A hybrid bionic membrane and its preparation method and application

Abstract

The present invention provides a hybrid biomimetic membrane, a preparation method thereof and an application. The hybrid membrane is formed by mixing phospholipids with an amphiphilic block copolymer. The hybrid membrane has good voltage tolerance (can withstand a voltage of 450 mV) and stability, and at the same time has easily achievable pore-inserting conditions, with low voltage stimulation and low protein concentration required for pore insertion; effectively solving the problems of poor voltage tolerance and poor membrane stability of the current lipid bilayer, and the harsh conditions for inserting channel proteins into the block copolymer membrane.

Description

Technical Field

[0001] The present invention belongs to the field of amphiphilic molecular membranes, and relates to a hybrid biomimetic membrane and its preparation method and application, in particular to a hybrid biomimetic membrane suitable for embedding channel proteins and its preparation method and application. Background Art

[0002] A lipid bilayer is a bilayer thin film formed by two layers of lipid molecules. Usually, the lipid bilayer is composed of phospholipids and can be found in the cell membranes of most living organisms. The lipid bilayer has selective permeability and is impermeable to most hydrophilic molecules and ions. As an excellent tool in experimental research, the lipid bilayer has broad application prospects in the field of biomedical technology. Due to its good compatibility with various channel proteins, it also has many applications in the field of nanopore detection. However, due to poor voltage tolerance and strong membrane fluidity, the lipid bilayer has disadvantages such as poor stability and difficulty in storage.

[0003] Amphiphilic block copolymers, usually obtained by chemical synthesis methods, have a certain range of applications in the field of nanopores. Block copolymer membranes usually have high voltage tolerance and low fluidity, and the membrane has strong stability. However, when embedding channel proteins into block copolymer membranes, more stringent conditions are often required compared to lipid bilayers, such as stronger electrical stimulation and higher protein concentration. Summary of the Invention

[0004] In order to improve the above technical problems, the present invention provides a hybrid membrane formed by mixing phospholipids and block copolymers.

[0005] According to an embodiment of the present invention, the hybrid membrane is a monolayer thin film.

[0006] According to an embodiment of the present invention, the hybrid membrane is a hybrid biomimetic membrane, specifically, a monolayer hybrid biomimetic membrane.

[0007] According to an embodiment of the present invention, in the hybrid membrane, the mass ratio of the phospholipid to the block copolymer is 1:(1 - 20), such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0008] According to an embodiment of the present invention, the block copolymer is an amphiphilic block copolymer. For example, the hydrophobic chain segment includes one or more of the following polymers: polysiloxane, polyolefin, polyalkyl acrylate, polyoxypropylene, polyacrylate, etc., and the hydrophilic chain segment is selected from one or more of the following polymers: polyacrylamide, polyacrylamide, polyC 1～6 alkylacrylamide, polyethoxyacrylate, polyethoxymethacrylate, polyethylene glycol, polymethyloxazoline, etc.

[0009] According to an embodiment of the present invention, the polyolefin may be one or more of polystyrene, polyisoprene, and polybutadiene.

[0010] According to an embodiment of the present invention, the weight-average molecular weight of the block copolymer is 1500 to 6000, such as 1800, 2500, 3000, 3500, 4000, 4500, 5000, 5500.

[0011] In some embodiments, the block copolymer is selected from the triblock copolymer poly(2-methyl oxazoline)-polydimethylsiloxane-poly(2-methyl oxazoline) (PMOXA-PDMS-PMOXA), preferably PMOXA-PDMS-PMOXA with a degree of polymerization of 6-35-6 and a weight-average molecular weight of 500-2500-500.

[0012] In some embodiments, the block copolymer is selected from the diblock copolymer poly(2-methyl oxazoline)-polydimethylsiloxane (PMOXA-PDMS), preferably PMOXA-PDMS with a degree of polymerization of 6-17 and a weight-average molecular weight of 500-1300.

[0013] It should be noted that in the above block copolymer, the degree of polymerization and the weight-average molecular weight correspond to the segments of the block copolymer.

[0014] According to an embodiment of the present invention, the thickness of the mixed film is 4 nm to 10 nm, such as 5 nm, 6 nm, 7 nm, 8 nm, 9 nm.

[0015] According to an embodiment of the present invention, in the mixed film, depending on the ratio of phospholipid to block copolymer, the phospholipid and block copolymer molecules may be uniformly dispersed and arranged, or may form local liquid-liquid phase separation.

[0016] The present invention also provides a method for preparing the above mixed film, and the preparation method includes: forming the mixed film by mixing phospholipid and block copolymer.

[0017] According to an embodiment of the present invention, the preparation method includes: forming the mixed film by the spontaneous arrangement of phospholipid and block copolymer in a non-polar solvent.

[0018] According to an embodiment of the present invention, in the preparation method, a film-forming device including a film-forming chip and a flow channel is used, and the film-forming chip includes a micro-well structure.

[0019] According to an embodiment of the present invention, the preparation method includes the following steps: First, fill the micro-wells with a first polar solvent, and then introduce a non-polar solvent dissolved with phospholipids and block copolymers into the flow channel. Use the non-polar solvent to drain the excess first polar solvent in the flow channel. Then, introduce a second polar solvent into the flow channel, and use the second polar solvent to drain the excess non-polar solvent in the flow channel. At this time, a non-polar solvent layer will be formed near the position of the micro-well opening. Wait for the phospholipids and block copolymers to spontaneously arrange to form the mixed film.

[0020] According to an embodiment of the present invention, the first polar solvent and the second polar solvent may be the same or different. For example, they may independently be selected from salt solutions containing one or several of KCl, K4[Fe(CN)6], K3[Fe(CN)6], HEPEs, and H3PO4. As an example, the first polar solvent is a salt solution containing KCl, K4[Fe(CN)6], K3[Fe(CN)6], and HEPEs.

[0021] According to an embodiment of the present invention, the non-polar solvent may be selected from one or more of silicone oil, mineral oil, long-chain alkanes, squalene, etc. For example, the silicone oil may be selected such as AR20 or dimethyl silicone oil, etc. For example, the long-chain alkane is C 10 -C 20 long-chain alkane, and as an example, it is dodecane, hexadecane, or octadecane. As an example, the non-polar solvent is a mixed solvent of silicone oil and long-chain alkane, for example, a mixed solvent of AR20 and hexadecane.

[0022] According to an embodiment of the present invention, the mass concentration of the phospholipids is 0.5 - 5 mg / ml, such as 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml.

[0023] According to an embodiment of the present invention, the mass concentration of the block copolymer is 2 - 25 mg / ml, such as 4 mg / ml, 5 mg / ml, 8 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml.

[0024] According to an embodiment of the present invention, the first polar solvent, the non-polar solvent, and the second polar solvent enter the flow channel in the same direction.

[0025] The present invention also provides a membrane, which includes a channel protein and the above-mentioned mixed film embedded in the channel protein.

[0026] According to an embodiment of the present invention, different applications are given to the membrane product according to the selection of the channel protein.

[0027] According to an embodiment of the present invention, the channel protein may be MspA.

[0028] The present invention also provides applications of the membrane in aspects such as nanopore sequencing and detection of transmembrane channel protein activity.

[0029] Beneficial effects

[0030] The hybrid membrane formed by mixing phospholipids and block copolymers provided by the present invention is specifically an amphiphilic molecular hybrid membrane, which has good voltage tolerance (can withstand a voltage of 450 mV) and stability; at the same time, it also has easily achievable pore-inserting conditions. Specifically, the voltage stimulation required for pore insertion is low and the protein concentration is low.

[0031] The present invention provides a membrane product containing the hybrid membrane, which effectively solves the problems of poor voltage tolerance and poor membrane stability of the current lipid bilayer, and the harsh conditions for inserting channel proteins into the block copolymer membrane, and provides a membrane suitable for nanopore sequencing, detection of transmembrane channel protein activity, etc. Description of the drawings

[0032] Figure 1 is a top view of the film-forming chip in Example 1 of the present invention;

[0033] Figure 2 is a schematic diagram of the film-forming flow channel in Example 1 of the present invention;

[0034] Figure 3 is a schematic diagram of the film-forming process in Example 1 of the present invention;

[0035] Figure 4 is a curve of the film-forming detection current changing with time in Example 1 of the present invention;

[0036] Figure 5 is a curve of the current versus time obtained by detecting the ionic current passing through MspA in Example 1 of the present invention;

[0037] Figure 6 is a top view of the micro-well and the hybrid membrane morphology in Example 1 of the present invention;

[0038] Figure 7 shows the process of the film-forming current change of the composite membrane in Example 2 of the present invention;

[0039] Figure 8 shows the current situation of the composite membrane in Example 2 of the present invention after MspA is inserted. Specific embodiments

[0040] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative and explanatory purposes of the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0041] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0042] The present invention provides a hybrid film and a method for forming the film. Refer to Figures 1 to 2 the film-forming device shown, which includes a chip micro-well and a film-forming channel. The micro-well is a structure for forming a film of amphiphilic molecules. The sequencing chip used includes a silicon wafer layer and a lithography layer. The lithography layer contains a micro-well structure as shown in Figure 3 The bottom of the well contains an electrode, which can be electrically connected to the liquid inside the well and can be connected to a micro-current detection amplifier outside the chip through the silicon wafer layer. The electrode is used to form a potential difference on both sides of the hybrid film. As shown in Figure 3 A in, a polar solvent 2 is introduced into the chip with the channels connected. Figure 3 As shown in B, after the polar solvent 2 enters the micro-well, a non-polar solvent 3 dissolved with phospholipids and block copolymers is introduced into the channel, and the polar solvent 2 is discharged from the channel. Figure 3 As shown in D, then a polar solvent 4 of the same kind or different from the polar solvent 2 is introduced into the channel to discharge the remaining non-polar solvent 3 in the channel. Thus, only a relatively thin liquid layer remains between the non-polar solvent 3 between the polar solvent 4 and the polar solvent 2, which is convenient for Figure 3 the formation of the composite film 5 in E, which is the hybrid film.

[0043] Example 1

[0044] In this example, the polar solvent 2 is a salt solution containing KCl, K4[Fe(CN)6], K3[Fe(CN)6] and HEPEs;

[0045] The non-polar solvent 3 is a mixed solution of AR20 and hexadecane, which is dissolved with phospholipids and block copolymers. The mass concentration of the phospholipids is 2 mg / ml, and the mass concentration of the block copolymer is 20 mg / ml. The block copolymer used is a triblock copolymer PMOXA-PDMS-PMOXA, with a degree of polymerization of 6-35-6 and a weight-average molecular weight of 500-2500-500. The degree of polymerization and the weight-average molecular weight correspond to the segments of the block copolymer;

[0046] The polar solvent 4 is the same solution as the polar solvent 2.

[0047] As Figure 3 shown in A, a polar solvent 2 is introduced into the chip with the channels connected. Figure 3 As shown in B, after the polar solvent 2 enters the micro-well, a non-polar solvent 3 dissolved with phospholipids and block copolymers is introduced into the channel, and the polar solvent 2 is discharged from the channel. Figure 3As shown in D, a polar solvent 4, which is the same as or different from the polar solvent 2, is then introduced into the flow channel to discharge the remaining non-polar solvent 3 in the flow channel, so that only a relatively thin liquid layer remains between the non-polar solvent 3 between the polar solvent 4 and the polar solvent 2. Then, wait for the spontaneous arrangement of the mixed film molecules.

[0048] After film formation, a micro-current signal detection and amplification device is used to detect the magnitude of the current. Since the current is proportional to the capacitance, it can be used to detect the size of the film.

[0049] Figure 4 It shows the process of the film formation current change of the composite film. The composite film completed spontaneous assembly and arranged into a monolayer film in a short time, with a speed far faster than that of the block copolymer film.

[0050] The current situation of the composite film after embedding MspA is as Figure 5 shown. After embedding MspA, the film state is stable, the magnitude of the current tends to a fixed value, and the required pore-forming voltage is between 140 mV and 400 mV, and the required channel protein concentration is lower than that of the block copolymer film. Generally, the voltage required for pore formation of the block copolymer film is between 250 mV and 500 mV, and the required channel protein concentration is 10 - 100 times that of the composite film.

[0051] The physical image of the composite film observed under an optical microscope is as Figure 6 shown. The fluidity of the film is lower than that of phospholipids. Under the same conditions, the phospholipid film can only maintain a stable size within 48 hours. Therefore, the composite film can maintain a stable size for a longer time and can maintain a state where the size hardly changes for a week.

[0052] Example 2

[0053] In this example, the polar solvent 2 is a salt solution containing KCl, K4[Fe(CN)6], K3[Fe(CN)6] and HEPEs;

[0054] The non-polar solvent 3 is AR20 and hexadecane, in which phospholipids and block copolymers are dissolved. The mass concentration of phospholipids is 2 mg / ml, and the mass concentration of block copolymers is 20 mg / ml; the block copolymer used is a diblock copolymer PMOXA-PDMS, with a degree of polymerization of 6 - 17 and a weight-average molecular weight of 500 - 1300, and the degree of polymerization and weight-average molecular weight correspond to the segments of the block copolymer;

[0055] The polar solvent 4 is the same solution as the polar solvent 2.

[0056] As Figure 3 shown in A, the polar solvent 2 is introduced into the chip with the flow channel connected. Figure 3After the polar solvent 2 shown in B enters the micro-well, the non-polar solvent 3 dissolved with phospholipids and block copolymers is introduced into the flow channel, and the polar solvent 2 is discharged from the flow channel. Figure 3 As shown in D, then a polar solvent 4 that is the same as or different from the polar solvent 2 is introduced into the flow channel, and the remaining non-polar solvent 3 in the flow channel is discharged, so that only a relatively thin liquid layer remains between the polar solvent 4 and the non-polar solvent 3. Then wait for the spontaneous arrangement of the mixed film molecules.

[0057] After film formation, use a micro-current signal detection and amplification device to detect the magnitude of the current. Since the current is proportional to the capacitance, an alternating current waveform, that is, a triangular wave, can be used to detect the size of the film.

[0058] Figure 7 The process of the film formation current change of the composite film is shown. The composite film completed spontaneous assembly in a short time, and the speed is much faster than that of the block copolymer film.

[0059] The current situation of the composite film after embedding MspA is as Figure 8 shown. After embedding MspA, the film state is stable, the magnitude of the current has slight fluctuations, but generally tends to a fixed value, and the required pore-forming voltage is between 120 mV and 260 mV, and the required channel protein concentration is lower than that of the block copolymer film. Generally, the voltage required for pore formation of the block copolymer film is between 250 mV and 500 mV, and the required channel protein concentration is 10 - 100 times that of the composite film.

[0060] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A membrane, characterized in that, The membrane includes channel proteins and a hybrid membrane embedded in the channel proteins; The channel protein is MspA; The hybrid membrane is formed by the spontaneous arrangement of phospholipids and block copolymers in a nonpolar solvent, and the hybrid membrane is a monolayer hybrid biomimetic membrane; The mass ratio of the phospholipids to the block copolymer is 1:(5 - 10); The hybrid membrane is formed by the spontaneous arrangement of the mixture of phospholipids and block copolymers; The block copolymer is selected from a triblock copolymer poly(2 - methyloxazoline)-polydimethylsiloxane-poly(2 - methyloxazoline) with a degree of polymerization of 6 - 35 - 6 and a weight - average molecular weight of 500 - 2500 - 500, and the degree of polymerization and the weight - average molecular weight correspond to the segments of the block copolymer; Alternatively, the block copolymer is selected from a diblock copolymer poly(2 - methyloxazoline)-polydimethylsiloxane with a degree of polymerization of 6 - 17 and a weight - average molecular weight of 500 - 1300, and the degree of polymerization and the weight - average molecular weight correspond to the segments of the block copolymer; The thickness of the hybrid membrane is 4 nm to 10 nm; Among them, the preparation of the membrane is to first prepare the hybrid membrane, and then embed the hybrid membrane into MspA to obtain the membrane; for the above - mentioned triblock copolymer, the pore - embedding voltage required for the composite membrane to embed MspA is between 140 mV and 400 mV; for the above - mentioned diblock copolymer, the pore - embedding voltage required for the composite membrane to embed MspA is between 120 mV and 260 mV.

2. The membrane according to claim 1, wherein The phospholipids and block copolymer molecules are uniformly dispersed and arranged or form local liquid - liquid phase separation; And / or, in the hybrid membrane, the mass ratio of the phospholipids to the block copolymer is 1:5, 1:6, 1:7, 1:8, 1:9 or 1:

10.

3. The membrane according to claim 1 or 2, characterized in that, In the preparation of the hybrid membrane, a film - forming device including a film - forming chip and a flow channel is used, and the film - forming chip includes a micro - well structure.

4. The membrane according to claim 3, wherein The hybrid membrane is prepared by a method including the following steps: First, fill a first polar solvent into the micro - wells, then introduce a nonpolar solvent dissolved with phospholipids and block copolymers into the flow channel, use the nonpolar solvent to drain the excess first polar solvent in the flow channel, then introduce a second polar solvent into the flow channel, use the second polar solvent to drain the excess nonpolar solvent in the flow channel, at this time, a layer of nonpolar solvent layer will be formed near the micro - well opening, and wait for the phospholipids and block copolymers to spontaneously arrange to form the hybrid membrane.

5. The membrane according to claim 4, wherein The first polar solvent and the second polar solvent are the same or different, and are independently selected from a salt solution containing one or several of KCl, K4[Fe(CN)6], K3[Fe(CN)6], HEPEs and H3PO4; And / or, the nonpolar solvent is selected from one or more of silicone oil, mineral oil, long - chain alkanes, and squalene.

6. The membrane according to claim 4, wherein The mass concentration of the phospholipids is 0.5 - 5 mg / ml; And / or, the mass concentration of the block copolymer is 2 - 25 mg / ml.

7. The membrane according to claim 4, characterized in that, The first polar solvent, the nonpolar solvent and the second polar solvent enter the flow channel in the same direction.

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