A solvent carrier for nanopore sequencing and methods of making and using the same

Abstract

This application provides a solvent carrier for nanopore sequencing, its preparation method, and its application. The solvent carrier for nanopore sequencing comprises a nanopore sequencing unit containing microwells, the upper end of which is covered with a nonpolar film-forming solution. The nonpolar film-forming solution includes a copolymer membrane and a nonpolar solvent. The end groups of the nonpolar solvent contain unsaturated bonds I, and the surface of the upper end of the microwell contains unsaturated bonds II. The nonpolar solvent is cross-linked with the surface of the upper end of the microwell to form the solvent carrier. This application cross-links the solvent of the film-forming solution to form a network structure, thereby allowing the formed membrane structure to be confined within this network structure and to exist stably, avoiding membrane rupture and other issues.

Description

Technical Field

[0001] This application relates to the field of nanopore sequencing technology, specifically to a solvent carrier for nanopore sequencing, its preparation method, and its application. Background Technology

[0002] Gene sequencing technology uses nanopores as biosensors, which are embedded in an insulating amphiphilic membrane. When a single-stranded DNA molecule passes through the nanopore, the different current changes caused by different base groups are read by the signal receiver in the nanopore, and then the corresponding base recognition is performed, thereby realizing the detection of gene sequences.

[0003] When preparing molecular membranes for gene sequencing chips, a first layer of electrolyte, a second layer of organic nonpolar film-forming solution, and a third layer of electrolyte polar solvent are sequentially introduced into the film-forming region of the gene sequencing chip, so that the nonpolar solvent of the amphiphilic material is sandwiched between the two polar solvent layers to form a molecular membrane.

[0004] Existing amphiphilic molecular films still have technical problems such as uneven film formation, low film formation stability, and large differences in film formation between various microsupport structures. Summary of the Invention

[0005] To solve the above problems, this application adopts the following technical solution: The inventive point of this application is to provide a solvent carrier for nanopore sequencing, wherein the nanopore sequencing unit contains microwells and the upper end of the microwells is covered with a non-polar film-forming liquid; the non-polar film-forming liquid includes a copolymer membrane and a non-polar solvent, the end groups of the non-polar solvent contain unsaturated bonds I, and the surface of the upper end of the microwell contains unsaturated bonds II; the non-polar solvent crosslinks with the surface of the upper end of the microwell to form a solvent carrier.

[0006] Optionally, unsaturated bond I and unsaturated bond II may be the same or different; unsaturated bond I and unsaturated bond II independently include any one or more of carbon-carbon double bonds, carbon-carbon triple bonds, carbonyl groups, ester groups, carboxyl groups, aldehyde groups, amide groups, cyano groups, and imine groups.

[0007] Optionally, the copolymer film may be cross-linked with a non-polar solvent and the surface of the microwell, or may not be cross-linked.

[0008] Another inventive point of this application is to provide a method for preparing the solvent carrier as described above.

[0009] Optionally, the preparation method includes: (1) adding a polar solution to a microwell; the non-polar film-forming liquid further includes an initiator; (2) adding a non-polar film-forming liquid to a microwell; (3) adding a polar solution to a microwell; (4) cross-linking to obtain a solvent carrier.

[0010] Optionally, the initiator includes a photoinitiator and / or a thermal initiator.

[0011] Optionally, before step (1), the method further includes: treating the surface of the upper end of the microwell to make the surface contain unsaturated bonds II; the treatment includes: modifying the surface of the upper end of the microwell to make the surface contain unsaturated bonds II.

[0012] Optionally, the modification includes hydrophilic modification and / or hydrophobic modification.

[0013] Optionally, the nonpolar solvent includes any one or more of isobornyl methacrylate, isobornyl acrylate, lauryl acrylate, lauryl methacrylate, dicyclopentadiene acrylate, and dipentaerythritol pentaacrylate.

[0014] Optionally, the photoinitiator includes any one or more of benzoin dimethyl ether, trimethylbenzoyl phenyl phosphate, hydroxycyclohexyl phenyl ketone, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide; the thermal initiator includes any one or more of azobisisobutyronitrile, cumene hydroperoxide, di-tert-butyl peroxide, and benzoyl peroxide.

[0015] Another inventive point of this application is to provide the application of the solvent carrier as described above or the solvent carrier prepared by any of the preparation methods described above in nanopore sequencing.

[0016] Compared with the prior art, this application has the following advantages: This application cross-links the solvent of the film-forming solution to form a network structure, thereby confining and stably maintaining the formed membrane structure within this network, preventing membrane rupture and other issues. Simultaneously, it does not restrict the membrane's flow state, avoiding any impact on sample delivery during subsequent sequencing. Furthermore, the cross-linking of the solvent with the microwells ensures a strong bond between the network structure and the microwells, preventing membrane position uncertainty caused by network movement. Attached Figure Description

[0017] Figure 1 This is a structural diagram of a gene sequencing chip provided in one embodiment of this application.

[0018] Figure 2 Another structural diagram of a gene sequencing chip provided in one embodiment of this application.

[0019] Figure 3 This is a structural diagram of a gene sequencing chip provided in an embodiment of this application, through which a first layer of electrolyte (polar solvent) is introduced.

[0020] Figure 4 Another structural diagram of a gene sequencing chip provided in one embodiment of this application, through which a first layer of electrolyte (polar solvent) is introduced.

[0021] Figure 5 This is a structural diagram of a gene sequencing chip provided in an embodiment of this application, through which a second layer of organic nonpolar film-forming solution is introduced.

[0022] Figure 6 Another structural diagram of a gene sequencing chip provided in one embodiment of this application, through which a second layer of organic nonpolar film-forming liquid is introduced.

[0023] Figure 7 This is a structural diagram of a gene sequencing chip provided in an embodiment of this application, through which a third layer of electrolyte (polar solvent) is introduced.

[0024] Figure 8 Another structural diagram of a gene sequencing chip provided in one embodiment of this application, through which a third layer of electrolyte (polar solvent) is introduced.

[0025] Figure 9 This is a structural diagram of a cross-linked gene sequencing chip provided in one embodiment of this application.

[0026] Figure 10 Another structural diagram of a cross-linked gene sequencing chip provided in one embodiment of this application.

[0027] Figure 11 This is a structural diagram of cross-linked and pore-forming proteins in a gene sequencing chip provided in an embodiment of this application.

[0028] Figure 12 Another structural diagram of cross-linked and porogen proteins in a gene sequencing chip provided in an embodiment of this application.

[0029] Figure 13 This is a current diagram for detecting the breakdown voltage of a membrane provided in one embodiment of this application.

[0030] Figure 14 A comparison diagram (a) and a diagram (b) illustrating the physical meaning of the opening current provided in an embodiment of this application.

[0031] Figure 15 This is a schematic diagram of a pre-crosslinking membrane provided in an embodiment of this application.

[0032] Figure 16 This is a schematic diagram of a cross-linked membrane provided in one embodiment of this application.

[0033] Figure 17 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0034] Figure 18 This is a schematic diagram of a pre-crosslinking membrane provided in an embodiment of this application.

[0035] Figure 19This is a schematic diagram of a cross-linked membrane provided in one embodiment of this application.

[0036] Figure 20 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0037] Figure 21 This is a schematic diagram of a pre-crosslinking membrane provided in an embodiment of this application.

[0038] Figure 22 This is a schematic diagram of a cross-linked membrane provided in one embodiment of this application.

[0039] Figure 23 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0040] Figure 24 This is a schematic diagram of a pre-crosslinking membrane provided in an embodiment of this application.

[0041] Figure 25 This is a schematic diagram of a cross-linked membrane provided in one embodiment of this application.

[0042] Figure 26 This is an example of an embodiment of the cross-linked porous protein provided in this application. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0045] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0046] The conventional preparation method for gene sequencing chip molecular membranes is as follows: first, obtain... Figure 1 or Figure 2 The chip shown has electrodes on its bottom, and a microwell structure is formed by photolithography using photoresist. Then, a first layer of electrolyte (polar solvent) is sequentially introduced into this microwell structure to wet the entire film-forming area, as shown below. Figure 3 or Figure 4As shown; then a second layer of organic nonpolar film-forming solution is introduced to displace part of the first layer of electrolyte, as shown. Figure 5 or Figure 6 As shown; then a third layer of electrolyte (polar solvent) is introduced, so that the nonpolar solvent of the amphiphilic material is sandwiched between the two polar solvent layers to form a molecular film, as shown. Figure 7 or Figure 8 As shown.

[0047] In conventional molecular film preparation methods, the non-polar solvent in the film-forming solution adheres to the photoresist through physical action, which cannot be fixed and easily causes the molecular film to expand and eventually break, resulting in a short lifespan.

[0048] A solvent carrier for nanopore sequencing is provided, wherein the nanopore sequencing unit contains microwells, and the upper end of each microwell is covered with a nonpolar film-forming solution. The nonpolar film-forming solution includes a copolymer membrane and a nonpolar solvent, wherein the end groups of the nonpolar solvent contain unsaturated bonds I, and the surface of the upper end of the microwell contains unsaturated bonds II. The nonpolar solvent crosslinks with the surface of the upper end of the microwell to form a solvent carrier. Figure 9 Or as shown in 10.

[0049] In conventional molecular membrane preparation methods, the nonpolar film-forming solution only comes into contact with the upper end of the microwell. Therefore, even if cross-linking occurs, it is the upper end of the microwell that cross-links with the nonpolar solvent.

[0050] Unsaturated bond I and unsaturated bond II may be the same or different; unsaturated bond I and unsaturated bond II independently include any one or more of the following: carbon-carbon double bond, carbon-carbon triple bond, carbonyl group, ester group, carboxyl group, aldehyde group, amide bond, cyano group, and imine group.

[0051] The copolymer film can be crosslinked with a non-polar solvent or with the surface of the microwell, or not crosslinked.

[0052] Since both the nonpolar solvent and the microwell surface have unsaturated bonds, when the copolymer film also has unsaturated bonds, cross-linking can occur between the three components; when the copolymer film does not contain cross-linkable unsaturated bonds, only the nonpolar solvent and the microwell surface undergo cross-linking.

[0053] Preferably, when the copolymer membrane does not contain crosslinkable unsaturated bonds, crosslinking occurs only between the nonpolar solvent and the microwell surface. This is because the membrane needs to have a certain degree of fluidity during sequencing, allowing the sample to pass near the nanopores more quickly during the sequencing process.

[0054] The copolymer film is preferably a diblock copolymer film or a triblock copolymer film that does not contain unsaturated bonds.

[0055] Copolymers that do not contain unsaturated bonds can be diblock copolymers or triblock copolymers containing polydimethylsiloxane; The hydrophilic end of the diblock copolymer includes one or more of polyethylene glycol, polyvinyl alcohol, polyethyleneamine, polyethyleneimine, and polyacrylamide.

[0056] The hydrophobic end includes one or more of polydimethylsiloxane, 100% hydrogenated poly1,2-butadiene, polypropylene, polyethylene, and 100% hydrogenated poly1,4-butadiene. That is, the hydrophobic end has no additional unsaturated bonds for covalent bonding; it cannot react with solvents or other substances.

[0057] Diblock copolymers can be represented by the following formula:

[0058] Where n ranges from 5 to 40; m ranges from 5 to 40; and p ranges from 5 to 40.

[0059] n can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; m can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; p can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0060] The concentration of the copolymer is 2 mg / mL to 100 mg / mL, and can be 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL or any value within this range.

[0061] The solvent for the copolymer is an alkane; preferably one or more of n-decane, n-tetradecane, and n-hexadecane.

[0062] The method for preparing the solvent carrier is an improvement on the conventional method for preparing molecular membranes.

[0063] The process includes: (1) adding a polar solution to a microwell; (2) adding a non-polar film-forming solution to a microwell; wherein the non-polar film-forming solution also includes an initiator; (3) adding a polar solution (200mM KCl, 150mM K3Fe(CN)6, 100mM K4Fe(CN)6) to a microwell; and (4) cross-linking to obtain a solvent carrier.

[0064] The polar solvent is an aqueous solution containing KCl, K3Fe(CN)6, and K4Fe(CN)6.

[0065] The preferred aqueous solution is 200 mM KCl, 150 mM K3Fe(CN)6, and 100 mM K4Fe(CN)6.

[0066] Initiators include one or more of photoinitiators, thermal initiators, and chemical initiators.

[0067] Non-polar solvents include non-polar crosslinkable solvents and inert solvents; wherein, the non-polar crosslinkable solvent is used to achieve surface crosslinking with the upper end of the microwell; the inert solvent is used to dissolve the copolymer film and the non-polar crosslinkable solvent. The inert solvent is preferably an alkane; preferably one or more of n-decane, n-tetradecane, and n-hexadecane.

[0068] The initiator accounts for 5 mg / mL to 20 mg / mL of the nonpolar crosslinkable solvent.

[0069] Photoinitiators are those that, under light irradiation, can cause nonpolar solvents to crosslink with the surface of the microstructure; the light can be ultraviolet light, infrared light, or visible light, etc.; ultraviolet light is preferred.

[0070] The conditions for light exposure are: The wavelength of light is 200 nm to 480 nm.

[0071] The light intensity is 10~100W; it can be 10W, 20W, 30W, 40W, 50W, 60W, 70W, 80W, 90W, 100W or any other value within this range.

[0072] The illumination time is 1 to 10 minutes; it can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes or any other value within this range.

[0073] Photoinitiators include any one or more of benzoin dimethyl ether, trimethylbenzoyl phenyl phosphate ethyl ester, 1-hydroxycyclohexylphenyl ketone, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide.

[0074] Thermal initiators are used to induce cross-linking between nonpolar solvents and the surface of the microwell under heating conditions.

[0075] The heating conditions are: The heating temperature is 30℃~50℃; it can be 30℃, 35℃, 40℃, 45℃, 50℃ or any other value within this range.

[0076] The heating time is 1 min to 2 h; it can be 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 0.5 h, 1 h, 1.5 h, 2 h or any other value within this range.

[0077] Thermal initiators include any one or more of azobisisobutyronitrile, cumene hydroperoxide, di-tert-butyl peroxide, and benzoyl peroxide.

[0078] The concentration of the thermal initiator is 2 mg / ml to 10 mg / ml, and can be 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL or any value within this range.

[0079] Chemical initiators initiate double bond polymerization reactions after the initiator is added.

[0080] Chemical initiators include weak reducing agents and peroxides; weak reducing agents include one or more of sodium metabisulfite, sodium sulfite, and tetramethylethylenediamine; peroxides include one or more of potassium persulfate, ammonium persulfate, and sodium persulfate.

[0081] Under the action of a weak reducing agent, the peroxide bond of the peroxide breaks to form a free radical, which initiates a double bond polymerization reaction.

[0082] Unsaturated bond I and unsaturated bond II may be the same or different; unsaturated bond I and unsaturated bond II independently include any one or more of carbon-carbon double bonds, carbon-carbon triple bonds, carbonyl groups, ester groups, carboxyl groups, aldehyde groups, amide groups, cyano groups, and imine groups; preferably any one or more of carbon-carbon double bonds, carbon-carbon triple bonds, carbonyl groups, ester groups, and carboxyl groups.

[0083] The unsaturated bond I is preferably a carbon-carbon double bond; more preferably an alkenyl group.

[0084] Preferably, the nonpolar solvent includes compounds containing an alkenyl group; In the preferred compounds containing alkenyl groups, the number of alkenyl groups is at least one; more preferably, the number of alkenyl groups is one, two, three, four, five or more.

[0085] The nonpolar solvent includes any one or more of isobornyl methacrylate, isobornyl acrylate, lauryl acrylate, lauryl methacrylate, dicyclopentadiene acrylate, difunctional polyurethane acrylate, dipentaerythritol pentaacrylate, and tricyclosepiacetic diacrylate. Preferably, the nonpolar solvent includes two or more of isobornyl methacrylate, isobornyl acrylate, lauryl acrylate, lauryl methacrylate, dicyclopentadiene acrylate, dipentaerythritol pentaacrylate, and tricyclosepiacetic diacrylate. Using a composite solvent can result in a more compact cross-linked structure and better membrane stability.

[0086] More preferably, the nonpolar solvent includes compounds containing different numbers of alkenyl groups. By varying the number of propylene groups, different cross-linking structures can be achieved, resulting in a more stable bond with the photoresist and better positional confinement of the film.

[0087] The surface at the upper end of the microwell contains unsaturated bond II; this unsaturated bond II matches unsaturated bond I and can undergo cross-linking. Preferably, it is any one or more of carbon-carbon double bonds, carbon-carbon triple bonds, carbonyl groups, ester groups, and carboxyl groups.

[0088] Currently, such as Figure 1 In the chip, the microwell structure is mostly formed by photolithography using photoresist; the photoresist is either negative or positive, preferably negative, and more preferably SU-8 photoresist.

[0089] SU-8 photoresist is a commonly used epoxy-based negative photoresist. "Negative" means that when the photoresist is exposed to ultraviolet light, the exposed portion forms crosslinks, while the remaining portion remains soluble and is washed away during development. The name SU-8 comes from the eight epoxy groups in its structure, which can crosslink to form the final structure.

[0090] The surface of the microwell is preferably modified to contain unsaturated bonds II.

[0091] Modifications include: (1) adding hydroxyl groups to the surface of the microwell; and (2) adding unsaturated II bonds.

[0092] Preferably, the solution is soaked in dilute sulfuric acid, cerium ammonium nitrate solution or 1-aminoglycerol solution, or plasma treatment is used.

[0093] More preferably, when the microwell is formed by photolithography using SU-8 photoresist, the epoxy groups on the surface of SU-8 are transformed into hydroxyl groups by soaking in 9M / L dilute sulfuric acid for half an hour, or by plasma treatment of the photoresist for 5 minutes, or by soaking in a 10% cerium ammonium nitrate aqueous solution for 30 minutes, or by soaking in a 20% aminoglycerol aqueous solution for 30 minutes.

[0094] Adding materials containing unsaturated II bonds allows them to combine with hydroxyl groups and adhere to the surface of the microwell.

[0095] Materials containing unsaturated bond II include those containing acrylates or olefins.

[0096] Modification includes hydrophilic modification and / or hydrophobic modification, meaning the modified microwell surface exhibits hydrophilic or hydrophobic properties. Preferably, the modification is hydrophobic, meaning the material containing unsaturated bond II has hydrophobic properties. This makes the microwell surface hydrophobic, allowing for better similarity-to-miscibility with nonpolar solvents, resulting in more contact, more complete subsequent binding, and a more stable solvent carrier.

[0097] Materials containing unsaturated bond II may include one or more of silanes, acyl chlorides, or carboxylic acids.

[0098] Silanes include one or more of trimethoxysilyl methacrylate, methoxysilyl acrylate, allyltrimethoxysilane, tris(2-methoxyethoxy)vinylsilane, vinyltrimethoxysilane, and triethoxyvinylsilane; acyl chlorides include one or more of 3,3-dimethylacryloyl chloride, crotonyl chloride, and 4-pentenoyl chloride; and carboxylic acids include acrylic acid and / or methacrylic acid.

[0099] For example, it can be done by dissolving 10% trimethoxysilyl acrylate in anhydrous ethanol and soaking for 12 hours.

[0100] The preparation method of this application further includes: embedding a porin between step (3) and step (4), or embedding a porin after step (4). The structure after embedding the porin is as follows: Figure 11 and Figure 12 As shown.

[0101] Method I: Porin is embedded between steps (3) and (4). That is, the porin is embedded after the nonpolar solvent crosslinks with the surface of the microwell.

[0102] Method II: Embedding the porin after step (4). That is, embedding the porin first, and then cross-linking the surface of the microwell with a non-polar solvent.

[0103] In this application, any suitable porin may be used; including but not limited to porins derived from Mycobacterium smegmatis A, Mycobacterium smegmatis B, Mycobacterium smegmatis C, Mycobacterium smegmatis D, hemolysin, cytolysin, interleukin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, WZA, or Neisseria autotransporter lipoprotein, etc.; for example, it may be a mutant of MspA, CsgG, FraC, ClyA, especially MspA or CsgG proteins; more specifically: CsgG nanopores (specifically CsgG-Y51A / F56Q / R97W in WO2017 / 149318A1) or MspA nanopores (MspA protein sequence is SEQ ID NO:31, according to Michael Faller et al., “The Structure of a Mycobacterial Outer-Membrane Channel”, Science). Preparation was carried out as described in 303,1189(2004); DOI:10.1126 / science.1094114.

[0104] Because copolymer films are fluid, they will rupture when the voltage across them reaches a critical value. This phenomenon can be observed under a microscope.

[0105] Test method for membrane breakdown voltage: initial voltage 140mV, voltage increased by 10mV every 3s; Method 1: Due to the fluidity of the copolymer film, it will rupture when the voltage across the copolymer film reaches a critical value. This phenomenon can be observed under a microscope.

[0106] Method 2: Because the membrane contains nanoporous proteins during testing, when the membrane is not broken down, the current detected by a single sequencing unit is around 1.0 nA when a voltage is applied. When the membrane breaks down, the current detected by a single sequencing unit increases by two orders of magnitude, reaching around 100 nA. The voltage applied across the membrane at this point is recorded; this voltage is the breakdown voltage. For example... Figure 13 After 14 pressurization cycles, the current rapidly increased (in the red box), indicating that the membrane ruptured. The breakdown voltage of this porous membrane was 480mV.

[0107] At a sampling rate of 5 kHz, 10,000 consecutive sampling points were selected, and the standard deviation σ was calculated. If σ < 16 / 6.6 pA, the pore opening current of the pore protein was considered normal.

[0108] Calculation formula:

[0109] Where, x iThe i-th sampling point; x (the horizontal line above) is the mean; N is the total number of sampling points, n = 6.6 × standard deviation σ.

[0110] According to the above formula, for example, in Figure 14 The state of the hole can be determined by the current signal of the hole. The smaller the value, the less noise, and the better the state of the hole.

[0111] Example 1

[0112] Pretreatment: Immerse the chip in 9M / L dilute sulfuric acid for half an hour, clean it with ethanol, then add anhydrous ethanol to dissolve 10% trimethoxysilyl acrylate and immerse for 12 hours. (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution is added to the microwell; the non-polar film-forming solution also includes an initiator; The nonpolar film-forming solution comprises: a block copolymer with a hydrophobic end of polydimethylsiloxane (degree of polymerization 10) and a hydrophilic end of polyethylene glycol (degree of polymerization 5), and a 10 mg / ml Ominirad 184 (1-hydroxycyclohexylphenyl ketone) photoinitiator; the solvent is: 40% isobornyl methacrylate, 40% light mineral oil (item number: M5904), and 20% tricyclosepiacetic acid diacrylate; (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the microwell; embed CsgG nanoporous protein; then add 600mM KCl solution; (4) Irradiate with 40W 254nm ultraviolet light at a distance of 5cm for 5min.

[0113] The obtained membrane and nanoporous protein system were tested; the breakdown voltage was 600mV, indicating that the cross-linked membrane has high stability. The morphology of the membrane before and after UV cross-linking was also compared. Figure 15 and Figure 16 As shown, after cross-linking, the membrane boundaries are clear and distinct, diffusion is not easily induced, and the stability is good. Furthermore, the pore-opening current of the cross-linked porin was tested, such as... Figure 17 As shown, after cross-linking, the porin still exhibits good pore opening current with a low range and a standard deviation σ < 16 / 6.6 pA; this indicates that the formation of the solvent carrier enhances the stability of the membrane without affecting the pore state or subsequent sequencing.

[0114] Example 2

[0115] Pretreatment: The chip was treated with a 100W plasma cleaner for 3 minutes, cleaned with ethanol, and then 10% of methoxysilyl acrylate was dissolved in anhydrous ethanol and soaked for 12 hours. (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution is added to the microwell; the non-polar film-forming solution also includes an initiator; The nonpolar film-forming solution includes: a block copolymer with 10 mg / ml of hydrophobic end polydimethylsiloxane (degree of polymerization 10) and hydrophilic end polyethylene glycol (degree of polymerization 10), and 10 mg / ml AIBN (azobisisobutyronitrile); the solvent is: 40% lauryl methacrylate, n-decane, and 20% tricyclic sebacate diacrylate; (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the microwell; embed MspA nanoporous protein; then add 600mM KCl solution; (4) Heat the membrane to 50 degrees Celsius and maintain for 30 minutes.

[0116] The obtained membrane and nanoporous protein system were tested; the breakdown voltage was 600mV, and the cross-linked membrane exhibited high stability. The morphology of the membrane before and after UV cross-linking was also compared. Figure 18 and Figure 19 As shown, after cross-linking, the membrane boundaries are clear and distinct, diffusion is not easily induced, and stability is good. Furthermore, the opening current of the cross-linked porin, such as... Figure 20 As shown, the range is low, with a standard deviation σ < 16 / 6.6 pA; this indicates that the formation of the solvent carrier enhances the stability of the membrane without affecting the state of the pores or subsequent sequencing.

[0117] Example 3

[0118] Pretreatment: The chip was soaked in a 10% (w / w) cerium ammonium nitrate aqueous solution for 30 min, cleaned with ethanol, and then 10% allyltrimethoxysilane was dissolved in anhydrous ethanol and soaked for 12 h. (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution is added to the microwell; the non-polar film-forming solution also includes an initiator; The nonpolar film-forming solution comprises: 10 mg / ml of a block copolymer with a hydrophobic end of 100% hydrogenated and reduced poly(1,2-butadiene) (degree of polymerization 20) and a hydrophilic end of polyethylene glycol (degree of polymerization 10), 5 mg / ml of AIBN (azobisisobutyronitrile), and 5 mg / ml of benzoin dimethyl ether photoinitiator; the solvent is: 40% isobornyl acrylate, 40% n-dodecane, and 20% tricyclosaccharide diacrylate. (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the microwell; embed CsgG nanoporous protein; then add 600mM KCl solution; (4) Irradiate the membrane with 40W 254nm ultraviolet light at a distance of 5cm for 3min; then heat the membrane to 50 degrees Celsius and maintain it for 15min.

[0119] The obtained membrane and nanoporous protein system were tested; the breakdown voltage was 600mV, and the cross-linked membrane exhibited high stability. The morphology of the membrane before and after UV cross-linking was also compared. Figure 21 and Figure 22 As shown, after crosslinking, the membrane boundaries are clear and distinct, diffusion is not easily induced, and the stability is good. Because both light and heat crosslinking were performed simultaneously, the degree of crosslinking of the solvent carrier was deepened, resulting in even better membrane stability. Furthermore, the pore current of the crosslinked porin, such as... Figure 23 As shown, the range is low, with a standard deviation σ < 16 / 6.6 pA; this indicates that the formation of the solvent carrier enhances the stability of the membrane without affecting the state of the pores or subsequent sequencing.

[0120] Example 4

[0121] Pretreatment: Immerse the chip in a 20% (w / w) aqueous solution of 1-aminoglycerol for half an hour, clean it with ethanol, then add anhydrous ethanol to dissolve 10% trimethoxysilyl acrylate and immerse for 12 hours. (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution is added to the microwell; the non-polar film-forming solution also includes an initiator; The nonpolar film-forming solution comprises: 10 mg / ml of 100% hydrogenated and reduced poly(1,2-butadiene) with a hydrophobic end (degree of polymerization 20) and a hydrophilic end of polyethylene glycol (degree of polymerization 15) block copolymer; 5 mg / ml of benzoyl peroxide; and 5 mg / ml of Ominirad 184 photoinitiator; the solvent comprises: 40% lauryl acrylate, 40% n-tetradecane, and 20% tricyclosepiacetic acid diacrylate. (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (4) Irradiate the membrane with 40W 254nm ultraviolet light at a distance of 5cm for 3min; then heat the membrane to 50 degrees Celsius and maintain it for 15min.

[0122] (5) Embed CsgG nanoporous protein; then add 600mM KCl solution.

[0123] The obtained membrane and nanoporous protein system were tested; the breakdown voltage was 600mV, and the cross-linked membrane exhibited high stability. The morphology of the membrane before and after UV cross-linking was also compared. Figure 24 and Figure 25 As shown, after cross-linking, the membrane boundaries are clear and distinct, diffusion is not easily induced, and stability is good. Furthermore, the opening current of the cross-linked porin, such as... Figure 26 As shown, the range is low, with a standard deviation σ < 16 / 6.6 pA; this indicates that the formation of the solvent carrier enhances the stability of the membrane without affecting the state of the pores or subsequent sequencing.

[0124] As demonstrated in Examples 3 and 4, the generation of the solvent carrier does not affect the embedding of nanoporous proteins, nor does it affect the performance of the pores or the subsequent sequencing results.

[0125] Comparative Example 1

[0126] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1 The micro well shown.

[0127] 2) A non-polar film-forming solution is added to the microwell; the non-polar film-forming solution also includes an initiator; The nonpolar film-forming solution comprises: a block copolymer with a hydrophobic end of polydimethylsiloxane (degree of polymerization 10) and a hydrophilic end of polyethylene glycol (degree of polymerization 5), and a 10 mg / ml Ominirad 184 (1-hydroxycyclohexylphenyl ketone) photoinitiator; the solvent is: 40% isobornyl methacrylate, 40% light mineral oil (item number: M5904), and 20% tricyclosepiacetic acid diacrylate; (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the microwell; embed CsgG nanoporous protein; then add 600mM KCl solution; The membrane and nanoporous protein system obtained above were tested; the breakdown voltage was 300mV, which is significantly lower than the breakdown voltage of Example 1; indicating that the solvent carrier of this application can effectively improve membrane stability.

[0128] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the system described above can be found in the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0129] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the storage device and processing device described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0130] The term "comprising" or any other similar term is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus / device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent in such process, method, article, or apparatus / device.

[0131] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A solvent carrier for nanopore sequencing, wherein the nanopore sequencing unit contains microwells, and the upper end of each microwell is covered with a nonpolar film-forming solution; the nonpolar film-forming solution comprises a copolymer membrane and a nonpolar solvent, characterized in that, The nonpolar solvent end group contains unsaturated bond I, and the surface of the microwell top contains unsaturated bond II; the nonpolar solvent crosslinks with the surface of the microwell top to form a solvent carrier.

2. The solvent carrier according to claim 1, characterized in that, Unsaturated bond I and unsaturated bond II may be the same or different; unsaturated bond I and unsaturated bond II independently include any one or more of the following: carbon-carbon double bond, carbon-carbon triple bond, carbonyl group, ester group, carboxyl group, aldehyde group, amide bond, cyano group, and imine group.

3. The solvent carrier according to claim 1, characterized in that, The copolymer film can be cross-linked with a non-polar solvent or with the surface of the microwell, or not cross-linked.

4. A method for preparing a solvent carrier as described in any one of claims 1 to 3, characterized in that, include: (1) Add the polar solution to the microwell; (2) Add the non-polar film-forming solution into the microwell; The non-polar film-forming liquid also includes an initiator (3) to add the polar solution into the microwell; (4) cross-linking occurs to obtain a solvent carrier.

5. The preparation method according to claim 4, characterized in that, The initiator includes one or more of photoinitiators, thermal initiators, and chemical initiators.

6. The preparation method according to claim 4, characterized in that, Before step (1), the method further includes: treating the surface of the upper end of the microwell to make it contain unsaturated bonds II; the treatment includes: modifying the surface of the upper end of the microwell to make it contain unsaturated bonds II.

7. The preparation method according to claim 6, characterized in that, The modification includes hydrophilic modification and / or hydrophobic modification; Materials containing unsaturated bond II may include one or more of silanes, acyl chlorides, or carboxylic acids.

8. The preparation method according to claim 4, characterized in that, The nonpolar solvent includes any one or more of isobornyl methacrylate, isobornyl acrylate, lauryl acrylate, lauryl methacrylate, dicyclopentadiene acrylate, and dipentaerythritol pentaacrylate.

9. The preparation method according to claim 4, characterized in that, The photoinitiator includes any one or more of benzoin dimethyl ether, trimethylbenzoyl phenyl phosphate ethyl ester, hydroxycyclohexylphenyl ketone, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide; the thermal initiator includes any one or more of azobisisobutyronitrile, cumene hydroperoxide, di-tert-butyl peroxide, and benzoyl peroxide.

10. The application of the solvent carrier as described in any one of claims 1 to 3 or the solvent carrier prepared by any one of claims 4 to 9 in nanopore sequencing.

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