WO2025054991A1 - Porin-membrane fusion method, porin insertion buffer solution, and use - Google Patents

Abstract

Provided in the present invention are a porin-membrane fusion method, a porin insertion buffer solution and the use. Said fusion method comprises: providing a first solution at a first side of a membrane and providing a second solution at a second side of the membrane; applying a voltage to both sides of the membrane to perform porin-membrane fusion; measuring a current in the solution; and when the current increases which indicates a porin has been inserted into the membrane, then adjusting the voltage to 0 V, thereby completing the porin-membrane fusion. The first solution comprises a first buffer solution containing the porin, and the second solution comprises a second buffer solution which does not contain the porin, the osmotic pressure of the first solution being greater than the osmotic pressure of the second solution. The present invention can resolve the issue of the single-pore ratio during porin-membrane fusion in the prior art, and is suitable for the field of biological analysis and detection.

Description

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

The 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

In 1996, scientists first used alpha-hemolysin protein to achieve recognition of different bases (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 such as nucleic acids, proteins, and polymers.

Principle of biological nanopore detection: A single biological nanopore protein is inserted into the phospholipid membrane. The substance to be tested passes through the nanopore channel under the action of the electric field force, and the substance to be tested is identified by the characteristic current signal generated. The pore protein and the membrane are immersed in the solution environment, and there is a pair of electrodes on both sides of the membrane.

The 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, etc. Different nanopore proteins have different properties such as amino acid composition, structure, charge, etc., and can be used for different types of objects to be detected, and their sensitivity and resolution are also different. 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 it easier to fuse with the phospholipid bilayer membrane. The hydrophobic area of the outer wall of viral motor channel proteins is small, and liposome vesicles are needed to promote the fusion of proteins with membranes.

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 characteristics or shortcomings of this method are: (1) This method is applicable to non-membrane protein channels; (2) The method of forming liposomes is relatively complicated, and special equipment (such as rotary evaporators, extruders, etc.) is required to prepare liposomes; (3) The prepared liposomes fuse with the polymer membrane while changing the composition of the polymer membrane. This patent describes the initiation of the fusion of nanochannel proteins and polymer membranes by liposomes; if the liposome method is not used, the nanochannel protein and the polymer membrane cannot be directly fused.

CN110023753A discloses a method for controlling the insertion of pores into a membrane using voltage. The potential difference of the membrane is controlled to prevent or reduce the insertion of another pore protein to form a porous nanopore channel. The method in the patent describes how to reduce the probability of multiple pore proteins inserting into the membrane, but does not explicitly involve how to increase the yield of pore insertion.

Oxford Nanopore, a British company, has launched a number of nanopore sequencers with different throughputs. Among them, a single chip of the small sequencer MinION contains 512 channels and 2048 nanopore channel slots or sequencing units (wells). If there are more than 800 single holes in the 2048 wells, the sequencing chip is qualified. The higher the single-hole rate of a single chip, the more effective nanopore single holes or effective sequencing units that can be used, and the greater the amount of data that can be generated. Therefore, improving the single-hole 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 For example, arrays ranging from a few thousand to a million cells. Each cell of the array contains membranes and porins. One of the challenges is to increase the yield of arrays with membranes and porins. The higher the yield, the higher the sequencing throughput. Therefore, it is necessary to increase the yield of porin plugs.

Summary of the invention

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

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, and the fusion method 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.

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.

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.

Furthermore, the concentration of the monovalent metal ions is 350 to 800 mM, and the concentration of the divalent metal ions is 250 to 550 mM.

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 first buffer and/or the second 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 250-550mM 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 comprises 470mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550mM sodium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0.

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.

Further, 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, phosphatidyl Inositol, 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)- 1,2-dipalmitoyl-sm-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.

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-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)- Acrylic and methacrylic esters, 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 cyclic ethylimines, β-lactones and β-lactams, vinyl ketone acetals, vinyl acetals and phosphoranes.

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, Aerobacterial lysin, plyAB, bacteriophage motor protein channel phi29, T3, T4, T7, SPP1 or gp20c.

Furthermore, the fusion method also includes: adjusting the voltage to 0V and incubating for 5-15 minutes, preferably 10 minutes; preferably, after incubation, using a second buffer solution that does not contain porins to replace the first buffer solution containing porins, and applying voltage to both sides of the membrane, detecting the current and counting whether a single porin is fused with the membrane.

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.

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.

Furthermore, the concentration of the monovalent metal ions is 350 to 800 mM, and the concentration of the divalent metal ions is 250 to 550 mM.

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, 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.

In order to achieve the above object, according to the third aspect of the present invention, a porin pore buffer combination is provided, 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 50mOsm/kg.

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.

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 250-550mM 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 comprises 470mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550mM sodium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0.

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

In order to achieve the above object, according to the fourth aspect of the present invention, a fusion method, or the above-mentioned porin pore buffer, or the above-mentioned porin pore buffer combination is provided for use in the preparation of a nanopore protein sequencing unit.

By applying the technical solution of the present invention and utilizing the above-mentioned method for fusing the porin to the membrane, the fusion of the porin to the membrane can be achieved by a simple buffer and applied voltage, and the porin can be embedded in the membrane. The porin can be embedded without the assistance of additional components such as liposomes, and the operation is simple, the fusion efficiency is high, and the single-hole ratio (single-hole rate) of the embedded hole is high, which can significantly improve the yield of the sequencing unit for nanoporin sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

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 of a single sequencing unit; 3 is a membrane; 4 is a buffer solution during the fluid storage period, located on the second side of the membrane; 5 is a working electrode; 6 is a chip pool shared by different sequencing units, located on the first side of the membrane.

FIG. 2 shows a graph of the opening current of a single CsgG pore embedded in a phospholipid membrane in a buffer according to Example 1 of the present invention.

FIG. 3 shows a graph showing the results of the number of embedded holes/number of membranes formed in different buffer systems according to an embodiment of the present invention.

FIG. 4 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.

FIG. 5 shows a result diagram of the number of single holes/number of membranes formed by embedding holes in different buffer systems according to an embodiment of the present invention.

FIG. 6 shows a comparison result of capacitance values under different buffer systems according to Example 2 of the present invention.

7 shows the result graphs of the single pore ratio (single pore number/total pore number) of embedded pores and the single pore number of embedded pores/film formation number at different potassium chloride concentrations according to Example 5 of the present invention.

8 shows the result graphs of the single pore ratio (single pore number/total pore number) of embedded holes and the single pore number of embedded holes/film formation number under different sodium chloride concentrations according to Example 5 of the present invention.

DETAILED DESCRIPTION

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 in conjunction with the embodiments.

As mentioned in the background art, the methods for fusing porins with membranes in the prior art are complex and have low fusion efficiency, which affects the throughput of subsequent high-throughput sequencing. In this application, the inventors attempt to develop a new method for fusing porins with membranes, and thus propose a series of protection schemes of this application.

In a first typical embodiment of the present application, a method for fusing porins with membranes is provided, and the fusion method 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 0 V, 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.

In the above fusion method, the membrane separates the first solution and the second solution, and by applying 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 realizing the fusion of the porin and the membrane. The above 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 porin to be inserted 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 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 produce a difference in osmotic pressure.

The prior art also discloses a method of using osmotic pressure difference to initiate pore and membrane fusion, but in this method, a method of inserting porins from a buffer with a lower osmotic pressure to a buffer with a higher osmotic pressure is disclosed. The insertion direction of the porins in the above fusion method of the present application is opposite to that of the prior art, and 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 for nanopore protein sequencing.

In the case of the above current increase, taking the voltage of 0.18V as an example, when the current value is 0-20pA, the channel is considered to be unfused with the membrane (not embedded in the hole), the hole with the current value concentrated in 180pA-250pA is called a single hole, and the hole with the current concentrated above 250pA is called a multi-hole. Through the above current changes, especially the increase in current, it can be judged whether fusion occurs, and further it can be judged whether a single or multiple porins fuse with the membrane. After the porins are inserted into the membrane, adjust the voltage to 0V to prevent more porins from continuing to insert into the membrane and affecting the performance of the product.

The above-mentioned first side and second side merely indicate the names of different sides of the membrane, and do not limit the direction, position, etc. of the membrane. The first solution may be located in any direction or position of the membrane, including but not limited to above or below the membrane.

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.

The above 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.

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.

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-hole ratio of the embedded holes, and the embedding process can be completed without the assistance of additional components such as liposomes.

In a preferred embodiment, the concentration of monovalent metal ions includes 350-800 mM, and the concentration of divalent metal ions includes 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.

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 includes: 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); or 250-550 mM chloride magnesium sulfate (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, 120, 140, 160, 180 or 200 mM) potassium ferrocyanide, 100-200 mM (including but not limited to 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.

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.

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.

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

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.

In a preferred embodiment, 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, 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 cyclic ethylimines, β-lactones and β-lactams, vinyl ketone acetals, vinyl acetals and phosphoranes.

In a preferred embodiment, the porin protein 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, Aerobacterial lysin, plyAB, bacteriophage motor protein channel phi29, T3, T4, T7, SPP1 or gp20c.

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 the porin with a second buffer not containing the porin, A voltage is applied to both sides of the membrane, the current is detected and the number of pores fused to the membrane is counted.

Through 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 be inserted 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 incubation for a period of time, the buffer containing 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 the voltage of 0.18V as an example, the pores with current values concentrated between 180pA-250pA are called single pores, which means that one porin is fused on the membrane; the pores with current values concentrated above 250pA are called multi-pores, which means that multiple porins are fused on the membrane.

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.

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.

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.

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. 8.2; or 250-550mM magnesium chloride, 10-50mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=7.0-8.2; or 150mM potassium ferrocyanide, 150mM potassium ferrocyanide, 25mM potassium phosphate, pH=7.0-8.2; preferably, the buffer comprises 470mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0; or 550mM sodium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0.

In a third typical embodiment of the present application, a porin pore buffer combination is provided, wherein 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.

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.

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.

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 ferrocyanide, 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.

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

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

The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

Embodiment 1:

This embodiment shows the results of a control experiment in which a buffer with the same osmotic pressure was used on both sides of the membrane, that is, buffer 1 (150mM potassium ferrocyanide, 150mM potassium ferrocyanide, 25mM potassium phosphate, pH=8.0) was used to embed holes on both sides of the membrane. The porin used in this example is a CsgG mutant (the wild-type amino acid sequence is shown by SEQ ID NO: 1, and the mutation site is: Y51A/F56Q/R97W/R192D). In the electrophysiological experiment, the porin was thoroughly mixed in 300μL buffer 1 and pushed into the upper side of the membrane in the chip array device (position 6 in Figure 1). The structure of the chip array device is shown in Figure 1, wherein 1 is a ground electrode; 2 is a fluid reservoir for a single sequencing unit; 3 is a membrane; 4 is a buffer for the fluid storage period, located on the second side of the membrane (the side below the membrane); 5 is a working electrode; 6 is a chip pool shared by different sequencing units, located on the first side of the membrane (the side above the membrane). The device used in the above fusion method includes but is not limited to the device shown in Figure 1.

Apply a voltage of 0.18V and wait for the porin to be inserted into the phospholipid membrane. When the porin is embedded in the phospholipid membrane, the current of a single channel will change. For the channel with a hole, a voltage of 0mV is applied to reduce the possibility of further holes in the channel. Wait for all channels for 10 minutes without the appearance of new holes, and use 2mL of buffer 1 to remove the excess porin above the membrane. Use 3mL of buffer (500mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) to flow through the system to completely replace the buffer 1 above the membrane. The hole state of different channels is counted at 0.18V, and the number of holes in each channel is determined according to the current value. Channels with current values of 0-20pA are considered to be un-hole-embedded, holes with current values concentrated between 180pA-250pA are called single holes (as shown in Figure 2), and holes with currents concentrated above 250pA are called multi-holes.

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

Results and Discussion:

Using buffer for embedding, the number of embedded holes/number of membranes can reach 78%±8% (as shown in Figure 3), but the single hole/total hole only accounts for 70%±15% (N=17) (as shown in Figure 4), so the number of single holes/number of membranes is only 54%±9% (N=17) (as shown in Figure 5). The membrane containing multiple nanopore channels cannot be used for subsequent single-molecule detection experiments. The array has a yield of membranes and single pores. The higher the yield, the higher the sequencing throughput. Therefore, improving the yield of pore protein plugs is of great significance for improving sequencing throughput.

When the osmotic pressure concentration on both sides of the membrane is consistent, the membrane area is large. When the pores are embedded, the porin will actively embed, the difficulty of embedding is reduced, and the probability of porous channels is greatly increased. In order to increase the single hole rate and reduce the probability of active embedding, the embedding method needs to be further optimized. By changing the osmotic pressure on both sides of the membrane to form different osmotic pressure differences, the membrane area can be effectively changed, thereby changing the difficulty of embedding.

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 plurality of non-isotonic embedding buffers that can effectively improve the single-pore rate are screened out.

Embodiment 2:

This embodiment shows the experiment of the pore optimization group, which shows that changing the buffer used for the pore on one side of the membrane can significantly improve the single-pore yield. The same CsgG porin mutant as in Example 1 was used in this example. In the electrophysiological experiment, 3 mL of buffer 2 containing potassium chloride (470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0) was pushed into the chip pool to fully replace the buffer 1 on the upper side of the membrane to avoid the influence of buffer 1 (buffer1) on the experiment. The porin was fully mixed in 300 μL of buffer 2 ((buffer2, 470 mM potassium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0) and pushed into the sequencing chip pool (on the upper side of the membrane). The lower side of the membrane is buffer 1, and the osmotic pressure of buffer 2 containing porin is greater than the osmotic pressure of buffer 1, and the osmotic pressure difference is 230 mOsm/kg.

Apply 0.18V voltage and wait for pores to be embedded. When pores are embedded in the phospholipid membrane, the current of a single channel will change. Apply 0mV voltage to the channel that has been embedded to reduce the possibility of further pores in the channel. Wait for 10 minutes until all channels have no new pores, and use 2mL buffer 2 (470mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) to remove excess pores above the membrane. Use 3mL buffer (500mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) to flow through the system to completely replace buffer 2 above the membrane. Count the pore states 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 to have no pores, pores with current values concentrated between 180pA-250pA are called single pores, and pores with currents concentrated above 250pA are called multi-pores.

Results and Discussion:

Using buffer 2 containing potassium chloride (470mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) for embedding, the number of embedded holes/number of membranes can reach 82%±5% (as shown in Figure 3), and the single hole/total hole is also greatly improved, reaching 92%±3%, which is 31% higher than the result of buffer 1 (as shown in Figure 4). The number of single holes/number of membranes is also greatly improved, which is 43% higher than the result of embedding in buffer 1, reaching 77%±8% (as shown in Figure 5).

It was unexpectedly found that buffer 2 can effectively reduce capacitance (as shown in Figure 6). When capacitance is low, noise is generally low. Under the experimental conditions of buffer 1, the peak capacitance is around 115pF; under the experimental conditions of buffer 2, the peak capacitance is effectively reduced to around 75pF.

Embodiment three:

This example demonstrates experiments with an optimized set of intercalation ions, showing that other monovalent ions besides potassium, such as sodium, also significantly improve intercalation yield and results.

In this example, the same CsgG porin mutant as in Example 1 was used. In the electrophysiological experiment, 3 mL of sodium chloride buffer was added. Flushing solution 3 (buffer3, 550mM sodium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) fully replaces buffer 1 on the upper side of the membrane. The porin is fully mixed in 300μL buffer 3 containing sodium chloride (550mM sodium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) and flows into the sequencing chip pool (on the upper side of the membrane). The lower side of the membrane is buffer 1. The osmotic pressure of buffer 3 containing porin is greater than the osmotic pressure of buffer 1, and the osmotic pressure difference is 250mOsm/kg.

Apply 0.18V voltage and wait for pores to be embedded. When the porin is embedded in the phospholipid membrane, the current of a single channel will change. For the channel with embedded holes, 0mV voltage protection is implemented to reduce the possibility of further pores in the channel. If no new pores appear within 10 minutes, use 2mL of sodium chloride buffer 3 (550mM sodium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) to remove the excess porin above the membrane. Use 3mL of buffer (500mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) to flow through the system to completely replace the buffer 3 above the membrane. The pore state of different channels is counted at 0.18V. The number of pores in each channel is determined by the current value. Channels with current values of 0-20pA are considered to be un-embedded, and pores with current values concentrated between 180pA and 250pA are called single pores, and pores with currents concentrated above 250pA are called multi-pores.

Results and Discussion:

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 formed can reach 92% ± 1% (as shown in Figure 3), and the single hole/total hole ratio 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 formed is the same as the embedding result of buffer 2, reaching 78% ± 4%, which is 44% higher than the single hole rate of the embedding in buffer 1 (as shown in Figure 5).

Embodiment 4:

This example shows the experiment of the embedding optimization group, which shows that other valence metal ions besides potassium and sodium, such as the divalent metal ion magnesium, can also significantly improve the embedding yield and results.

The same CsgG porin mutant as in Example 1 was used in this example. In the electrophysiological experiment, 3 mL of magnesium chloride buffer 4 (buffer 4, 320 mM magnesium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0) was pushed to fully replace the buffer 1 on the upper side of the membrane. The porin was fully mixed in 300 μL of buffer 4 containing magnesium chloride (320 mM magnesium chloride, 25 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 8.0) and flowed into the sequencing chip pool (on the upper side of the membrane). The lower side of the membrane was buffer 1, and the osmotic pressure of buffer 4 containing porin was greater than the osmotic pressure of buffer 1, and the osmotic pressure difference was 260 mOsm/kg.

Apply 0.18V voltage and wait for pores to be embedded. When the porin is embedded in the phospholipid membrane, the current of a single channel will change. For the channel with embedded holes, 0mV voltage protection is implemented to reduce the possibility of further pores in the channel. If no new pores appear within 10 minutes, use 2mL magnesium chloride buffer 4 (320mM magnesium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) to remove excess porin above the membrane. Use 3mL buffer (500mM potassium chloride, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=8.0) to flow through the system to completely replace the buffer 4 above the membrane. The pore state of different channels is counted at 0.18V. The number of pores in each channel is determined according to the current value. Channels with current values of 0-20pA are considered to be un-embedded, and pores with current values concentrated between 180pA and 250pA are called single pores, and pores with currents concentrated above 250pA are called multi-pores.

Results and Discussion:

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/film formation can reach 82% ± 6% (as shown in Figure 3), and the single hole/total hole result is improved compared with the embedding result of buffer 1. The single hole number/film formation number is the same as the results of buffer 2 and buffer 3, reaching 78%±8%, which is 44% higher than the single hole rate of buffer 1 (as shown in Figure 5).

Embodiment five:

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

As shown in Figure 7, the ratio of single pore number to total pore number is best in 0.47M potassium chloride buffer and 0.5M potassium chloride buffer, but the ratio of single pore number to membrane number is poor in 0.5M potassium chloride buffer. In summary, the best potassium chloride concentration range in potassium chloride buffer is selected between 0.35M and 0.5M, among which 0.47M performs best.

For sodium chloride buffer, 0.47M-0.55M sodium chloride buffer was tested based on potassium chloride buffer. As shown in FIG8 , sodium chloride buffers above 0.5M performed better than isotonic buffer 1 (single pore/total pores: 0.70±0.15; single pore/number of membranes formed: 0.54±0.09).

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: using the above-mentioned method for fusing the porin with the membrane, the fusion of the porin with the membrane can be achieved by a simple buffer and applied voltage, and the porin embedded in the membrane can be completed. The porin embedded can be achieved without the assistance of additional components such as liposomes, the operation is simple, the fusion efficiency is high, and the single hole ratio of the embedded hole is high, which can significantly improve the yield of the sequencing unit for nanoporin sequencing. And the sequencing unit obtained by the fusion of the porin with the membrane using the above-mentioned buffer has a 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.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (19)

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. The fusion method according to claim 1 is characterized in that the osmotic pressure of the first solution and the osmotic pressure of the second solution have a difference of at least 50mOsm/kg, preferably a difference of at least 200mOsm/kg. The fusion method according to claim 1, characterized in that the first buffer and/or the second buffer contains 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. The fusion method according to claim 3 is characterized in that the concentration of the monovalent metal ions is 350 to 800 mM, and the concentration of the divalent metal ions is 250 to 550 mM. The fusion method according to claim 4, characterized in that the concentration of potassium ions is 350 to 500 mM, the concentration of sodium ions is 470 to 550 mM, and the concentration of magnesium ions is 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. The fusion method according to claim 1 is characterized in that the membrane comprises a diblock phospholipid membrane, a diblock polymer membrane, a triblock phospholipid membrane or a triblock polymer membrane. The fusion method according to claim 6, characterized in that 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 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. The fusion method according to claim 6, characterized in that 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 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. The fusion method according to claim 1, characterized in that the porin comprises one or more proteins having at least 70% homology with the following proteins: bacterial amyloid secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, OmpG, InvG, GspD, Frac, PA63, SP1, Aerobacterial lysin, plyAB, bacteriophage motor protein channel phi29, T3, T4, T7, SPP1 or gp20c. 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. A porin pore buffer, characterized in that the porin pore buffer contains monovalent metal ions and/or divalent metal ions. The porin pore 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. The porin pore buffer according to claim 12, characterized in that the concentration of the monovalent metal ions is 350 to 800 mM, and the concentration of the divalent metal ions is 250 to 550 mM. The porin pore buffer according to claim 13, characterized in that the concentration of potassium ions is 350 to 500 mM, the concentration of sodium ions is 470 to 550 mM, and the concentration of magnesium ions is 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. 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 50mOsm/kg. The porin pore buffer combination according to claim 15, characterized in that 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. The porin pore buffer according to claim 16, characterized in that the concentration of potassium ions is 350 to 500 mM, the concentration of sodium ions is 470 to 550 mM, and the concentration of magnesium ions is 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. The porin pore buffer combination according to claim 15, characterized in that the osmotic pressure difference between the two porin pore buffers is greater than or equal to 200 mOsm/kg. 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.