of clauses 19-23, further comprising using an alkyne-conjugated primer as the target reactant. 25. The method of any one of clauses 19-23, further comprising using an alkyne-linked fluorophore as the target reactant. 26. The method of any one of clauses 19 to 25, further comprising using a laser as the light source. 27. The method of any one of clauses 19 to 26, further comprising using a material having a refractive index in the range of 1.0 to 1.3 for the layers of the optical coupling grating and a material having a refractive index in the range of 2.0 to 2.15 for the core layer. 28. The method of any one of the preceding clauses 1 to 18, wherein the silanization is carried out by chemical vapor deposition of norbornene silane. 29. The method according to any one of preceding clauses 4 to 9 and 13 to 18, wherein poly(N-(5-azidoacetamidylpentyl)acrylamide) is spin-coated onto the silanized layer by the following procedure: step 1: 600 rpm, 5 seconds, acceleration 1500 rpm / sec; step 2: 1500 rpm, 30 seconds, acceleration 5000 rpm / sec; step 3: 4000 rpm, 5 seconds, acceleration 5000 rpm / sec; step 4: 600 rpm, 5 seconds, acceleration 5000 rpm / sec, followed by heating, preferably at 65 to 75°C, for 1 hour. 30. The method of any one of the preceding clauses 21 to 27, wherein the silanization is carried out by vapor deposition of 3-azidopropyltrimethoxysilane, after which the poly(N-(5-azidoacetamidylpentyl)acrylamide) is crosslinked to the azido groups, preferably using a photoinitiated reaction using a bifunctional crosslinker such as NH-bis(PEG-2 propargyl), a photoinitiator (e.g., CQ at 470 nm), and copper sulfate with a ligand, e.g., PMDTA. 31. The method of any one of the preceding clauses 1 to 30, wherein poly(N-(5-azidoacetamidylpentyl)acrylamide) is covalently attached to the surface at the underside of the nanowell using light, preferably laser light. 32. The method according to clause 30 or 31, wherein the copper is then removed using a dilute solution of EDTA (0.1 M). 33. The method of any one of the preceding clauses 1-32, wherein there is no polishing step to remove any pre-existing surface chemistry found in the interstitial areas between the nanowells. 34. The method of any one of the preceding clauses 1 to 33, wherein light is directed through an optical coupling grating only to the bottom of each nanowell to photo-initiate a chemical reaction between a first group of reactants and a second group of reactants. 35. A flow cell (10) for photoinitiated chemical reactions, the flow cell comprising: A substrate (100); an optical coupling grating layer (200) disposed on the substrate (100), the optical coupling grating layer (200) having a refractive index; a core layer (300) disposed on the grating layer (200), the core layer (300) having a refractive index greater than the refractive index of the optical coupling grating layer (200); a silanized layer (400) disposed on the core layer (300), wherein a plurality of nanowells (500) are present in the silanized layer (400); A flow cell (10) in which nanowells (500) are separated by interstitial areas (600). 36. The flow cell (10) according to conclusion 35, wherein the substrate (100) is glass. 37. The flow cell according to clause 35 or 36, wherein the optical coupling grating layer (200) and the core layer (300) are formed from resin. 38. The flow cell of clause 37, wherein the optical coupling grating layer (200) and / or the core layer (300) are formed from tantalum pentoxide. 39. The flow cell of any one of clauses 35 to 38, wherein the silanized layer (400) is formed from a water buffer or a polymer cladding layer. 40. A flow cell described in any one of the preceding clauses 35 to 39, wherein the refractive index of the optical coupling grating layer (200) is in the range of 0.5 to 2.0, 0.8 to 1.5, or 1.0 to 1.3, and the refractive index of the core layer (300) is in the range of 1.5 to 2.5, 1.8 to 2.3, or 2.0 to 2.15. 41. The flow cell according to any one of preceding clauses 35 to 40, wherein poly(N-(5-azidoacetamidylpentyl)acrylamide) is covalently bound to the surface at the lower portion of the nanowell (500). 42. The flow cell of clause 41, further comprising a first group of reactants disposed on poly(N-(5-azidoacetamidylpentyl)acrylamide) covalently bound to the surface at the lower portion of the nanowell (500). 43. The flow cell of clause 42, wherein a second group of reactants is disposed on the first group of reactants, the second group of reactants comprising at least one target reactant, a copper chelating ligand, and a light-sensitive photoinitiator system. 44. A method of sequencing nucleic acids using a flow cell manufactured according to any one of the preceding clauses 1 to 34 and / or a flow cell according to any one of clauses 35 to 43. 45. A system for photoinitiated chemical reactions, comprising a flow cell according to any one of the preceding clauses 35 to 43 and a light source, preferably a laser light source.

[0013] Further features and aspects of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the embodiments. As will be appreciated by those of ordinary skill in the art, further embodiments of the present invention are possible without departing from the scope and spirit of the present invention. Accordingly, the drawings and associated description are to be regarded as illustrative and not restrictive in nature. [Brief explanation of the drawings]

[0014] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, drawings, and claims.

[0015] [Figure 1A] 1A and 1B illustrate the structure of a planar waveguide flow cell according to one embodiment of the disclosed systems and methods.

[0016] [Figure 1B] FIG. 1B shows the flow cell of FIG. 1A, in one embodiment, where the top surface of the flow cell is hydrogel coated with azide moieties using poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM).

[0017] [Figure 1C] FIG. 1C illustrates the flow cell of FIG. 1B, in one embodiment, where reactants are introduced into the flow cell, the reactants including an alkyne-conjugated primer, a copper-chelating ligand, and a light-sensitive photoinitiator system.

[0018] [Figure 1D] FIG. 1D shows the flow cell of FIG. 1C, in one embodiment, with a planar waveguide coupled to the flow cell and directing light into the flow cell.

[0019] [Figure 1E] FIG. 1 is a magnified view of a nanowell in a flow cell showing the area of ​​the nanowell to which a primer is covalently attached, in one embodiment.

[0020] [Figure 2A] FIG. 1 shows the chemical reaction in which a PAZAM-azide polymer is attached to the surface of a planar waveguide flow cell in one embodiment of the disclosed method.

[0021] [Figure 2B] FIG. 1 shows the chemistry for covalently attaching a target reactant to a nanowell substrate using a photoinitiated alkyne-azide click reaction in one embodiment of the disclosed method.

[0022] [Figure 3A] FIG. 10 shows a chemical reaction in which a PAZAM-azide polymer coated on the surface of a planar waveguide flow cell is patterned in a first photoinitiated click reaction in another embodiment of the disclosed method.

[0023] [Figure 3B] FIG. 3B shows the chemistry of patterning a primer (fluorophore) onto the PAZAM layer of FIG. 3A using a second photoinitiated click reaction.

[0024] [Figure 4] 1 is a flow chart illustrating an embodiment of a first method for patterning a flow cell substrate.

[0025] [Figure 5] 10 is a flow chart illustrating an embodiment of a second method for patterning a flow cell substrate.

[0026] [Figure 6] 10 is a flow chart illustrating an embodiment of a third method for patterning a flow cell substrate. DETAILED DESCRIPTION OF THE INVENTION

[0027] Embodiments of the disclosed systems and methods utilize planar waveguides to spatially control the chemical functionalization of nanowells used in microfluidic devices, such as flow cells used in sequencing-by-synthesis. Planar waveguides (i.e., waveguides with a planar geometry that guides light in only one direction) direct excitation light only to the lower region of the nanowell, where the excitation light photo-initiates a chemical reaction that covalently attaches a target reactant to the lower portion of the nanowell. Unreacted molecules are removed during a washing process, thereby localizing the target chemical to the lower portion of the nanowell.

[0028] Embodiments of the disclosed systems and methods may include photoinitiated azide-alkyne click reactions, such as those used in sequencing-by-synthesis techniques. In this type of reaction, an azide functional group is attached to a hydrogel layer formed across the surface of a microfluidic channel, followed by the addition of an alkyne-primer reactant. The azide-alkyne click reaction, in certain examples, is photoinitiated using a copper compound and a Type II photoinitiator system, such as camphorquinone. This photoinitiator system can use blue light at approximately 470 nm as the excitation source. Mixed chemistry can be achieved by incorporating alternative functional groups into the PAZAM by adding alternative functional groups to the hydrogel layer. For example, azide and tetrazole can be incorporated into PAZAM. The azide-alkyne click reaction is photoinitiated with light having a wavelength between 450 nm and 495 nm (e.g., blue), while the tetrazole-alkene reaction is photoinitiated with light having a different wavelength between 520 nm and 560 nm (e.g., green). In one embodiment, the blue light used has a wavelength of approximately 470 nm.

[0029] In general, the click reaction (click chemistry) involves biocompatible small molecule reactions commonly used in bioconjugation reactions to attach various substances to specific biomolecules. The term "click chemistry" does not refer to a single specific reaction, but rather to a chemical method for generating substances by joining small modular units together. In many applications, the click reaction is used to attach biomolecules to reporter molecules. Click chemistry is not limited to biological applications; the click reaction concept has been used in pharmacological applications. The azide-alkyne click reaction involves the copper-catalyzed reaction of an azide with an alkyne to form a five-membered heteroatom ring, i.e., Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Photoinitiated reactions of this nature have been described in Chen et al., Photoinitiated Alkyne-Azide Click and Radical Cross-Linking Reactions for the Patterning of PEG Hydrogels, BioMacromolecules, 2012, 13:889-895; Shete and Kloxin, One-pot blue light triggered tough interpenetrating polymeric network (IPN) using CuAAC and methacrylate reactions, Polym. Chem., 2017, 8(24):3668-3673; and Shete et al., Blue-light activated rapid polymerization for defect-free bulk Cu(i)-catalyzed azide-alkene cycloaddition (CuAAC) crosslinked networks, Chem. Commun. (Camb.), 2016, 52(69):10574-10577.

[0030] 1A-1E, the figures illustrate the fabrication of a flow cell including a planar waveguide. An exemplary planar waveguide flow cell 10 includes a substrate 100, which may be glass; an optical coupling grating layer 200, which may be resin; a core layer 300, which may be resin having a refractive index higher than that of the resin used for the optical coupling grating layer 200 (e.g., tantalum pentoxide); and a water buffer or patterned polymer cladding layer 400 in which multiple nanowells 500 are formed (see FIG. 1A). The top surface of the cladding layer 400 is then coated with a hydrogel (e.g., PAZAM) to which azide moieties 700 are attached (see FIG. 1B). Next, various reactants 800 are introduced into the flow cell 10, including alkyne-conjugated primers, copper-chelating ligands, and a photosensitive photoinitiator system (see FIG. 1C). Light is then directed into the planar waveguide flow cell 10 using optical focusing optics so that the optical coupling grating 200 and core layer 300 internally reflect the light within the flow cell 10. The evanescent wave penetrates the lower portion of each nanowell 500, initiating the desired chemical reaction. Sequencing primers or other molecules are covalently bound only to the lower region of each nanowell 500 and spatially excluded from the gap area 600. A washing step then removes unreacted components, such as any unbound hydrogel or unbound primers, from the nanowells. Suitable washing solutions include alkaline buffers with a pH of at least 10 and sodium hydroxide. Figure 1E provides a close-up view of the nanowell 500, with area 502 in Figure 1E indicating the location of the covalently bound sequencing primer (or other molecule) after the photoinitiated reaction is complete.

[0031] In various embodiments, the refractive index of the material of the optical coupling grating layer 200 ranges from 0.5 to 2.0, 0.8 to 1.5, or 1.0 to 1.3, and the refractive index of the material of the core layer (300) ranges from 1.5 to 2.5, 1.8 to 2.3, or 2.0 to 2.15. Increasing the contrast between the refractive index values ​​of the optical coupling grating layer 200 and the core layer 300 can improve the coupling efficiency of light into the planar waveguide flow cell 10, provided that the refractive index of the upper layer (i.e., core layer 300) remains greater than the refractive index of the lower layer (i.e., optical coupling grating layer 200). In one embodiment, the core layer 300 includes or is fabricated from a metal oxide, such as, for example, tantalum pentoxide (TaO).

[0032] The following commercially available materials can be used in embodiments of the disclosed method: (i) norbornene silane: [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane, tech-95, endo / exo isomer (Gelest Inc.), (ii) PAZAM: poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) at any acrylamide to Azapa ratio, (iii) Azapa: N-(5-azidoacetamidylpentyl)acrylamide, (iv) copper(II) sulfate pentahydrate (CuSO · 5H2O) (Sigma-Aldrich), (v) N,N,N',N",N"-pentamethyldiethylenetriamine (PMDTA) (Sigma-Aldrich), (vi) Alexa Fluor® 488 alkyne (AF-488 alkyne) (Invitrogen), (vii) camphorquinone (CQ) photoinitiator (Sigma-Aldrich), (viii) ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich), (ix) NH-bis(PEG-2 propargyl) (BroadPharm), BP-2313, (x) 3-azidopropyltrimethoxysilane (Gelest Inc.). Scheme I: One-step photoinitiated click reaction

[0033] Referring to Figures 2A-2B, one embodiment of the disclosed method utilizes a photoinitiation mechanism for the azide-alkyne click reaction involving a camphorquinone-amine photosensitizer system using approximately 470 nm light. A planar waveguide flow cell is fabricated as described above, and the cladding layer 400 is further treated by utilizing a PAZAM polymer to which azide moieties are attached. As shown in Figure 2A, the top surface of the cladding layer 400 is first silanized with a norbornene silane derivative using a chemical vapor deposition process. The process may be standard in the industry. The surface is coated and then thermally crosslinked with PAZAM. This can be achieved according to the following method (described in U.S. Patent Application Publication No. 2015 / 0005447(A1)): 500 μL of aqueous PAZAM (0.25% + 5% ethanol) is deposited onto a norbornene silane-treated glass substrate and spread over the entire surface. A thin film of PAZAM is obtained by spin-coating using the following procedure: Step 1: 600 rpm, 5 seconds, acceleration 1500 rpm / sec, Step 2: 1500 rpm, 30 seconds, acceleration 5000 rpm / sec, Step 3: 4000 rpm, 5 seconds, acceleration 5000 rpm / sec, Step 4: 600 rpm, 5 seconds, acceleration 5000 rpm / sec. After spin coating, the substrate is heated in an oven or on a hot plate at 65-75°C for 1 hour.

[0034] As shown in Figure 2B, a polymer solution containing potassium carbonate (35 mM) and the same concentration of CuSO4·5H2O, Alexa Fluor® 488 (AF-488 alkyne) (30% w / w) with PMDTA and the photoinitiator camphorquinone is prepared (Note: A modification of this solution contains a sequencing primer instead of AF-488 alkyne). The solution may be sonicated to facilitate dissolution. The solution is then introduced into the flow cell channel and retained within the channel for subsequent photoinitiation of the click reaction using the excitation laser optics of the planar waveguide. After the photoinitiation click reaction is complete, a solution of EDTA (0.1 M), which complexes with copper, is used to flush the copper from the flow cell channel. Fluorescence images can be collected using a confocal fluorescence microscope to confirm the presence of the fluorophore within the nanowells of the flow cell. As previously mentioned, light is directed exclusively into the nanowells via the planar waveguide grating, so the reaction occurs only in the lower region of the nanowell. Polishing was not used because the dye-labeled molecules bind only within the nanowells and not in the interstitial spaces or interstitial regions between the nanowells. Scheme II: Two-step photoinitiated click reaction

[0035] Referring to Figures 3A-3B, another embodiment of the disclosed method provides a two-step photoinitiated click reaction that also uses a camphorquinone-amine photosensitizer system using light at approximately 470 nm. This two-step process minimizes diffusion of reagents from the desired target area, thereby reducing functionalization of the interstitial spaces or interstitial regions between the nanowells of the flow cell. Planar waveguides are fabricated as described above, and in the first step of this embodiment (see Figure 3A), PAZAM is photopatterned within the nanowells using a first photoinitiated click reaction. This first step involves attaching azide groups to the surface of the flow cell using 3-azidopropyltrimethoxysilane in a chemical vapor deposition process, which may be standard in the industry. The PAZAM is then crosslinked to the azide groups using a photoinitiated reaction that uses a bifunctional crosslinker such as NH-bis(PEG-2 propargyl), a photoinitiator (e.g., CQ at 470 nm), and copper sulfate with a ligand (e.g., PMDTA), and light. PAZAM is covalently attached to the surface at the bottom of the nanowell using laser light directed into a planar waveguide. After the reaction is complete, the copper is removed using a dilute solution (0.1 M) of EDTA, which complexes with the copper.

[0036] In the second step (see Figure 3B), a fluorescent tag (or another molecule) is patterned using a second photoinitiated click reaction. A polymer solution containing potassium carbonate (35 mM) and the same concentration of CuSO4·5H2O, PMDTA, and Alexa Fluor® 488 (AF-488 alkyne) (30% w / w) with camphorquinone is prepared. The solution may be sonicated to facilitate dissolution. The solution is then introduced into the flow cell channel and retained there for subsequent photoinitiation using the planar waveguide excitation laser optics. After the photoinitiated click reaction is complete, the copper is washed out of the channel using a solution of EDTA (0.1 M), which complexes with copper. Fluorescence images are collected using a confocal fluorescence microscope to confirm the presence of the fluorophore within the nanowells of the flow cell. As previously mentioned, light is directed exclusively into the nanowells via the planar waveguide grating, so the reaction occurs only in the underside of the nanowells. Polishing is not required because the Alexa-labeled dye molecules are bound only in the nanowells and not in the interstitial spaces or interstitial regions between the nanowells.

[0037] 4 is a flow chart illustrating an embodiment of a first method for patterning a flow cell substrate. The first method 400 for patterning a flow cell substrate includes, in block 402, preparing a flow cell for photoinitiated chemical reactions, the flow cell including a substrate having an optical coupling grating formed thereon, a first material layer disposed on the substrate, a second material layer disposed on the first material layer, and nanowells formed in the second material layer, each nanowell including an upper portion and a lower portion. The preparing the flow cell includes, in block 404, silanizing the second material layer, and, in block 406, silanizing the silanized second material layer and the nanowells. and coating each nanowell with a first group of reactants; and at block 408, introducing a second group of reactants into the nanowells, the second group of reactants including at least one target reactant, a copper chelating ligand, and a photosensitive photoinitiator system; and at block 410, directing light inwardly within the flow cell through the light coupling grating to only the lower portion of each nanowell to photoinitiate a chemical reaction between the first group of reactants and the second group of reactants, the photoinitiated chemical reaction covalently binding the target reactant only to the lower portion of each nanowell.

[0038] With respect to the embodiment shown in FIG. 4, the following are non-limiting examples of various aspects of the described method. Exemplary photoinitiated chemical reactions include azide-alkyne chemistry using blue light at a predetermined wavelength between about 450 nm and 495 nm, tetrazole-alkene chemistry using green light at a predetermined wavelength between about 520 nm and 560 nm, and metal-free azide / acetylene cycloaddition reactions utilizing triple bond masking using dibenzocyclooctyne as the cyclopropenone (see, e.g., JACS 2009, 131, 15769-15776). Exemplary techniques for forming optical coupling gratings on a substrate (which may be glass) include photolithographic patterning of silicon dioxide (SiO) gratings, lift-off processes, laser etching, and nanoimprinting. Exemplary materials for the first material layer include low-refractive-index nanoimprint lithography (NIL) resins and low-refractive-index polymers. Exemplary techniques for depositing a first material layer on a substrate include sputter coating and spin coating. Exemplary materials for the second material layer include high-refractive-index resins, high-refractive-index polymers, and metal oxides such as tantalum pentoxide (Ta2O5). Exemplary techniques for depositing a second material layer on a first material layer include vacuum thin-film deposition, sputter coating, and spin coating. Exemplary techniques for forming nanowells in the second material layer include nanoimprint lithography patterning. Exemplary techniques for coating the silanized second material layer and nanowells with a first group of reactants include sputter coating and spin coating. Exemplary techniques for introducing a second group of reactants into the nanowells include using a microfluidic pumping system, such as a peristaltic pump. Exemplary techniques for directing light inwardly through a light coupling grating within a flow cell include using focusing optics to direct light from an external light source into the flow cell.

[0039] 5 is a flow chart illustrating an embodiment of a second method for patterning a flow cell substrate. The second method 500 for patterning a flow cell substrate includes fabricating a planar waveguide flow cell at block 502 by forming a layer of optical coupling grating on a glass substrate layer at block 504, depositing a core layer on the layer of optical coupling grating at block 506, depositing a cladding layer on the core layer at block 508, and forming nanowell substrates within the cladding layer at block 510, each nanowell substrate including an upper and lower portion and defining a gap region therebetween; silanizing the cladding layer at block 512; and silanizing the cladding layer at block 514. coating the coated cladding layer and nanowell substrate with a first group of reactants; introducing a second group of reactants into the nanowells at block 516, the second group of reactants including at least one target reactant, a copper chelating ligand, and a photosensitive photoinitiator system; and directing light inwardly within the planar waveguide flow cell through the optical coupling grating to only a lower portion of each nanowell substrate to photoinitiate a chemical reaction between the first group of reactants and the second group of reactants at block 518, the photoinitiated chemical reaction covalently binding the target reactants only to the lower portion of each nanowell substrate.

[0040] With respect to the embodiment shown in FIG. 5, the following are non-limiting examples of various aspects of the described method. Exemplary techniques for forming a layer of an optical coupling grating on a substrate (which may be glass) include photolithographic patterning of a silicon dioxide (SiO) grating, lift-off processes, laser etching, and nanoimprinting. Exemplary materials for the core layer include low-refractive-index nanoimprint lithography (NIL) resins and low-refractive-index polymers. Exemplary techniques for depositing the core layer on the substrate include sputter coating and spin coating. Exemplary materials for the cladding layer include high-refractive-index resins, high-refractive-index polymers, and metal oxides such as tantalum pentoxide (TaO). Exemplary techniques for depositing the cladding layer on the core layer include vacuum thin-film deposition, sputter coating, and spin coating. Exemplary techniques for forming nanowells in the second material layer include nanoimprint lithography patterning. Exemplary techniques for coating the silanized cladding layer and nanowells with the first group of reactants include sputter coating and spin coating. An exemplary technique for introducing the second group of reactants into the nanowells includes using a microfluidic pumping system, such as a peristaltic pump. An exemplary technique for directing light inward through a light coupling grating into the flow cell includes using focusing optics to direct light from an external light source into the flow cell. Exemplary photoinitiated chemical reactions include azide-alkyne chemistry using blue light at a predetermined wavelength of approximately 450 nm to 495 nm, tetrazole-alkene chemistry using green light at a predetermined wavelength of approximately 520 nm to 560 nm, and metal-free azide / acetylene cycloaddition reactions utilizing triple bond masking using dibenzocyclooctyne as the cyclopropenone (see, e.g., JACS 2009, 131, 15769-15776).

[0041] 6 is a flow chart illustrating an embodiment of a third method for patterning a flow cell substrate. The third method 600 for patterning a flow cell substrate includes fabricating a planar waveguide flow cell at block 602 by forming a layer of optical coupling grating on a glass substrate layer at block 604, depositing a core layer on the layer of optical coupling grating at block 606, depositing a cladding layer on the core layer at block 608, and forming nanowell substrates within the cladding layer at block 610, each nanowell substrate including an upper and lower portion and defining a gap region therebetween, silanizing the cladding layer at block 612, and coating the cladding layer and nanowell substrates with a first group of reactants at block 614, the first group of reactants further including a polymer, an azide moiety attached to the polymer, a copper ligand, and a photosensitive photoinitiator system. At block 616, directing light of a predetermined wavelength inwardly within the planar waveguide flow cell only toward a lower portion of each nanowell substrate to photoinitiate a chemical reaction between reactants in a first group of reactants, the photoinitiated chemical reaction covalently bonding a polymer to only the lower portion of each nanowell substrate; at block 618, introducing a second group of reactants into the nanowell substrate, the second group of reactants including at least one target reactant, a copper ligand, and a photosensitive photoinitiator system; and at block 620, directing light of a predetermined wavelength inwardly within the planar waveguide flow cell only toward a lower portion of each nanowell substrate to photoinitiate a chemical reaction between the covalently bonded polymer and the second group of reactants, the photoinitiated chemical reaction covalently bonding a target reactant to only the lower portion of each nanowell substrate.

[0042] With respect to the embodiment shown in FIG. 6 , the following are non-limiting examples of various aspects of the described method. Exemplary techniques for forming an optical coupling grating on a substrate (which may be glass) include photolithographic patterning of a silicon dioxide (SiO 2 ) grating, lift-off processes, laser etching, and nanoimprinting. Exemplary materials for the core layer include low-refractive-index nanoimprint lithography (NIL) resins and low-refractive-index polymers. Exemplary techniques for depositing the core layer on the substrate include sputter coating and spin coating. Exemplary materials for the cladding layer include high-refractive-index resins, high-refractive-index polymers, and metal oxides such as tantalum pentoxide (Ta 2 O 5 ). Exemplary techniques for depositing the cladding layer on the core layer include vacuum thin-film deposition, sputter coating, and spin coating. Exemplary techniques for forming nanowells in the second material layer include nanoimprint lithography patterning. Exemplary techniques for coating the silanized cladding layer and nanowells with the first group of reactants include sputter coating and spin coating. An exemplary technique for introducing the second group of reactants into the nanowells includes using a microfluidic pumping system, such as a peristaltic pump. An exemplary technique for directing light inward through a light coupling grating into the flow cell includes using focusing optics to direct light from an external light source into the flow cell. Exemplary photoinitiated chemical reactions include azide-alkyne chemistry using blue light at a predetermined wavelength of approximately 450 nm to 495 nm, tetrazole-alkene chemistry using green light at a predetermined wavelength of approximately 520 nm to 560 nm, and metal-free azide / acetylene cycloaddition reactions utilizing triple bond masking using dibenzocyclooctyne as the cyclopropenone (see, e.g., JACS 2009, 131, 15769-15776).

[0043] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and web pages, regardless of the form such literature and similar materials may be in, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application in terms of, but not limited to, defined terms, term usage, described techniques, etc., this application controls.

[0044] The foregoing description is provided to enable one skilled in the art to practice the various configurations described herein. While the subject technology has been particularly described with reference to various diagrams and configurations, it should be understood that these are for illustrative purposes only and should not be construed as limiting the scope of the subject technology.

[0045] As used herein, elements or steps described in the singular and preceded by the words "a" or "an" should be understood as not excluding a plurality of such elements or steps, unless such exclusion is expressly stated. Furthermore, references to "one embodiment" are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Furthermore, unless expressly stated to the contrary, embodiments that "comprise" or "have" an element or elements having a particular characteristic may include the additional elements, whether or not they have that characteristic.

[0046] As used throughout this specification, the terms "substantially" and "about" are used to describe and account for small variations such as processing variations. For example, they can refer to ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0047] There may be many other ways to implement the subject technology. The various functions and elements described herein may be divided differently than shown without departing from the scope of the subject technology. Various modifications to these embodiments may be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments. Accordingly, many changes and modifications may be made to the subject technology by those skilled in the art without departing from the scope of the subject technology. For example, a different number of given modules or units may be used, different types or multiple types of given modules or units may be used, given modules or units may be added, or given modules or units may be omitted.

[0048] Underlined and / or italicized headings and subheadings are used for convenience only, do not limit the subject technology, and are not referred to in connection with interpreting the description of the subject technology. All structural and functional equivalents to the elements of the various embodiments described throughout this disclosure that are known, or that later become known, to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the subject technology. Furthermore, nothing disclosed herein is intended to be publicly exclusive, regardless of whether such disclosure is expressly recited in the description above.

[0049] It is understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (provided that such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

Claims

1. 1. A method for patterning a flow cell substrate, comprising: preparing a flow cell for a photoinitiated chemical reaction; The flow cell comprises: a substrate having an optical coupling grating formed thereon; a first layer of material disposed over the substrate; a second layer of material disposed on the first layer of material; nanowells formed in the second layer of material, each nanowell including an upper portion and a lower portion; Preparing the flow cell comprises: silanizing the second layer of material; coating the silanized second material layer and nanowells with a first group of reactants; introducing a second group of reactants into the nanowell, wherein the second group of reactants comprises at least one target reactant, a copper chelating ligand, and a light-sensitive photoinitiator system; directing light inwardly within the flow cell through the optical coupling grating only to the lower portion of each nanowell to photoinitate a chemical reaction between the first group of reactants and the second group of reactants; Including, wherein the photoinitiated chemical reaction covalently attaches the target reactant only to the bottom portion of each nanowell.

2. The method of claim 1 , further comprising washing unreacted materials from the nanowells.

3. 3. The method of claim 1 or 2, further comprising using a polymer as the first group of reactants and an azide moiety attached to the polymer.

4. The method of claim 3 further comprising using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer.

5. 5. The method of any one of claims 1 to 4, further comprising using a camphorquinone-amine photosensitizer system using a light wavelength of 470 nm as the photosensitive photoinitiator system.

6. The method of any one of claims 1 to 5, further comprising using an alkyne-conjugated primer as the target reactant.

7. 7. The method of any one of claims 1 to 6, further comprising using an alkyne-linked fluorophore as the target reactant.

8. The method of any one of claims 1 to 7, further comprising using a laser as the light source.

9. 9. The method according to any one of claims 1 to 8, wherein the material used for the substrate has a refractive index in the range of 1.0 to 1.3 and the material used for the first material layer has a refractive index in the range of 2.0 to 2.

15.

10. 1. A method for patterning a flow cell substrate, comprising: fabricating a planar waveguide flow cell; Fabricating the planar waveguide flow cell comprises: forming a layer of an optical coupling grating on a glass substrate layer; depositing a core layer on the optical coupling grating layer; depositing a cladding layer on the core layer; forming nanowell substrates within the cladding layer, each nanowell substrate including an upper portion and a lower portion, the nanowell substrates defining a gap area therebetween; silanizing the cladding layer; coating the silanized cladding layer and nanowell substrate with a first group of reactants; introducing a second group of reactants into the nanowell substrate, wherein the second group of reactants comprises at least one target reactant, a copper chelating ligand, and a light-sensitive photoinitiator system; directing light inwardly within the planar waveguide flow cell through the optical coupling grating only to the lower portion of each nanowell substrate to photoinitate a chemical reaction between the first group of reactants and the second group of reactants; Including, wherein the photoinitiated chemical reaction covalently attaches the target reactant to only the lower portion of each nanowell substrate.

11. The method of claim 10 , further comprising washing unreacted materials from the nanowell substrate.

12. 12. The method of claim 10 or 11, further comprising using a polymer as the first group of reactants and an azide moiety attached to the polymer.

13. The method of claim 12, further comprising using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer.

14. 14. The method of any one of claims 10 to 13, further comprising using a camphorquinone-amine photosensitizer system using a light wavelength of 470 nm as the photosensitive photoinitiator system.

15. The method of any one of claims 10 to 14, further comprising using an alkyne-conjugated primer as the target reactant.

16. 16. The method of any one of claims 10 to 15, further comprising using an alkyne-linked fluorophore as the target reactant.

17. The method of any one of claims 10 to 16, further comprising using a laser as the light source.

18. 18. The method according to any one of claims 10 to 17, wherein the material used for the layer of the optical coupling grating has a refractive index in the range of 1.0 to 1.3, and the material used for the core layer has a refractive index in the range of 2.0 to 2.

15.

19. 1. A method for patterning a flow cell substrate, comprising: fabricating a planar waveguide flow cell; Fabricating the planar waveguide flow cell comprises: forming a layer of an optical coupling grating on a glass substrate layer; depositing a core layer on the optical coupling grating layer; depositing a cladding layer on the core layer; forming nanowell substrates within the cladding layer, each nanowell substrate including an upper portion and a lower portion, the nanowell substrates defining a gap area therebetween; silanizing the cladding layer; coating the cladding layer and the nanowell substrate with a first group of reactants, wherein the first group of reactants further comprises a polymer, an azide moiety attached to the polymer, a copper ligand, and a light-sensitive photoinitiator system; directing light of a predetermined wavelength inwardly within the planar waveguide flow cell only to the lower portion of each nanowell substrate to photoinitiate a chemical reaction between reactants in the first group of reactants, wherein the photoinitiated chemical reaction covalently bonds the polymer to only the lower portion of each nanowell substrate; introducing a second group of reactants into the nanowell substrate, wherein the second group of reactants comprises at least one target reactant, a copper ligand, and a light-sensitive photoinitiator system; directing light of a predetermined wavelength inwardly within the planar waveguide flow cell only to the lower portion of each nanowell substrate to photoinitiate a chemical reaction between the covalently attached polymer and the second group of reactants, wherein the photoinitiated chemical reaction covalently attaches the target reactants only to the lower portion of each nanowell substrate; A method comprising:

20. 20. The method of claim 19, further comprising washing unreacted materials from the nanowell substrate after each photoinitiated chemical reaction.

21. The method of claim 19 or 20, further comprising using 3-azidopropyltrimethoxysilane to silanize the cladding layer.

22. 22. The method of any one of claims 19 to 21, further comprising using poly(N-(5-azidoacetamidylpentyl)acrylamide) as the polymer.

23. 23. The method of any one of claims 19 to 22, further comprising using a camphorquinone-amine photosensitizer system using a light wavelength of 470 nm as the photosensitive photoinitiator system.

24. 24. The method of any one of claims 19 to 23, further comprising using an alkyne-conjugated primer as the target reactant.

25. 24. The method of any one of claims 19 to 23, further comprising using an alkyne-linked fluorophore as the target reactant.

26. The method of any one of claims 19 to 25, further comprising using a laser as the light source.

27. 27. The method according to claim 19, wherein the material used for the layer of the optical coupling grating has a refractive index in the range of 1.0 to 1.3, and the material used for the core layer has a refractive index in the range of 2.0 to 2.

15.

28. The method of any one of claims 1 to 18, wherein the silanization is carried out by chemical vapor deposition of norbornene silane.

29. 19. The method of any one of claims 4 to 9 and 13 to 18, wherein poly(N-(5-azidoacetamidylpentyl)acrylamide) is spin coated onto the silanized layer by the following procedure: step 1—600 rpm, 5 seconds, acceleration 1500 rpm / sec; step 2—1500 rpm, 30 seconds, acceleration 5000 rpm / sec; step 3—4000 rpm, 5 seconds, acceleration 5000 rpm / sec; step 4—600 rpm, 5 seconds, acceleration 5000 rpm / sec.

30. 30. The method of claim 29, further comprising step 5, subsequently heating at 65-75°C for 1 hour.

31. A method according to any one of claims 21 to 27, wherein the silanization is carried out by vapor deposition of 3-azidopropyltrimethoxysilane.

32. 32. The method of claim 31 , wherein after vapor deposition of 3-azidopropyltrimethoxysilane, the poly(N-(5-azidoacetamidylpentyl)acrylamide) is crosslinked to the azido groups using a photoinitiated reaction using a bifunctional crosslinker.

33. 33. The method of any one of claims 4, 13, 22, 29 and 32, wherein the poly(N-(5-azidoacetamidylpentyl)acrylamide) is covalently attached to a surface at the underside of the nanowell using the light.

34. 33. The method of claim 32, wherein the light is laser light.

35. A method according to any one of claims 31 to 33, wherein the copper is then removed using a dilute solution of EDTA (0.1 M).

36. 20. The method of claim 10 or 19, wherein there is no polishing step to remove existing surface chemistry found in the interstitial areas between the nanowells.

37. 37. The method of any one of claims 1 to 36, wherein light is directed through the optical coupling grating only to a lower portion of each nanowell to photoinitate the chemical reaction between the first group of reactants and the second group of reactants.

38. 1. A flow cell for photoinitiated chemical reactions, comprising: The flow cell comprises: A substrate; an optical coupling grating layer disposed on the substrate, wherein the optical coupling grating layer has a refractive index; a core layer disposed on the grating layer, wherein the core layer has a refractive index, the refractive index of the core layer being greater than the refractive index of the optical coupling grating layer; a silanized layer disposed on the core layer, wherein a plurality of nanowells in the silanized layer are present; Equipped with A flow cell, wherein the nanowells are separated by interstitial areas.

39. 39. The flow cell of claim 38, wherein the substrate is glass.

40. 40. The flow cell of claim 38 or 39, wherein the optical coupling grating layer and the core layer are formed from resin.

41. 41. The flow cell of claim 40, wherein the optical coupling grating layer and / or the core layer are formed from tantalum pentoxide.

42. The flow cell of any one of claims 38 to 41, wherein the silanized layer is formed from a polymer clad layer.

43. 43. The flow cell of any one of claims 38 to 42, wherein the refractive index of the optical coupling grating layer is in the range of 0.5 to 2.0, and the refractive index of the core layer is in the range of 1.5 to 2.

5.

44. 44. The flow cell of any one of claims 38 to 43, wherein poly(N-(5-azidoacetamidylpentyl)acrylamide) is covalently attached to the surface at the lower side of the nanowell.

45. 45. The flow cell of claim 44, further comprising a first group of reactants disposed on the poly(N-(5-azidoacetamidylpentyl)acrylamide) covalently attached to a surface at the lower portion of the nanowell.

46. 46. ​​The flow cell of claim 45, wherein a second group of reactants is disposed on the first group of reactants and comprises at least one target reactant, a copper chelating ligand, and a light-sensitive photoinitiator system.

47. 38. A method of sequencing nucleic acids using a flow cell manufactured according to any one of claims 1 to 37.

48. A system for photoinitiated chemical reactions, comprising a flow cell according to any one of claims 38 to 46 and a light source.

49. 49. The system of claim 48, wherein the light source is a laser light source.

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