WO2021028815A1 - Système et procédé pour former des motifs sur des substrats de cellules d'écoulement - Google Patents

Système et procédé pour former des motifs sur des substrats de cellules d'écoulement Download PDF

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WO2021028815A1
WO2021028815A1 PCT/IB2020/057507 IB2020057507W WO2021028815A1 WO 2021028815 A1 WO2021028815 A1 WO 2021028815A1 IB 2020057507 W IB2020057507 W IB 2020057507W WO 2021028815 A1 WO2021028815 A1 WO 2021028815A1
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Prior art keywords
layer
light
flow cell
reactants
nanowell
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PCT/IB2020/057507
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English (en)
Inventor
Steven Modiano
Dajun Yuan
Randall Smith
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Illumina Inc.
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Priority claimed from NL2023679A external-priority patent/NL2023679B1/en
Priority to CA3122944A priority Critical patent/CA3122944A1/fr
Priority to KR1020217020370A priority patent/KR20220038578A/ko
Priority to JP2021538044A priority patent/JP2022543176A/ja
Priority to EP20754048.5A priority patent/EP3880356A1/fr
Priority to BR112021012898A priority patent/BR112021012898A2/pt
Application filed by Illumina Inc. filed Critical Illumina Inc.
Priority to US17/414,612 priority patent/US20220134333A1/en
Priority to CN202080007617.XA priority patent/CN113286652A/zh
Priority to AU2020328254A priority patent/AU2020328254A1/en
Priority to MX2021006295A priority patent/MX2021006295A/es
Publication of WO2021028815A1 publication Critical patent/WO2021028815A1/fr
Priority to IL284388A priority patent/IL284388A/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J19/12—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electromagnetic waves
    • B01J19/122—Incoherent waves
    • B01J19/127—Sunlight; Visible light
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00427—Means for dispensing and evacuation of reagents using masks
    • B01J2219/00432—Photolithographic masks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00644—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being present in discrete locations, e.g. gel pads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J2219/0873—Materials to be treated
    • B01J2219/0879—Solid

Definitions

  • Nanowell substrates used in industrial processes may involve a complex process that includes surface functionalization of a microfluidic device such as, for example, a flow cell that houses or otherwise contains the nanowell substrates.
  • a particularly difficult aspect of nanowell substrate fabrication involves removing existing surface chemistry found in interstitial areas between nanowells by polishing these interstitial spaces. Problematic issues associated with this type of surface preparation include over polishing, under polishing, and scratching or damaging of patterned surfaces. Accordingly, there is a need for method for fabricating nanowell substrates that does not suffer from the aforementioned complexities and difficulties.
  • Implementations of the described system and methods have the benefits of enabling spatially resolved chemical patterning of nanometer scaled features within microfluidic devices such as flow cells and improving nanowell substrate fabrication by eliminating the polishing step completely.
  • Described methods utilize planar waveguides or similar devices to spatially control chemical functionalization of specific, predetermined areas of each nanowell substrate fabricated into a flow cell. Nanowell substrates are patterned into microfluidic flow cell devices using known flow cell fabrication processes.
  • the planar waveguide directs excitation light into only the bottom of each nanowell substrate where it photoinitiates a chemical reaction that covalently binds a target reactant to the bottom region of the nanowell substrate.
  • a first method for patterning flow cell substrates is provided.
  • This method comprises preparing a flow cell for a photoinitiated chemical reaction, wherein the flow cell includes a substrate having light coupling gratings formed thereon; a first layer of material disposed over the substrate; a second layer of material disposed over the first layer of material; and nanowells formed in the second layer of material, wherein each nanowell includes a top portion and a bottom portion, and wherein preparing the flow cell includes silanizing the second layer of material; and coating the silanized second layer of material and nano wells with a first group of reactants; introducing a second group of reactants into the nano wells, wherein the second group of reactants includes at least one target reactant, a copper chelated ligand, and a light- sensitive photoinitiator system; and directing light internally within the flow cell through the light coupling gratings to only the bottom portion of each nanowell for photo-initiating a chemical reaction between the first and second groups of reactants, wherein the photo-initiated chemical reaction covalently
  • the method further comprises washing unreacted reactants out of the nanowells; using a polymer and azide moieties that are bound to the polymer as the first group of reactants; using poly(N-(5-azidoacetamidylpentyl) acrylamide as the polymer; using a camphorquinone-amine photosensitizing system using a light wavelength of about 470 nm as the light-sensitive photoinitiator system; using an alkyne-linked primer as the target reactant; using an alkyne-linked fluorophore as the target reactant; using a laser as a source of light; 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 layer, although other values are possible.
  • a second method for patterning flow cell substrates comprises fabricating a planar waveguide flow cell by forming a layer of light coupling gratings on a glass substrate layer; depositing a core layer on the layer of light coupling gratings; depositing a cladding layer on the core layer; and forming nano well substrates in the cladding layer, wherein each nanowell substrate includes a top portion and a bottom portion, and wherein the nanowell substrates define interstitial regions therebetween; silanizing the cladding layer; coating the silanized cladding layer and nanowell substrates with a first group of reactants; introducing a second group of reactants into the nanowell substrates, wherein the second group of reactants includes at least one target reactant, a copper chelated ligand, and a light-sensitive photoinitiator system; directing light internally within the planar waveguide flow cell through the light coupling gratings to only the bottom portion of each nanowell substrate
  • the method further comprises washing unreacted reactants out of the nano wells; using a polymer and azide moieties that are bound to the polymer as the first group of reactants; using poly(N-(5-azidoacetamidylpentyl) acrylamide as the polymer; using a camphorquinone-amine photosensitizing system using a light wavelength of about 470 nm as the light-sensitive photoinitiator system; using an alkyne-linked primer as the target reactant; using an alkyne-linked fluorophore as the target reactant; using a laser as a source of light; and using a material having a refractive index in the range of 1.0 to 1.3 for the layer of light coupling gratings and a material having refractive index in the range of 2.0 to 2.15 for the core layer, although other values are possible.
  • a third method for spatially patterning flow cell substrates comprises fabricating a planar waveguide flow cell by forming a layer of light coupling gratings on a glass substrate layer; depositing a core layer on the layer of light coupling gratings; depositing a cladding layer on the core layer; and forming nano well substrates in the cladding layer, wherein each nanoweh substrate includes a top portion and a bottom portion, and wherein the nanoweh substrates define interstitial regions therebetween; and silanizing the cladding layer; coating the cladding layer and nanoweh substrates with a first group of reactants, wherein the first group of reactants further includes a polymer, azide moieties bound to the polymer, a copper ligand and a light-sensitive photoinitiator system; directing light of a predetermined wavelength internally within the planar waveguide flow cell to only the bottom portion of each nanoweh substrate for photo-initiating a chemical reaction
  • the method further comprises washing unreacted reactants out of the nanoweh substrates after each photo-initiated chemical reaction; using 3-azidopropyltrimethoxysilane for silanizing the cladding layer; using poly(N-(5-azidoacetamidylpentyl) acrylamide as the polymer; using a camphorquinone-amine photosensitizing system using a light wavelength of about 470 nm as the light-sensitive photoinitiator system; using an alkyne-linked primer as the target reactant; using an alkyne-linked fluorophore as the target reactant; using a laser as a source of light; and using a material having a refractive index in the range of 1.0 to 1.3 for the layer of light coupling gratings and a material having refractive index in the range of 2.0 to 2.15 for the core layer, although other values are possible.
  • the methods herein include silanizing carried out by way of vapor deposition of norbornene silane.
  • poly(N-(5-azidoacetamidylpentyl) acrylamide is spin coated on the silanized layer by way of the following procedure: Step 1—600 rpm, 5 seconds, acceleration 1500 rpm/second; Step 2—1500 rpm, 30 seconds, acceleration 5000 rpm/second; Step 3—4000 rpm, 5 seconds, acceleration 5000 rpm/second; Step 4—600 rpm, 5 seconds, acceleration 5000 rpm/second, and subsequently preferably heating at 65-75°C for 1 hour.
  • silanizing is carried out by vapor deposition of 3-azidopropyltrimethoxysilane, whereafter poly(N-(5-azidoacetamidylpentyl) acrylamide is preferably crosslinked to azide groups using a photo-initiated reaction that uses a bi-functional crosslinker such as NH-bis (PEG-2 Propargyl), a photo-initiator, (e.g., camphorquinone at 470 nm), and copper sulfate with a ligand, for example PMDTA.
  • a bi-functional crosslinker such as NH-bis (PEG-2 Propargyl)
  • a photo-initiator e.g., camphorquinone at 470 nm
  • copper sulfate with a ligand for example PMDTA.
  • poly(N-(5-azidoacetamidylpentyl) acrylamide is covalently bound to surfaces at the bottom of the nanowells using the light, preferably laser light.
  • the methods herein include light directed through the light coupling gratings to only a bottom portion of each nanowell for photo-initiating the chemical reaction between the first and second groups of reactants.
  • copper is subsequently removed using a dilute solution of EDTA (0.1M).
  • the method is absent a polishing step to remove any existing surface chemistry found in interstitial areas between the nano wells.
  • a flow cell comprises: a substrate, a light coupling grating layer arranged on the substrate, which light coupling grating layer has a refractive index, a core layer arranged on the light coupling grating layer, which core layer has a refractive index, wherein the refractive index of the core layer is greater than the refractive index of the light coupling grating layer, a silanized layer arranged on the core layer in which silanized layer multiple nano wells are separated by interstitial areas.
  • the substrate is glass.
  • the light coupling grating layer and core layer are formed from a resin.
  • the light coupling grating layer and/or the core layer are formed from a tantalum pentoxide.
  • the flow cell includes poly(N-(5-azidoacetamidylpentyl) acrylamide covalently bound to surfaces at the bottom of the nano wells.
  • the flow cell includes the second group of reactants arranged on the first group of reactants, and includes at least one target reactant, a copper chelated ligand, and a light sensitive photoinitiator system.
  • the flow cell is a planar waveguide flow cell. This flow cell may be used for nucleic acid sequencing including high-volume sequencing-by-synthesis and may be made in accordance with any method disclosed herein for manufacturing flow cells and spatially patterning flow cell substrates.
  • a method for patterning flow cell substrates comprising: preparing a flow cell for a photoinitiated chemical reaction, wherein the flow cell includes a substrate having light coupling gratings formed thereon; a first layer of material disposed over the substrate; a second layer of material disposed over the first layer of material; and nano wells formed in the second layer of material, wherein each nanoweh includes a top portion and a bottom portion, and wherein preparing the flow cell includes: silanizing the second layer of material; coating the silanized second layer of material and nano wells with a first group of reactants; introducing a second group of reactants into the nanowehs, wherein the second group of reactants includes at least one target reactant, a copper chelated ligand, and a light- sensitive photoinitiator system; and directing light internally within the flow cell through the light coupling gratings to only the bottom portion of each nanoweh for photo-initiating a chemical reaction between the first and second groups of reactants, wherein
  • a method for patterning flow cell substrates comprising: fabricating a planar waveguide flow cell, wherein fabricating the planar waveguide flow cell includes: forming a layer of light coupling gratings on a glass substrate layer; depositing a core layer on the layer of light coupling gratings; depositing a cladding layer on the core layer; and forming nanowell substrates in the cladding layer, wherein each nanowell substrate includes a top portion and a bottom portion, and wherein the nanowell substrates define interstitial regions therebetween; silanizing the cladding layer; coating the silanized cladding layer and nano well substrates with a first group of reactants; introducing a second group of reactants into the nanowell substrates, wherein the second group of reactants includes at least one target reactant, a copper chelated ligand, and a light-sensitive photoinitiator system; and directing light internally within the planar waveguide flow cell through the light coupling gratings to only the bottom portion of each nanowell substrate
  • a method for patterning flow cell substrates comprising: fabricating a planar waveguide flow cell, wherein fabricating the planar waveguide flow cell includes: forming a layer of light coupling gratings on a glass substrate layer; depositing a core layer on the layer of light coupling gratings; depositing a cladding layer on the core layer; and forming nanowell substrates in the cladding layer, wherein each nanowell substrate includes a top portion and a bottom portion, and wherein the nanowell substrates define interstitial regions therebetween; and silanizing the cladding layer; coating the cladding layer and nanowell substrates with a first group of reactants, wherein the first group of reactants further includes a polymer, azide moieties bound to the polymer, a copper ligand, and a light-sensitive photoinitiator system; directing light of a predetermined wavelength internally within the planar waveguide flow cell to only the bottom portion of each nanoweh substrate for photo-initiating a chemical reaction between the react
  • (5-azidoacetamidylpentyl) acrylamide is spin coated on the silanized layer by way of the following procedure: Step 1—600 rpm, 5 seconds, acceleration 1500 rpm/second; Step 2—1500 rpm, 30 seconds, acceleration 5000 rpm/second; Step 3—4000 rpm, 5 seconds, acceleration 5000 rpm/second; Step 4—600 rpm, 5 seconds, acceleration 5000 rpm/second, and subsequently preferably heating at 65-75°C for 1 hour.
  • a flow cell (10) for a photoinitiated chemical reaction which flow cell comprises: a substrate (100), a light coupling grating layer (200) arranged on the substrate (100), which light coupling grating layer (200) has a refractive index, a core layer (300) arranged on the grating layer (200), which core layer (300) has a refractive index, wherein the refractive index of the core layer (300) is greater than the refractive index of the light coupling grating layer (200), a silanized layer (400) arranged on the core layer (300) in which silanized layer (400) multiple nanowehs (500) are present, and wherein the nano wells (500) are separated by interstitial areas (600).
  • a system for photoinitiated chemical reactions comprising a flow cell according to any of the preceding clauses 35-43 and a light source, preferably a laser light source.
  • FIG. 1A depicts the structure of a planar waveguide flow cell in accordance with one implementation of the disclosed system and method
  • FIG. IB depicts, in one implementation, the flow cell of FIG. 1A, wherein the upper surface of the flow cell has been hydrogel coated with azide moieties using poly(N-(5- azidoacetamidylpentyl) acrylamide -co-acrylamide) (PAZAM);
  • FIG. 1C depicts, in one implementation, the flow cell of FIG. IB, wherein reactants have been introduced into the flow cell, wherein the reactants include alkyne-linked primers, a copper chelated ligand, and a light sensitive photoinitiator system;
  • FIG. ID depicts, in one implementation, the flow cell of FIG. 1C, wherein a planar waveguide has been coupled to the flow cell and is directing light into the flow cell;
  • FIG. IE depicts, in one implementation, a close-up view of a nanowell within the flow cell showing the areas of the nanowell onto which primers have been covalently bound;
  • FIG. 2A depicts a chemical reaction wherein a PAZAM-azide polymer is attached to the surface of a planar waveguide flow cell in one implementation of the disclosed method
  • FIG. 2B depicts a chemical reaction wherein a photo-initiated alkyne-azide click reaction is used to covalently bind a target reactant to a nanowell substrate in one implementation of the disclosed method;
  • FIG. 3A depicts a chemical reaction wherein a PAZAM-azide polymer coated onto the surface of a planar waveguide flow cell is patterned with a first photo-initiated click reaction in another implementation of the disclosed method;
  • FIG. 3B depicts a chemical reaction wherein primers (fluorophores) are patterned onto the PAZAM layer of FIG. 3A using a second photo-initiated click reaction.
  • FIG. 4 is a flowchart depicting an implementation of a first method for patterning flow cell substrates
  • FIG. 5 is a flowchart depicting an implementation of a second method for patterning flow cell substrates.
  • FIG. 6 is a flowchart depicting an implementation of a third method for patterning flow cell substrates.
  • Implementations of the disclosed system and method utilize planar waveguides to spatially control the chemical functionalization of nanowells used in microfluidic devices such as flow cells used for sequencing-by-synthesis.
  • a planar waveguide i.e., a waveguide having a planar geometry, which guides light in only one direction
  • Implementations of the disclosed system and method may include a photoinitiated azide - alkyne click reaction, such as that used in sequencing-by-synthesis technologies.
  • azide functional groups are bound to a hydrogel layer formed across the surface of a microfluidic channel and alkyne -primer reactants are then added.
  • the azide-alkyne click reaction is photo-initiated using, in certain examples, a copper compound and a photoinitiator system such as a Type II photoinitiator system, e.g., camphorquinone. This photoinitiator system can use blue light at about 470 nm as the excitation source.
  • Mixed chemistries can be realized by adding alternate functional groups into the hydrogel layer by incorporating them into PAZAM.
  • azides and tetrazoles 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) and the tetrazole-alkene reaction is photoinitiated with light having a different wavelength between 520 nm and 560 nm (e.g., green).
  • the blue light employed has a wavelength of about 470 nm.
  • click reactions involve biocompatible small molecule reactions commonly used in bioconjugation for joining various substrates with specific biomolecules.
  • click chemistry does not refer to a single specific reaction, but rather refers to chemical methods for generating substances by joining small modular units to one another.
  • click reactions are used to join a biomolecule and a reporter molecule.
  • Click chemistry is not limited to biological applications and the concept of a click reaction has been used in pharmacological applications.
  • the azide-alkyne click reaction involves the copper-catalyzed reaction of an azide with and alkyne to form a 5-membered heteroatom ring: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC).
  • Cu(I)-catalyzed azide-alkyne cycloaddition CuAAC
  • Photoinitiated reactions of this nature are 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.
  • An example planar waveguide flow cell 10 includes substrate 100, which may be glass; light coupling grating layer 200, which may be a resin; core layer 300, which may be a resin having a refractive index higher than the resin used for light coupling grating layer 200 (e.g., tantalum pentoxide); and water buffer or patterned polymer cladding layer 400 (see FIG. 1A) in which multiple nanowells 500 are formed.
  • substrate 100 which may be glass
  • light coupling grating layer 200 which may be a resin
  • core layer 300 which may be a resin having a refractive index higher than the resin used for light coupling grating layer 200 (e.g., tantalum pentoxide)
  • water buffer or patterned polymer cladding layer 400 see FIG. 1A
  • the upper surface of cladding layer 400 is coated with a hydrogel (e.g., PAZAM) to which azide moieties 700 have been bound (see FIG. IB).
  • a hydrogel e.g
  • various reactants 800 are introduced into flow cell 10, wherein the reactants include alkyne -linked primers, a copper chelated ligand, a light sensitive photoinitiator system (see FIG. 1C).
  • the reactants include alkyne -linked primers, a copper chelated ligand, a light sensitive photoinitiator system (see FIG. 1C).
  • light is directed into planar waveguide flow cell 10 using a light-focusing optic such that light coupling gratings 200 and core layer 300 reflect the light internally within flow cell 10.
  • Evanescence waves penetrate the bottom of each nanowell 500 to initiate the desired chemical reaction.
  • Sequencing primers or other molecules are covalently bound only to the bottom region of each nanowell 500 and are spatially excluded from interstitial areas 600.
  • a washing step subsequently removes unreacted constituents such as any unbound hydrogel or unbound primers from the nano wells.
  • Suitable washing solutions include alkaline buffers having a pFi of at least 10 and sodium hydroxide.
  • FIG. IE provides a close-up of a nanowell 500 and areas 502 of FIG. IE indicate the location of the covalently bound sequencing primers (or other molecules) after the photoinitiated reaction is complete.
  • the refractive index of the material of light 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 material of core layer 300 is in the range of 1.5 to 2.5; 1.8 to 2.3; or 2.0 to 2.15.
  • Increasing the contrast difference between the refractive index values of light coupling grating layer 200 and core layer 300 may improve coupling efficiency of light into 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., light coupling grating layer 200).
  • core layer 300 includes or is made from a metal oxide such as, for example, tantalum pentoxide (Ta 2 0 5 ) .
  • a metal oxide such as, for example, tantalum pentoxide (Ta 2 0 5 ) .
  • norbornene silane [(5-bicycIo[2.2.1]HEPT-2- ENYL)ETHYL]TRIMETHOXYSILANE, tech-95, endo/exo isomers (Gelest Inc.);
  • PAZAM poly(N-(5-azidoacetamidylpentyl) acrylamide -co-acrylamide) of any acrylamide to Azapa ratio;
  • Azapa N-(5-azidoacetamidylpentyl) acrylamide;
  • copper(II) sulfate pentahydrate (CUS0 4 5H 2 0) (Sigma- Aldrich);
  • one implementation of the disclosed method utilizes a photoinitiation mechanism for the azide-alkyne click reaction that includes a camphorquinone- amine photosensitizing system using about 470 nm light.
  • a planar waveguide flow cell is fabricated as described above and cladding layer 400 is further processed by utilizing the PAZAM polymer, to which azide moieties have been bound.
  • the upper surface of cladding layer 400 is first silanized with a norbornene silane derivative using a chemical vapor deposition process; the process may be one that is standard in the industry. The surface is coated and then thermally cross-linked with PAZAM.
  • aqueous PAZAM 0.25%+5% ethanol
  • a thin film of PAZAM is obtained by spin coating with the following procedure: Step 1—600 rpm, 5 seconds, acceleration 1500 rpm/second; Step 2—1500 rpm, 30 seconds, acceleration 5000 rpm/second; Step 3—4000 rpm, 5 seconds, acceleration 5000 rpm/second; Step 4—600 rpm, 5 seconds, acceleration 5000 rpm/second.
  • substrates are heated at 65-75°C in oven or hot plate for 1 hour.
  • a polymer solution containing Alexa Fluor ® 488 (AF-488 alkyne) (30% w/w) with potassium carbonate (35 mM) and equal concentrations of CuS0 4 -5H 2 0, (PMDTA) and the photo-initiator camphorquinone is prepared (note: a variant of this solution includes sequencing primers rather than AF-488 alkyne).
  • the solution may be sonicated to facilitate dissolution.
  • the solution is then introduced into the channels of the flow cell and held there for subsequent photo-initiation of the click reaction using the excitation laser optics of the planar waveguide.
  • the copper is washed away from the flow cell channels using a solution of EDTA (0.1M), which forms a complex with the copper.
  • Fluorescence images may be collected using a confocal fluorescence microscope to verify the presence of the fluorophore within the nanowells of the flow cell.
  • the reaction occurs only in the bottom region of the nanowells because light is directed only into the nanowells by way of the planar waveguide grating. No polishing was used because dye-labeled molecules are bound only in the nanowells and not in interstitial spaces or regions between nano wells.
  • another implementation of the disclosed method provides a two-step photo-initiated click reaction that also uses a camphorquinone-amine photosensitizing system using about 470 nm light. This two-step process minimizes diffusion of reagents away from desired target regions, thereby leading to less functionalization of interstitial spaces or regions between flow cell nanowells.
  • a planar waveguide is fabricated as described above, and in a first step of this implementation (see FIG. 3A), PAZAM is photo-patterned in the nanowehs using a first photo-initiated click reaction.
  • This first step includes attaching azide groups to the surface of the flow cell using 3-azidopropyltrimethoxysilane with chemical vapor deposition process; the process may be one that is standard in the industry.
  • PAZAM is then crosslinked to azide groups using a photo-initiated reaction that uses a bi-functional crosslinker such as NH-bis (PEG-2 Propargyl), a photo-initiator, (e.g., CQ, Ex. 470 nm), and copper sulfate with a ligand (e.g., PMDTA), and light.
  • PAZAM is covalently bound to surfaces at the bottom of the nanowehs using laser light directed into the planar wave guide.
  • the copper is removed using a dilute solution of EDTA (0.1M), which forms a complex with the copper.
  • a fluorescent tag (or another molecule) is patterned using a second photo-initiated click reaction.
  • a polymer solution containing Alexa Fluor ® 488 (AF-488 alkyne) (30% w/w) with potassium carbonate (35 mM) and equal concentrations of CuS0 4 -5H 2 0, PMDTA, and the photo-initiator camphorquinone is prepared.
  • the solution may be sonicated to facilitate dissolution.
  • the solution is then introduced into the channels of the flow cell and held there for subsequent photo-initiation using the excitation laser optics of the planar waveguide.
  • the copper is washed away from the channels using a solution of EDTA (0.1M), which forms a complex with the copper.
  • Fluorescence images are collected using a confocal fluorescence microscope to verify the presence of the fluorophore within the nanowehs of the flow cell.
  • the reaction occurs only at the bottom of the nanowehs because light is directed only into the nanoweh by way of the planar waveguide grating. No polishing is necessary because Alexa-labeled dye molecules are bound only in the nanowehs and not in interstitial spaces or regions between nanowehs.
  • FIG. 4 is a flowchart depicting an implementation of a first method for patterning flow cell substrates.
  • First method for patterning flow cell substrates 400 comprises preparing a flow cell for a photoinitiated chemical reaction at block 402, wherein the flow cell includes a substrate having light coupling gratings formed thereon; a first layer of material disposed over the substrate; a second layer of material disposed over the first layer of material; and nanowehs formed in the second layer of material, wherein each nanowell includes a top portion and a bottom portion, and wherein preparing the flow cell includes silanizing the second layer of material at block 404; and coating the silanized second layer of material and nanowells with a first group of reactants at block 406; introducing a second group of reactants into the nano wells at block 408, wherein the second group of reactants includes at least one target reactant, a copper chelated ligand, and a light- sensitive photoinitiator system; and directing light internally within the flow cell through the
  • Example photoinitiated chemical reactions include azide-alkyne chemical reactions using blue light of a predetermined wavelength between about 450 nm and 495 nm; tetrazole-alkene chemical reactions using green light of a predetermined wavelength between about 520 nm and 560 nm; and metal free azide/acetylene cycloaddition reactions utilizing triple bond masking with dibenzocyclooctynes as cyclopropenone (see, for example, JACS 2009, 131, 15769-15776).
  • Example techniques for forming light coupling gratings on the substrate include photolithographic patterning of a silicon dioxide (Si0 2 ) grating, lift-off processes, laser etching, and nanoimprinting.
  • Example materials for the first layer of material include low refractive index nanoimprint lithography (NIL) resins and low refractive index polymers.
  • Example techniques for depositing the first layer of material on the substrate include sputter coating and spin coating.
  • Example materials for the second layer of material include high refractive index resins, high refractive index polymers, and metal oxides such as, for example, tantalum pentoxide (Ta 2 Os) .
  • Example techniques for depositing the second layer of material on the first layer of material include vacuum thin film vapor deposition, sputter coating and spin coating.
  • Example techniques for forming the nanowells in the second layer of material include nanoimprinting lithographic patterning.
  • Example techniques for coating the silanized second layer of material and nanowells with a first group of reactants include sputter coating and spin coating.
  • Example techniques for introducing a second group of reactants into the nanowells include using a microfluidic pump system such as, for example, a peristaltic pump.
  • Example techniques for directing light internally within the flow cell through the light coupling gratings include using a focusing optic to direct light from an external source into the flow cell.
  • FIG. 5 is a flowchart depicting an implementation of a second method for patterning flow cell substrates.
  • Second method for patterning flow cell substrates 500 comprises fabricating a planar waveguide flow cell at block 502 by forming a layer of light coupling gratings on a glass substrate layer at block 504; depositing a core layer on the layer of light coupling gratings at block 506; depositing a cladding layer on the core layer at block 508; and forming nano well substrates in the cladding layer at block 510, wherein each nano well substrate includes a top portion and a bottom portion, and wherein the nanowell substrates define interstitial regions therebetween; silanizing the cladding layer at block 512; coating the silanized cladding layer and nano well substrates with a first group of reactants at block 514; introducing a second group of reactants into the nanowell substrates at block 516, wherein the second group of reactants includes at least one target reactant, a copper chelated ligand
  • Example techniques for forming a layer of light coupling gratings on the substrate include photolithographic patterning of a silicon dioxide (Si0 2 ) grating, lift-off processes, laser etching, and nanoimprinting.
  • Example materials for the core layer include low refractive index nanoimprint lithography (NIL) resins and low refractive index polymers.
  • Example techniques for depositing the core layer on the substrate include sputter coating and spin coating.
  • Example materials for the cladding layer include high refractive index resins, high refractive index polymers, and metal oxides such as, for example, tantalum pentoxide (Ta 2 0 5 ) .
  • Example techniques for depositing the cladding layer on the core layer include vacuum thin film vapor deposition, sputter coating and spin coating.
  • Example techniques for forming the nanowells in the second layer of material include nanoimprinting lithographic patterning.
  • Example techniques for coating the silanized cladding layer and nanowehs with a first group of reactants include sputter coating and spin coating.
  • Example techniques for introducing a second group of reactants into the nanowehs include using a microfluidic pump system such as, for example, a peristaltic pump.
  • Example techniques for directing light internally within the flow cell through the light coupling gratings include using a focusing optic to direct light from an external source into the flow cell.
  • Example photoinitiated chemical reactions include azide-alkyne chemical reactions using blue light of a predetermined wavelength between about 450 nm and 495 nm; tetrazole-alkene chemical reactions using green light of a predetermined wavelength between about 520 nm and 560 nm; and metal free azide/acetylene cycloaddition reactions utilizing triple bond masking with dibenzocyclooctynes as cyclopropenone (see, for example, JACS 2009, 131, 15769- 15776).
  • FIG. 6 is a flowchart depicting an implementation of a third method for patterning flow cell substrates.
  • Third method for patterning flow cell substrates 600 comprises fabricating a planar waveguide flow cell at block 602 by forming a layer of light coupling gratings on a glass substrate layer at block 604; depositing a core layer on the layer of light coupling gratings at block 606; depositing a cladding layer on the core layer at block 608; and forming nanoweh substrates in the cladding layer at block 610, wherein each nano well substrate includes a top portion and a bottom portion, and wherein the nanowell substrates define interstitial regions therebetween; and silanizing the cladding layer at block 612; coating the cladding layer and nano well substrates with a first group of reactants at block 614, wherein the first group of reactants further includes, a polymer, azide moieties bound to the polymer, a copper ligand, and a light-sensitive photoinitiator system
  • Example techniques for forming light coupling gratings on the substrate include photolithographic patterning of a silicon dioxide (Si0 2 ) grating, lift-off processes, laser etching, and nanoimprinting.
  • Example materials for the core layer include low refractive index nanoimprint lithography (NIL) resins and low refractive index polymers.
  • Example techniques for depositing the core layer on the substrate include sputter coating and spin coating.
  • Example materials for the cladding layer include high refractive index resins, high refractive index polymers, and metal oxides such as, for example, tantalum pentoxide (Ta 2 Os) .
  • Example techniques for depositing the cladding layer on the core layer include vacuum thin film vapor deposition, sputter coating and spin coating.
  • Example techniques for forming the nanowells in the second layer of material include nanoimprinting lithographic patterning.
  • Example techniques for coating the silanized cladding layer and nanowehs with a first group of reactants include sputter coating and spin coating.
  • Example techniques for introducing a second group of reactants into the nanowehs include using a microfluidic pump system such as, for example, a peristaltic pump.
  • Example techniques for directing light internally within the flow cell through the light coupling gratings include using a focusing optic to direct light from an external source into the flow cell.
  • Example photoinitiated chemical reactions include azide-alkyne chemical reactions using blue light of a predetermined wavelength between about 450 nm and 495 nm; tetrazole-alkene chemical reactions using green light of a predetermined wavelength between about 520 nm and 560 nm; and metal free azide/acetlylene cycloaddition reactions utilizing triple bond masking with dibenzocyclooctyynes as cyclopropenone (see, for example, JACS 2009, 131, 15769-15776).

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Abstract

L'invention concerne également un procédé de formation de motifs sur des substrats de cellules d'écoulement à l'aide de réactions chimiques photo-initiées qui comprend la fabrication d'une cellule d'écoulement de guide d'ondes planaire par formation d'une couche de réseaux de couplage de lumière sur une couche de substrat de verre ; le dépôt d'une couche centrale sur la couche de réseaux de couplage de lumière ; le dépôt d'une couche de gainage sur la couche centrale ; et formation des nano-puits dans la couche de gainage ; silanisation de la couche de gainage ; le revêtement de la couche de gainage silanisée et des nano-puits avec un premier groupe de réactifs ; l'introduction d'un second groupe de réactifs dans les nano-puits, le second groupe de réactifs comprenant un réactif cible et un système photo-initiateur sensible à la lumière ; couplage d'une source de lumière aux réseaux de couplage de lumière et diriger la lumière à l'intérieur de la cellule d'écoulement de guide d'ondes planaire pour photo-initier une réaction chimique entre les premier et second groupes de réactifs, la réaction chimique initiée par la lumière se liant de manière covalente au réactif cible uniquement à la partie inférieure de chaque nanopuits.
PCT/IB2020/057507 2019-08-09 2020-08-10 Système et procédé pour former des motifs sur des substrats de cellules d'écoulement WO2021028815A1 (fr)

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MX2021006295A MX2021006295A (es) 2019-08-09 2020-08-10 Sistema y metodo para estampar sustratos de celda de flujo.
KR1020217020370A KR20220038578A (ko) 2019-08-09 2020-08-10 플로우 셀 기재를 패턴화하기 위한 시스템 및 방법
JP2021538044A JP2022543176A (ja) 2019-08-09 2020-08-10 フローセル基板をパターン化するためのシステム及び方法
EP20754048.5A EP3880356A1 (fr) 2019-08-09 2020-08-10 Système et procédé pour former des motifs sur des substrats de cellules d'écoulement
BR112021012898A BR112021012898A2 (pt) 2019-08-09 2020-08-10 Métodos para padronizar substratos de célula de fluxo, célula de fluxo para uma reação química fotoiniciada, método de sequenciamento de ácidos nucleicos e sistema para reações químicas fotoiniciadas
CA3122944A CA3122944A1 (fr) 2019-08-09 2020-08-10 Systeme et procede pour former des motifs sur des substrats de cellules d'ecoulement
US17/414,612 US20220134333A1 (en) 2019-08-09 2020-08-10 System and method for patterning flow cell substrates
CN202080007617.XA CN113286652A (zh) 2019-08-09 2020-08-10 用于使流通池基板图案化的系统和方法
AU2020328254A AU2020328254A1 (en) 2019-08-09 2020-08-10 System and method for patterning flow cell substrates
IL284388A IL284388A (en) 2019-08-09 2021-06-26 Method and system for designing flow cell substrates

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