Fusion method of pore protein and membrane, pore protein jack buffer solution and application

Abstract

The invention provides a fusion method of pore protein and a membrane, a pore protein jack buffer solution and application. The fusion method comprises the following steps: distributing a first solution on the first side of the membrane, and distributing a second solution on the second side of the membrane; applying voltage to two sides of the membrane, fusing the pore protein and the membrane, and detecting current in the solution; when the current is increased, the pore protein is inserted into the membrane, the voltage is adjusted to 0V, and the fusion of the pore protein and the membrane is completed; the first solution comprises a first buffer solution containing pore protein, and the second solution comprises a second buffer solution without pore protein; the osmotic pressure of the first solution is greater than that of the second solution. The porous protein and membrane fusion single porosity problem in the prior art can be solved, and the method is suitable for the field of biological analysis and detection.

Description

Porin and membrane fusion method, porin pore buffer and application Technical Field

[0001] The present invention relates to the field of biological analysis and detection, and in particular to a method for fusing a porin with a membrane, a porin pore buffer and an application thereof. Background Art

[0002] In 1996, scientists first demonstrated the recognition of different bases using the alpha-hemolysin protein (Kasianowicz, John J., et al. Proceedings of the National Academy of Sciences 93, 24 (1996): 13770-13773), revealing the potential of using biological nanopore sensors for DNA sequencing. With the rapid development of nanotechnology in this field, nanopore detection technology has been gradually applied to the detection of a variety of macromolecules, including nucleic acids, proteins, and polymers.

[0003] The biological nanopore detection principle: A single biological nanopore protein is inserted into a phospholipid membrane. The analyte passes through the nanopore channel under the influence of an electric field, and the analyte is identified by the characteristic current signal generated. The pore protein and membrane are immersed in a solution environment, and each side of the membrane contains a pair of electrodes.

[0004] Research on biological nanopore detection mainly involves the following aspects: (1) the discovery and modification of different biological pore proteins; (2) the development of detection methods based on nanopore platforms; (3) basic theoretical research on nanopore detection. Different nanopore proteins, due to their different amino acid composition, structure, charge and other properties, can be used for different types of objects to be detected, and their sensitivity and resolution also vary. The way nanopore proteins fuse with membranes is also slightly different. For example, membrane proteins have a large hydrophobic area on the outer wall, which makes them easier to fuse with the phospholipid bilayer membrane. The hydrophobic area on the outer wall of viral motor channel proteins is small, and liposome vesicles are needed to promote protein fusion with the membrane.

[0005] CN112119033A discloses a method for preparing a phage DNA-packaged motor protein channel into a liposome and fusing it with a polymer to promote the fusion of the pore protein with the membrane. The advantages and disadvantages of this method are: (1) it is applicable to non-membrane protein channels; (2) the method for forming liposomes is relatively complex, requiring the use of specialized equipment (e.g., rotary evaporators, extruders, etc.) to prepare liposomes; and (3) the fusion of the prepared liposomes with the polymer membrane changes the composition of the polymer membrane. This patent describes the use of liposomes to initiate the fusion of nanochannel proteins with polymer membranes; if liposomes are not used, the nanochannel proteins and polymer membranes cannot directly fuse.

[0006] CN110023753A discloses a method for voltage-controlled pore insertion into a membrane. By controlling the potential difference in the membrane, the insertion of additional porins into the membrane to form multi-porous nanopore channels is prevented or reduced. While the patent describes how to reduce the probability of multiple porins inserting into the membrane, it does not explicitly address how to increase the insertion rate.

[0007] Oxford Nanopore, a British company, has launched a number of nanopore sequencers with different throughputs. Among them, the small sequencer MinION contains 512 channels on a single chip, 2048 nanopore channel slots or sequencing units (wells). If there are more than 800 single pores in 2048 wells, the sequencing chip is qualified. The higher the single-pore rate of a single chip, the more effective nanopore single pores or effective sequencing units that can be used, and the greater the amount of data that can be generated. Therefore, improving the single-pore rate on a single chip is of great significance to improving sequencing throughput. Nanopore-based sequencing chips contain a large number of arrays of sensor units, such as arrays ranging from several thousand to one million units. Each unit of the array contains membranes and pore proteins. One of the challenges is to increase the yield of membranes and pore proteins in the array. The higher the yield, the higher the sequencing throughput. Therefore, it is necessary to increase the yield of pore protein pores.

[0008] Summary of the Invention

[0009] The main purpose of the present invention is to provide a method for fusing porin with a membrane, a porin pore buffer and an application thereof, so as to solve the problem of low efficiency of fusing porin with a membrane in the prior art.

[0010] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a method for fusing porins with membranes is provided, which comprises: distributing a first solution on a first side of the membrane and distributing a second solution on a second side of the membrane; applying voltage on both sides of the membrane to fuse the porins with the membrane, and detecting the current in the solution; when the current increases, it indicates that the porins are inserted into the membrane, and the voltage is adjusted to 0V, and the fusion of the porins with the membrane is completed; the first solution comprises a first buffer solution containing porins, and the second solution comprises a second buffer solution not containing porins; the osmotic pressure of the first solution is greater than the osmotic pressure of the second solution.

[0011] Furthermore, the osmotic pressure of the first solution and the osmotic pressure of the second solution differ by at least 50 mOsm / kg, preferably by at least 200 mOsm / kg.

[0012] Furthermore, the first buffer and / or the second buffer contain monovalent metal ions and / or divalent metal ions; preferably, the monovalent metal ions include one or more of sodium ions, potassium ions or lithium ions, and the divalent metal ions include calcium ions and / or magnesium ions.

[0013] Furthermore, the concentration of the monovalent metal ion is 350-800 mM, and the concentration of the divalent metal ion is 250-550 mM.

[0014] Further, the concentration of potassium ions includes 350-500mM, the concentration of sodium ions includes 470-550mM, and the concentration of magnesium ions includes 250-550mM; preferably, the first buffer and / or the second buffer include: 350-500mM potassium chloride, 10-50mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=7.0-8.2; or 470-550mM sodium chloride, 10-50mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=7.0-8.2; or 250-550mM magnesium chloride, 10-50mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=7.0-8.2; or 100-200mM potassium ferrocyanide, 100-200mM potassium ferrocyanide, 10-50mM potassium phosphate; preferably, the first buffer includes 470mM potassium chloride, 25mM 4-Hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550 mM sodium chloride, 25 mM 4-Hydroxyethylpiperazineethanesulfonic acid, pH=8.0.

[0015] Furthermore, the membrane includes a diblock phospholipid membrane, a diblock high molecular polymer membrane, a triblock phospholipid membrane, or a triblock high molecular polymer membrane.

[0016] Furthermore, the phospholipid membrane includes a membrane composed of one or more of the following: diphytanoyl-phosphatidylcholine, 1,2-diphytanoyl-sn-glycero-3-phosphocholine, 1,2-di-O-phytanoyl-sn-glycero-3-phosphocholine, palmitoyl-oleoyl-phosphatidylcholine, dioleoyl-phosphatidyl-methyl ester, dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanoyl-sn-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-35 0], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dipalmitoyl-sm-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dipalmitoyl-sm-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dioleoyl-sm-glycero-3-phosphoethanolamine-N-lactosyl, GM1 ganglioside, or lysophosphatidylcholine.

[0017] Further, the high molecular polymer film includes any one or more of the following: copolymers of one or more of polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene, polybutadiene, polyvinyl chloride, polyalkyl acrylate, polyalkyl methacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylate, polyacrylate, polysulfone, polyethylene ether, poly(propylene oxide), C1-C6 alkyl acrylate and methacrylate, acrylamide, methacrylamide, (C1-C6 alkyl) acrylamide and methacrylamide, N,N-dialkyl-acrylamide, ethoxy acrylate and methacrylate, polyethylene glycol monomethacrylate and polyethylene glycol monomethyl ether methacrylate, hydroxy-substituted (C1-C6 alkyl) acrylamide and methacrylamide, hydroxy-substituted C1-C6 alkyl vinyl ether, sodium vinyl sulfonate, styrene sodium sulfonate, 2-acrylamido-2-methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl-2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4′-bisalkyloxazolinyl-5-one, 2,4-vinylpyridine, ethylenically unsaturated carboxylic acids having 3 to 5 carbon atoms, amino(C1-C6 alkyl)-, mono(C1-C6 alkylamino)(C1-C6 alkyl)- and bis(C1-C6 alkylamino)(C1-C6 alkyl)- Acrylates and methacrylates, allyl alcohol, 3-trimethylammonium 2-hydroxypropyl methacrylate chloride, dimethylaminoethyl methacrylate, dimethylaminoethyl methacrylamide, glycerol methacrylate, N-(1,1-dimethyl-3-oxobutyl)acrylamide, cyclic imino ethers, vinyl ethers, cyclic ethers including epoxy derivatives, cyclic unsaturated ethers, N-substituted ethylimines, β-lactones and β-lactams, ketene acetals, vinyl acetals and phosphoranes.

[0018] Further, the porin protein includes one or more proteins having at least 70% homology to the following proteins: bacterial amyloid secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, OmpG, InvG, GspD, Frac, PA63, SP1, aerobic bacterial lysis protein, plyAB, phage motor protein channel phi29, T3, T4, T7, SPP1 or gp20c.

[0019] Furthermore, the fusion method further comprises: adjusting the voltage to 0 V and incubating for 5-15 minutes, preferably 10 minutes; preferably, after incubation, replacing the first buffer containing porins with a second buffer not containing porins, applying voltage to both sides of the membrane, detecting the current and counting whether a single porin is fused with the membrane.

[0020] In order to achieve the above object, according to a second aspect of the present invention, a porin pore buffer is provided, wherein the porin pore buffer contains monovalent metal ions and / or divalent metal ions.

[0021] Furthermore, the monovalent metal ions include one or more of sodium ions, potassium ions or lithium ions, and the divalent metal ions include calcium ions and / or magnesium ions.

[0022] Furthermore, the concentration of the monovalent metal ion is 350-800 mM, and the concentration of the divalent metal ion is 250-550 mM.

[0023] Further, the concentration of potassium ions includes 350-500 mM, the concentration of sodium ions includes 470-550 mM, and the concentration of magnesium ions includes 250-550 mM; preferably, the porin pore buffer includes: 350-500 mM potassium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 470-550 mM sodium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 250-550 mM magnesium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 100-200 mM potassium ferrocyanide, 100-200 mM potassium ferrocyanide, 10-50 mM potassium phosphate, pH = 7.0-8.2; preferably, The buffer included 470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550 mM sodium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0.

[0024] To achieve the above object, according to a third aspect of the present invention, a porin pore buffer combination is provided, which comprises two porin pore buffers, and the osmotic pressure difference between the two porin pore buffers is greater than or equal to 50 mOsm / kg.

[0025] Furthermore, the porin pore buffer contains monovalent metal ions and / or divalent metal ions; preferably, the concentration of the monovalent metal ions is 350-800 mM, and the concentration of the divalent metal ions is 250-550 mM.

[0026] Further, the concentration of potassium ions includes 350-500mM, the concentration of sodium ions includes 470-550mM, and the concentration of magnesium ions includes 250-550mM; preferably, the porin pore buffer includes: 350-500mM potassium chloride, 10-50mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=7.0-8.2; or 470-550mM sodium chloride, 10-50mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=7.0-8.2; or 250-550mM magnesium chloride, 10-50mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=7.0-8.2; or 100-200mM potassium ferrocyanide, 100-200mM potassium ferrocyanide, 10-50mM potassium phosphate; preferably, the buffer includes 470mM potassium chloride, 25mM 4-Hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550 mM sodium chloride, 25 mM 4-Hydroxyethylpiperazineethanesulfonic acid, pH=8.0.

[0027] Furthermore, the osmotic pressure difference between the two porin plug buffers is greater than or equal to 200 mOsm / kg.

[0028] To achieve the above objectives, according to a fourth aspect of the present invention, a fusion method, or the porin pore buffer, or the porin pore buffer combination is provided for use in the preparation of a nanopore sequencing unit.

[0029] By applying the technical solution of the present invention and utilizing the aforementioned porin-membrane fusion method, fusion of the porin and membrane can be achieved using a simple buffer and applied voltage, completing the porin embedding in the membrane. This method eliminates the need for additional components such as liposomes, allowing for simple operation, high fusion efficiency, and a high single-pore ratio (single-pore rate) in the embedded pores, significantly improving the yield of sequencing units used for nanoporin sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 shows a schematic diagram of a chip array device according to Example 1 of the present invention, wherein 1 is a ground electrode; 2 is a fluid reservoir for a single sequencing unit; 3 is a membrane; 4 is a buffer solution for the fluid storage period, located on the second side of the membrane; 5 is a working electrode; and 6 is a chip pool shared by different sequencing units, located on the first side of the membrane.

[0032] FIG2 shows a graph of the pore opening current of a single CsgG pore embedded in a phospholipid membrane in a buffer according to Example 1 of the present invention.

[0033] FIG3 shows a graph showing the results of the number of embedded pores / number of membranes formed under different buffer systems according to an embodiment of the present invention.

[0034] FIG4 shows a graph showing the single-well ratio (number of single-wells / total number of wells) under different buffer systems according to an embodiment of the present invention.

[0035] FIG5 shows a graph showing the result of single pore number / film formation number obtained by embedding pores in different buffer systems according to an embodiment of the present invention.

[0036] FIG6 shows a comparison result of capacitance values ​​under different buffer systems according to Example 2 of the present invention.

[0037] FIG7 shows the result graph of the ratio of single pores of embedded holes (number of single pores / total number of pores) and the number of single pores of embedded holes / number of formed films at different concentrations of potassium chloride according to Example 5 of the present invention.

[0038] FIG8 shows the result graph of the ratio of single pores of embedded holes (number of single pores / total number of pores) and the number of single pores of embedded holes / number of formed films at different concentrations of sodium chloride according to Example 5 of the present invention. DETAILED DESCRIPTION

[0039] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0040] As mentioned in the background, existing methods for fusing porins to membranes are complex and have low fusion efficiency, which affects the throughput of subsequent high-throughput sequencing. In this application, the inventors attempted to develop a new method for fusing porins to membranes, and thus proposed a series of protection schemes in this application.

[0041] In a first typical embodiment of the present application, a method for fusing porins with membranes is provided, which comprises: distributing a first solution on a first side of the membrane and a second solution on a second side of the membrane; applying voltage to both sides of the membrane to fuse the porins with the membrane, and detecting the current in the solution; when the current increases, it indicates that the porins are inserted into the membrane, and the voltage is adjusted to 0V, and the fusion of the porins with the membrane is completed; the first solution comprises a first buffer solution containing porins, and the second solution comprises a second buffer solution not containing porins; the osmotic pressure of the first solution is greater than the osmotic pressure of the second solution.

[0042] In the above-mentioned fusion method, a membrane separates the first solution and the second solution. By applying a voltage to both sides of the membrane, namely the first solution and the second solution, the porin is promoted to be inserted into the membrane from the first solution, thereby achieving the fusion of the porin and the membrane. The above-mentioned first solution and the second solution are both buffers containing monovalent metal ions and / or divalent metal ions. There is a difference in osmotic pressure between the first solution and the second solution. The osmotic pressure of the first solution is greater than the osmotic pressure of the second solution. This difference in osmotic pressure can promote the insertion of the porin into the membrane from the area with higher osmotic pressure, thereby improving the efficiency of the fusion of the porin and the membrane. The osmotic pressure of the above-mentioned first solution and the second solution can be derived from the porin or from the difference in the components of the first buffer and the second buffer. The different ionic strengths in different buffers result in a difference in osmotic pressure.

[0043] Prior art also discloses a method for initiating pore-membrane fusion using an osmotic pressure difference, but this method discloses a method for inserting the porin from a buffer with lower osmotic pressure to a buffer with higher osmotic pressure. The porin insertion direction in the above-mentioned fusion method of the present application is opposite to that of the prior art. However, the fusion method of the present application has high fusion efficiency and a high proportion of single pores in the embedded pores, which can significantly improve the yield of sequencing units used for nanopore protein sequencing.

[0044] For the aforementioned current increase, using a voltage of 0.18V as an example, channels with current values ​​between 0-20pA are considered unfused (not embedded) with the membrane. Pores with current values ​​between 180pA and 250pA are considered single pores, and those with currents above 250pA are considered multi-porous. By observing these current changes, particularly the increase, it is possible to determine whether fusion has occurred and, furthermore, whether it is a single or multiple porins fusing with the membrane. Once the porins have inserted into the membrane, the voltage is adjusted to 0V to prevent further insertion of porins, which could affect product performance.

[0045] The above-mentioned first side and second side only represent the names of different sides of the membrane, and do not limit the direction, position, etc. of the membrane. The first solution can be located in any direction or position of the membrane, including but not limited to above or below the membrane.

[0046] In a preferred embodiment, the osmotic pressure of the first solution differs from the osmotic pressure of the second solution by at least 50 mOsm / kg, preferably by at least 200 mOsm / kg.

[0047] The osmotic pressure difference includes, but is not limited to, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450 or 500 mOsm / kg.

[0048] In a preferred embodiment, the first buffer and / or the second buffer contain monovalent metal ions and / or divalent metal ions; preferably, the monovalent metal ions include one or more of sodium ions, potassium ions or lithium ions, and the divalent metal ions include calcium ions and / or magnesium ions.

[0049] The monovalent metal ions and / or divalent metal ions in the buffer can promote the fusion efficiency of the porin and the membrane, increase the single pore ratio of the embedded pores, and the embedding process can be completed without the assistance of additional components such as liposomes.

[0050] In a preferred embodiment, the concentration of the monovalent metal ion is 350-800 mM, and the concentration of the divalent metal ion is 250-550 mM. The above monovalent metal ion concentration includes but is not limited to 350, 400, 450, 470, 500, 550, 600, 650, 700, 750 or 800 mM, and the above divalent metal ion concentration includes but is not limited to 250, 300, 350, 400, 450, 500 or 550 mM.

[0051] In a preferred embodiment, the concentration of potassium ions includes 350-500 mM, the concentration of sodium ions includes 470-550 mM, and the concentration of magnesium ions includes 250-550 mM; preferably, the first buffer and / or the second buffer include: 350-500 mM (including but not limited to 350, 375, 400, 425, 450, 475 or 500 mM) potassium chloride, 10-50 mM (including but not limited to 10, 20, 30, 40 or 50 mM) 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2 (including but not limited to 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or 8.2); or 470-550 mM sodium chloride (including but not limited to 470, 480, 490, 500, 510, 520, 530, 540 or 550 mM), 10-50 mM (including but not limited to 10, 20, 30, 40 or 50 mM) 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2 (including but not limited to 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or 8.2); 6, 7.8, 8.0 or 8.2); or 250-550 mM magnesium chloride (including but not limited to 250, 300, 350, 400, 450, 500 or 550 mM), 10-50 mM (including but not limited to 10, 20, 30, 40 or 50 mM) 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2 (including but not limited to 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or 8.2); or 100-200 mM (including but not limited to 100, 12 0, 140, 160, 180 or 200 mM) potassium ferrocyanide, 100-200 mM (including but not limited to 100, 120, 140, 160, 180 or 200 mM) potassium ferrocyanide, 100-50 mM (including but not limited to 10, 20, 30, 40 or 50 mM) potassium phosphate, pH = 7.0-8.2 (including but not limited to 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or 8.2); preferably, the first buffer comprises 470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0; or 550 mM sodium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0.

[0052] The above-mentioned potassium ion concentration includes but is not limited to 350, 370, 400, 420, 450, 470 or 500 mM, the sodium ion concentration includes but is not limited to 470, 480, 490, 500, 510, 520, 530, 540 or 550 mM, and the magnesium ion concentration includes but is not limited to 250, 300, 350, 400, 450, 500 or 550 mM.

[0053] In the present application, the inventors unexpectedly discovered that the use of the above-mentioned buffer for the fusion of pore proteins and membranes can improve the embedding rate, single pore ratio (number of single pores / total number of pores), number of single pores / number of membranes, and other data. Compared with other embedding buffers used in the prior art, it has better fusion efficiency and can significantly improve the yield of sequencing units used for nanopore protein sequencing.

[0054] In a preferred embodiment, the membrane comprises a diblock phospholipid membrane, a diblock polymer membrane, a triblock phospholipid membrane, or a triblock polymer membrane.

[0055] In a preferred embodiment, the phospholipid membrane comprises a membrane composed of one or more of the following: diphytanoyl-phosphatidylcholine, 1,2-diphytanoyl-sn-glycero-3-phosphocholine, 1,2-di-O-phytanoyl-sn-glycero-3-phosphocholine, palmitoyl-oleoyl-phosphatidylcholine, dioleoyl-phosphatidyl-methyl ester, dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanoyl-sn-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] -350], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl, GM1 ganglioside, or lysophosphatidylcholine.

[0056] In a preferred embodiment, the polymer film comprises any one or more of the following: copolymers of one or more of polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene, polybutadiene, polyvinyl chloride, polyalkyl acrylate, polyalkyl methacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylate, polyacrylate, polysulfone, polyethylene ether, poly(propylene oxide), C1-C6 alkyl acrylate and methacrylate, acrylamide, methacrylamide, (C1-C6 alkyl) acrylamide and methacrylamide, N,N-dialkyl-acrylamide, ethoxy acrylate and methacrylate, polyethylene glycol monomethacrylate and polyethylene glycol monomethyl ether methacrylate, hydroxy-substituted (C1-C6 alkyl) acrylamide and methacrylamide, hydroxy-substituted C1-C6 alkyl vinyl ether, sodium vinyl sulfonate, Sodium styrenesulfonate, 2-acrylamido-2-methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl-2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4′-bisalkyloxazolinyl-5-one, 2,4-vinylpyridine, ethylenically unsaturated carboxylic acids having 3 to 5 carbon atoms, amino(C1-C6 alkyl)-, mono(C1-C6 alkylamino)(C1-C6 alkyl)- and bis(C1-C6 alkylamino)(C1-C6 alkyl )-acrylates and methacrylates, allyl alcohol, 3-trimethylammonium 2-hydroxypropyl methacrylate chloride, dimethylaminoethyl methacrylate, dimethylaminoethyl methacrylamide, glycerol methacrylate, N-(1,1-dimethyl-3-oxobutyl)acrylamide, cyclic imino ethers, vinyl ethers, cyclic ethers including epoxy derivatives, cyclic unsaturated ethers, N-substituted ethylimines, β-lactones and β-lactams, ketene acetals, vinyl acetals and phosphoranes.

[0057] In a preferred embodiment, the porin comprises one or more proteins having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8% or 99.9% homology to the bacterial amyloid secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, OmpG, InvG, GspD, Frac, PA63, SP1, aerobic bacterial lysis protein, plyAB, bacteriophage motor protein channel phi29, T3, T4, T7, SPP1 or gp20c.

[0058] In a preferred embodiment, the fusion method further comprises: adjusting the voltage to 0 V and incubating for 5-15 minutes, preferably 10 minutes; preferably, after incubation, replacing the first buffer containing porins with a second buffer not containing porins, applying a voltage on both sides of the membrane, detecting the current and counting whether a single porin is fused with the membrane.

[0059] By observing the above-mentioned changes in current, especially the increase in current, it is possible to determine whether fusion has occurred. After the porin is inserted into the membrane, the voltage is adjusted to 0V to prevent more porins from continuing to insert into the membrane and affecting the performance of the product. Incubating for a period of time under the condition of voltage 0V can improve the stability of the obtained product, i.e., the sequencing unit. After a period of incubation, the buffer containing the porin is replaced with a buffer containing no porin, and voltage is applied to prevent more porins from continuing to fuse with the membrane, and the number of porins inserted into the membrane is determined by detecting the current. Taking a voltage of 0.18V as an example, a pore with a current value concentrated between 180pA and 250pA is called a single pore, which means that one porin is fused to the membrane; a pore with a current concentrated above 250pA is called a multi-pore, which means that multiple porins are fused to the membrane.

[0060] In a second typical embodiment of the present application, a porin pore buffer is provided, wherein the porin pore buffer contains monovalent metal ions and / or divalent metal ions.

[0061] In a preferred embodiment, the monovalent metal ions include one or more of sodium ions, potassium ions or lithium ions, and the divalent metal ions include calcium ions and / or magnesium ions.

[0062] In a preferred embodiment, the concentration of monovalent metal ions is 350-800 mM, and the concentration of divalent metal ions is 250-550 mM.

[0063] In a preferred embodiment, the concentration of potassium ions includes 350-500 mM, the concentration of sodium ions includes 470-550 mM, and the concentration of magnesium ions includes 250-550 mM; preferably, the porin pore buffer includes: 350-500 mM potassium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 470-550 mM sodium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 250-550 mM magnesium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 150 mM potassium ferricyanide, 150 mM potassium ferrocyanide, 25 mM potassium phosphate, pH = 7.0-8.2; preferably, the buffer includes 470 mM potassium chloride, 25 mM 4-Hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550 mM sodium chloride, 25 mM 4-Hydroxyethylpiperazineethanesulfonic acid, pH=8.0.

[0064] In a third typical embodiment of the present application, a porin pore buffer combination is provided, wherein the porin pore buffer combination includes two porin pore buffers, and the osmotic pressure difference between the two porin pore buffers is greater than or equal to 50 mOsm / kg.

[0065] The above osmotic pressure difference includes but is not limited to greater than or equal to 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450 or 500 mOsm / kg.

[0066] In a preferred embodiment, the porin pore buffer contains monovalent metal ions and / or divalent metal ions; preferably, the concentration of the monovalent metal ions is 350-800 mM, and the concentration of the divalent metal ions is 250-550 mM.

[0067] In a preferred embodiment, the concentration of potassium ions includes 350-500 mM, the concentration of sodium ions includes 470-550 mM, and the concentration of magnesium ions includes 250-550 mM; preferably, the porin pore buffer includes: 350-500 mM potassium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 470-550 mM sodium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 250-550 mM magnesium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 150 mM potassium ferricyanide, 150 mM potassium ferrocyanide, 25 mM potassium phosphate, pH = 7.0-8.2; preferably, the buffer includes 470 mM potassium chloride, 25 mM 4-Hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550 mM sodium chloride, 25 mM 4-Hydroxyethylpiperazineethanesulfonic acid, pH=8.0.

[0068] In a preferred embodiment, the osmotic pressure difference between the two porin plug buffers is greater than or equal to 200 mOsm / kg.

[0069] In a fourth typical embodiment of the present application, there is provided an application of the above-mentioned fusion method, or the above-mentioned porin pore buffer, or the above-mentioned porin pore buffer combination in the preparation of a nanopore sequencing unit.

[0070] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0071] Example 1:

[0072] This example demonstrates the results of a control experiment in which buffers with the same osmotic pressure were used on both sides of the membrane, namely, buffer 1 (150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 25 mM potassium phosphate, pH 8.0) was used on both sides of the membrane. The porin used in this example was a CsgG mutant (the wild-type amino acid sequence is shown in SEQ ID NO: 1, with the mutations Y51A / F56Q / R97W / R192D). In electrophysiological experiments, the porin was thoroughly mixed in 300 μL of buffer 1 and introduced into the chip array device on the upper side of the membrane (position 6 in Figure 1). The structure of the chip array device is shown in Figure 1, where 1 is the ground electrode; 2 is the fluid reservoir for a single sequencing unit; 3 is the membrane; 4 is the buffer for the fluid storage period, located on the second side of the membrane (below the membrane); 5 is the working electrode; and 6 is the chip reservoir shared by different sequencing units, located on the first side of the membrane (above the membrane). The devices used in the above-described fusion method include, but are not limited to, the device shown in Figure 1.

[0073] A voltage of 0.18V was applied, and the porin was allowed to penetrate the phospholipid membrane. Once the porin was embedded in the membrane, the current of a single channel would change. A voltage of 0mV was applied to the already embedded channel to reduce the possibility of further pores forming in the channel. After waiting for 10 minutes without the appearance of new embedded pores in all channels, 2mL of buffer 1 was used to remove excess porin above the membrane. 3mL of buffer (500mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) was passed through the system to completely replace the buffer 1 above the membrane. The embedded state of different channels was statistically analyzed at 0.18V, and the number of pores in each channel was determined based on the current value. Channels with current values ​​between 0-20pA were considered to be unincorporated, pores with current values ​​concentrated between 180pA and 250pA were considered single pores (as shown in Figure 2), and pores with current values ​​concentrated above 250pA were considered multi-pores.

[0074] The amino acid sequence of the wild-type CsgG transmembrane protein is represented by SEQ ID NO: 1:

[0075] Results and Discussion

[0076] Using buffer for embedding, the number of embedded pores / number of membranes can reach 78% ± 8% (as shown in Figure 3), but the ratio of single pores to total pores is only 70% ± 15% (N = 17) (as shown in Figure 4), so the number of single pores / number of membranes is only 54% ± 9% (N = 17) (as shown in Figure 5). Membranes containing multiple nanopore channels cannot be used for subsequent single-molecule detection experiments. The yield of membranes and single pores in the array is high, and the higher the yield, the higher the sequencing throughput. Therefore, improving the yield of pores in porin is of great significance for improving sequencing throughput.

[0077] When the osmotic pressure concentration on both sides of the membrane is consistent, the membrane area is large. This leads to active pore formation, which reduces the difficulty of pore formation and significantly increases the probability of porous channels. To increase the single-pore ratio and reduce the probability of active pore formation, pore formation methods need to be further optimized. By varying the osmotic pressure on both sides of the membrane, creating a differential osmotic pressure, the membrane area can be effectively altered, thereby adjusting the difficulty of pore formation.

[0078] In Examples 2 to 4, the single-pore embedding effects of a series of embedding buffers with similar osmotic pressures but containing different monovalent or divalent metal cations are demonstrated, and finally a number of non-isotonic embedding buffers that can effectively improve the single-pore rate are screened out.

[0079] Example 2:

[0080] This example demonstrates experiments in a pore optimization group, demonstrating that changing the buffer used for the pores on one side of the membrane can significantly improve single-pore yield. The same CsgG porin mutant used in Example 1 was used in this example. In electrophysiological experiments, 3 mL of buffer 2 (470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 8.0) containing potassium chloride was pumped into the chip pool to fully displace buffer 1 on the upper side of the membrane to prevent buffer 1 from affecting the experiment. The porin was thoroughly mixed in 300 μL of buffer 2 (470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 8.0) and pumped into the sequencing chip pool (on the upper side of the membrane). Buffer 1 was placed on the lower side of the membrane. The osmotic pressure of buffer 2 containing the porin was greater than that of buffer 1, with a difference of 230 mOsm / kg.

[0081] Apply a voltage of 0.18V and wait for pore formation. Once porins are embedded in the phospholipid membrane, the current of individual channels will change. Apply a voltage of 0mV to channels that have already been pore-embedded to reduce the possibility of further pore formation in the channel. Wait 10 minutes until all channels show no new pores. Remove excess porins above the membrane using 2mL of buffer 2 (470mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0). Flow 3mL of buffer (500mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) through the system to completely replace the buffer 2 above the membrane. Count the pore formation status of different channels at 0.18V. Determine the number of pores in each channel based on the current value. Channels with current values ​​between 0-20pA are considered unpore-embedded, pores with current values ​​concentrated between 180pA and 250pA are considered single pores, and pores with current values ​​concentrated above 250pA are considered multi-pores.

[0082] Results and Discussion

[0083] Using buffer 2 containing potassium chloride (470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0) for embedding, the number of embedded pores / number of formed membranes reached 82% ± 5% (as shown in Figure 3), and the ratio of single pores to total pores also increased significantly, reaching 92% ± 3%, a 31% increase over the result of buffer 1 (as shown in Figure 4). The number of single pores / number of formed membranes also increased significantly, increasing by 43% over the result of embedding in buffer 1 to 77% ± 8% (as shown in Figure 5).

[0084] Unexpectedly, buffer 2 was found to effectively reduce capacitance (as shown in Figure 6). Lower capacitance generally results in lower noise. Under the experimental conditions of buffer 1, the peak capacitance was around 115pF; under the experimental conditions of buffer 2, the peak capacitance was effectively reduced to around 75pF.

[0085] Example 3:

[0086] This example presents experiments with a pore optimization group, showing that other monovalent ions besides potassium, such as sodium, also significantly improve pore yield and results.

[0087] In this example, the same CsgG porin mutant as in Example 1 was used. During electrophysiological experiments, 3 mL of sodium chloride buffer 3 (550 mM sodium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 8.0) was added to fully displace buffer 1 on the upper side of the membrane. The porin was thoroughly mixed in 300 μL of buffer 3 (550 mM sodium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 8.0) and then flowed into the sequencing chip pool (on the upper side of the membrane). Buffer 1 was placed on the lower side of the membrane. The osmotic pressure of buffer 3 containing the porin was greater than that of buffer 1, with a differential osmotic pressure of 250 mOsm / kg.

[0088] Apply a voltage of 0.18V and wait for pore formation. Once porins are embedded in the phospholipid membrane, the current of individual channels will change. Apply a 0mV voltage protection to channels that have already been embedded to reduce the possibility of further pore formation in the channel. If no new pores appear within 10 minutes, remove excess porins above the membrane using 2mL of sodium chloride buffer 3 (550mM sodium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0). Flow 3mL of buffer (500mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) through the system to completely replace the buffer 3 above the membrane. Count the pore formation status of different channels at 0.18V. Determine the number of pores in each channel based on the current value. Channels with current values ​​between 0-20pA are considered un-poreed, pores with current values ​​concentrated between 180pA and 250pA are considered single pores, and pores with currents concentrated above 250pA are considered multi-pores.

[0089] Results and Discussion

[0090] When using buffer 3 containing sodium chloride (550 mM sodium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0) for embedding, the number of embedded holes / number of membranes can reach 92% ± 1% (as shown in Figure 3), and the ratio of single holes / total holes is improved compared with the embedding result of buffer 1, reaching 85% ± 4%, an increase of 20% (as shown in Figure 4); the number of single holes / number of membranes is the same as the embedding result of buffer 2, reaching 78% ± 4%, which is a 44% increase in the single hole rate compared with the embedding result of buffer 1 (as shown in Figure 5).

[0091] Example 4:

[0092] This example presents experiments on the pore optimization group, which show that other valence metal ions besides potassium and sodium, such as the divalent metal ion magnesium, can also significantly improve the pore yield and results.

[0093] In this example, the same CsgG porin mutant as in Example 1 was used. During electrophysiological experiments, 3 mL of magnesium chloride buffer 4 (320 mM magnesium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 8.0) was added to fully displace buffer 1 on the upper side of the membrane. The porin was thoroughly mixed in 300 μL of magnesium chloride-containing buffer 4 (320 mM magnesium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 8.0) and then flowed into the sequencing chip pool (on the upper side of the membrane). Buffer 1 was placed on the lower side of the membrane. The osmotic pressure of buffer 4 containing the porin was greater than that of buffer 1, with a differential osmotic pressure of 260 mOsm / kg.

[0094] Apply a voltage of 0.18V and wait for pore formation. Once porins are embedded in the phospholipid membrane, the current of individual channels will change. Apply a 0mV voltage protection to channels that have already been pore-embedded to reduce the possibility of further pore formation in the channel. If no new pores appear within 10 minutes, remove excess porins above the membrane using 2mL of magnesium chloride buffer 4 (320mM magnesium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0). Flow 3mL of buffer (500mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) through the system to completely replace the buffer 4 above the membrane. Count the pore formation status of different channels at 0.18V. Determine the number of pores in each channel based on the current value. Channels with current values ​​between 0-20pA are considered unpore-embedded, pores with current values ​​concentrated between 180pA and 250pA are considered single pores, and pores with currents concentrated above 250pA are considered multi-pores.

[0095] Results and Discussion

[0096] When using buffer 4 containing magnesium chloride (320 mM magnesium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0) for embedding, the number of embedded holes / number of membranes can reach 82% ± 6% (as shown in Figure 3), and the single hole / total pore ratio is improved compared with the embedding result of buffer 1, reaching 96% ± 2%, an increase of 38% (as shown in Figure 4); the number of single holes / number of membranes is the same as the embedding results of buffer 2 and buffer 3, reaching 78% ± 8%, which is a 44% increase in the single hole rate compared with the embedding result of buffer 1 (as shown in Figure 5).

[0097] Embodiment 5:

[0098] The results obtained in different concentrations of the same metal ion embedding buffer solution were different. We tried potassium chloride solution and sodium chloride solution with different ion concentrations and selected the experimental group with the best embedding effect.

[0099] As shown in Figure 7, the ratio of single pores to total pores is best in 0.47 M potassium chloride buffer and 0.5 M potassium chloride buffer, but the ratio of single pores to membrane formation is poor in 0.5 M potassium chloride buffer. In summary, the best potassium chloride concentration range among potassium chloride buffers is selected to be between 0.35 M and 0.5 M, among which 0.47 M performs the best.

[0100] For sodium chloride buffer, 0.47M-0.55M sodium chloride buffer was tested based on potassium chloride buffer. As shown in Figure 8, sodium chloride buffers above 0.5M performed better than isotonic buffer 1 (single pore / total pores: 0.70±0.15; single pore / film formation number: 0.54±0.09).

[0101] From the above description, it can be seen that the above-mentioned embodiment of the present invention achieves the following technical effects: using the above-mentioned method of fusion of porin and membrane, it is possible to achieve fusion of porin and membrane by a simple buffer and applied voltage, and complete the embedding of porin on the membrane. Porin embedding can be achieved without the assistance of additional components such as liposomes, and the operation is simple, the fusion efficiency is high, and the ratio of single pores in the embedding is high, which can significantly improve the yield of this sequencing unit for nanoporin sequencing. The sequencing unit obtained by the fusion of porin and membrane using the above-mentioned buffer has lower capacitance than the sequencing unit of the prior art, and thus has lower noise when sequencing, which is conducive to improving the performance of subsequent high-throughput sequencing.

[0102] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for fusing a porin to a membrane, characterized in that: The fusion method comprises: Distributing a first solution on a first side of the membrane and a second solution on a second side of the membrane; applying voltage on both sides of the membrane to fuse the porin with the membrane, and detecting the current in the solution; When the current increases, it indicates that the porin is inserted into the membrane, and the voltage is adjusted to 0 V, and the fusion of the porin and the membrane is completed; The first solution includes a first buffer containing the porin, and the second solution includes a second buffer not containing the porin; The osmotic pressure of the first solution is greater than the osmotic pressure of the second solution.

2. The fusion method according to claim 1, characterized in that: The osmotic pressure of the first solution differs from the osmotic pressure of the second solution by at least 50 mOsm / kg, preferably by at least 200 mOsm / kg.

3. The fusion method according to claim 1, characterized in that: The first buffer and / or the second buffer contain monovalent metal ions and / or divalent metal ions; Preferably, the monovalent metal ions include one or more of sodium ions, potassium ions or lithium ions, and the divalent metal ions include calcium ions and / or magnesium ions.

4. The fusion method according to claim 3, characterized in that: The concentration of the monovalent metal ions is 350-800 mM, and the concentration of the divalent metal ions is 250-550 mM.

5. The fusion method according to claim 4, characterized in that: The concentration of potassium ions includes 350 to 500 mM, the concentration of sodium ions includes 470 to 550 mM, and the concentration of magnesium ions includes 250 to 550 mM; Preferably, the first buffer and / or the second buffer comprises: 350-500 mM potassium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 470-550 mM sodium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 250-550 mM magnesium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 100-200 mM potassium ferrocyanide, 100-200 mM potassium ferrocyanide, 10-50 mM potassium phosphate, pH = 7.0-8.2; Preferably, the first buffer comprises 470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550 mM sodium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.

0.

6. The fusion method according to claim 1, characterized in that: The membrane includes a diblock phospholipid membrane, a diblock high molecular polymer membrane, a triblock phospholipid membrane or a triblock high molecular polymer membrane.

7. The fusion method according to claim 6, characterized in that: The phospholipid membrane includes one or more of the following membranes: Diphytanoyl-phosphatidylcholine, 1,2-diphytanoyl-sn-glycero-3-phosphocholine, 1,2-di-O-phytanoyl-sn-glycero-3-phosphocholine, palmitoyl-oleoyl-phosphatidylcholine, dioleoyl-phosphatidyl-methyl ester, dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanoyl-sn-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dipalmitoyl 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl, GM1 ganglioside or lysophosphatidylcholine.

8. The fusion method according to claim 6, characterized in that: The polymer film includes any one or more of the following: Copolymers of one or more of polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene, polybutadiene, polyvinyl chloride, polyalkyl acrylate, polyalkyl methacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylate, polyacrylate, polysulfone, polyethylene ether, poly(propylene oxide), C1-C6 alkyl acrylate and methacrylate, acrylamide, methacrylamide, (C1-C6 alkyl) acrylamide and methacrylamide, N,N-dialkyl-acrylamide, ethoxy acrylate and methacrylate, polyethylene glycol monomethacrylate and polyethylene glycol monomethyl ether methacrylate, hydroxy-substituted (C1-C6 alkyl) acrylamide and methacrylamide, hydroxy-substituted C1-C6 alkyl vinyl ether, sodium vinyl sulfonate, sodium styrene sulfonate, 2-acrylamide- 2-Methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl-2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4′-bisalkyloxazolinyl-5-one, 2,4-vinylpyridine, ethylenically unsaturated carboxylic acids having 3 to 5 carbon atoms, amino(C1-C6-alkyl)-, mono(C1-C6-alkylamino)(C1-C6-alkyl)- and bis(C1-C6-alkylamino)(C1-C6-alkyl)-acrylates and methacrylates, allyl alcohol, 3-trimethylammonium methacrylate 2-hydroxypropyl chloride, dimethylaminoethyl methacrylate, dimethylaminoethyl methacrylamide, glycerol methacrylate, N-(1,1-dimethyl-3-oxobutyl)acrylamide, cyclic imino ethers, vinyl ethers, cyclic ethers including epoxy derivatives, cyclic unsaturated ethers, N-substituted ethylimines, β-lactones and β-lactams, vinyl ketone acetals, vinyl acetals and phosphoranes.

9. The fusion method according to claim 1, characterized in that: The porins include one or more proteins having at least 70% homology to the bacterial amyloid secretion channel CsgG, Mycobacterium smegmatis porins, α-hemolysin, OmpG, InvG, GspD, Frac, PA63, SP1, Aerobacterial lysin, plyAB, bacteriophage motor protein channel phi29, T3, T4, T7, SPP1 or gp20c.

10. The fusion method according to claim 1, characterized in that: The fusion method further comprises: Adjust the voltage to 0 V and incubate for 5-15 minutes, preferably 10 minutes; Preferably, after the incubation, the first buffer containing the porin is replaced with the second buffer not containing the porin, and a voltage is applied to both sides of the membrane to detect the current and count whether a single porin is fused with the membrane.

11. A porin plug buffer, characterized in that The porin plug buffer contains monovalent metal ions and / or divalent metal ions.

12. The porin plug buffer according to claim 11, characterized in that The monovalent metal ions include one or more of sodium ions, potassium ions or lithium ions, and the divalent metal ions include calcium ions and / or magnesium ions.

13. The porin pore buffer according to claim 12, characterized in that The concentration of the monovalent metal ions is 350-800 mM, and the concentration of the divalent metal ions is 250-550 mM.

14. The porin pore buffer according to claim 13, characterized in that The concentration of potassium ions includes 350 to 500 mM, the concentration of sodium ions includes 470 to 550 mM, and the concentration of magnesium ions includes 250 to 550 mM; Preferably, the porin plug buffer comprises: 350-500 mM potassium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 470-550 mM sodium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 250-550 mM magnesium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 100-200 mM potassium ferrocyanide, 100-200 mM potassium ferrocyanide, 10-50 mM potassium phosphate, pH = 7.0-8.2; Preferably, the buffer comprises 470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550 mM sodium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.

0.

15. A porin pore buffer combination, characterized in that: The porin pore buffer combination comprises two porin pore buffers, and the osmotic pressure difference between the two porin pore buffers is greater than or equal to 50 mOsm / kg.

16. The porin pore buffer combination according to claim 15, characterized in that: The porin plug buffer contains monovalent metal ions and / or divalent metal ions; Preferably, the concentration of the monovalent metal ions is 350-800 mM, and the concentration of the divalent metal ions is 250-550 mM.

17. The porin plug buffer according to claim 16, characterized in that The concentration of potassium ions includes 350 to 500 mM, the concentration of sodium ions includes 470 to 550 mM, and the concentration of magnesium ions includes 250 to 550 mM; Preferably, the porin plug buffer comprises: 350-500 mM potassium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 470-550 mM sodium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 250-550 mM magnesium chloride, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.0-8.2; or 100-200 mM potassium ferrocyanide, 100-200 mM potassium ferrocyanide, 10-50 mM potassium phosphate, pH = 7.0-8.2; Preferably, the buffer comprises 470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550 mM sodium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.

0.

18. The porin pore buffer combination according to claim 15, characterized in that: The osmotic pressure difference between the two porin plug buffers is greater than or equal to 200 mOsm / kg.

19. Use of the fusion method according to any one of claims 1 to 10, or the poron pore buffer according to any one of claims 11 to 14, or the poron pore buffer combination according to any one of claims 15 to 18 in the preparation of a nanoporin sequencing unit.

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