RU2785680C2 - Flow cells - Google Patents

Abstract

FIELD: biology; chemistry.

SUBSTANCE: group of inventions relates to the field of biological and chemical researches. A flow cell is disclosed, containing a base; a selectively removable self-assembled porous molecular mesh located on the base, defining a pattern of open sections of the base; and a nano-structure located at least on some of open sections, and polymerase attached to the nano-structure. The nano-structure is an electroconductive channel including material selected from a group consisting of a conductor and a semiconductor, and it has a geometrical shape selected from a group consisting of a tube, wire, and band, where the selectively removable self-assembled porous molecular mesh is a planar supra-molecular mesh of amine and diimide. A sequencing system and a method for the production of the above-mentioned flow cell are also disclosed.

EFFECT: group of inventions provides improved specificity of attachment of biomolecules and better sensitivity of sensors.

13 cl, 11 dwg

Description

Cross-references to related applications

This application claims the benefit of Provisional Patent Application US 62/715177, filed August 6, 2018, the contents of which are hereby incorporated by reference in their entirety.

Prior Art

Various biological or chemical research protocols involve conducting a large number of controlled reactions on local surfaces of substrates or inside pre-installed reaction chambers. In this case, certain reactions can be observed or detected, and subsequent analysis may allow the identification or determination of the properties of the chemicals involved in the reaction. In some examples, controlled reactions generate fluorescence and thus an optical system can be used for detection. In other examples, controlled reactions change charge, conductivity, or some other electrical property, and thus an electronic system can be used for detection.

The essence of the invention

The first aspect of the present invention relates to a flow cell. The flow cell includes a substrate; located on the basis of a selectively removable porous molecular network that defines the open areas of the base; and a surface chemical for sequencing located on at least some of the exposed areas, where the surface chemical is selected from the group consisting of: i) an activated pad, a polymer layer attached to the activated substrate and a primer attached to the polymer layer; or ii) a nanostructure and an enzyme attached to the nanostructure.

In one example of a flow cell, the selectively removable porous molecular network is a planar supramolecular network of amine and diimide. In one example, the amine is melamine or an analogue thereof, and the diimide is perylenetetracarboxylic acid diimide or an analogue thereof.

In one example of a flow cell, the surface chemical is ii) a nanostructure and an enzyme attached to the nanostructure, where in ii): the nanostructure is an electrically conductive channel comprising a material selected from the group consisting of a conductor and a semiconductor and has a geometric a shape selected from the group consisting of tube, wire and strip; and the enzyme is a polymerase. In one example, one polymerase is attached to one nanostructure. In one example, the nanostructure is electrically connected to two electrodes. In one example, the flow cell further includes a connector that attaches the polymerase to the nanostructure. In one example, the nanostructure is selected from the group consisting of silicon nanowire and carbon nanotube.

In one example of a flow cell, the surface chemical is i) an activated layer, a polymer layer, and a primer; where in i) the polymer layer is:

where: R ¹ represents H or optionally substituted alkyl; R ^A is selected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, and thiol; each of R ⁵ , R ₆ and R ₈ is independently selected from the group consisting of H and optionally substituted alkyl; each of the -(CH ₂ ) _p - groups may be optionally substituted; p is an integer from 1 to 50; n is an integer from 1 to 50000; and m is an integer from 1 to 100000.

It should be understood that any features of the flow cell discussed in this work can be combined with each other in any desired manner and/or with the formation of any desired configuration.

The second aspect of the present invention relates to a system for sequencing. The sequencing system includes a removable flow cell including: a base; located on the base selectively removable porous molecular network that defines the open areas of the base; nanostructure attached to each of the open areas of the base; an enzyme attached to at least some of the nanostructures; and openings for inlet and outlet of fluids; a reservoir for i) permanently accommodating a removable flow cell or ii) for inserting a removable flow cell; and a fluidic system for selectively supplying a de-patterning reagent to a removable flow cell in order to remove a selectively removable porous molecular network from the base and to supply a patterning reagent to a removable flow cell ) in order to apply a new (fresh) selectively removable porous molecular network to the substrate.

In one example of a sequencing system, each of the nanostructures is an electrically conductive channel, including a material selected from the group consisting of a conductor and a semiconductor, and has a geometric shape selected from the group consisting of a tube, wire, and strip; and each of the nanostructures is available for individual electroaddressing.

In one example of a sequencing system, the fluid system includes a cartridge reservoir for housing a reagent cartridge including at least a disruptive reagent and a structurant reagent. In some examples, the sequencing system further includes a reagent cartridge, and the reagent cartridge includes: a fluid storage system, including: a reservoir for a disruptive reagent; a destructuring agent contained in the destructuring agent reservoir; a structurant reservoir comprising two separate compartments; a first structurant component contained in a first of two separate compartments; and a second structurant component contained in a second of two separate compartments. In one example, the destructuring agent includes an oxidizing agent; the first structurant component is perylenetetracarboxylic acid diimide or an analog thereof, and the second structurant component is melamine or an analog thereof. In one example, the oxidizing agent is selected from the group consisting of permanganates, perborates, halogens, perchlorates, and peroxides.

It should be understood that any features of the sequencing system may be combined with each other in any desired manner. In addition, it should be understood that any combination of features of the system for sequencing and/or flow cell can be used together and/or combined with any of the examples discussed in this work.

The third aspect of the present invention relates to a method. The method includes forming a masked substrate by applying a porous molecular network to the surface of the substrate on which the nanostructures are located, so that at least a portion of each nanostructure remains open; and exposing the screened substrate to an enzyme solution that is inert to the porous molecular network, resulting in selective attachment of the appropriate enzyme to at least some of the exposed portions of the nanostructures.

In one example of this method, applying a porous molecular network includes: heating a saturated mixture of perylenetetracarboxylic acid diimide or an analogue thereof and melamine or an analogue thereof; depositing the saturated mixture on the substrate to which the nanowire was attached; and cooling the precipitated saturated mixture.

It should be understood that any features of this method may be combined with each other in any desired manner. In addition, it should be understood that any combination of features of this method and/or system for sequencing and/or flow cell can be used together and/or combined with any of the examples discussed in this work.

The fourth aspect of the present invention relates to another method. This method includes applying a porous molecular network to the base to determine the structure (or pattern (English pattern)) of open areas of the base; activation of exposed areas of the base, leading to the formation of activated substrates; removing the porous molecular network, leaving undamaged activated substrates separated by intermediate regions of the substrate; applying an appropriate polymer layer to each of the activated substrates; and grafting a primer onto each of the respective polymer layers.

In one example of this method, removing the porous molecular network includes exposing the porous molecular network to an oxidizing agent.

In one example of this method, applying a porous molecular network includes: heating a saturated mixture of perylenetetracarboxylic acid diimide or an analogue thereof and melamine or an analogue thereof; depositing the saturated mixture on the substrate to which the nanowire was attached; and cooling the precipitated saturated mixture.

It should be understood that any features of this method may be combined with each other in any desired manner. In addition, it should be understood that any combination of features of this method and/or system for sequencing and/or flow cell can be used together, and/or combined with any of the examples discussed in this work.

It should also be understood that any features of any of the methods and/or any of the sequencing systems and/or any of the flow cells may be combined with each other in any desired manner, and/or may be combined with any of the examples discussed herein. .

Brief description of graphic materials

Features of the exemplary embodiments of the present invention will become more apparent upon consideration of the following detailed description in conjunction with the figures, in which like reference numerals refer to similar but possibly not identical components. For brevity, numerals or features having the functions previously described may or may not be described in the description of other figures in which they are depicted.

On FIG. 1A and 1B are schematic plan views of an exemplary process for preparing an example flow cell discussed in this paper;

On FIG. 2 is a sectional view of the flow cell along line 2-2 shown in FIG. 1B;

On FIG. 3A is a block diagram of another example of the method discussed in this paper;

On FIG. 3B is a sectional view of one example of a flow cell obtained by the method shown in FIG. 3A;

On FIG. 4 is a block diagram of another example of the method discussed in this paper;

On FIG. 5A-5C are schematic plan views of an example of the method shown in FIG. 4;

On FIG. 6 is a sectional view along the line 6-6 shown in FIG. 5C, except that the porous molecular network shown in FIG. 6 is different from the example shown in FIG. 5C; and

On FIG. 7 shows a perspective view of a portion of one example of the sequencing system discussed in this paper.

Information confirming the possibility of carrying out the invention

In the examples considered in this work, the creation of a nanometric structure is possible due to the use of a porous molecular network. The porous molecular network self-assembles on the substrate surface and forms a structure that exposes i) portions of the underlying substrate or ii) nanostructures previously applied to the substrate. The extremely hydrophobic porous molecular network covers at least substantially the entire remaining surface of the substrate and thus forms a stencil (mask) on the surface of the substrate. This stencil may minimize and in some cases even completely eliminate the attachment of the subsequently applied chemical to the surface, but does not prevent the attachment of the subsequently applied chemical to i) exposed portions of the underlying substrate or ii) exposed nanostructures. Thus, the porous molecular network allows functionalization of only the required areas.

The porous molecular network is also easily removed from the surface of the substrate under mild conditions that do not adversely affect the chemicals applied to the surface.

In some of the examples discussed in this work, the porous molecular network can also remain on the surface of the support during sequencing. In these examples, an extremely hydrophobic porous molecular network can enhance the interaction between the injected reagents and the surface chemical.

It should be understood that, unless otherwise indicated, the terms used in the present description have their usual meanings known in the relevant field of technology. Some of the terms used in the present description and their meanings are given below.

The singular forms include the plural unless the context makes it clear otherwise.

The terms "comprising", "comprising" and various forms of these terms are synonymous with each other and have the same broad meaning.

The terms "top", "bottom", "top", "bottom", "on", etc. are used in this work to describe the flow cell and/or the various components of the flow cell. It should be understood that such direction designations do not refer to a specific orientation, but are used to indicate the orientation of components relative to each other. The use of directional designations should not be construed as limiting the examples discussed herein to any particular orientation(s).

Used in the present description, the term "attached" means the state of two items that are combined, fastened, glued, connected or attached to each other. For example, the nucleic acid may be chemically attached to the polymeric coating by a covalent or non-covalent bond. A covalent bond is characterized by the socialization of a pair of electrons by a set of atoms. A non-covalent bond is a physical bond that does not involve the sharing of electron pairs and may include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

As used herein, the term "flow cell" means a container including a chamber (i.e., a flow channel) in which a reaction can be carried out, an inlet for supplying a reagent(s) to the chamber, and an outlet for removing the reagent(s) from the cameras. In some examples, the camera allows you to observe the progress of the reaction in the chamber. For example, the camera may include one or more transparent surfaces through which optical detection of arrays, optically labeled molecules, or the like located in the recess can be made. In another example, the camera/flow channel may include electronic circuitry that allows electrical signals to be detected.

As used herein, the term "deposition" refers to any suitable deposition technique, which may be manual or automated, and its application results in a modification of surface properties. Typically, the deposition can be performed using vapor deposition techniques, coating techniques, grafting techniques, or similar techniques. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dip or dip coating, blade coating, solution distribution coating, continuous flow coating, by spray printing, screen printing, microcontact printing, inkjet printing, or the like.

As used herein, the term "nucleotide" includes a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of a nucleic acid sequence. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, i.e. a sugar that does not have a hydroxyl group that is present at the 2' position of the ribose. The nitrogen-containing heterocyclic base (ie, nucleic base) may be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G) and modified derivatives or analogues thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U) and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is linked to the N-1 of the pyrimidine or N-9 of the purine. In the nucleic acid analog, both the phosphate backbone and the sugar or nucleic base can be changed. Examples of nucleic acid analogs include, for example, universal bases or analogs having a similar phosphate-sugar backbone, such as peptide nucleic acids (PNA for short).

Used in the present description, the term "primer" means a single-stranded nucleic acid sequence (for example, single-stranded DNA or single-stranded RNA). Some primers, which may be referred to as amplification primers, serve as the starting point for template amplification and cluster generation. Other primers, which may be referred to as sequencing primers, serve as the starting point for DNA or RNA synthesis. The 5' end of, for example, the amplification primer can be modified to carry out an addition reaction to the functional group of the polymer coating. The primer may be any number of bases in length, and the primer may include various non-naturally occurring nucleotides. In one example, the sequencing primer is a short chain of 10 to 60 bases or 20 to 40 bases.

The term "substrate" means a support on which the various components of the flow cell can be placed. The carrier may be a laminated plate, panel, rectangular sheet, matrix, or any other suitable configuration. The carrier is usually rigid and insoluble in an aqueous liquid. The carrier may be inert to the chemicals used to modify the recesses. For example, the carrier may be inert to the chemicals used to form the block copolymer, to attach the primer(s), and so on. Examples of suitable carriers will be discussed further below.

Used in the present description, the term "surfaced (applied to the surface) chemical substance (substances)" in some aspects refers to the activated substrate, the polymer layer attached to the activated substrate, and the primer (primers) attached to at least part of the polymer layer. In other aspects, the term "surface chemical" refers to a nanostructure and an enzyme attached to the nanostructure.

porous molecular network

In the examples considered in this work, the porous molecular network is a planar supramolecular network of amine and diimide. The selected amine and diimide more preferably form intermolecular hydrogen bonds between dissimilar molecules rather than intermolecular hydrogen bonds between like molecules. Below is an example of intermolecular hydrogen bonding between melamine and perylene tetra-carboxylic di-imide (PTCDI for short):

As shown in the image above, three hydrogen bonds form between these molecules. Melamine includes three functional units (H ₂ NCNC-NH ₂ ) that can participate in the formation of a hydrogen bond network, and perylenetetracarboxylic acid diimide includes two functional units (O=CR-NR-CR=O) that participate in the formation of a network linked by hydrogen bonds. The symmetry of the third and second order, respectively, of melamine and perylenetetracarboxylic acid diimide results in the formation of a hexagonal network, shown for example in FIG. 1A. The bimolecular assembly of these molecules defines the grid pattern (eg, the hexagonal grid shown in Fig. 1A) and results in the formation of pores (eg, at the center of each hexagon shown in Fig. 1A) that are defined by the contours of the pattern formed.

The presence of a plurality of donors and/or acceptors of hydrogen bonds allows the formation of a two-dimensional network on the surface of the substrate. Thus, while melamine and FTCDI are two examples of molecules that can form a porous molecular network, it should be understood that other combinations of amine and diimide can be used to form structures having a different geometry pattern. For example, the use of an amine molecule containing four functional units of melamine (H ₂ NCNC-NH ₂ ) and a 4th order axis of symmetry allows the design of rectangular, eg square pores, instead of the hexagonal pores obtained with FTCDI and melamine. This allows the design of the pore size through the correct selection of the constituent molecules from which the molecular network is formed.

Examples of suitable amines include melamine:

or its analogues. Melamine analogues include at least a functional unit (H ₂ NCNC-NH ₂ ), which, together with the diimide, participates in the formation of a hydrogen bonded network. One example of a melamine analog includes 4,4',4'-(1,3,5-benzenetriyl)tris-2,6-pyridinediamine:

as well as its options. In one possible embodiment, the central benzene ring is substituted with another aromatic or other cyclic system that allows the addition of another number (other than 3), for example, 2 or 4, 2,6-diaminopyridyl fragments (or other fragments containing H ₂ NCNC-NH ₂ ). In some embodiments, one or more separating units, such as alkynylene, bis(alkynylene), or arylene diradicals, may be located between the central structure and side 2,6-diaminopyridyl fragments (or other fragments containing H ₂ NCNC-NH ₂ ). Another example of a melamine analog includes:

which includes four H ₂ NCNC-NH ₂ fragments. Although some analogues of melamine are described here, it is believed that other aromatic amines or non-aromatic conjugated amines may also be used, provided that the chosen amine preferably forms intermolecular hydrogen bonds with the chosen diimide.

Examples of suitable diimides include perylenetetracarboxylic acid diimide (PTCDI):

or its analogs PTCDI analogs include at least two diimide functional units (O=CR-NR-CR=O) that participate in the formation of hydrogen bonds with amine molecules to form a network.

An example of a PTCDI analog includes:

It is believed that any of the following aromatic moieties can be used as a PTCDI analog after they have been converted to the diimide:

The porous molecular network can be pre-assembled in solution and then deposited onto the desired substrate surface. Since the molecules are capable of self-assembly with the formation of a supramolecular structure, the solution does not include any reagents (for example, a catalyst), except for the solvent and the two components that make up the resulting matrix/network. Examples of the solvent include dimethylsulfoxide (DMSO) and dimethylformamide (DMF). Other solvents may be chosen, the choice of which may depend in part on the type of the two components that make up the resulting matrix/network.

The assembled porous molecular network can form in solution upon reaching thermodynamic equilibrium. One way to achieve thermodynamic equilibrium is to heat a saturated mixture of perylenetetracarboxylic acid diimide or an analogue thereof and melamine or an analogue thereof. The rich mixture comprises perylenetetracarboxylic acid diimide or an analogue thereof and melamine or an analogue thereof in a suitable stoichiometric ratio (molar ratio). In one example, the diimide and amine are present in a 1:1 molar ratio. However, the stoichiometric ratio depends on the type of molecules chosen and their affinity for the surface of a particular substrate. If one of the molecules has a higher affinity for the substrate surface, then the amount of this molecule in solution may be less than the amount of the other molecule in solution.

The temperature to which the solution is heated depends in part on the type of the two constituents of the solution. In one example, heating is performed to a temperature of from about 51°C (~325 K) to about 127°C (~400K).

Then the heated saturated mixture can be deposited on the substrate (for example, by any of the methods discussed in this paper) and cooled. Cooling can be done slowly, for example at a rate of up to about 10° C. per minute. In one example, the cooling rate is from about 0.1°C per minute to about 10°C per minute. In another example, the cooling rate is from about 0.5°C per minute to about 5°C per minute. In another example, the cooling rate is approximately 1°C per minute.

The porous molecular network formed on the substrate surface is a non-reactive pi-(π)-system. In one example, this type of system is essentially chemically inert and thus will not react with certain reagents and/or undergo activation used to functionalize the surface of a substrate having a chemical deposited thereon; thus, this type of system successfully passivates the surface of the substrate to which it is applied. Due in part to this passivating ability, a porous molecular network can be used as a pattern for selectively functionalizing the surface of a substrate with a chemical present on the surface. Examples of the possible use of a porous molecular network in creating the structure of various flow cells are discussed in more detail below.

In addition, the non-reactive pi-(π)-system of the porous molecular network means that the porous molecular network is not covalently attached to the underlying surface of the support. Thus, the porous molecular network, if necessary, can be easily removed from the surface of the substrate. To remove the porous molecular network, a reagent with a higher affinity for the diimide moiety than for the substrate surface itself can be used. The presence of such a reagent shifts the thermodynamic equilibrium, and the porous molecular network is desorbed from the substrate surface. Examples of removing reagents include an oxidizing reagent that can oxidize the cyclic diimide moiety. The oxidized diimide has a nonplanar structure and is easily desorbed from the base surface. Suitable oxidizing agents are selected from the group consisting of permanganates, perborates, halogens, perchlorates and peroxides. Examples of porous molecular network removal options are discussed in more detail below.

Flow cell for optical detection

In one of the examples discussed in this work, the flow cell includes a base; located on the basis of a selectively removable porous molecular network that defines the open areas of the base; and surface sequencing chemicals located in at least some of the exposed areas, where the surface chemicals include an activated layer, a polymer layer attached to the activated layer, and a primer attached to the polymer layer.

One example of a method for producing such a flow cell is shown schematically in FIG. 1A and Fig. 1B. The method includes applying a porous molecular network 14 to the substrate 12 to determine the pattern of open areas 18 of the substrate (Fig. 1A); and the formation of a surface chemical 20 on at least some of the exposed portions 18 of the base (FIG. 1B). As shown in FIG. 2, in this method example, the formation of a chemical 20 on the surface includes the activation of exposed areas 18 of the substrate to form activated substrates 22; applying an appropriate polymer layer 24 to each of the activated substrates; and grafting primer 26 onto each of the respective polymer layers 24.

On FIG. 1A is a schematic plan view of a support 12 on which a porous molecular network 14 is self-assembled.

Examples of suitable bases 12 include epoxysiloxane, glass and modified or functionalized glass, polymers (including acrylic polymers, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON® supplied by Chemours), cyclic olefins/ cycloolefin polymers (abbreviated COP) (such as ZEONOR® supplied by Zeon), polyimides, etc.), nylon, ceramic materials/ceramic oxides, silicon oxide, fused silica or materials based on silicon oxide, aluminum silicate, silicon and modified silicon (for example, boron-doped p+ silicon), silicon nitride (Si ₃ N ₄ ), silicon oxide (SiO ₂ ), tantalum pentoxide (TaO ₅ ), or other tantalum oxide(s) (TaO _x ) , hafnium oxide (HaO ₂ ), carbon, metals, inorganic glasses or the like. The support may also be made of glass or silicon and have a tantalum oxide or other ceramic oxide coating on the surface.

In one example, the diameter of the base 12 may be from about 2 mm to about 300 mm, or the base 12 may be a rectangular sheet or panel, the largest dimension of which is up to about 10 feet (~ 3 meters). In one example, the substrate is a laminated plate having a diameter of from about 200 mm to about 300 mm. In another example, the carrier is a matrix (cube, English die), the width of which is from about 0.1 mm to about 10 mm. While exemplary sizes are provided herein, it should be understood that any suitable carrier size may be used. In another example, a panel may be used which is a rectangular carrier having a larger surface area than a 300 mm diameter circular sandwich plate.

The porous molecular network 14 includes a structure 16, which is formed by self-assembled amine and diimide molecules, and pores, which are defined by the structure 16. The structure 16 covers certain parts of the base 12, and the resulting pores correspond to the open areas 18 of the base. The porous molecular network 14 can be pre-assembled and deposited on the substrate 12, as discussed in this work. Rinsing and drying can be done prior to applying the chemical 20 to the surface.

As noted above, the formation of the surface chemical 20 in this exemplary method initially includes the activation of exposed areas 18 of the base. In some examples, the activation includes silanization of the exposed areas of the substrate by the action of a silane or a silane derivative. The silane or silane derivative used for silanization has no affinity for the expanded pi system of the porous molecular network 14, and thus the porous molecular network 14 effectively defines isolated anchoring points on the basis 12 (for example, open areas 18 of the support) at which the silane or silane derivative will react.

By activating the exposed base portions 18 through silanization, the silanization can be done with any silane or silane derivative. The choice of silane or silane derivative may depend in part on the type of polymer layer 24 formed, since it may be desirable to form a covalent bond between the silane or silane derivative and polymer layer 24. vary depending on the type of silane or silane derivative used. Below are some examples.

Examples of suitable silanization methods include vapor deposition (eg, YES method (method developed by Yield Engineering Systems)), spin coating, or other precipitation methods.

In one example using a YES CVD oven, a substrate 12 is placed in the CVD oven, onto which a porous molecular network 14 is deposited. The chamber can be equilibrated with the atmosphere, after which the silanization cycle begins. When running cycles, a suitable temperature may be maintained in the silane or silane derivative vessel (eg, approximately 120°C for norbornene silane), a suitable temperature may be maintained in the silane or silane derivative vapor lines (eg, approximately 125°C for norbornene silane), and a suitable temperature can be maintained in the vacuum lines (eg, approximately 145°C).

In another example, a silane or silane derivative (e.g., liquid norbornene silane) can be deposited inside a glass vial that is placed in a glass vacuum desiccator containing a base 12 coated with a porous molecular network 14. The desiccator can then be evacuated until pressure is reached, from about 15 mTorr to about 30 mTorr (which is about 0.13 to 0.26 Pa), and placed in an oven at a temperature of from about 60°C to about 125°C. After the silanization has taken place, the desiccator is removed from the oven, cooled and equilibrated with the atmosphere.

Vapor deposition, the YES process and/or the vacuum desiccator process can be carried out using various silanes or silane derivatives, such as silanes or silane derivatives containing a cycloalkene unsaturated moiety, such as norbornene, a norbornene derivative (e.g., heteronorbornene , including an oxygen or nitrogen atom instead of one of the carbon atoms), trans-cyclooctene, trans-cyclooctene derivatives, trans-cyclopentene, trans-cycloheptene, trans-cyclononene, bicyclo[3,3,1] non-1-ene, bicyclo[ 4,3,1]dec-1(9)-ene, bicyclo[4,2,1]non-1(8)-ene and bicyclo[4,2,1]non-1-ene. Any of these cycloalkenes may be substituted, for example, with an R group such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or heteroalicyclylalkyl. One example of a norbornene derivative includes [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. In other examples, these methods can be used if the silane or silane derivative includes an unsaturated cycloalkyne moiety, such as cyclooctyne, a cyclooctyne derivative, or bicyclononines (e.g., bicyclo[6.1.0]non-4-yne or derivatives thereof, bicyclo[6 ,1,0]non-2-yne or bicyclo[6,1,0]non-3-yne). Such cycloalkynes may be substituted by any of the R groups discussed in this work.

As a result of the addition of the silane or silane derivative to at least some of the exposed areas 18 of the substrate, activated substrates 22 are formed. In this example, the activated substrates 22 are isolated from each other by the structure 16.

The polymer layer 24 can then be applied to the activated substrates 22. The polymer layer 24 can be formed from a semi-rigid polymer material that is permeable to liquids and gases. The type of molecules used to form the polymer layer 24 may be chosen such that they preferentially react with the activated substrates 22 rather than the porous molecular network 14. In some cases, the molecules may be highly reactive with the activated substrates 22 and have very little or no affinity for the porous molecular network. This improves the specificity of the reactivity of the molecules and allows the molecules to form the respective polymeric layers 24 on the activated supports 22 rather than on the porous molecular network 14.

One example of polymer layer 24 includes an acrylamide copolymer such as N-(5-azidoacetamidylpentyl)acrylamide-acrylamide (poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide, abbreviated as PAZAM) and some other forms. acrylamide copolymers have the following structure (I):

where:

R ¹ is H or optionally substituted alkyl;

R ^A is selected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, and thiol;

each of R ⁵ , R ₆ and R ₈ is independently selected from the group consisting of H and optionally substituted alkyl;

each of the -(CH ₂ ) _p - groups may be optionally substituted;

p is an integer from 1 to 50;

n is an integer from 1 to 50000; and

m is an integer ranging from 1 to 100000.

One skilled in the art will appreciate that the arrangement of the repeating elements "n" and "m" in structure (I) is a non-limiting example, and the monomeric subunits may be present in the polymer structure in any order (e.g., as a random polymer, block polymer, structured polymer, or a combination of such polymers).

The molecular weight of PAZAM may be from about 10 kDa to about 1500 kDa, or in one specific example, it can be about 312 kDa.

In some examples, PAZAM is a linear (unbranched) polymer. In some other examples, PAZAM is a low cross-linked polymer.

В этом примере акриламидная единица в структуре (I) может быть заменена на звено

в котором каждый из R₆, R₇ и R₈ представляет собой Н, и каждый из R₉ и R₁₀ представляет собой метильную группу (вместо Н в случае акриламида). В этом примере q может представлять собой целое число, составляющее от 1 до 100000. В другом примере наряду с акриламидным звеном может быть применен N,N-диметилакриламид. В этом примере наряду с повторяющимися звеньями "n" и "m" структура (I) может включать единицу

в которой каждый из R₆, R₇ и R₈ представляет собой Н, и каждый из R₉ и R₁₀ представляет собой метильную группу. В этом примере q может представлять собой целое число, составляющее от 1 до 100000.In other examples, the polymer layer 24 may be formed by a variant of structure (I). In one example, the acrylamide unit can be replaced by N,N-dimethylacrylamide

In this example, the acrylamide unit in structure (I) can be replaced by a unit

in which each of R ₆ , R ₇ and R ₈ represents H, and each of R ₉ and R ₁₀ represents a methyl group (instead of H in the case of acrylamide). In this example, q may be an integer from 1 to 100,000. In another example, N,N-dimethylacrylamide may be used along with the acrylamide unit. In this example, along with the repeating units "n" and "m", the structure (I) can include a unit

in which each of R ₆ , R ₇ and R ₈ represents H, and each of R ₉ and R ₁₀ represents a methyl group. In this example, q may be an integer ranging from 1 to 100000.

It should be understood that other molecules can be used to form polymer layer 24, provided that their functional groups are capable of interacting with activated substrates 22 and subsequently applied primer(s) 26. Other examples of molecules suitable for forming polymer layer 24 include materials , having a colloidal structure, such as agarose, or a polymer network, such as gelatin, or a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane free acrylamide (English silane free acrylamide, abbreviated SFA) or an azidolized version of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or acrylic acid containing a vinyl group, or from monomers that undergo [2+2] photocycloaddition reactions. Other examples of suitable molecules to form the polymeric layer 24 include mixed copolymers of acrylamides and acrylates.

As stated in this work, the molecule used to form the polymer layer 24 has no affinity (or very little affinity) for the extended pi system of the porous molecular network 14, and thus the polymer layer 24 is formed on the activated substrate 22, rather than on a porous molecular network 14. The molecule (eg, PAZAM) can be deposited by spin coating or dip or dip coating or by passing the functionalized molecule under positive or negative pressure, or other suitable method. The functionalized molecule may be present in a mixture. In one example, the mixture includes PAZAM in water or in a mixture of ethanol and water. The molecule reacts to form a polymer layer 24 on the activated substrates 22 rather than on the porous molecular network 14.

After being coated, the molecule may also be cured, which results in the formation of a polymer layer 24 on each of the activated substrates 22. ) to about 95°C, for a time ranging from about 1 millisecond to about several days. In another example, the curing time may be from 10 seconds to at least 24 hours. In another example, the curing time may be from about 5 minutes to about 2 hours.

The attachment of the polymer layer 24 to the activated substrates 22 can be done by covalent bonding. The covalent bonding of the polymer layer 24 to the activated substrates 22 allows the surface chemical 20 to be retained in appropriate isolated areas for the life of the finished flow cell in various applications. The following are some examples of reactions that can take place between the activated substrates 22 and the polymer layer 24.

If the silane or silane derivative includes norbornene or norbornene derivative as an unsaturated moiety, then norbornene or norbornene derivative can: i) undergo a 1,3-bipolar cycloaddition reaction with the PAZAM azide/azido group; ii) couple with a tetrazine group attached to PAZAM; enter into a cycloaddition reaction with a hydrazone group attached to PAZAM; enter into a photo-click reaction with a tetrazole group attached to PAZAM; or enter into a cycloaddition reaction with a nitrile oxide group attached to PAZAM.

If the silane or silane derivative contains cyclooctyne or a cyclooctyne derivative as an unsaturated moiety, then the cyclooctyne or cyclooctyne derivative could: i) undergo strain-promoted azide-alkyne 1,3-cycloaddition, abbreviated as SPAAC) with an azide/azido group of PAZAM, or ii) undergo a stress-induced alkine-nitrile oxide cycloaddition reaction with a nitrile oxide group attached to PAZAM.

If the silane or silane derivative includes bicyclononine as an unsaturated moiety, then bicyclononine, under the influence of tension in the bicyclic ring system, can enter into a similar alkyne cycloaddition reaction according to the SPAAC mechanism with azides or nitrile oxides attached to PAZAM.

To fix the primer 26 on the polymer layers 24 perform grafting. Primer 26 can be any forward amplification primer or reverse amplification primer that includes an alkyne functionality, or a primer with a different end group. Other examples of primers with a different end group that can be used according to the invention include a tetrazine-terminated primer, an azido-terminated primer, an amino-terminated primer, an epoxy- or glycidyl-terminated primer, a thiophosphate-terminated primer, a thiol-terminated primer. group, an aldehyde-terminated primer, a hydrazine-terminated primer, and a thiazolinedione-terminated primer. A mixture of primers may also be used. Specific examples of suitable primers include primers P5 and/or P7, which are available on the surface of commercially available flow cells supplied by Illumina inc. for sequencing platforms HISEQ™, HISEQX™, MISEQ™, MISEODX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, ISEQ™, GENOME ANALYZERS, etc. and other tool platforms.

In one example, grafting can be done by flow deposition (e.g., using a temporarily attached cap), dip coating, spray coating, solution spread coating, or any other suitable method that allows the primer(s) 26 to be fixed to the polymer layers 24. In each of the exemplary procedures, a primer solution or primer-containing mixture may be used, which may include primer(s), water, buffer, and catalyst.

Dip coating may involve immersing the substrate 12 (which has activated substrates 22 and polymer layers 24) in a series of temperature controlled baths. The baths may also have flow control and/or be under a nitrogen atmosphere. Baths may contain a solution or mixture including a primer. By passing through the various baths, the primer(s) 26 are attached to the functional group(s) of the polymer layers 24. ) 26, after which the base is transferred to the next washing baths. Moving from one bath to another can be done with an automatic manipulator or manually. In dip coating, a drying system can also be used.

Spray coating can be performed by spraying the primer solution or mixture containing the primer directly onto the substrate 12 (on which the activated substrates 22 and polymer layers 24 are placed). The spray-coated multilayer plate may be incubated for about 4 minutes to about 60 minutes at a temperature of about 0°C to about 70°C. After incubation, the primer solution or mixture containing the primer can be diluted and removed using, for example, a spin coater.

Coating by spreading the solution can be performed by a method including pouring and spin coating, and thus it can be performed using a spin coater. The primer solution or mixture containing the primer may be applied (manually or automatically) to the substrate 12 (on which the activated substrates 22 and polymer layers 24 are placed). The applied primer solution or mixture containing the primer may be applied to or spread over the entire surface, including the structure 16. The coated substrate 12 containing the primer may be incubated for a time ranging from about 2 minutes to about 60 minutes, at a temperature of from about 0°C to about 80°C. After incubation, the primer solution or primer-containing mixture can be diluted and removed using, for example, a spin coater.

Primers 26 react with the functional groups of polymer layers 24 and have no affinity for the extended pi system of porous molecular network 14. Thus, primers 26 are grafted to polymer layer 24 and not to porous molecular network 14.

In another example of the method, the application of the surface chemical 20 can be performed before and after the application of the porous molecular network. In such examples, activation of the surface of the substrate occurs prior to deposition of the porous molecular network 14, and formation of polymer layer 24 and grafting of primer 26 occurs after deposition of the porous molecular network.

In this example, the (exposed) surface of substrate 12 is subjected to plasma etching, which results in the generation of surface activating agent(s) (eg, -OH groups) on the surface of substrate 12. It is noted that the addition of -OH groups can reduce the affinity of backing 12 for the porous molecular network (which is hydrophobic), and thus the plasma etch conditions and duration can be controlled so that the surface is not too hydrophilic.

In this example, a porous molecular network 14 is applied to the activated substrate surface, leaving discrete areas of the activated substrate surface exposed. As a result of applying a porous molecular network 14 to an activated base, activated substrates 22 are formed.

In addition, in this example, polymer layer 24 can then be selectively applied to activated substrates 22 as described herein, and primers 26 can be grafted onto polymer layer 24 as indicated herein.

On FIG. 2 shows an example of a flow cell 10 obtained by the method shown in FIG. 1A and Fig. 1B (eg, structuring and applying surface chemical 20). While a single kind of primer 24 is shown, it should be understood that two or more different primers 24 may be grafted.

In this example, the X and Y dimensions of each of the discrete regions coated with the surface chemical 20 substantially correspond to the X and Y dimensions of each of the pores of the porous molecular network 14. up to a nanometer.

In other examples, the selectively removable porous molecular network 14 may be removed from the substrate 12 during the execution of the method and no longer be present as part of the flow cell during a subsequent sequencing operation. An example of such a method 100 is shown in FIG. 3A and an example of a resulting flow cell 10' is shown in FIG. 3B. The method 100 includes applying a porous molecular network 14 to the substrate 12 to determine the pattern of open areas 18 of the substrate (symbol 102); activation of open areas 18 of the base with the formation of activated substrates 22 (symbol 104); removal of porous molecular network 14, in which the activated substrate 22 remain intact and separated by intermediate areas 28 of the base (symbol 104); applying an appropriate polymer layer 24 to each of the activated substrates 22 (symbol 106); and grafting primer 26 onto each of the respective polymer layers 24 (reference 108). The use of the method 100 may be desirable if the molecules of the porous molecular network 14 can interact with the fluorophores used in the sequencing operation. Some fluorophores may adhere to some examples of the porous molecular network 14. Such interaction disrupts or has other undesirable effects on the sequencing operation because fluorophore-labeled analytes attached to the porous molecular network 14 cannot interact with the sequencing template. In these examples, a method 100 can be used in which the porous molecular network 14 is effectively used to create activated substrates 22 (sites for anchoring the remaining surface chemical 20) and then the porous molecular network 14 is removed so that it does not interfere with the subsequent sequencing operation. In these examples, the imprint of the porous molecular network 14 is removed, eliminating its potential influence.

In method 100, porous molecular network 14 may be formed as discussed herein and activation may be performed as discussed herein using a silane or silane derivative. In this example, with the activation of exposed areas 18 of the substrate in the pores by a silane or a silane derivative, the X and Y dimensions of each of the discrete activated substrates 22 (and thus each of the areas on the surface of which the chemical agent 20 is applied) correspond to the X and Y dimensions of each of pores of a porous molecular network 14.

Upon completion of the formation of activated substrates 22, the porous molecular network 14 is removed in the method 100. The chemical nature of the porous molecular network 14 allows it to be exposed to any of the oxidizing reagents discussed in this work. The oxidizing agent may be applied to the porous molecular network 14 (and to the remainder of the substrate 12) using any suitable application technique. The action of an oxidizing agent removes the porous molecular network 14 from the substrate 12 (for example, by desorption) as a result of the oxidation of the diimide. The oxidizing agent does not react with the activated substrates 22 and thus these activated portions of the substrate 12 remain intact to participate in subsequent reactions. After removal of the porous molecular network 14, the substrate 12 and the activated substrates 22 thereon can be washed (eg with water).

The removal of the porous molecular network 14 exposes portions of the substrate 12, which are referred to herein as intermediate regions 28. The intermediate regions 18 may be continuous (and have the same pattern as the porous molecular network 14), while the silanized regions 22 are discrete.

In method 100, polymer layers 24 may be formed as discussed herein. In these examples, the molecule used to form the polymer layer 24 has no affinity for the substrate 12, and thus the polymer layer(s) 24 is selectively formed on the silanized substrate(s) 22 rather than on the intermediate regions 28 of the substrate 12. B method 100, primer grafting 26 can also be performed as discussed in this paper.

The flow cells 10, 10' discussed in this work can be used to implement many different sequencing techniques or methods, including techniques often referred to as sequencing-by-synthesis (English sequencing-by-synthesis, abbreviated as SBS), cycle sequencing of arrays, sequencing by ligation , pyrosequencing, etc. In either of these techniques, primer(s) 26 is present as part of the surface chemical 20 not on structure 16 or intermediate regions 28, and therefore sequencing is limited to the area covered by surface chemical 20.

By way of example, a sequencing-by-synthesis (SBS) reaction can be carried out on a system such as the HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NOVASEQ™, NEXTSEQDX™ or NEXTSEQ™ sequencing systems, or any other system sequencing supplied by Illumina, Inc. (San Diego, CA). When conducting SBS to determine the sequence of nucleotides in the matrix track the extension (extension) of the sequencing primer along the template nucleic acid (ie template sequencing). The chemical process underlying this method may be polymerization (eg catalyzed by a polymerase enzyme) or ligation (eg catalyzed by a ligase enzyme).

In one particular polymerase SBS method, a nucleic acid library template is introduced into the flow cell 10, 10' and the nucleic acid library template is hybridized to primer 26. The nucleic acid library template array is hybridized to, for example, one of two types of primers 26 immobilized on the flow cell 10, 10'. Cluster generation can then be performed. In one example of template cluster generation, nucleic acid libraries are copied from hybridized primers 26 by 3' extension by high-fidelity DNA polymerase. The original templates of the nucleic acid library are denatured, which leaves copies immobilized at the localization of primers 26. Isothermal bridged amplification can be used to amplify the immobilized copies. For example, the copied templates are folded to hybridize with the adjacent complementary primer 26, and the polymerase copies the copied templates to form double-stranded bridges, which are denatured to form two single-stranded strands. These two strands are twisted and hybridized with adjacent complementary primers 26 and are completed again, forming two new double-stranded loops. The process is repeated on each copy of the template during cycles of isothermal denaturation and amplification in order to obtain dense clonal clusters. Each cluster of double-stranded bridges undergoes denaturation. In one example, the reversed strand is removed by specific base cleavage, leaving straight oriented template strands.

The ends of the 3' templates and any primers 26 could/t be blocked to prevent unwanted initiation (priming, English priming). A sequencing primer may be introduced into the flow cell 10, 10'. Because the sequencing primer is complementary to the nucleic acid template strand adapter, it will hybridize to the adapter (eg read 1 of the template sequencing primer).

Fluorescently labeled nucleotides are attached to the template (filling in the sequencing primer) according to the structure of the template, and thus determining the order and type of nucleotides added to the primer can be used to determine the sequence of the template. For example, one or more labeled nucleotides, DNA polymerase, etc. can be delivered to initiate the first cycle of SBS into/through the flow channel, etc., in which the primer array 26 is located, to which the template strands are attached. The completion of the sequencing primer causes insertion of the labeled nucleotide, and this insertion can be detected by imaging. When rendered, an illumination system (not shown) may emit radiation that excites a surface chemical 20.

In some examples, the nucleotides may additionally have a reversible terminator property that prevents subsequent primer extension after the addition of the nucleotide. For example, a nucleotide analog containing a reversible terminator fragment can be added to the sequencing primer along the template strand, such that subsequent extension cannot occur until a deblocking agent is added that can remove the fragment. Thus, in those examples where reversible terminators are used, into the flow channel, etc. a deblocking reagent may be sent (before or after detection).

Flushing(s) may be performed between the various fluid supply steps. The SBS cycle can then be repeated n times to extend the sequencing primer by n nucleotides, whereby a sequence of length n can be determined.

While the SBS method has been discussed in detail, it should be understood that the flow cells 10, 10' discussed herein may be applied in other sequencing protocols, for genotyping, or in other chemical and/or biological assays. In another example, the flow cells 10, 10' discussed in this work can be used to generate a library on cells.

Flow cell for electrical detection

In another example discussed in this paper, the flow cell includes a base; located on the basis of a selectively removable porous molecular network that defines the open areas of the base; and a surface-based sequencing chemical located in at least some of the exposed areas, where the surface-based chemical includes a nanostructure and an enzyme attached to the nanostructure.

One example of a method for producing such a flow cell is shown in FIG. 4 and in FIG. 5A - Fig. 5C.

As shown in FIG. 4, method 200 includes forming a stenciled substrate by applying a porous molecular network 14 to the surface of substrate 12 on which nanostructures are assembled such that at least a portion of each nanostructure remains exposed (label 202); and exposing the screened substrate to an enzyme solution that is inert to the porous molecular network 14, resulting in selective attachment of the appropriate enzyme to at least some of the exposed portions of the nanostructure (reference 204).

On FIG. 5A is a schematic plan view of a support 12 on which the nanostructures are assembled. Base 12 can be any of the examples of the base discussed in this paper.

On FIG. 5A, the nanostructure is indicated by the symbol 30. The nanostructure 30 is an electrically conductive channel of the charge sensor 32, which also includes electrodes 34, 34'. The term "charge sensor" as used herein means a detection device that converts perturbations on its surface or in the electric field surrounding it into an electrical signal. For example, the charge sensor 32 may convert the delivery or exit of the reaction component into an electrical signal. In some examples, charge sensor 32 may also convert an interaction between two reaction components (eg, nucleic acid template and nucleotide) into an electrical signal. An example of a charge sensor 32 is a field effect transistor (also called a field effect transistor, FET for short), such as a carbon nanotube transistor FET, CNT for short, a single-walled carbon nanotube FET ( single-wailed carbon nanotube transistor, abbreviated as SWNT), silicon nanowire (SiNW) FET, polymer nanowire FET and graphene nanostrip FET (and corresponding nanostrip FETs made from 2D materials such as MoS ₂ , silicene etc.). In a FET, current flows in a conductive path (conductive channel/nanostructure 30) that is connected to two electrodes 34, 34' (source and drain). The conductivity of the channel between the source and the drain is blocked and renewed using a third (valve) electrode, which can be capacitively connected through a thin dielectric layer.

Any type of nanostructure 30 may be used, provided that it can serve as an electrically conductive channel for charge sensor 32. Examples of materials suitable for making electrically conductive channels include conductors (eg, metals, carbon-based materials, certain metal oxides, electrically conductive polymers, etc.) or semiconductors (eg, silicon, other metal oxides, etc.). Nanostructure 30 may have any suitable geometric shape such as a tube, wire, strip, etc., and at least one dimension (eg, length, width, diameter, etc.) of nanostructure 30 may be in the nanorange. In various examples, the nanostructure 30 is an electrically conductive channel, including a material selected from the group consisting of a conductor and a semiconductor, and has a geometric shape selected from the group consisting of a tube, wire, and strip. Some specific examples of the nanostructure 30 include carbon nanotube (CNT), single shift carbon nanotube (SWCN), silicon nanowire (SiNW), polymer nanowire, nanostrip (e.g., graphene nanostrip, nanostrip made from 2D materials such as MoS ₂ , silicene etc.) or any other nanostructure that can serve as an electrically conductive channel of the charge sensor 32. In one example, the nanostructure 30 is a silicon nanowire. In another example, the nanostructure is a carbon nanotube.

As shown in FIG. 5A, opposite ends of the nanostructure 30 are in electrical connection with the respective electrodes 34, 34'. Any suitable electrode material may be used that can be chemically and electrically bonded to the nanostructure 30. Examples of suitable electrode materials include gold, platinum, carbon, indium tin oxide, and the like.

In some examples, the assembly of charge sensor(s) 32 on base 12 may include forming electrodes 34, 34' and forming nanostructure 30 between electrodes 34, 34' by any suitable technique such as direct growth, solution dropping, or various printing techniques. In one example, a carbon nanotube can be produced by arc discharge, laser ablation, high pressure disproportionation of carbon monoxide, or chemical vapor deposition (CVD). In another example, silicon nanowire (SiNW) can be obtained from an organosilicon precursor by solid etching or catalyzed vapor or liquid phase growth.

Each charge sensor 32 can be connected individually (and thus tracked individually). In other words, the electrodes 34, 34' of any individual charge sensor 32 can be connected to an electronic circuit that regulates its operation (for example, after being connected to a detector and a power source). The electronic circuit may be electrically connected to the detection means and to the power supply.

The location of charge sensors 32 on substrate 12 may depend in part on the geometry of structure 16 and in particular the pores of the porous molecular network 14 to be formed on substrate 12. This is partly due to the fact that porous molecular network 14 self-assembles on substrate 12 and on charge sensors 32, and therefore it is desirable that at least a portion of the nanostructure 30 be localized in the pores of the porous molecular network 14. maintaining the proper distance between the biomolecules attached to the nanostructures 30, and/or for passivation of areas of the charge sensor 32 in order to prevent the interaction of reagents (eg, salt solutions, etc.) with the passivated areas.

As shown at 202 in FIG. 4, method 200 includes forming a stenciled substrate by applying a porous molecular network 14 to the surface of substrate 12 on which nanostructures 30 are placed such that at least a portion of each nanostructure 30 remains exposed. The porous molecular network 14 can be pre-assembled in solution and then deposited onto the substrate 12, as discussed in this work.

The porous molecular network 14 self-assembles on the surface of the substrate 12 and in some cases on the components (30, 34, 34) of the charge sensor 32 depending at least in part on the size of the pores and the size and location of the charge sensor 32. After deposition, the porous molecular network 14 self-assembles in such a way that the parts of the substrate 12 and/or the parts of the charge sensors 32 that are in the pores of the substrate 12 are exposed. This forms the stenciled base shown in FIG. 5V. To simply and clearly illustrate porous molecular network 14 and charge sensors 32, each charge sensor 32 in FIG. 5B is shown open and located in a corresponding pore of the porous molecular network 14. However, it should be understood that the individual pores of the porous molecular network 14 are smaller than the charge sensor 32 and in some cases smaller than the nanostructure 30. Thus, the porous molecular network 14 may cover the electrodes 34, 34' and/or some or all of the nanostructure 30 of the charge sensors 32, not just the exposed base 12. An example of a porous molecular network 14 overlaying the electrodes 34, 34' and some of the nanostructure 30 is shown in FIG. 6. It should be understood that the areas that are covered by the porous molecular network 14 and the areas that open in the pores of the porous molecular network 14 depend in part on the size and location of the charge and self-assembly sensors 32 of the porous molecular network 14. In the examples discussed in this work , at least some of the pores of the porous molecular network 14 remain open areas of at least some fraction of the nanostructure 30; however, it should be understood that some of the nanostructures 30 may be completely covered by the porous molecular network 14 and/or that portions of the substrate 12 may also remain open in some of the pores.

Thereafter, method 200 includes exposing the stenciled substrate to an enzyme solution that is inert to the porous molecular network 14. The term "inert" means that the enzyme solution does not combine with or react with the porous molecular network, and thus the enzymes that are in solution, selectively attach to at least some of the exposed parts of the nanostructure 30. The inertness of the enzyme solution may be due to the hydrophilicity of the solution and the hydrophobicity of the porous molecular network 14. The porous molecular network 14 is conjugated and thus very hydrophobic. The enzyme solution is hydrophilic (compared to the porous molecular network 14) and thus does not combine with or react with the porous molecular network 14. Thus, when the stenciled substrate is exposed to the enzyme solution, the enzymes present in the solution can attach to any of the open parts of the nanostructure 30, but not to the porous molecular network 14. Since the porous molecular network 14 may cover some areas of the surface of some or all of the nanostructure 30, only small areas of this nanostructure (s) 30 may remain open in the pores. The functionalization that can be performed on any of the nanostructures 30 is limited to these small exposed portions, which can increase the specificity of the attachment of biomolecules. In some examples, a single enzyme can be attached to a small exposed portion of the nanostructure, which can improve the sensitivity of the charge sensor 32 .

The enzyme solution may include enzyme 36 (as such or in combination with coupler 38 attached thereto) and an enzyme carrier liquid. The liquid carrier is an aqueous solution. In one example, a saline solution may be used as the liquid carrier.

In the examples discussed in this work, it is desirable that one enzyme 36 was attached to one nanostructure 30 (as shown in Fig. 5C and Fig. 6). To fulfill this condition, the enzyme 36 used can be chosen so that its hydrodynamic radius in solution is close to the size of the region of the nanostructure 30 that can remain open in the pore of the structure 16. By "close" is meant that the hydrodynamic radius can be 50% larger than or 50% smaller than the pore size of the porous molecular network. An example of a suitable enzyme 36 is a polymerase. Examples of suitable DNA polymerases include polymerases classified according to structural homology to the families designated A, B, C, D, X, Y, and RT. Family A DNA polymerases include, for example, T7 DNA polymerase, eukaryotic mitochondrial DNA polymerase y, E. coli DNA Pol I, Thermys aquaticus Pol I, and Bacillus stearothermophilus Pol I. Family B DNA polymerases include, for example, eukaryotic DNA- polymerases α, δ and ε; DNA polymerase ζ; T4 DNA polymerase, Phi29 DNA polymerase and RB69 bacteriophage DNA polymerase. Examples of Family C include, for example, the alpha subunit of E. coli DNA polymerase III. Examples of Family D include, for example, polymerases derived from the Euryarchaeota subdomain of the Archaea domain. Family X DNA polymerases include, for example, the eukaryotic polymerases Pol β, Pol σ, Pol λ and Pol μ and S. cerevisiae Pol4. Family Y DNA polymerases include, for example, Pol η., Pol iota, Pol kappa, E. coli Pol IV (DINB) and E. coli Pol V (UmuD'2C). The RT (reverse transcriptase) family of DNA polymerases includes, for example, retrovirus reverse transcriptases and eukaryotic telomerase.

Enzyme 36 may be directly attached to the exposed area of the nanostructure 30 or indirectly attached to the exposed area of the nanostructure 30, for example, through a connector 38 (shown in FIG. 6). The connection between the enzyme 36 and the exposed portion of the nanostructure 30 or between the enzyme 36 and the connector 38 and between the connector 38 and the exposed portion of the nanostructure 30 may be covalent or non-covalent, which may depend on the type of material of the nanostructure 30, the enzyme 36 and any connector 38 used. In one example, the connection is made by the nickel nitrile acetic acid/histidine tag (nickel NTA/His tag) method.

The connector 38 can be used as an anchor for an enzyme 36 (eg polymerase). Examples of suitable connectors 38 include polyethylene glycol (PEG), a DNA strand, a peptide nucleic acid (PNA) strand, an aliphatic hydrocarbon strand, and the like. In some instances, the length of the connector 38 is sufficient to keep the enzyme 36 at least 10 nm away from the nanostructure 30. This may be desirable, for example, so that conformational changes in the polymerase (or other enzyme 36), 36) and/or the charges of the target/template polynucleotide strand held by the polymerase (or other enzyme 36) did not interfere with sensor detection by the nanostructure 30.

Exposure of the screened substrate to an enzyme solution may be accompanied by heating to couple the enzyme 36 or connector 38 to the exposed portion(s) of any nanostructure(s) 30. However, it should be understood that some coupling reactions (e.g., amide coupling) may proceed with room temperature (for example, from about 18°C to about 25°C), and thus the need for additional heating may be determined by the type of enzyme 36 or connector 38, nanostructure 30 and the type of coupling reaction occurring between components 36 and 30 or 38 and 30.

The example flow cell 10'' shown in FIG. 6 obtained by method 200. Although each of the nanostructures 30 of the array can be functionalized with enzyme 36, it should be understood that in some examples the coupling reaction may not occur over the entire nanostructure 30, and thus only some (but not all) of the nanostructure 30 in the array can be functionalized with enzyme 36. In this example flow cell 10'', the porous molecular mesh 14 can remain on the base 12 and on the covered areas of the charge sensor 32 during a subsequent sequencing operation. Because the detection is done electronically and does not involve the use of fluorophores, the hydrophobic porous molecular network 14 can direct injected reagents (which are hydrophilic) to the enzyme-functionalized nanostructure 30.

Although not shown in FIG. 6, it should be understood that each charge sensor 32 may also include detection means that can sense the electrical response of the individual sensor 32. In one example, each of the detection means is an ammeter.

The 10'' flow cell discussed in this paper can be used to detect polymerase-induced incorporation into a single molecule (ie, to detect an event of nucleotide insertion into a growing strand). In particular, charge sensors 32 could track the dynamics of reactions of a single molecule.

To study a single molecule, the template polynucleotide chain can be introduced into the 10'' flow cell. The template polynucleotide strand may be any sample to be sequenced and may be composed of DNA, RNA, or analogs thereof (eg, peptide nucleic acids). The source of the template (or target) polynucleotide strand can be genomic DNA, messenger RNA, or other nucleic acids derived from natural sources.

The template polynucleotide strand can be held in place by a polymerase (one example of enzyme 36) that is directly or indirectly attached to the nanostructure 30. The template polynucleotide strand can be introduced into a biologically stable solution along with reagents such as nucleotide(s) inserted into the growing strand formed along the template polynucleotide chain. The biologically stable solution can be any buffer suitable for polymerase base insertion reactions such as polymerase chain reaction (PCR) or linear amplification. In one example, a biologically stable solution may include a buffer whose pH is about 7, the salt concentration is more than a few millimoles, and the concentration of Mg ^{2+ ions} is on the order of millimoles.

At least some of the nucleotides introduced may include a base that is complementary to the target nucleic acid of the template polynucleotide strand. Part of the nucleotide is held in place by the polymerase, which is also linked to the template polynucleotide chain. The bound nucleotide changes the charge sensor signal 32, which is read by the detection means.

It should be understood that after analyzing a single molecule (nucleotide) with the charge sensor(s) 32, the flow cell 10'' may be exposed to a destructuring agent, such as the oxidizing agents discussed herein. In this example, the destructuring agent cleaves the bound nucleotide from the polymerase and also oxidizes the diimide of the porous molecular network 14, which leads to the cleavage of the porous molecular network 14 from the backbone 12.

The flow cell 10'' can be reused after the introduction of a structurant (comprising a porous molecular network pre-assembled in solution) which forms a new porous molecular network 14 on the base 12 around the charge sensors 32, after which a sample is introduced into the cell, which includes a nucleotide (nucleotides), which are subsequently inserted into the next target nucleic acid of the template polynucleotide chain. The detection of a single molecule, the removal of the structure, and the re-creation of the structure can be repeated many times to analyze the entire template polynucleotide chain.

The considered flow cell 10'' can also be reused in other sequencing operations with a different template polynucleotide chain.

Covers for flow cells

The examples shown in FIG. 2, Fig. 3B and FIG. 6 are examples of flow cells 10, 10', 10'' on which the lids are not attached. Although not shown, the flow cells 10, 10', 10'' may have lids attached to at least a portion of the base 12. The lid may be positioned on the base 12 such that it defines a single flow channel or collection fluidly separated flow channels. It should be understood that in the flow channel(s) the chemicals 20, 20' are present on the surface (either singly or as an array).

Covers for flow cells 10, 10' (which are used for optical detection) can be made of any material that is transparent to excitation radiation directed at the surface chemical 20. For example, the cover can be made of glass (for example, borosilicate glass , fused silica, etc.), polymer or similar material. A commercially available example of a suitable borosilicate glass is D 263® glass available from Schott North America, Inc. Commercially available examples of suitable polymeric materials, namely cycloolefin polymers, are the ZEONOR® products supplied by Zeon Chemicals L.P. Covers for flow cells 10'' (which are used for electronic detection) can be made of any material, including transparent materials or any other material required. The lid may be attached using any suitable technique such as laser welding, diffusion welding, anode welding, eutectic alloy soldering, plasma activated bonding, glass solder, or other methods known in the art.

Detection system

One example of a detection system 40 discussed in this paper is shown in FIG. 7. This system 40 includes a flow cell 10'' and is an electronic detection system 40. In this example, the flow cell 10'' is a removable flow cell 10'', but it should be understood that the flow cell 10'' may be permanently included in the system 40 or may be removable from the system 40. In the example of a removable cell, the removable the flow cell 10'' can be removed and replaced with a new removable flow cell 10'', for example, for other sequencing operations. The detection system 40 is one example, and it should be understood that other system configurations may be applied.

An example of the detection system 40 is an electronic detection system that includes a housing 42 that contains various fixed components including, for example, electrical components, computer components, a power supply, a fan (blower), and the like. The system may include a screen 44, such as on the front surface of the housing 42, which may serve as a graphical user interface and may provide various information such as operating status, status of an ongoing analytical procedure (e.g., sequencing run), status of data transfer to or from the device. 40, instructions for use, warnings or similar information. Also located on the front surface of housing 42 is reservoir 46. Reservoir 46 may include two sections, a compartment related to maintenance of the flow cell, and a compartment related to the reservoir for the reagent cartridge. Removable flow cell 10'' may reside in reservoir compartment 46 to service the flow cell, or removable flow cell 10'' may be placed in (and removed from) reservoir compartment 46. If the removable flow cell 10'' resides in the flow cell service reservoir compartment 46, then the reservoir 46 may include one reagent cartridge accommodating port 50 in the reagent cartridge reservoir compartment. If a replaceable flow cell 10″ is placed in the compartment of the reservoir 46 for servicing the flow cell, then the reservoir 46 may include two ports 48, 50, one for receiving the removable flow cell 10″ and the other for receiving the reagent cartridge. The window(s) 48, 50 may have a protective door(s).

The detection system 40 may include a status indicator 52, which in this example is an indicator light on the frame of the reservoir 46. The status indicator 52 may indicate the presence or absence of a reagent cartridge and/or a replaceable flow cell 10''.

As shown, the flow cell 10'' includes a base 12 and a lid 54. Both the fixed and replaceable removable flow cell 10'' may also include fluid inlets and outlets 56, 58 that allow a volume of reagents to be delivered to the sensor(s). ) 32 charges (FIG. 5A) of the 10'' removable flow cell.

The sequencing system 40 may also include a reagent delivery system for selectively introducing reagent(s) into the fluid inlet 56 of the removable flow cell 10″ to flow over the sensors 32 and then withdraw through the fluid outlets 58 provided in the removable flow cell. cell 10''. The reagent delivery system may include tubing or other fluid systems that can be permanently or removably attached to openings 56, 58. The reagent delivery system may also include tubing or other fluid systems that can be operably connected to the reagent cartridge 60. The reagent delivery system may also include a pump or other suitable equipment to remove the reagent(s) from the reagent cartridge 60 and deliver it to the fluid inlet 56 in accordance with sequencing and structure removal/structure generation protocols (e.g., according to the method of using a removable flow cell 10'', considered in this work).

Various reagents can be supplied from the reagent cartridge 60 to the removable flow cell 10″. The reagent cartridge 60 includes a housing that protects various components of the fluid handling system, such as reservoirs, fluid connections, pumps, valves, and the like. In one example, the reagent cartridge 60 includes a fluid storage system that includes a destructuring agent reservoir 62 and a destructuring agent (eg, an oxidizing agent) contained in the destructuring agent reservoir 62; a structurant reservoir including two separate compartments 64, 66, where the first structurant component (eg, melamine or equivalent) is contained in the first of two separate compartments 64 and the second structurant component (eg, PTCDI or equivalent) is contained in the second of two separate compartments 66. The reagent cartridge 60 may also include a sample compartment 68, which may include sub-compartments for a template polynucleotide strand (which is subjected to sequencing) and for samples including nucleotides (which are inserted into a growing chain that is formed along the template polynucleotide strand ). The reagent cartridge 60 can be programmed to release the template polynucleotide strand and sample(s) during sequencing, to release a destructuring agent to remove the anchored nucleotide and porous molecular network 14, and to release structurants to form a new porous molecular network 14. Cartridge The reagent 60 may also include a mixing chamber into which the structurant reagents are sent for pre-assembly prior to being fed to the base 12 of the detachable flow cell 10″.

Additional Notes

It should be understood that all combinations of the above features and additional features discussed in more detail below (provided that these features are not mutually exclusive) form part of the subject matter of the invention discussed in this work. In particular, all combinations of the disclosed subjects of the invention, obvious after reading the proposed description, form part of the subject matter of the invention discussed in this work. It should also be understood that the terminology used in the present description, which can also be used in any document included in the present description by reference, should have the meaning that is most consistent with the specific features discussed in this work.

Reference in the present description to "one example", "another example", "example", etc. means that a specific element (eg, element, structure and/or characteristic) disclosed in the description of this example is included in at least one example discussed in this work, and may or may not be present in other examples. In addition, it should be understood that, unless otherwise clear from the context, the discussed elements of any example can be combined in various examples in any suitable way.

The terms "substantially" and "approximately" as used herein, including the claims, are used to describe and explain minor variations, such as those due to variations in processing. For example, these deviations may be less than or equal to plus/minus 5%, such as less than or equal to plus/minus 2%, such as less than or equal to plus/minus 1%, such as less than or equal to plus/minus 1%. /minus 0.5% e.g. less than or equal to plus/minus 0.2% e.g. less than or equal to plus/minus 0.1% e.g. less than or equal to plus/minus 0.05 %.

In addition, it should be understood that the ranges given in the present description include the indicated ranges and any values or subranges that are within the specified range, as if such values or subranges were clearly indicated. For example, a range of 1 to 50 includes not only the explicitly mentioned ranges of 1 to 50, but also includes individual values such as about 15, 22.5, 45, etc., and subranges such as from about 20 to about 48, etc.

Although some of the examples have been described in detail, it should be understood that the discussed examples can be modified. Thus, the above description is not limiting.

Claims (39)

1. Flow cell, including: basis; located on the basis of a selectively removable self-assembled porous molecular network that determines the pattern of open areas of the base; and a nanostructure located on at least some of the open areas, and a polymerase attached to the nanostructure, and the nanostructure is an electrically conductive channel, including a material selected from the group consisting of a conductor and a semiconductor, and has a geometric shape selected from the group consisting of tubes, wires and strips, where the selectively removable self-assembled porous molecular network is a planar supramolecular network of amine and diimide. 2. The flow cell of claim 1 wherein the amine is melamine or an analogue thereof and the diimide is perylenetetracarboxylic acid diimide or an analogue thereof. 3. The flow cell according to claim 1, in which one polymerase is attached to one nanostructure. 4. The flow cell according to claim 1, wherein the nanostructure is electrically connected to two electrodes. 5. The flow cell according to claim 1, additionally including a connecting element that attaches the polymerase to the nanostructure. 6. The flow cell according to claim 1, wherein the nanostructure is selected from the group consisting of a silicon nanowire and a carbon nanotube. 7. System for sequencing, including: removable flow cell including: basis; located on the basis of selectively removable self-assembled porous molecular network that determines the pattern of open areas of the base, where the selectively removable self-assembled porous molecular network is a planar supramolecular network of amine and diimide; a nanostructure attached to each of the open portions of the substrate, the nanostructure being an electrically conductive channel comprising a material selected from the group consisting of a conductor and a semiconductor and having a geometric shape selected from the group consisting of a tube, a wire and a strip; a polymerase attached to at least some of the nanostructures; and openings for inlet and outlet of fluids; a reservoir for i) permanently accommodating a removable flow cell or ii) for inserting a removable flow cell; and a liquid system for selectively supplying a destructuring agent to the removable flow cell in order to remove a selectively removable self-assembled porous molecular network from the substrate and selectively supply a structure-forming reagent to the removable flow cell in order to apply a new selectively removable self-assembled porous molecular network to the substrate. 8. The sequencing system of claim 7, wherein the fluidic system includes a cartridge reservoir for housing a reagent cartridge including at least a disruptive reagent and a structurant reagent. 9. The sequencing system of claim 8, further comprising a reagent cartridge, wherein the reagent cartridge includes: a fluid storage system comprising: reservoir for destructuring reagent; a destructuring agent contained in the destructuring agent reservoir; a structurant reservoir comprising two separate compartments; the first component of the structurant reagent contained in the first of two separate compartments; and the second component of the structurant contained in the second of two separate compartments. 10. A sequencing system according to claim 9, wherein: the destructuring agent includes an oxidizing agent; the first component of the structure-forming reagent is perylenetetracarboxylic acid diimide or an analogue thereof; and the second component of the structure-forming reagent is melamine or its equivalent. 11. The sequencing system of claim 10 wherein the oxidizing agent is selected from the group consisting of permanganates, perborates, halogens, perchlorates, and peroxides. 12. A method for obtaining a flow cell according to claim 1, including: formation of a stencilled backing by applying a self-assembling porous molecular network to the surface of the support on which the nanostructures are located, where the self-assembling porous molecular network is a planar supramolecular network of amine and diimide, and where the self-assembled porous molecular network self-assembles to form a pattern of open areas of the support, so that at least a portion of each nanostructure remains open; and exposing the screened backing to a polymerase solution that is inert to the self-assembled porous molecular network, resulting in the selective attachment of the appropriate polymerase to at least some of the exposed parts of the nanostructures. 13. The method according to p. 12, in which the application of a self-assembled porous molecular network includes: heating a saturated mixture of perylenetetracarboxylic acid diimide or an analogue thereof and melamine or an analogue thereof; depositing the saturated mixture on the substrate to which the nanowire was attached; and cooling the precipitated saturated mixture.

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