Ionic cross-linked biomimetic membrane as well as preparation method and application thereof
Patent Information
- Application Number
- CN202280099838.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-16
- Publication Date
- 2025-04-11
AI Technical Summary
The molecular film formed by the self-assembly of existing polymers in biosensors has low mechanical strength, large leakage, and insufficient impedance, which limits its application.
The amphiphilic block copolymer is cross-linked through a cross-linking agent with opposite charge to form an ionically cross-linked biomimetic membrane, which reduces the gaps between molecules and improves the stability and resistance of the membrane.
It achieves higher mechanical strength and lower leakage current, extends the service life of the bionic membrane, and is suitable for biosensors.
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Figure CN119816360A_ABST
Abstract
Description
Ion-crosslinked biomimetic membrane, preparation method and application thereof Technical Field
[0001] The invention belongs to the field of membrane technology, and in particular relates to an ion-crosslinked biomimetic membrane, a preparation method and application thereof. Background Art
[0002] The phospholipid bilayer is the primary structure of most biological cell membranes. Furthermore, the cell membranes of biological cells contain transmembrane proteins with diverse functions, which play a vital role in the exchange of substances and the transmission of information. Inspired by the membrane-protein system of organisms, biosensors based on this system have been designed. These sensors can be used to analyze various biological and chemical components, such as ions, small molecules, nucleic acids, and amino acids.
[0003] Phospholipid bilayers can be prepared using chemically extracted or synthesized phospholipids, but the low chemical stability and mechanical strength of phospholipid bilayers limit their application in biosensing. The molecular membranes formed by the self-assembly of synthetic amphiphilic polymers have higher chemical stability and higher mechanical strength, are more resistant to external interference, and are excellent substitutes for phospholipid membranes. The Chinese patent with publication number CN104936682B discloses a droplet interface, which discloses a technology for forming an amphiphilic molecular membrane by self-assembly of amphiphilic block polymers at the interface of two polar solution droplets. Both diblock and triblock amphiphilic block copolymers can self-assemble to form an amphiphilic molecular layer, wherein the diblock polymer contains a hydrophilic segment and a hydrophobic segment, the middle segment of the triblock polymer is a hydrophobic segment, and the two ends are hydrophilic segments. In the process of forming the amphiphilic molecular membrane, the hydrophobic segments of the block copolymers are arranged close to each other under the hydrophobic force to form a non-polar inner layer, and the hydrophilic segments are arranged toward the polar solvent side to form a hydrophilic outer layer. Although polymer membranes are more stable than phospholipid bilayers, their stability still has significant room for improvement. Furthermore, many biosensors designed based on membrane-protein systems require the membrane to have a very high impedance to obtain a lower-noise electrical signal. However, when many polymers self-assemble into membranes, the gaps between the molecules are large, resulting in loose packing of the membrane molecules, low mechanical strength, and a high degree of leakage when a voltage is applied across the membrane, indicating that the membrane impedance is not high enough.
[0004] Summary of the Invention
[0005] The purpose of the present invention is to provide an ion-crosslinked biomimetic membrane with strong mechanical strength and low leakage current, and a preparation method and application thereof.
[0006] The present invention provides an ionically cross-linked biomimetic membrane, which is formed by self-assembly of membrane-forming molecules; the membrane-forming molecules include amphiphilic block copolymers; the hydrophilic chains of the amphiphilic block copolymers are cross-linked by a cross-linking agent; and the hydrophilic segments of the amphiphilic block copolymers have opposite charges to those of the cross-linking agent in aqueous solution.
[0007] Preferably, the hydrophilic segment of the amphiphilic block copolymer is formed by a hydrophilic monomer; the hydrophilic monomer includes a substituted and / or unsubstituted oxazoline monomer; and the substituent in the substituted oxazoline monomer is selected from a C1 to C10 alkyl group.
[0008] Preferably, the hydrophilic monomer includes one or more of oxazoline, 2-methyloxazoline and 2-ethyloxazoline.
[0009] Preferably, the hydrophobic segment of the amphiphilic block copolymer is selected from one or more of polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene, polybutadiene, polyalkyl acrylate, polyacrylonitrile, polytetrahydrofuran, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, polypropylene oxide, polycaprolactone and the above copolymers;
[0010] Preferably, the degree of polymerization of the hydrophilic chain of the amphiphilic block polymer is 1-20, preferably 3-15, most preferably 3-10; the degree of polymerization of the hydrophobic chain of the amphiphilic block polymer is 10-60, preferably 20-40, most preferably 25-35.
[0011] Preferably, the polymerization degree ratio of the hydrophilic segment to the hydrophobic segment of the amphiphilic block copolymer is (2-20): (20-60), preferably (2-18): (25-50), more preferably (2-16): (25-45), and even more preferably (2-16): (28-42).
[0012] Preferably, the cross-linking agent is an anionic compound; the anionic compound is selected from one or more of polyphosphates, polyphosphonic acid polymers, citrates, ethylenediaminetetraacetates, hexametaphosphates, polyacrylates, polysilicates and polyolefin sulfonates.
[0013] The present invention also provides a method for preparing an ion-crosslinked biomimetic membrane, comprising:
[0014] dissolving the amphiphilic block copolymer in a solvent to obtain a membrane solution;
[0015] The membrane solution is self-assembled under the condition that a first buffer solution and a second buffer solution are respectively set on both sides to obtain an ion-crosslinked biomimetic membrane; the first buffer solution and / or the second buffer solution contain a crosslinking agent; the hydrophilic segment of the amphiphilic block copolymer has an opposite charge to that of the crosslinking agent in aqueous solution.
[0016] Preferably, the concentration of the amphiphilic block copolymer in the membrane solution is 10 to 50 mg / mL; and the concentration of the cross-linking agent in the first buffer solution and / or the second buffer solution is 0.1 to 100 mM.
[0017] Preferably, the solvent is selected from alkanes and / or silicone oil; the first buffer solution and the second buffer solution are each independently selected from one of HEPES buffer solution, Tris buffer solution and PBS buffer solution.
[0018] The present invention also provides a biosensor comprising the ion-crosslinked biomimetic membrane.
[0019] The present invention provides an ionically crosslinked biomimetic membrane formed by the self-assembly of an amphiphilic block copolymer; the hydrophilic chains of the amphiphilic block copolymer are cross-linked via a crosslinking agent; the hydrophilic segments of the amphiphilic block copolymer have an opposite charge to that of the crosslinking agent in aqueous solution. Compared to the prior art, the present invention uses a crosslinking agent with an opposite charge to that of the amphiphilic block copolymer to form ionic crosslinks between adjacent hydrophilic segments through electrostatic adsorption to create a bridging effect. This reduces the intermolecular gaps within the amphiphilic molecular membrane formed by the self-assembly of the amphiphilic block copolymer, improves the membrane's stability, and simultaneously increases the membrane's electrical resistance and mechanical strength, resulting in lower leakage current and longer service life when used in biosensors. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG1 is a schematic diagram of the transmembrane current of the biomimetic membrane obtained in 1.4 of Example 1 of the present invention;
[0021] FIG2 is a schematic diagram of the transmembrane current of the biomimetic membrane obtained in 1.6 of Example 1 of the present invention;
[0022] FIG3 is a schematic diagram of the transmembrane current of the biomimetic membrane obtained in 1.7 of Example 1 of the present invention;
[0023] FIG4 is a schematic diagram of the transmembrane current of the biomimetic membrane obtained in 2.4 of Example 2 of the present invention;
[0024] FIG5 is a schematic diagram of the transmembrane current of the biomimetic membrane obtained in 2.6 of Example 2 of the present invention;
[0025] FIG6 is a schematic diagram of the transmembrane current of the biomimetic membrane obtained in 2.7 of Example 2 of the present invention;
[0026] FIG7 is a graph showing a trend of the sequencing channel ratio obtained in 3.6 of Example 3 of the present invention as a function of sequencing time. DETAILED DESCRIPTION
[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0028] The present invention provides an ionically cross-linked biomimetic membrane, which is formed by self-assembly of membrane-forming molecules; the membrane-forming molecules include amphiphilic block copolymers; the hydrophilic chains of the amphiphilic block copolymers are cross-linked by a cross-linking agent; and the hydrophilic segments of the amphiphilic block copolymers have opposite charges to those of the cross-linking agent in aqueous solution.
[0029] The biomimetic membrane provided by the present invention is formed by self-assembly of an amphiphilic block copolymer; wherein the amphiphilic block copolymer is an amphiphilic block copolymer well known to those skilled in the art and is not particularly limited; in the present invention, the amphiphilic block copolymer is selected from one or more of an amphiphilic diblock copolymer, an amphiphilic triblock copolymer and an amphiphilic pentablock copolymer; the configuration of the amphiphilic diblock copolymer is AB type; the configuration of the amphiphilic triblock copolymer is AB-A1 type; the configuration of the amphiphilic pentablock copolymer is B1-A1-B-A2-B2 type; wherein A, A1 and A2 is a hydrophilic segment, which may be the same or different; B, B1 and B2 are hydrophobic segments, which may be the same or different; in the present invention, the hydrophilic segment of the amphiphilic block copolymer is formed by a hydrophilic monomer; the hydrophilic monomer preferably includes a substituted and / or unsubstituted oxazoline monomer; the substituent in the substituted oxazoline monomer is preferably a C1-C10 alkyl group, more preferably a C1-C8 alkyl group, more preferably a C1-C6 alkyl group, more preferably a C1-C4 alkyl group, and most preferably a methyl or ethyl group; most preferably, the hydrophilic monomer includes an oxazoline monomer. One or more of oxazoline, 2-methyloxazoline and 2-ethyloxazoline; the hydrophobic segment of the amphiphilic block copolymer is a hydrophobic segment well known to those skilled in the art, and there is no special restriction. In the present invention, the hydrophobic segment is preferably polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene, polybutadiene, polyalkyl acrylate, polyacrylonitrile, polytetrahydrofuran, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, polypropylene oxide, polycaprolactone and the above copolymer. The invention relates to one or more polymers; the degree of polymerization of the hydrophilic chain of the amphiphilic block polymer is 1-20, preferably 3-15, and most preferably 3-10; the degree of polymerization of the hydrophobic chain of the amphiphilic block polymer is 10-60, preferably 20-40, and most preferably 25-35; the polymerization degree ratio of the hydrophilic segment to the hydrophobic segment of the amphiphilic block copolymer is preferably (2-20): (20-60), more preferably (2-18): (25-50), further preferably (2-16): (25-45), and most preferably (2-16): (28-42).
[0030] The hydrophilic chains of the amphiphilic block copolymer are cross-linked by a cross-linking agent; the hydrophilic segments of the amphiphilic block copolymer have an opposite charge to that of the cross-linking agent in aqueous solution; in the present invention, the hydrophilic segments of the amphiphilic block copolymer preferably have a positive charge in aqueous solution, and therefore the cross-linking agent is preferably an anionic compound, i.e., a compound having a negative charge in aqueous solution; further preferably, the anionic compound is selected from one or more of polyphosphates, polyphosphonic acid polymers, citrates, ethylenediaminetetraacetates, hexametaphosphates, polyacrylates, polysilicates and polyolefin sulfonates; in the present invention, the cross-linking agent is preferably in contact with the amphiphilic block copolymer in a buffer solution to cross-link its hydrophilic chains; the concentration of the cross-linking agent in the buffer solution is preferably 0.1-100 mM, more preferably 1-50 mM, and most preferably 5-30 mM.
[0031] In the present invention, pore proteins can preferably be inserted into the amphiphilic molecular membrane, so that the biomimetic membrane carries transmembrane proteins for biosensing. In the present invention, the channel protein is preferably inserted into the biomimetic membrane by voltage drive, autonomous fusion or protein vesicle fusion, and then used for biosensing.
[0032] The present invention adopts a cross-linking agent with a charge opposite to that of the amphiphilic block copolymer to generate a bridging effect through electrostatic adsorption to cross-link adjacent hydrophilic segments in the form of ionic bonds, thereby reducing the intermolecular gaps in the amphiphilic molecular membrane formed by self-assembly of the amphiphilic block copolymer, improving the stability of the membrane, and increasing the resistance and mechanical strength of the membrane, so that the membrane has lower leakage current and longer service life when used for biosensing.
[0033] The present invention also provides a method for preparing the above-mentioned ion-crosslinked biomimetic membrane, comprising: dissolving an amphiphilic block copolymer in a solvent to obtain a membrane solution; self-assembling the membrane solution under the condition that a first buffer solution and a second buffer solution are respectively provided on both sides to obtain an ion-crosslinked biomimetic membrane; the first buffer solution and / or the second buffer solution include a crosslinker; and the hydrophilic segment of the amphiphilic block copolymer has an opposite charge to that of the crosslinker in aqueous solution.
[0034] The present invention has no particular limitation on the sources of all raw materials, and any commercially available raw materials are sufficient. The types and proportions of the amphiphilic block copolymer and the cross-linking agent are the same as those described above and will not be repeated here.
[0035] The amphiphilic block copolymer is dissolved in a solvent to obtain a membrane solution. The present invention has no particular limitation on the type of the solvent, and any solvent well known to those skilled in the art can be used. In the present invention, alkanes and / or silicone oils are preferred. The concentration of the amphiphilic block copolymer in the membrane solution is preferably 0.1 to 50 mg / mL, more preferably 1 to 40 mg / mL, and even more preferably 1 to 30 mg / mL.
[0036] The membrane solution is self-assembled under the condition that the first buffer solution and the second buffer solution are respectively set on both sides to obtain an ion-crosslinked biomimetic membrane; in the present invention, this self-assembly process is preferably carried out in an open hole array chip or a patch clamp; taking the open hole array chip as an example, the polymer solution is brushed into the open hole array chip, and the first buffer solution and the second buffer solution are used to self-assemble, or the first buffer solution, the membrane solution and the second buffer solution are sequentially introduced into the channel of the open hole array chip to self-assemble to form an ion-crosslinked biomimetic membrane; the first buffer solution and / or the second buffer solution include a crosslinking agent; the crosslinking agent is preferably an anionic compound, that is, a compound with a negative charge in an aqueous solution; further preferably, the anionic compound is selected from polyphosphate, polyphosphonic acid polymer, citrate, ethylenediaminetetraethyl or the like. one or more of phosphate, hexametaphosphate, polyacrylate, polysilicate and polyolefin sulfonate; the concentration of the crosslinking agent in the first buffer and / or the second buffer is preferably 0.1-100 mmol / L, more preferably 0.1-50 mmol / L, further preferably 1-30 mmol / L, and most preferably 5-30 mmol / L; in the present invention, the first buffer and the second buffer may both contain the crosslinking agent or only one of them may contain the crosslinking agent, and the hydrophilic segments of the amphiphilic molecules on the side to which the crosslinking agent is added will be crosslinked; the crosslinking of the hydrophilic segments of the membrane-forming molecules of the biomimetic membrane provided by the present invention is reversible, that is, the crosslinking degree of the biomimetic membrane can be adjusted by changing the content of the crosslinking agent in the first buffer and / or the second buffer on both sides of the membrane.The cross-linking reaction balance between membrane molecules is determined by the content of the cross-linking agent in the first buffer and / or the second buffer on both sides of the membrane. Increasing the content of the cross-linking agent in the first buffer and / or the second buffer increases the degree of cross-linking of the membrane, the more stable the membrane is, and the smaller the leakage current is. Conversely, reducing the content of the cross-linking agent in the first buffer and / or the second buffer makes the membrane more unstable and the leakage current greater. The first buffer and the second buffer can be buffers well known to those skilled in the art without any special restrictions. In the present invention, the first buffer and the second buffer are each independently preferably one of HEPES buffer, Tris buffer, and PBS buffer; the concentration of the HEPES buffer is preferably specifically 10 mmol / L; the concentration of the PBS buffer is specifically 10 mmol / L; the first buffer and the second buffer preferably also include a stabilizer; the stabilizer is preferably specifically EDTA; the concentration of the buffer in the first buffer and the second buffer is preferably 10 mmol / L. The concentration of each of the first and second buffer solutions is independently 1 to 10 mmol / L, more preferably independently 2 to 8 mmol / L, further preferably independently 4 to 6 mmol / L, and most preferably 5 mmol / L; the first buffer solution and the second buffer solution may further include other substances depending on the purpose of the biomimetic membrane. For example, if the biomimetic membrane is used for biosensing, the first buffer solution and the second buffer solution preferably further include potassium ferrocyanide and potassium ferrocyanide; the concentration of potassium ferrocyanide in the first and second buffer solutions is preferably 10 to 1000 mmol / L, more preferably 30 to 800 mmol / L, further preferably 40 to 600 mmol / L, and most preferably 150 mmol / L; the concentration of potassium ferrocyanide in the first and second buffer solutions is preferably 10 to 1000 mmol / L, more preferably 30 to 800 mmol / L, further preferably 40 to 600 mmol / L, and most preferably 150 mmol / L.
[0037] The present invention also provides an application of the above-mentioned ion-crosslinked biomimetic membrane in carrying transmembrane proteins; it can be used for biosensing, such as nanopore gene sequencing, protein sequencing and detection of other components.
[0038] The present invention also provides a biosensor comprising the above-mentioned ion-crosslinked biomimetic membrane; the biosensor is preferably a nanopore gene sequencer or a protein sequencer.
[0039] In order to further illustrate the present invention, an ion-crosslinked biomimetic membrane, a preparation method thereof and applications provided by the present invention are described in detail below with reference to examples.
[0040] The reagents used in the following examples are all commercially available. The data in these examples are primarily based on the biomimetic membrane described herein, applied to a 256-channel nanopore sequencing system. This system consists of a microstructured chip with an open-pore array, a control circuit system, and control software. Specifically, the biomimetic membrane is formed within a microstructured chip with an open-pore array containing 256 channels.
[0041] Example 1
[0042] 1.1 Dissolve the triblock copolymer (6PMOXZ-33PDMS-6PMOXZ) in silicone oil AR-20 to obtain a 20 mg / ml membrane solution.
[0043] 1.2 Use ultrapure water as the solvent to prepare polar solution 1 according to the following formula: 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 5 mM EDTA, 10 mM HEPES, and 5 mM sodium citrate.
[0044] 1.3 Polar solution 1, membrane solution 1, and polar solution 1 are introduced into the chip channel in sequence to form an ion-crosslinked biomimetic membrane on the chip array.
[0045] 1.4 A voltage of 0.18 V was applied to both sides of the membrane by the sequencer, and the transmembrane current was obtained as shown in Figure 1. The transmembrane current was concentrated below 20 pA.
[0046] 1.5 Use ultrapure water as the solvent to prepare polar solution 2 according to the following formula: 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 5 mM EDTA, and 10 mM HEPES.
[0047] 1.6 200 μl of polar solution 2 was introduced into the fluid channel of the sequencing chip. A voltage of 0.18 V was applied to both sides of the membrane by the sequencer. The transmembrane current was obtained as shown in FIG2 . The transmembrane current was concentrated below 35 pA.
[0048] 1.7 400 μl of polar solution 2 was introduced into the fluid reservoir of the sequencing chip again. A voltage of 0.18 V was applied to both sides of the membrane by the sequencer. The transmembrane current was obtained as shown in FIG3 . The transmembrane current was concentrated below 68 pA.
[0049] By comparing the transmembrane current size of steps 1.4, 1.6, and 1.7, it can be found that due to containing sodium citrate in polar solution one, when both sides of biomimetic membrane are polar solution one, transmembrane leakage current is minimum. And after the biomimetic membrane upper layer is progressively replaced by polar solution two that does not contain sodium citrate, the sodium citrate concentration in the polar solution of the biomimetic membrane upper layer decreases gradually, and transmembrane leakage current increases gradually thereupon. Therefore, by the result comparison of the present embodiment, it can be proved that sodium citrate has a cross-linking effect on the biomimetic membrane formed by membrane solution one, and this cross-linking effect can reduce membrane leakage, and this cross-linking is reversible and regulatable, and the cross-linking degree of membrane can be adjusted by adjusting the cross-linking agent concentration in the polar solution on both sides of biomimetic membrane.
[0050] Example 2
[0051] 2.1 Dissolve the triblock copolymer (8PEtOXZ-42PDMS-8PMOXZ) in silicone oil AR-20 to obtain a 20 mg / ml membrane solution.
[0052] 2.2 Use ultrapure water as the solvent to prepare polar solution 2 according to the following formula: 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 5 mM EDTA, and 10 mM HEPES.
[0053] 2.3 Polar solution 1, membrane solution 1, and polar solution 1 are introduced into the chip channel in sequence to form an uncross-linked biomimetic membrane on the chip array.
[0054] 2.4 A voltage of 0.18 V was applied to both sides of the membrane by the sequencer, and the transmembrane current was obtained as shown in Figure 4. The transmembrane current was concentrated below 55 pA.
[0055] 2.5 Use ultrapure water as the solvent to prepare polar solution three according to the following formula: 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 5 mM EDTA, 10 mM HEPES, and 10 mM sodium tripolyphosphate.
[0056] 2.6 200 μl of polar solution 3 was introduced into the fluid channel of the sequencing chip. A voltage of 0.18 V was applied to both sides of the membrane by the sequencer. The transmembrane current was obtained as shown in FIG5 . The transmembrane current was concentrated in the range of 6 to 27 pA.
[0057] 2.7 400 μl of polar solution 3 was introduced into the fluid reservoir of the sequencing chip again. A voltage of 0.18 V was applied to both sides of the membrane by the sequencer. The transmembrane current was obtained as shown in FIG6 . The transmembrane current was concentrated in the range of 0 to 25 pA.
[0058] By comparing the transmembrane current size of steps 2.4, 2.6, and 2.7, it can be found that due to the absence of sodium citrate in polar solution two, when both sides of the biomimetic membrane are polar solution two, the transmembrane leakage current is the largest. And when the biomimetic membrane upper layer is progressively replaced by polar solution three containing sodium tripolyphosphate, the sodium citrate concentration in the polar solution on one side of the membrane gradually increases, and the membrane molecules are gradually cross-linked, causing the transmembrane leakage current to gradually decrease. Therefore, it can be proved by the result of the present embodiment that sodium tripolyphosphate has a cross-linking effect on the biomimetic membrane formed by 8PEtOXZ-42PDMS-8PMOXZ, and this cross-linking effect can reduce membrane leakage, and this cross-linking is regulatable.
[0059] Example 3
[0060] 3.1 Dissolve the triblock copolymer (6PMOXA-33PDMS-6PMOXA) in silicone oil AR-20 to obtain a 20 mg / ml membrane solution.
[0061] 3.2 Use ultrapure water as the solvent to prepare polar solution three according to the following formula: 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 5 mM EDTA, 10 mM HEPES, and 10 mM sodium tripolyphosphate.
[0062] 3.3 Polar solution 3, membrane solution 1, and polar solution 3 are introduced into the chip channel in sequence to form an ion-crosslinked biomimetic membrane on the chip array.
[0063] 3.4 Dilute the hemolysin protein with polar solution III to obtain 200 μl of 0.01 μg / ml hemolysin protein solution and inject it into the fluid tank of the sequencing chip.
[0064] 3.5 Apply a voltage of 0.3 V to both sides of the membrane using the sequencer. After 30 min, stop applying the voltage and count the number of embedded holes and membrane ruptures. The data are recorded in Table 1.
[0065] 3.6 Add 10 mM sodium tripolyphosphate to the sequencing buffer (480 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0), then add the library to start sequencing. The change in the number of sequencing channels over sequencing time is counted. The data is recorded as the ratio of the number of sequencing channels to the total number of channels at the start of sequencing. The ratio of sequencing channels at the start is recorded as 100%. The data are plotted in Figure 7.
[0066] Comparative Example 1
[0067] 1.1 Dissolve the triblock copolymer (6PMOXZ-33PDMS-6PMOXZ) in silicone oil AR-20 to obtain a 20 mg / ml membrane solution.
[0068] 1.2 Use ultrapure water as the solvent to prepare polar solution 2 according to the following formula: 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 5 mM EDTA, and 10 mM HEPES.
[0069] 1.3 Polar solution 2, membrane solution 1, and polar solution 2 are introduced into the chip channel in sequence to form an ion-crosslinked biomimetic membrane on the chip array.
[0070] 1.4 Dilute the hemolysin protein with polar solution 2 to obtain 200 μl of 0.01 μg / ml hemolysin protein solution 1 and inject it into the fluid cell of the sequencing chip.
[0071] 1.5 Apply a voltage of 0.3 V to both sides of the membrane through the sequencer. After applying the voltage for 30 min, stop applying the voltage and count the number of embedded holes and membrane ruptures. The data are recorded in Table 1.
[0072] 1.6 Add 10 mM sodium tripolyphosphate to the sequencing buffer (480 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0), then add the library to start sequencing. The change in the number of sequencing channels over sequencing time is counted. The data is recorded as the ratio of the number of sequencing channels to the total number of channels at the start of sequencing. The ratio of sequencing channels at the start is recorded as 100%. The data are plotted in Figure 7.
[0073] By comparing the data in Table 1, it is obvious that Example 3 has more embedded holes and less membrane ruptures than Comparative Example 1. This shows that the ion-crosslinked biomimetic membrane has stronger mechanical strength and better stability.
[0074] The data in Figure 7 show that the number of sequencing channels in Example 3 decreases more slowly than in Comparative Example 1, meaning that the sequencing channels in Example 3 can operate for a longer period of time. This suggests that the crosslinker, through electrostatic adsorption, creates a bridging effect that ionically crosslinks adjacent hydrophilic segments, improving the stability of the amphiphilic molecular membrane formed by self-assembly of the amphiphilic block copolymer and extending its service life for biosensing applications.
[0075] Table 1 Number of embedded holes and broken membranes in Example 3 and Comparative Example 1
[0076] Experimental name Number of embedded holes Number of broken membranes Example 3 160 24 Comparative example 165 113
Claims
1. An ion-crosslinked biomimetic membrane, characterized in that: The biomimetic membrane is formed by self-assembly of membrane-forming molecules; the membrane-forming molecules include amphiphilic block copolymers; the hydrophilic chains of the amphiphilic block copolymers are cross-linked by a cross-linking agent; and the hydrophilic segments of the amphiphilic block copolymers have opposite charges to those of the cross-linking agent in aqueous solution.
2. The biomimetic membrane according to claim 1, characterized in that The hydrophilic segment of the amphiphilic block copolymer is formed by a hydrophilic monomer; the hydrophilic monomer includes a substituted and / or unsubstituted oxazoline monomer; and the substituent in the substituted oxazoline monomer is selected from a C1 to C10 alkyl group.
3. The biomimetic membrane according to claim 2, characterized in that The hydrophilic monomer includes one or more of oxazoline, 2-methyloxazoline and 2-ethyloxazoline.
4. The biomimetic membrane according to claim 1, characterized in that The hydrophobic segment of the amphiphilic block copolymer is selected from one or more of polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene, polybutadiene, polyalkyl acrylate, polyacrylonitrile, polytetrahydrofuran, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, polypropylene oxide, polycaprolactone and the above copolymers.
5. The biomimetic membrane according to claim 1, characterized in that The degree of polymerization of the hydrophilic chain of the amphiphilic block polymer is 1-20, preferably 3-15, and most preferably 3-10; the degree of polymerization of the hydrophobic chain of the amphiphilic block polymer is 10-60, preferably 20-40, and most preferably 25-35.
6. The biomimetic membrane according to claim 1, characterized in that The polymerization degree ratio of the hydrophilic segment to the hydrophobic segment of the amphiphilic block copolymer is (2-20): (20-60), preferably (2-18): (25-50), more preferably (2-16): (25-45), and even more preferably (2-16): (28-42).
7. The biomimetic membrane according to claim 1, characterized in that The cross-linking agent is an anionic compound; the anionic compound is selected from one or more of polyphosphates, polyphosphonic acid polymers, citrates, ethylenediaminetetraacetates, hexametaphosphates, polyacrylates, polysilicates and polyolefin sulfonates.
8. A method for preparing an ion-crosslinked biomimetic membrane, characterized in that: include: dissolving the amphiphilic block copolymer in a solvent to obtain a membrane solution; The membrane solution is self-assembled under the condition that a first buffer solution and a second buffer solution are respectively set on both sides to obtain an ion-crosslinked biomimetic membrane; the first buffer solution and / or the second buffer solution contain a crosslinking agent; the hydrophilic segment of the amphiphilic block copolymer has an opposite charge to that of the crosslinking agent in aqueous solution.
9. The preparation method according to claim 8, characterized in that The concentration of the amphiphilic block copolymer in the membrane solution is 0.1-50 mg / mL; the concentration of the cross-linking agent in the first buffer solution and / or the second buffer solution is 0.1-100 mM.
10. The preparation method according to claim 8, characterized in that The solvent is selected from alkanes and / or silicone oil; the first buffer and the second buffer are each independently selected from one of HEPES buffer, Tris buffer or PBS buffer.
11. A biosensor, characterized in that: The invention comprises the ionically cross-linked biomimetic membrane according to any one of claims 1 to 7 or the ionically cross-linked biomimetic membrane prepared by the preparation method according to any one of claims 8 to 10.