Nanopore sequencing polymeric membrane and membrane forming method

Abstract

The invention relates to the field of nanopore sequencing, and particularly discloses a nanopore sequencing polymeric membrane and a membrane forming method.The polymeric membrane is a single-layer molecular membrane and is formed by a block polymer, the block polymer comprises a hydrophilic-end polymer and a hydrophobic-end polymer, the polymerization degree of the hydrophilic-end polymer is 4-6, and the hydrophobic-end polymer is 2-6. The polymerization degree of the hydrophobic end polymer is between 18 and 33; the preparation method of the polymeric membrane comprises the following steps: S1, adding a first buffer solution into a microstructure of a chip, introducing a non-polar solvent dissolved with a block polymer into a runner of the microstructure, and pushing away the redundant first buffer solution to form a lower interface of the polymeric membrane; s2, introducing gas into the microstructure to push away the redundant non-polar solvent to form an upper interface of the polymeric membrane; and S3, adding a second buffer solution, emptying upper-layer gas, and forming a polymeric membrane on the surface of the microstructure of the chip after the block polymer is stably arranged. The film forming process provided by the invention is easy to operate in film forming, low in film rupture rate, and relatively good in stability and voltage tolerance.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a nanopore sequencing polymer membrane and a membrane forming method. Background Art

[0002] Nanopore sequencing technology has the characteristics of real-time sequencing, fast detection speed, high throughput, and long read length. Unlike the previous two generations of sequencing technologies, nanopore sequencing technology does not require PCR amplification, and uses the special structure of nanopores to achieve real-time sequencing of single DNA / RNA molecules, thus overcoming the problems of PCR amplification errors, short sequencing read lengths, and DNA / RNA methylation modifications in the second-generation sequencing technology (NGS), and is widely used in multiple research fields such as genome sequencing and mutation identification.

[0003] Nanopore sequencing technology is based on channel proteins embedded in a monolayer polymer membrane. DNA or RNA molecules are transferred from the negatively charged side to the positively charged side through the channel proteins under the action of a constant voltage added at both ends. Under the action of the rate-controlling protein, the double-stranded DNA molecules unwind into single-stranded DNA molecules and pass through the protein at a stable rate. When passing through the protein, the protein recognition region is deformed due to different base groups, resulting in changes in the generated current. Later, an algorithm is used to analyze the current data and output the algorithm model to identify the bases.

[0004] The polymer membranes currently used are mainly phospholipid bilayers and block polymers. The voltage tolerance of phospholipid bilayers is poor, and the membrane stability is poor. It is easy to break the membrane during the embedding of channel proteins and sequencing. Block polymers have greatly improved membrane stability and voltage tolerance compared to phospholipid bilayers, but they are also difficult to embed in channel proteins, requiring higher protein concentrations and higher voltages. If the concentration of embedded pore proteins is too high, more than one channel protein may be embedded in the polymer layer, resulting in the inability to detect current signals during later sequencing. Higher embedded pore voltages may cause the polymer membrane to be broken by current during the embedding process, affecting the final embedding rate, resulting in a lower number of available pores during sequencing. Moreover, the preparation process of this type of polymer membrane is generally relatively complex. In order to achieve a higher flux in actual applications, more array structures are generally required, and there are more variables. The membrane formation process is too complicated, which can easily lead to the inability to achieve a higher membrane formation rate when moving from single-channel membrane formation to multi-channel membrane formation, which in turn leads to substandard flux. For example, the cell-like membrane for nanopore sequencing with publication number CN112831395A uses a bubbling process to prepare a block-structured double-layer polymer membrane, which works well in single-channel nanopore tests. However, due to the complexity of the process, the film formation rate decreases when forming a multi-channel film, affecting the flux. Based on this, it is necessary to provide a simplified process to achieve a higher film formation rate when forming a film on the surface of a multi-channel chip, so as to facilitate its application in later production conversion. Summary of the invention

[0005] In view of the problems and defects of the current phospholipid bilayer nanosequencing polymer membrane, such as poor stability and voltage tolerance, difficulty in embedding block polymers into channel proteins, complex membrane-forming process and low membrane-forming rate of multi-channel membrane, the present invention provides a nanopore sequencing polymer membrane and a membrane-forming method.

[0006] The solution adopted by the present invention to solve its technical problem is: a nanopore sequencing polymer membrane, which is a single-layer polymer membrane formed by a block polymer, wherein the block polymer includes a hydrophilic end polymer and a hydrophobic end polymer, the degree of polymerization of the hydrophilic end polymer is between 4-6, and the degree of polymerization of the hydrophobic end polymer is between 18-33.

[0007] In the above-mentioned nanopore sequencing polymer membrane, the hydrophilic end polymer is one or more of polymethyloxazoline, polystyrene, polybutadiene, and polymethacrylate; the hydrophobic end polymer is one or more of polydimethylsiloxane, polyethylene, polyethylene oxide, and polyalkyl acrylate.

[0008] The above-mentioned nanopore sequencing polymer membrane is a triblock polymer, which is a hydrophilic end polymer-hydrophobic end polymer-hydrophilic end polymer, and the corresponding degree of polymerization is 6-33-6.

[0009] The above-mentioned nanopore sequencing polymer membrane is poly(2-methyloxazoline)-polydimethylsiloxane-poly(2-methyloxazoline) with a degree of polymerization of 6-33-6.

[0010] The present invention also provides a method for forming a nanopore sequencing polymer membrane, comprising the following steps: S1, adding the first buffer solution into the microstructure of the chip, passing the non-polar solvent containing the block polymer into the flow channel of the microstructure at a rate of 1000-2000 μL / min, and pushing away the excess first buffer solution to form the lower interface of the polymer film; S2, passing gas into the microstructure at a rate of 20-200 μL / min to push away excess non-polar solvent to form an upper interface of the polymer film; S3, adding the second buffer solution to exhaust the upper gas, and waiting for the block polymer to be arranged stably to form a polymer film on the microstructure surface of the chip.

[0011] In the above-mentioned nanopore sequencing polymer membrane formation method, the first buffer solution and the second buffer solution are one or more of KCl, K4[Fe(CN)6], K3[Fe(CN)6], HEPEs, K2HPO4, and KH2PO4.

[0012] In the above-mentioned nanopore sequencing polymer membrane forming method, the non-polar solvent is silicone oil, C 10 -C 20 One or more of long-chain alkanes, squalane, and squalene, among which C 10 -C 20 Long chain alkanes include dodecane, tetradecane, hexadecane and their halides.

[0013] In the above-mentioned nanopore sequencing polymer membrane forming method, the gas is a gas that does not react with the buffer solution and the block polymer.

[0014] In the above-mentioned nanopore sequencing polymer membrane forming method, the mass concentration of the block polymer is 5-30 mg / ml.

[0015] In the above-mentioned nanopore sequencing polymer membrane forming method, the mass concentration of the block polymer is 10-20 mg / ml.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention controls the content of block polymers and the introduction rate of solvents and gases during the preparation process, so that the self-assembly of the polymer as a whole presents a relatively controllable state to form a single-layer polymer film.

[0017] 2. The polymer film prepared by the preparation method of the present invention is a single-layer molecular film. The block polymer used can be arranged stably and spontaneously in the system. After stabilization, the capacitance value is tested. The overall capacitance fluctuation range is within 1pF, and the film uniformity is good.

[0018] 3. The polymer membrane prepared by the present invention uses MspA as the channel protein, and the protein is embedded in the polymer membrane. The membrane rupture ratio in this process is less than 5%, the partial ratio is less than 3%, and the embedding voltage is between 240-320mV, which can easily embed the channel protein.

[0019] 4. The single-layer polymer membrane prepared by the present invention can also withstand a voltage of 380-480 mV when no channel protein is embedded.

[0020] 5. The protein concentration required for the polymer membrane prepared by the present invention is only about 2-5 times the embedding concentration of phospholipids, but the embedding capacity is equivalent to that of phospholipids.

[0021] 6. The polymer membrane prepared by the present invention is a kind of biological membrane, which is formed by spontaneous arrangement of hydrophilic ends and hydrophobic ends. It does not need photocuring cross-linking, nor does it need cross-linking with other monomer molecules, and can still achieve good stability and voltage tolerance.

[0022] 7. The present invention ensures the performance of the polymer membrane by optimizing the mass concentration and degree of polymerization of the hydrophilic and hydrophobic polymers when preparing the block polymer, which can effectively avoid the problems of multiple channel proteins being embedded in the membrane due to the hydrophilic end being too short, the solubility being reduced due to the hydrophilic end being too long, and the membrane being easily broken due to the hydrophobic end being too short, and the channel protein being ineffective due to the hydrophobic end being too long and exceeding the length of the channel protein.

[0023] 8. In the process of preparing the polymer membrane, the present invention optimizes the gas introduction rate to obtain a suitable single-layer polymer membrane, thereby avoiding the problem that the membrane is too thick due to too high a rate, resulting in easy failure of protein embedding, and the problem that the polymer membrane is difficult to form due to too low a rate.

[0024] 9. The single-layer polymer membrane prepared by the present invention has a small change in the number of membranes and no obvious change in capacitance after the chip is stored in a refrigerator at 2-8°C for one week. MspA is used as a channel protein and embedded in the polymer membrane. It can still withstand the voltage that the initial membrane can withstand, the required protein concentration remains unchanged, and the membrane rupture rate does not change significantly during the pore embedding process.

[0025] 10. The film forming process of the present invention is simple and easy to operate, and can achieve a higher film forming rate when forming a film on the surface of a multi-channel chip, thereby facilitating achieving a higher flux. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the polymer film formation process of the present invention, in which (a) is the chip before film formation, (b) is the state of introducing the first buffer solution, (c) is the state of introducing a non-polar solvent containing block polymers to form the lower interface of the polymer film; (d) is the state of introducing an inert gas to form the upper interface of the polymer film; and (e) is the state of introducing the second buffer solution to form the polymer film. DETAILED DESCRIPTION

[0027] In view of the problem that the current process of preparing polymer membranes with block polymers is relatively complex, resulting in a decrease in the multi-channel film formation rate, the present invention provides a simple process for forming polymer membranes for nanopore sequencing. The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0028] Example 1: This example provides a method for forming a nanopore sequencing polymer membrane, wherein the polymer membrane is formed by a block polymer, wherein the block polymer is a triblock polymer, specifically poly(2-methyloxazoline)-polydimethylsiloxane-poly(2-methyloxazoline), and the degree of polymerization is 6-33-6.

[0029] In this example, the block polymer poly (2-methyloxazoline) 6-polydimethylsiloxane is selected 33-poly (2-methyloxazoline) 6, dissolve it in a non-polar solvent to make its mass concentration 10mg / ml. The non-polar solvent used in this embodiment is a mixed solvent of silicone oil AR20 and hexadecane. In addition, C 10 -C 20 One or more of long chain alkanes, squalane, squalene, etc., wherein C 10 -C 20 Long chain alkanes include dodecane, tetradecane, hexadecane and their halides.

[0030] During preparation, the first buffer solution is first added to the microstructure of the chip. The first buffer solution used in this embodiment is KCl. In addition, an aqueous solution containing any one or more of K4[Fe(CN)6], K3[Fe(CN)6], HEPEs, K2HPO4, and KH2PO4 can also be selected; a non-polar solvent containing a block polymer is introduced into the flow channel of the microstructure at a rate of 2000 μL / min, and excess first buffer solution is pushed away to form the lower interface of the polymer membrane.

[0031] Then, gas is introduced into the microstructure at a rate of 100 μL / min to push away excess non-polar solvent to form the upper interface of the polymer film; the gas used in this embodiment is air, and other gases that may be selected include but are not limited to nitrogen, air, inert gas, and other gases that do not react with solutions or solvents.

[0032] Finally, a second buffer solution is added to exhaust the upper gas. The second buffer solution used here can be the same as or different from the first buffer solution. In this embodiment, the second buffer solution is consistent with the first buffer solution. After the block polymer arrangement is stabilized, a polymer film is formed on the microstructure surface of the chip.

[0033] The film capacitance after film formation was measured, and the film capacitance was measured multiple times and was all between 15±1pF, indicating that the film formation uniformity of the process of this embodiment is good.

[0034] Then, using MspA as a channel protein, the protein was embedded into the polymer membrane, and the polymer membrane rupture rate in this process was counted. The results showed that the membrane rupture rate was less than 3%, and the embedding voltage was between 240-280mV, and it could withstand a voltage of 380-400mV. The required protein concentration was only about twice the phospholipid embedding concentration.

[0035] After the film-formed chip was stored in a refrigerator at 2-8°C for one week, the number of membranes changed less than 3% and the capacitance did not change significantly. MspA was used as a channel protein and embedded in a polymer membrane, and it was still able to withstand a voltage of 380-400mV. The required protein concentration remained unchanged, and the membrane rupture rate during the embedding process was less than 4%.

[0036] Example 2: The nanopore sequencing polymer membrane formation method of this example is to use a triblock polymer poly (2-methyloxazoline) 6-polydimethylsiloxane 33 -Poly (2-methyloxazoline) 6 was prepared by dissolving it in a mixed solution of AR20 and dodecane bromide at a mass concentration of 20 mg / ml.

[0037] During preparation, an aqueous solution containing KCl and HEPEs is first added to the microstructure of the chip; then a mixed solution of AR20 and dodecane bromide containing block polymer is passed into the flow channel of the microstructure at a rate of 1500 μL / min to push away excess first buffer solution to form the lower interface of the polymer membrane.

[0038] Then, air was introduced into the microstructure at a rate of 150 μL / min to push away the excess nonpolar solvent and form the upper interface of the polymer film; Finally, an aqueous solution containing KCl and HEPEs is added to exhaust the upper gas, and a polymer film is formed on the microstructure surface of the chip after the block polymer arrangement is stabilized.

[0039] The film capacitance after film formation was measured, and the film capacitance was measured multiple times and was all between 14±1 pF, indicating that the film formation uniformity of the process of this embodiment is good.

[0040] Then, using MspA as a channel protein, the protein was embedded into the polymer membrane, and the polymer membrane rupture rate in this process was counted. The results showed that the membrane rupture rate was less than 5%, and the embedding voltage was between 260-320mV, and it could withstand a voltage of 440-480mV. The required protein concentration was only about 3-4 times the phospholipid embedding concentration.

[0041] After the film-formed chip was stored in a refrigerator at 2-8°C for one week, the membrane number changed less than 1.5% and the capacitance did not change significantly. MspA was used as a channel protein and embedded in a polymer membrane, and it could still withstand a voltage of 440-480mV. The required protein concentration remained unchanged, and the membrane rupture rate during the embedding process was less than 5%.

[0042] Example 3: The nanopore sequencing polymer membrane formation method of this example is to use a triblock polymer poly (2-methyloxazoline) 6-polydimethylsiloxane 33 -Poly (2-methyloxazoline) 6 was prepared by dissolving it in a mixed solution of AR20 and dodecane bromide at a mass concentration of 15 mg / ml.

[0043] During preparation, first add KCl, KH2PO 4，An aqueous solution of K2HPO4 was added to the microstructure of the chip; then a mixed solution of AR20 and bromododecane containing block polymer was introduced into the flow channel of the microstructure at a rate of 1000 μL / min to push away excess first buffer solution to form the lower interface of the polymer membrane.

[0044] Then nitrogen gas was introduced into the microstructure at a rate of 80 μL / min to push away excess non-polar solvent to form the upper interface of the polymer film;.

[0045] Finally, an aqueous solution containing KCl and HEPEs is added to exhaust the upper gas, and a polymer film is formed on the microstructure surface of the chip after the block polymer arrangement is stabilized.

[0046] The film capacitance after film formation was measured, and the film capacitance was measured multiple times and was all between 15±1pF, indicating that the film formation uniformity of the process of this embodiment is good.

[0047] Then, using MspA as a channel protein, the protein was embedded into the polymer membrane, and the polymer membrane rupture rate in this process was counted. The results showed that the membrane rupture rate was less than 5%, and the embedding voltage was between 280-300mV, and it could withstand a voltage of 380-420mV. The required protein concentration was only about 2-3 times the phospholipid embedding concentration.

[0048] After the film-formed chip was stored in a refrigerator at 2-8°C for one week, the number of membranes changed by less than 2% and the capacitance did not change significantly. MspA was used as a channel protein and embedded in a polymer membrane, and it was still able to withstand a voltage of 380-420mV. The required protein concentration remained unchanged, and the membrane rupture rate during the embedding process was less than 5%.

[0049] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanopore sequencing polymer membrane, characterized in that: The polymer membrane is a single-layer polymer membrane formed by block polymers, wherein the block polymers include a hydrophilic end polymer and a hydrophobic end polymer, the degree of polymerization of the hydrophilic end polymer is between 4-6, and the degree of polymerization of the hydrophobic end polymer is between 18-33.

2. The nanopore sequencing polymer membrane according to claim 1, characterized in that: The hydrophilic end polymer is one or more of polymethyloxazoline, polystyrene, polybutadiene, and polymethacrylate; the hydrophobic end polymer is one or more of polydimethylsiloxane, polyethylene, polyethylene oxide, and polyalkyl acrylate.

3. The nanopore sequencing polymer membrane according to claim 1, characterized in that: The polymer membrane is a triblock polymer, which is a hydrophilic end polymer-hydrophobic end polymer-hydrophilic end polymer, and the corresponding degree of polymerization is 6-33-6.

4. The nanopore sequencing polymer membrane according to claim 3, characterized in that: The polymer film is poly(2-methyloxazoline)-polydimethylsiloxane-poly(2-methyloxazoline) with a polymerization degree of 6-33-6.

5. A method for forming a nanopore sequencing polymer membrane according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, adding the first buffer solution into the microstructure of the chip, passing the non-polar solvent containing the block polymer into the flow channel of the microstructure at a rate of 1000-2000 μL / min, and pushing away the excess first buffer solution to form the lower interface of the polymer film; S2, passing gas into the microstructure at a rate of 20-200 μL / min to push away excess non-polar solvent to form an upper interface of the polymer film; S3. Add the second buffer solution to exhaust the upper gas, and wait overnight to form a polymer film on the microstructure surface of the chip.

6. The method for forming a nanopore sequencing polymer membrane according to claim 5, characterized in that: The first buffer solution and the second buffer solution are one or more of KCl, K4[Fe(CN)6], K3[Fe(CN)6], HEPEs, K2HPO4, and KH2PO4.

7. The method for forming a nanopore sequencing polymer membrane according to claim 5, characterized in that: The non-polar solvent is silicone oil, C 10 -C 20 One or more of long-chain alkanes, squalane, and squalene, among which C 10 -C 20 Long chain alkanes include dodecane, tetradecane, hexadecane and their halides.

8. The method for forming a nanopore sequencing polymer membrane according to claim 5, characterized in that: The gas is a gas that does not react with the buffer solution and the block polymer.

9. The method for forming a nanopore sequencing polymer membrane according to claim 5, characterized in that: The mass concentration of the block polymer is 5-30 mg / ml.

10. The method for forming a nanopore sequencing polymer membrane according to claim 9, characterized in that: The mass concentration of the block polymer is 10-20 mg / ml.

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