Membrane structure solvent for nanopore sequencing and preparation method and application thereof

Abstract

This application provides a membrane structure solvent for nanopore sequencing, its preparation method, and its application. The membrane structure solvent for nanopore sequencing has the membrane structure located within a crosslinkable solvent; the crosslinkable solvent self-crosslinks to form a network structure, encapsulating the membrane structure. This application crosslinks the solvent of the membrane-forming solution to form a network structure, thereby confining the formed membrane structure within this network structure and ensuring its stability, preventing membrane rupture and other issues.

Description

Technical Field

[0001] This application relates to the field of nanopore sequencing technology, specifically to a membrane structure solvent 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 typically introduced sequentially 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 membrane structure solvent for nanopore sequencing, wherein the membrane structure is located inside a crosslinkable solvent; the crosslinkable solvent self-crosslinks to form a network structure, thereby forming a coating structure on the membrane structure.

[0006] Optionally, the crosslinkable solvent comprises a monomer containing unsaturated bonds; the crosslinkable solvent is obtained by crosslinking through unsaturated bonds; the conditions for self-crosslinking include light, heating, or a chemical initiator.

[0007] Optionally, the unsaturated bond includes 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.

[0008] Optionally, the crosslinkable solvent includes a monomer or polymer containing an alkenyl group; wherein the number of alkenyl groups in the monomer or polymer is at least one.

[0009] Optionally, the crosslinkable solvent includes compounds containing different numbers of alkenyl groups.

[0010] Optionally, the crosslinkable solvent is a non-polar solvent; the non-polar solvent includes any one or more of isobornyl methacrylate, isobornyl acrylate, lauryl acrylate, lauryl methacrylate, dicyclopentadiene acrylate, difunctional polyurethane acrylate, dipentaerythritol pentaacrylate, and tricyclosediacrylate.

[0011] Another inventive point of this application is to provide a method for preparing the membrane structure solvent as described above.

[0012] Optionally, the preparation method includes: (1) adding the raw materials of the membrane structure to a crosslinkable solvent to self-assemble into a membrane structure; (2) performing self-crosslinking on the solvent to form a membrane structure solvent.

[0013] Optionally, the raw materials of the membrane structure include phospholipids and / or copolymers; the membrane structure includes hydrophilic ends and hydrophobic ends; the hydrophilic ends and hydrophobic ends form a bilayer structure through self-assembly.

[0014] Optionally, the solvent may also include an initiator; the initiator may include one or more of photoinitiators, thermal initiators, and chemical initiators.

[0015] Optionally, the initiator includes a photoinitiator and / or a thermal initiator.

[0016] 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.

[0017] Another inventive point of this application is to provide the application of membrane structure solvents as described above or membrane structure solvents prepared by any of the preparation methods described above in nanopore sequencing.

[0018] Compared with the prior art, this application has the following advantages: This application cross-links the membrane with a cross-linkable solvent to form a network structure, thereby confining and stably maintaining the membrane structure within the network structure and preventing membrane rupture; at the same time, it does not restrict the flow state of the membrane, thus avoiding any impact on the subsequent transport of samples during sequencing. Attached Figure Description

[0019] Figure 1 This is a structural diagram of a gene sequencing chip provided in one embodiment of this application.

[0020] Figure 2 Another structural diagram of a gene sequencing chip provided in one embodiment of this application.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Figure 9 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 10 This is a current diagram for detecting the breakdown voltage of a membrane provided in one embodiment of this application.

[0029] Figure 11 A comparison diagram of the opening current provided in one embodiment of this application.

[0030] Figure 12 This is a schematic diagram of the crosslinked membrane provided in Example 1 of this application at a voltage of 600mV.

[0031] Figure 13 This is an opening current diagram of the cross-linked porous protein provided in Example 1 of this application.

[0032] Figure 14 This is a schematic diagram of the crosslinked membrane provided in Example 2 of this application at a voltage of 600mV.

[0033] Figure 15 This is an opening current diagram of the cross-linked porous protein provided in Example 2 of this application.

[0034] Figure 16 This is a schematic diagram of the crosslinked membrane provided in Example 2 of this application at a voltage of 600mV.

[0035] Figure 17 This is an opening current diagram of the cross-linked porous protein provided in Example 2 of this application.

[0036] Figure 18 This is a schematic diagram of the crosslinked membrane provided in Example 2 of this application at a voltage of 600mV.

[0037] Figure 19This is an opening current diagram of the cross-linked porous protein provided in Example 2 of this application.

[0038] Figure 20 This is a schematic diagram of the crosslinked membrane provided in Example 3 of this application at a voltage of 600mV.

[0039] Figure 21 This is an opening current diagram of the cross-linked porous protein provided in Example 3 of this application.

[0040] Figure 22 This is a schematic diagram of the crosslinked membrane provided in Example 4 of this application at a voltage of 600mV.

[0041] Figure 23 This is an opening current diagram of the cross-linked porous protein provided in Example 4 of this application.

[0042] Figure 24 This is a schematic diagram of the crosslinked membrane provided in Example 4 of this application at a voltage of 600mV.

[0043] Figure 25 This is an opening current diagram of the cross-linked porous protein provided in Example 4 of this application.

[0044] Figure 26 This is a schematic diagram of the crosslinked membrane provided in Example 5 of this application at a voltage of 600mV.

[0045] Figure 27 This is an opening current diagram of the cross-linked porous protein provided in Example 5 of this application.

[0046] Figure 28 This is a schematic diagram of the crosslinked membrane provided in Example 6 of this application at a voltage of 600mV.

[0047] Figure 29 This is an open-pore current diagram of the cross-linked porous protein provided in Example 6 of this application.

[0048] Figure 30 This is a schematic diagram of the cross-linked membrane provided in Comparative Example 1 of this application at a voltage of 300mV. Detailed Implementation

[0049] 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.

[0050] 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.

[0051] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0052] In the nonpolar film-forming solution, the copolymer film component accounts for 1%, and the nonpolar solvent accounts for 99%. Solvent crosslinking has a more significant effect on improving film performance than copolymer crosslinking.

[0053] This application provides a solvent carrier for nanopore sequencing, wherein the membrane structure is located inside a crosslinkable solvent; the crosslinkable solvent self-crosslinks to form a network structure, thereby encapsulating the membrane structure.

[0054] Generally, because the solvent constitutes a large proportion of the film-forming solution and has high fluidity, solvent fluctuations occur when subjected to external forces or over time. Meanwhile, the membrane component constitutes a small proportion, and the membrane's self-assembly stability is limited, leading to problems such as membrane structure rupture or short storage time, significantly impacting long-term sequencing. Therefore, this application reduces solvent fluidity by cross-linking the solvent, thereby spatially restricting the membrane and preventing its arbitrary movement.

[0055] The preferred crosslinkable solvent is a nonpolar solvent; only nonpolar solvents can serve as solvents for nonpolar film-forming solutions in nanopore sequencing systems.

[0056] If a crosslinkable solvent is to undergo self-crosslinking, then the basis for self-crosslinking must exist, namely, unsaturated bonds.

[0057] Unsaturated bonds include any one or more of the following: carbon-carbon double bonds, carbon-carbon triple bonds, carbonyl groups, ester groups, carboxyl groups, aldehyde groups, amide groups, cyano groups, and imine groups.

[0058] There are no restrictions on the specific type of crosslinkable solvent. The crosslinkable solvent can be a monomer or a polymer, as long as there are unsaturated bonds.

[0059] Preferred crosslinkable solvents include monomers or polymers containing alkenyl groups; that is, unsaturated bonds are preferably carbon-carbon double bonds; more preferably acrylic groups.

[0060] Preferred crosslinkable solvents include any one or more of isobornyl methacrylate, isobornyl acrylate, lauryl acrylate, lauryl methacrylate, dicyclopentadiene acrylate, difunctional polyurethane acrylate, dipentaerythritol pentaacrylate, and tricyclosediol diacrylate.

[0061] In monomers or polymers, there is at least one carbon-carbon double bond.

[0062] Cross-linking can only occur when unsaturated bonds are present. Preferably, there are two or more unsaturated bonds to generate complex network structures.

[0063] Preferably, the crosslinkable solvent includes compounds containing different numbers of carbon-carbon double bonds.

[0064] By using different numbers of carbon-carbon double bonds, the structure becomes more complex and compact, providing better confinement of the membrane and making the membrane structure more stable.

[0065] Preferably, the crosslinkable solvent includes compounds containing one carbon-carbon double bond and compounds containing two carbon-carbon double bonds. Examples of compounds containing one carbon-carbon double bond include isobornyl methacrylate, isobornyl acrylate, lauryl acrylate, and lauryl methacrylate. Examples of compounds containing two carbon-carbon double bonds include dicyclopentadiene acrylate, dipentaerythritol pentaacrylate, and tricyclosepiacetic acid diacrylate.

[0066] The crosslinkable solvent can also be a compound containing 3, 4, 5, 6, 7, 8, 9, 10 or more carbon-carbon double bonds. There are no restrictions on this, as long as crosslinking can be achieved.

[0067] The conditions for cross-linking unsaturated bonds can include light, heat, or chemical initiation.

[0068] Crosslinking can be carried out under conditions of light, heat, or chemical initiation.

[0069] The light can be ultraviolet light, infrared light, or visible light, etc.; ultraviolet light is preferred.

[0070] The conditions for light exposure are: 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.

[0071] 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.

[0072] Photoinitiated crosslinking also requires a photoinitiator; photoinitiators include any one or more of benzoin dimethyl ether, trimethylbenzoyl phenyl phosphate, 1-hydroxycyclohexylphenyl ketone, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide.

[0073] 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.

[0074] 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.

[0075] Crosslinking initiated by heating also requires a thermal initiator; the initiator includes any one or more of azobisisobutyronitrile, cumene hydroperoxide, di-tert-butyl peroxide, benzoyl peroxide, potassium persulfate, sodium persulfate, and ammonium persulfate.

[0076] Chemical initiation is the process by which a chemical initiator initiates a double bond polymerization reaction after its addition.

[0077] 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.

[0078] 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.

[0079] Because copolymer molecules are arranged at the interface between polar and non-polar solvents, the chemical initiators used for chemical crosslinking are generally dissolved in polar media and cannot effectively contact the non-polar solvent components in the film-forming solution. Therefore, crosslinking by light and heat is preferred.

[0080] The initiator is in the proportion of the nonpolar crosslinkable solvent of 5 mg / mL to 20 mg / mL; for example, it can be 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL or any other value within this range.

[0081] The preparation method of the membrane structure solvent is an improvement based on the conventional molecular membrane preparation method.

[0082] This includes: (1) adding the raw materials of the membrane structure to a crosslinkable solvent to self-assemble into a membrane structure; and (2) performing self-crosslinking on the crosslinkable solvent to form a membrane structure solvent.

[0083] The raw materials of the membrane structure include phospholipids and / or copolymers; the membrane structure includes hydrophilic ends and hydrophobic ends; the hydrophilic ends and hydrophobic ends form a bilayer structure through self-assembly.

[0084] Preferably, when the copolymer membrane does not contain crosslinkable unsaturated bonds, only the crosslinkable solvent itself undergoes crosslinking. 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.

[0085] The copolymer film is preferably a block copolymer film that does not contain unsaturated bonds.

[0086] Block copolymers that do not contain unsaturated bonds can be diblock copolymers or triblock copolymers containing polydimethylsiloxane; The hydrophilic end of the block copolymer includes one or more of polyethylene glycol, polyvinyl alcohol, polyethyleneamine, polyethyleneimine, polyacrylamide, and polyacrylamide; the hydrophobic end includes one or more of polydimethylsiloxane, 100% hydrogenated reduced 1,2-butadiene, polypropylene, polyethylene, and 100% hydrogenated reduced 1,4-butadiene. That is, the hydrophobic end lacks additional unsaturated bonds for covalent bonding and cannot react with solvents or other substances.

[0087] Diblock copolymers can be represented by the following formula:

[0088] Where n ranges from 5 to 40; m ranges from 5 to 40; and p ranges from 5 to 40.

[0089] 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.

[0090] 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.

[0091] The solvent for the copolymer is an alkane; preferably one or more of n-decane, n-tetradecane, and n-hexadecane.

[0092] 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 4 As shown; then a second layer of non-polar 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.

[0093] The preparation method of the membrane structure solvent in the sequencing system includes: (1) adding a polar solution to a microwell; (2) adding a non-polar film-forming liquid (the raw material of the membrane structure is added to a crosslinkable solvent to form a non-polar film-forming liquid) to the microwell and self-assembling into a membrane structure; (3) adding a polar solution to the microwell; and (4) performing self-crosslinking on the crosslinkable solvent to form a membrane structure solvent.

[0094] The polar solvent is an aqueous solution containing KCl, K3Fe(CN)6, and K4Fe(CN)6.

[0095] The preferred aqueous solution is 200 mM KCl, 150 mM K3Fe(CN)6, and 100 mM K4Fe(CN)6.

[0096] The nonpolar film-forming solution also includes an inert solvent; this inert solvent is used to dissolve the copolymer film and the nonpolar crosslinkable solvent. The inert solvent is preferably an alkane; preferably one or more of n-decane, n-tetradecane, and n-hexadecane, or a commercially available blended oil; such as white oil.

[0097] The method for preparing the membrane structure solvent in the sequencing system further includes: embedding porin between step (3) and step (4), or embedding porin after step (4).

[0098] Method I: Embed the porin between steps (3) and (4). That is, embed the porin first, and then perform self-crosslinking of the solvent.

[0099] Method II: Porin embedding after step (5). That is, pore embedding is performed after solvent self-crosslinking.

[0100] The results are as follows Figure 9 As shown, the cross-linked solvent fixes the membrane, restricting its movement.

[0101] 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). The preparation was carried out as described in 303,1189(2004); DOI:10.1126 / science.1094114.

[0102] The method for testing membrane breakdown voltage is as follows: An initial voltage of 140 mV is applied, with the voltage increased by 10 mV every 3 seconds. Because the membrane contains nanoporous proteins, when the membrane is not yet broken down, the current detected by a single sequencing unit is approximately 1.0 nA. When the membrane breaks down, the current detected by a single sequencing unit increases by two orders of magnitude, reaching approximately 100 nA. The voltage applied across the membrane at this point is recorded; this voltage is the breakdown voltage.

[0103] For example Figure 10 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.

[0104] Alternatively, due to the fluidity of the copolymer film, it may rupture when the voltage across the copolymer film reaches a critical value. This phenomenon can be observed under a microscope.

[0105] 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.

[0106] Calculation formula:

[0107] Where, x i The 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.

[0108] According to the above formula, for example, in Figure 11The 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.

[0109] Example 1

[0110] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) Add a non-polar film-forming solution (the raw materials of the membrane structure are added to a crosslinkable solvent to form a non-polar film-forming solution) into the microwell to self-assemble into a membrane structure; The nonpolar film-forming solution includes: a block copolymer with a hydrophobic end of polydimethylsiloxane (degree of polymerization 10) and a hydrophilic end of polyacrylamide (degree of polymerization 5), and 5 mg / ml of Ominirad 184 (1-hydroxycyclohexylphenyl ketone) photoinitiator; the solvent is: 50% isobornyl methacrylate and 50% white oil; (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.

[0111] This indicates that the cross-linked membrane has high stability. For example... Figure 12 After cross-linking, the membrane boundaries are clear and distinct, diffusion is not easy, and stability is good. After cross-linking, such as... Figure 13 As shown, the porin still exhibits good pore opening current with a low range and a standard deviation σ < 16 / 6.6 pA; its breakdown voltage is approximately 830 mV, indicating no protein damage.

[0112] Example 2

[0113] The solvent in step (2) of Example 1 is simply changed to: A. 40% isobornyl methacrylate, 40% white oil, 20% tricyclosaccharide diacrylate: breakdown voltage 900mV, no protein damage; B. 30% isobornyl methacrylate, 50% white oil, 20% tricyclosaccharide diacrylate: breakdown voltage 850mV, no protein damage; C. 20% isobornyl methacrylate, 60% white oil, 20% tricyclosperidic acid diacrylate: breakdown voltage 800mV, no protein damage.

[0114] The cross-linked films described above are respectively as follows: Figure 14 , Figure 16 and Figure 18As shown; Figure 15 , Figure 17 and Figure 19 Both studies demonstrated good orifice current signal and low rang.

[0115] Example 3

[0116] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) Add a non-polar film-forming solution (the raw materials of the membrane structure are added to a crosslinkable solvent to form a non-polar film-forming solution) into the microwell to self-assemble into a membrane structure; The nonpolar film-forming solution includes: a block copolymer with 10 mg / ml of hydrophobic end polydimethylsiloxane (degree of polymerization 10) and hydrophilic end polyacrylamide (degree of polymerization 5), and 5 mg / ml AIBN (azobisisobutyronitrile) thermal initiator; the solvent is: 40% isobornyl methacrylate, 40% white oil, 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.

[0117] This indicates that the cross-linked membrane has high stability. Figure 20 The edges are clear and stable, and it is not easily flowable. The breakdown voltage is 900mV. After cross-linking, the protein is not damaged, and the opening current is low. Figure 21 ), and the range is relatively small.

[0118] Example 4

[0119] The solvent in step (2) of Example 3 is simply changed to: A. 30% isobornyl methacrylate, 50% white oil, 20% tricyclosperidic acid diacrylate: breakdown voltage 850mV, no protein damage; B. 20% isobornyl methacrylate, 60% white oil, 20% tricyclosperidic acid diacrylate: breakdown voltage 800mV, no protein damage.

[0120] The cross-linked films described above are respectively as follows: Figure 22 and Figure 24 As shown; Figure 23 and Figure 25 Both studies demonstrated that they have good aperture current signals and small range.

[0121] Example 5

[0122] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) Add a non-polar film-forming solution (the raw materials of the membrane structure are added to a crosslinkable solvent to form a non-polar film-forming solution) into the microwell to self-assemble into a membrane structure; The nonpolar film-forming solution includes: a block copolymer with a hydrophobic end of polydimethylsiloxane (degree of polymerization 20) and a hydrophilic end of polyacrylamide (degree of polymerization 10), 5 mg / ml AIBN (azobisisobutyronitrile), and 5 mg / ml Ominirad 184 (1-hydroxycyclohexylphenyl ketone) photoinitiator; the solvent is: 40% isobornyl acrylate, 40% white oil, 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 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.

[0123] The cross-linked membrane exhibits high stability. The simultaneous application of light and heat cross-linking deepens the cross-linking of the solvent support, resulting in improved membrane stability. Figure 26 The orifice current signal is low ( Figure 27 Its breakdown voltage increased to approximately 950 mV. Furthermore, the cross-linked porin remained undamaged, and the sequencing current signal was normal.

[0124] Example 6

[0125] (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 a hydrophobic end of polydimethylsiloxane (degree of polymerization 20) and a hydrophilic end of polyacrylamide (degree of polymerization 10), 5 mg / ml AIBN (azobisisobutyronitrile), and 5 mg / ml Ominirad 184 (1-hydroxycyclohexylphenyl ketone) photoinitiator; the solvent is: 40% isobornyl acrylate, 40% white oil, 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.

[0126] (5) Embed Csgg nanoporous protein; then add 600mM KCl solution.

[0127] like Figure 28 and 29 As shown, the membrane state, breakdown voltage, and pore opening current of the pore protein are similar to those in Example 5. It can be seen from Examples 5 and 6 that the generation of membrane structure solvent does not affect the embedding of nanoporin, nor does it affect the pore performance or subsequent sequencing results.

[0128] Comparative Example 1

[0129] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) Add a non-polar film-forming solution (the raw materials of the membrane structure are added to a crosslinkable solvent to form a non-polar film-forming solution) into the microwell to self-assemble into a membrane structure; 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 5 mg / ml AIBN (azobisisobutyronitrile) thermal initiator; the solvent is: 40% isobornyl methacrylate, 40% white oil, 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.

[0130] Compared to Example 3, the heating step is missing.

[0131] The membrane and nanoporous protein system obtained above were tested; the membrane state was as follows. Figure 30 As shown, its morphology differs slightly from other embodiments; and its breakdown voltage of 370mV is significantly lower than that of Example 3; indicating that the crosslinking of the solvent in this application can effectively improve the stability of the film.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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 membrane-structured solvent for nanopore sequencing, characterized in that, The membrane structure is located inside the crosslinkable solvent; the crosslinkable solvent self-crosslinks to form a network structure, which encapsulates the membrane structure.

2. The membrane structure solvent according to claim 1, characterized in that, The crosslinkable solvent comprises monomers or polymers containing unsaturated bonds; the crosslinkable solvent is obtained by crosslinking through unsaturated bonds; the conditions for self-crosslinking include light, heating, or chemical initiators.

3. The membrane structure solvent according to claim 1, characterized in that, Unsaturated bonds include any one or more of the following: carbon-carbon double bonds, carbon-carbon triple bonds, carbonyl groups, ester groups, carboxyl groups, aldehyde groups, amide groups, cyano groups, and imine groups.

4. The membrane structure solvent according to claim 1, characterized in that, The crosslinkable solvent includes monomers or polymers containing carbon-carbon double bonds; wherein the number of carbon-carbon double bonds in the monomers or polymers is at least one.

5. The membrane solvent according to claim 1, characterized in that, The crosslinkable solvent includes compounds containing different numbers of carbon-carbon double bonds.

6. The membrane structure solvent according to claim 1, characterized in that, The crosslinkable solvent is a non-polar solvent; the non-polar solvent includes any one or more of isobornyl methacrylate, isobornyl acrylate, lauryl acrylate, lauryl methacrylate, dicyclopentadiene acrylate, dipentaerythritol pentaacrylate, and tricyclosepiacetic acid diacrylate.

7. A method for preparing a film structure solvent as described in any one of claims 1 to 6, characterized in that, include: (1) Add the raw materials of the membrane structure to the crosslinkable solvent and self-assemble them into a membrane structure; (2) Perform self-crosslinking on the crosslinkable solvent to form a membrane structure solvent.

8. The preparation method according to claim 7, characterized in that, The raw materials of the membrane structure include phospholipids and / or copolymers; the membrane structure includes hydrophilic ends and hydrophobic ends; the hydrophilic ends and hydrophobic ends form a bilayer structure through self-assembly.

9. The preparation method according to claim 8, characterized in that, The conditions for cross-linking unsaturated bonds can include light, heat, or chemical initiation; The crosslinkable solvent also includes an initiator; the initiator includes one or more of photoinitiators, thermal initiators, and chemical initiators; 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 membrane structure solvent according to any one of claims 1 to 6 or the membrane structure solvent prepared by any one of claims 7 to 9 in nanopore sequencing.

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