Amphiphilic block copolymer self-assembly biomimetic membrane and molecular membrane anchoring method and application

Abstract

This invention relates to the field of biomaterials, specifically to a self-assembled biomimetic membrane of an amphiphilic block copolymer and a method and application of molecular membrane anchoring. The biomimetic membrane is formed by the self-assembly of membrane-forming molecules, including an amphiphilic triblock copolymer with a configuration of hydrophilic segment A-hydrophobic segment B-hydrophilic segment A. The copolymer is modified with an organosilicon surfactant before self-assembly into the amphiphilic block copolymer biomimetic membrane. A molecular membrane anchoring method is then used to promote the embedding of biomimetic nanoporous proteins into the biomimetic membrane, thereby enabling its application in the fabrication of biosensors. The addition of an organosilicon surfactant acts as a "bridge," eliminating the polar repulsion between the substrate and the polymer, allowing the triblock polymer to stably adhere to the substrate surface. Anchoring molecules achieve anchoring through covalent bonds with the membrane surface and biomolecules, resulting in extremely high stability, strong resistance to elution and environmental interference, making it suitable for biosensors.

Description

Technical Field

[0001] This invention relates to the field of polymer materials, specifically to a self-assembled biomimetic membrane of amphiphilic block copolymer and a method and application for anchoring the molecular membrane. Background Technology

[0002] Existing membrane-forming technologies primarily utilize the self-assembly properties of phospholipid molecules or amphiphilic block copolymers at the water-oil interface to form amphiphilic molecular membranes. Because phospholipid bilayers (phospholipid membranes) have a natural affinity for biomolecules, exhibiting excellent affinity for various channel proteins (nanoporins) and high nanoporin embedding efficiency, phospholipid membranes are often used in nanopore sequencing to mimic cell membrane structures and support nanoporins for sequencing. However, due to the weak mechanical strength of phospholipid molecules, phospholipid membranes suffer from poor stability. While this meets the needs of most biological, physical, and physiological experiments, it is far from sufficient for the technological applications of channel proteins.

[0003] Existing polymers used for biomimetic membranes are synthetic amphiphilic block copolymers. Their intermolecular forces are stronger than those of phospholipid molecules, resulting in high molecular packing density and thus high mechanical strength and stability in the formed polymer membranes. However, typical amphiphilic block copolymers exhibit problems such as poor interfacial compatibility, low order, and structural inhomogeneity during the self-assembly process on biochip surfaces. Traditional biomimetic membranes are bonded to the chip substrate only by weak hydrogen bonds. Biochip substrates (such as glass, silicon wafers, and graphene oxide-modified substrates) are mostly strongly polar / hydrophilic surfaces. Triblock polymers (such as PS-b-PEO-b-PS and PMOXA-b-PDMS-b-PMOXA) typically contain hydrophobic blocks (such as polydimethylsiloxane PDMS) and hydrophilic blocks (such as poly(2-methyloxazoline)PMOXA). When these two blocks are in direct contact, the polar repulsion between the hydrophobic blocks and the polar substrate makes it difficult for the polymer to adhere or for the assembled structure to detach easily.

[0004] Existing block polymer biomimetic membranes lack structural compatibility with biological protein pores and specific interactions with biomolecules, resulting in high non-specific protein adsorption, low intercalation efficiency, and poor activity. On one hand, protein intercalation conditions are demanding; the hydrophobic core of the polymer membrane has poor compatibility with the hydrophobic band of the protein (e.g., the hydrophobic band thickness of PDMS is 10 nm, far exceeding the 4-5 nm thickness of proteins), requiring the application of high-intensity voltages (>200 mV) or the use of high-concentration protein solutions (>1 mg / mL) to achieve intercalation, which not only increases costs but also easily leads to protein conformational damage. On the other hand, maintaining biological activity is difficult: the rigidity of polymer chains and the lack of specific interactions with proteins result in a shortened half-life of intercalated aquaporins and ion channel proteins to less than 24 hours, with an activity loss rate >50% under high-salt environments. Summary of the Invention

[0005] The purpose of this invention is to provide a self-assembled biomimetic membrane using amphiphilic block copolymers and a method and application for anchoring the molecular membrane. By adding an organosilicon surfactant during the self-assembly of the biomimetic membrane into a nanopore sequencing chip using an amphiphilic block copolymer, the amphiphilic structure of this surfactant (polysiloxane hydrophobic chain + polyether / carboxyl hydrophilic group) acts as a "bridge," eliminating the polar repulsion between the substrate and the polymer, allowing the triblock polymer to stably adhere to the substrate surface. Simultaneously, anchoring molecules promote the embedding of biomimetic nanoporous proteins into the biomimetic membrane. These anchoring molecules achieve anchoring through covalent bonds with the membrane surface and biomolecules, exhibiting extremely high stability, strong resistance to elution and environmental interference, and are suitable for biosensors.

[0006] The first aspect of the present invention is to provide a self-assembled biomimetic membrane of an amphiphilic block copolymer, the biomimetic membrane being formed by the self-assembly of film-forming molecules; the film-forming molecules include an amphiphilic triblock copolymer, the configuration of which is hydrophilic segment A-hydrophobic segment B-hydrophilic segment A; and the amphiphilic triblock copolymer is modified by an organosilicon surfactant and then self-assembled into an amphiphilic block copolymer biomimetic membrane.

[0007] Using the above technical solution, an organosilicon surfactant is added during the self-assembly of the biomimetic membrane of the nanopore sequencing chip using the amphiphilic block copolymer. The amphiphilic structure of the organosilicon surfactant (hydrophobic polysiloxane chain + hydrophilic polyether / carboxyl group) can act as a "bridge". Its hydrophilic groups (such as PEO chain, PMOXA chain) are combined with the polar groups such as hydroxyl (-OH) and amino (-NH2) groups of the nanopore sequencing chip substrate through hydrogen bonding and electrostatic interaction, realizing the anchoring and adsorption of the surfactant on the substrate. The hydrophobic polysiloxane chain faces the solution phase and is combined with the hydrophobic blocks of the triblock polymer (such as PS, PDMS) through van der Waals forces and hydrophobic interactions, thereby eliminating the polar repulsion between the substrate and the polymer, allowing the triblock polymer to be stably attached to the substrate surface, laying the foundation for self-assembly. Compared with the existing technology where hydrophilic blocks of triblock copolymers are cross-linked by cross-linking agents to promote self-assembly into membranes via ionic bonding, this invention achieves strong binding with the substrate at one end using organosilicon surfactants, while enhancing the biocompatibility of the biomimetic membrane at the other end. Through interface compatibility optimization, energy barrier reduction, and precise template guidance, it solves the assembly problem of amphiphilic block copolymers on nanopore sequencing chip substrates. The hydrophilic and hydrophobic segments of the amphiphilic block copolymers are regulated by surfactants to self-assemble into biomimetic membranes, with different types corresponding to different interface interaction mechanisms and application scenarios.

[0008] Preferably, the concentration of the organosilicon surfactant in the amphiphilic triblock copolymer solution is 0.5-10%; the organosilicon surfactant includes one or more of the following types: polyether type, carboxyl / sulfonic acid type, amino / quaternary ammonium salt type, and multi-functional hydrophilic group type.

[0009] When the organosilicon surfactant is a polyether type, it includes one or a mixture of polyether-modified polydimethylsiloxane, polyether-modified silicone oil, polyether-modified heptamethyltrisiloxane, and polyethoxylated trisiloxane.

[0010] When the organosilicon surfactant is of the carboxyl / sulfonic acid type, it includes carboxypropyl polydimethylsiloxane and / or sulfonic acid-modified polysiloxane;

[0011] When the organosilicon surfactant is of the amino / quaternary ammonium salt type, it includes aminopropyl polydimethylsiloxane and / or quaternized polysiloxane;

[0012] When the organosilicon surfactant is of the multi-component hydrophilic group type, it includes polyethoxylated trisiloxane and / or 3-[methoxypoly(ethoxy)]propyl-methyl-bis(trimethylsiloxy)silane.

[0013] Organosilicon surfactants possess bifunctional hydrophilic blocks, serving as the "regulatory core" for the self-assembly of triblock polymers. One end achieves strong binding to the substrate, while the other end enhances biocompatibility. Through interface compatibility optimization, energy barrier reduction, and precise template guidance, they solve the assembly challenges of amphiphilic block copolymers on nanopore sequencing chip substrates. Furthermore, different types correspond to different interfacial interaction mechanisms and application scenarios. The organosilicon surfactants described in this invention are classified into the following categories based on the type of hydrophilic group (which determines their interfacial interaction characteristics and applicable scenarios):

[0014] 1) Polyether type

[0015] PDMS-b-PEO (polydimethylsiloxane-polyoxyethylene block copolymer) and PMPS-PEO (methylphenylsiloxane-polyoxyethylene) have PEO chains as their hydrophilic groups, exhibiting excellent biocompatibility and good water solubility. They are highly compatible with triblock polymers containing PEO blocks, exhibiting mild interfacial interactions. Triblock polymers containing PEO hydrophilic blocks (such as PS-b-PEO-b-PS and PDMS-b-PEO-b-PDMS) are used in nanopore sequencing chips for "molecular recognition arrays" (such as antibody / antigen immobilization) and for microfluidic channel inner wall modification.

[0016] 2) Carboxyl / sulfonic acid type

[0017] Carboxypropyl polydimethylsiloxane and sulfonic acid-modified polysiloxane have negatively charged hydrophilic groups -COOH / -SO3H. They are strongly adsorbed to polar substrates (such as amino-modified silicon wafers) through electrostatic interactions, resulting in strong interfacial bonding. In addition, triblock polymers containing cationic or hydrophobic blocks (such as P2VP-b-PS-b-P2VP, where P2VP is poly-2-vinylpyridine) are also included.

[0018] 3) Amino / quaternary ammonium salt type

[0019] Aminopropyl polydimethylsiloxane and quaternized polysiloxane, with hydrophilic groups of -NH2 / -N. + (CH3)3, positively charged; readily binds to negatively charged triblock polymers (such as those containing sulfonic acid groups), and can interact with phosphate groups of DNA / RNA. Triblock polymers containing anionic blocks (such as PS-b-PSS-b-PS, where PSS is sodium polystyrene sulfonate).

[0020] 4) Multi-group hydrophilic group type

[0021] PDMS-PEO-COOH (polydimethylsiloxane-polyoxyethylene-carboxyl group) and siloxane-polyether-phosphate ester contain two or more hydrophilic groups (such as PEO+COOH, PEO+phosphate ester), combining "mild compatibility" and "strong adsorption", with high flexibility in interface regulation; in addition, there are triblock polymers with complex structures (such as PEO-b-PS-b-PDMS, containing multiple polar blocks), which can be used for multi-component nanopore sequencing chips (such as simultaneous detection of protein + nucleic acid) and high-sensitivity sensor surface assembly.

[0022] The organosilicon surfactant can be one or more of the following: polyether type, polyether-modified polydimethylsiloxane, polyoxyethylene and polypropylene copolymer, polyether-modified heptamethyltrisiloxane, polyether-modified silicone oil (polyether-siloxane), ethoxy-modified trisiloxane, 3-[methoxypoly(ethoxy)]propyl-methyl-bis(trimethylsiloxy)silane (polyethoxy-siloxane). It can also be a multi-functional hydrophilic group type, such as polyethoxy-modified trisiloxane, 3-[methoxypoly(ethoxy)]propyl-methyl-bis(trimethylsiloxy)silane; or a mixture of the above organosilicon surfactants, such as a mixture of one or more of polyoxyethylene and polypropylene copolymer (PEO).

[0023] Preferably, the organosilicon surfactant includes one or more of polyether-modified polydimethylsiloxane, polyether-modified silicone oil, and polyether-modified heptamethyltrisiloxane.

[0024] Preferably, the concentration of the organosilicon surfactant in the amphiphilic triblock copolymer solution is 1-5%.

[0025] Preferably, the concentration of the organosilicon surfactant in the amphiphilic triblock copolymer solution is 1-3%.

[0026] Preferably, the hydrophilic segment A comprises one or more of the following: polyacrylamide, polyacrylamide, polyalkylacrylamide, polyethoxyacrylate, polyethoxymethacrylate, polyethylene glycol, polymethyloxazoline, and polyhexyloxazoline.

[0027] The hydrophobic segment B includes one or more of the following: polysiloxane, polyolefin, polyalkyl acrylate, polyoxypropylene, polyacrylate, and polylactic acid.

[0028] Preferably, the amphiphilic triblock copolymer is a polyoxazoline-based triblock copolymer comprising:

[0029] One of the following: PEOXA-PEO-PEOXA, PMOXA-PB-PMOXA, PMOXA-PE-PMOXA, PMOXA-PEO-PMOXA, PMOXA-PDMS-PMOXA, or PMOXA-PLA-PMOXA. Specifically:

[0030] (1) PEOXA-PEO-PEOXA [poly(2-ethyloxazoline)-b-poly(epoxyalkyl)-b-poly(2-ethyloxazoline)];

[0031] (2)PMOXA-PB-PMOXA [poly(2-methyloxazoline)-b-poly(1,-butadiene)-b-poly(2-methyloxazoline)];

[0032] (3) PMOXA-PE-PMOXA [poly(2-methyloxazoline)-b-poly(ethylene)-b-poly(2-methyloxazoline)];

[0033] (4)PMOXA-PEO-PMOXA [poly(2-methyloxazoline)-b-poly(cycloalkane)-b-poly(2-methyloxazoline)]; its blocks are linked by ethyl- or propyl- or propyl-ethoxy groups;

[0034] (5) PMOXA-PDMS-PMOXA [poly(2-methyl)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline)]

[0035] (6)PMOXA-PLA-PMOXA[poly(2-methyloxazoline)-b-poly(lactic acid)-b-poly(2-methyloxazoline)].

[0036] Preferably, the amphiphilic triblock copolymer is PMOXA. n -PDMS m -PMOXA n Or PMOXA n -PEO m -PMOXA n Or PMOXA n -PLA m -PMOXA nThe weight-average molecular weight of the triblock copolymer is 3500-10000 Da. The degree of polymerization of the hydrophilic segment A is n; the degree of polymerization of the hydrophobic segment B is m, where m+n≤55 and the ratio of n:m is 1:(6-8). The weight-average molecular weight of the triblock copolymer mainly affects the thickness of the membrane. A suitable membrane thickness is beneficial for the embedding of bio-nanoporotic proteins. By controlling the degree of polymerization and its ratio of polymer blocks (e.g., the total degree of polymerization of hydrophilic blocks, the ratio of the degree of polymerization of hydrophilic blocks to that of hydrophobic blocks) and the number-average molecular weight at appropriate levels, this invention not only ensures a high film formation rate but also ensures good membrane-protein interactions. That is, the amphiphilic triblock copolymer provided by this invention can be adapted to different bio-nanoporotic proteins, and the bio-nanoporotic proteins can exist stably on the biomimetic membrane formed thereon for a long time (e.g., more than 90 days). The biocompatibility is higher than that of the amphiphilic triblock copolymer without organosilicon surfactant modification to form a biomimetic membrane, and it requires less bio-protein and has a lower embedding voltage.

[0037] Preferably, the siloxane triblock copolymer is HO-PMOXA. n -PDMS m -PMOXA n -OH or MA-PMOXA n -PDMS m -PMOXA n -MA, where n and m represent the degree of polymerization, m+n≤55 and the ratio of n:m is 1:(6-8). The hydrophilic segment A is capped with maleic anhydride or hydroxyl groups. The anhydride group generates a carboxyl group after ring opening, which can enhance the hydrophilicity, water solubility, or ionization ability of the polymer. The hydroxyl end group serves as an active site, enabling block copolymerization, graft modification, and cross-linking curing of the polymer. At the same time, it imparts a certain degree of hydrophilicity to the polymer (hydroxyl groups can form hydrogen bonds).

[0038] A second aspect of the present invention is to provide an application of the self-assembled biomimetic membrane of the amphiphilic block copolymer as described in the first aspect of the present invention in the preparation of a biosensor.

[0039] Preferably, a biomimetic nanoporous protein is embedded in the biomimetic membrane; specifically, a molecular membrane anchoring method is used to promote the embedding of the biomimetic nanoporous protein into the biomimetic membrane by anchoring molecules; wherein, the anchoring molecule is an aptamer-long siloxane chain structure.

[0040] This anchoring molecule is an aptamer-long siloxane chain covalent type. It achieves anchoring by forming covalent bonds with the membrane surface and biomolecules, exhibiting extremely high stability and strong resistance to elution and environmental interference, making it suitable for biosensors. A significant feature of the anchoring molecule in this invention is its core structure, which consists of a long siloxane chain (hydrophobic end) at the center and specific aptamers at both ends. The siloxane chain is embedded in the hydrophobic microregions of the membrane (such as PDMS segments) and fixed through hydrophobic interactions. The aptamers and biomolecules (such as the active sites of specific proteins and enzymes) bind in a spatially complementary manner. In the anchoring system of triblock polymer membranes with biomolecules such as proteins and enzymes, the selection of the anchoring molecule must match the surface properties of the membrane (such as hydrophilicity / hydrophobicity, active groups) with the structure of the biomolecule (such as surface functional groups, tag sequences).

[0041] Preferably, the anchoring molecule is polydimethylsiloxane bis(4-aminobutyric acid) ester, with the following general structural formula:

[0042] H2N-(CH2)3-C(=O)-O-(CH2)3-[Si(CH3)2-O] n -Si(CH3)2-(CH2)3-OC(=O)-(CH2)3-NH2, with a polymer range of n of 12-44. To ensure that the length of the long siloxane chain matches the thickness of the hydrophobic segment of the triblock copolymer (such as PDMS), a polydimethylsiloxane chain containing 12-44 siloxane units is typically used to achieve sufficient embedding and anchoring stability of the hydrophobic block. This structure, through a dual mechanism of "hydrophobic anchoring-specific recognition," ensures the stable fixation of anchored molecules in the membrane while achieving precise capture of biomolecules.

[0043] Preferably, the specific structural formula of polydimethylsiloxane bis(4-aminobutyric acid) ester is as follows:

[0044] m represents the degree of polymerization.

[0045] Preferably, the bio-nanoporin is α-hemolysin, Mycobacterium smegmatis porin A (MspA), or Escherichia coli curly hair protein G (CsgG); the biosensor is a nanopore sequencing chip.

[0046] A second aspect of the present invention is to provide a molecular membrane anchoring method as described in the second aspect of the present invention, the specific steps of which are:

[0047] S1 Preparation of anchoring molecules: Hydroxyl-terminated polydimethylsiloxane (HO-PDMS-OH) and 4-aminobutyric acid or its active ester derivatives are used as raw materials, and di-tert-butyl dicarbonate (Boc2O) is used as an amino protecting agent; a condensing agent is added, and an esterification reaction is carried out in an organic solvent to generate polydimethylsiloxane bis(4-aminobutyric acid) ester.

[0048] S2 is embedded in bio-nanoporin:

[0049] S21: First, a nanopore sequencing chip is used to self-assemble into an amphiphilic block copolymer biomimetic membrane;

[0050] S22: After the amphiphilic block copolymer biomimetic membrane assembled in step S21 has stabilized, anchoring molecules and biological nanoporous protein solution are added; then, the nanoporous protein is embedded into the amphiphilic block copolymer self-assembled biomimetic membrane by voltage driving, autonomous fusion or protein vesicle fusion.

[0051] Among them, the stability of the amphiphilic block copolymer biomimetic membrane refers to the time when the transmembrane current is stable and basically without fluctuation after 10-15 minutes of self-assembly into the biomimetic membrane.

[0052] In step S1, the preparation of anchor molecules requires strict control of reaction temperature, time, and raw material molar ratio to ensure the full progress of the esterification reaction and the purity of the product.

[0053] Preferably, the specific steps of step S21 are as follows:

[0054] S211: Dissolve the triblock polymer in a nonpolar solvent to obtain a polymer membrane solution, and add an organosilicon surfactant at a concentration of 0.5-10% of the polymer membrane solution to obtain a nonpolar polymer membrane solution; the mass concentration of the triblock copolymer in the nonpolar polymer membrane solution is 10-20 mg / mL;

[0055] S212: Prepare polar solutions according to the specified proportions;

[0056] S213: After pretreatment of the nanopore sequencing chip, the polar solution prepared in step S212 is first introduced into the nanopore sequencing chip, and then the nonpolar polymer membrane solution prepared in step S211 is added into the micropore channels of the nanopore sequencing chip. After waiting for 15-20 minutes, the polar solution is added, thereby forming a polar-nonpolar-polar system in the micropore channels of the nanopore sequencing chip, so that the amphiphilic polymer self-assembles into an amphiphilic block copolymer biomimetic membrane, and the membrane formation process is completed.

[0057] In some specific embodiments, a polar-nonpolar-polar system is formed in the gear structure region of the microporous channel of the nanopore sequencing chip.

[0058] Preferably, the pretreatment of the nanopore sequencing chip specifically involves: pre-coating 2-3 μL of a nonpolar polymer film solution onto the surface of the micropores of the nanopore sequencing chip, and baking it on a hot plate for 3-5 minutes.

[0059] In some specific embodiments, the non-polar solvent in step S211 is silicone oil, which includes at least one of methylphenyl silicone oil, PDMS, n-decane, and hexadecane;

[0060] The triblock polymer is one or more of the siloxane triblock copolymers HO-PMOXA-PDMS-PMOXA-OH and / or MA-PMOXA-PDMS-PMOXA-MA;

[0061] The organosilicon surfactant is added to the prepared amphiphilic triblock copolymer membrane solution.

[0062] Preferably, the polar solution in step S212 is an aqueous buffer solution, including one or more of the following: phosphate buffer solution, HEPES buffer solution containing KCl or NaCl, CAPS buffer solution containing KCl or NaCl, and CAPS buffer solution containing KCl, H3PO4, K2[Fe(CN)6], and K3[Fe(CN)6].

[0063] Preferably, the film formation effect is detected by electrical characterization of the membrane capacitance. Each structural unit has a first electrode at its bottom, which can contact a polar solvent in a polar-nonpolar-polar solvent-polar solvent configuration. A second electrode is located at the other end of the nanopore sequencing chip structure, away from the bottom of the structural unit, which can contact another polar solvent. Therefore, each structural unit is actually a membrane capacitor, and the electrical characterization will differ depending on the thickness of the thin film layer. Each rectangular block represents a membrane capacitor, corresponding to one structural unit. The value in the rectangular block represents the capacitance value of that membrane capacitor. A larger value indicates a relatively smaller membrane thickness.

[0064] Preferably, in step S22, after the amphiphilic block copolymer biomimetic membrane is stabilized, anchoring molecules are added, and biological protein nanopores are inserted into the biomimetic membrane through voltage-driven, autonomous fusion, or protein vesicle fusion, thereby enabling biosensing applications such as nanopore gene sequencing, drug molecule detection, and protein detection. The nanopore sequencing chip involved is a 256-channel chip.

[0065] Preferably, in step S22, the pore-embedding voltage applied to both ends of the amphiphilic polylactic acid block copolymer biomimetic membrane is 350-400mV, which can embed nanoscale protein pores into the amphiphilic polylactic acid block copolymer biomimetic membrane.

[0066] Beneficial technical effects:

[0067] (1) In the process of self-assembling biomimetic membranes of amphiphilic block copolymers on biochips (nanopore sequencing chips), organosilicon surfactants are added. Organosilicon surfactants can effectively promote the self-assembly of amphiphilic block copolymers into biomimetic membranes on the micropores of nanopore sequencing chips. The bifunctional hydrophilic blocks of the organosilicon surfactants are the core regulators of the self-assembly of triblock polymers. One end achieves strong binding with the substrate, and the other end enhances the biocompatibility with the biomimetic membrane. The polysiloxane chains of the surfactants form a tight hydrophobic binding region with the hydrophobic blocks of the triblock polymers, reducing the penetration of water molecules into the hydrophobic blocks. On the other hand, the hydrophilic groups of the surfactants form irreversible adsorption (such as covalent bonds and strong hydrogen bonds) with the substrate, adsorbing the triblock polymer assemblies onto the substrate and preventing them from falling off or migrating in the solution. By optimizing interface compatibility, reducing energy barriers, and guiding precise templates, the assembly problem of amphiphilic block copolymers on nanopore sequencing chip substrates is solved; and different types correspond to different interface interaction mechanisms and application scenarios. According to this method, based on the block composition (hydrophobic / hydrophilic properties) of the triblock polymer, the substrate material of the nanopore sequencing chip, and the final functional requirements, a matching type of organosilicon surfactant can be selected to form a highly ordered, highly stable, and highly specific self-assembled biomimetic membrane. The resulting biomimetic membrane has high formation quality, consistent uniformity, good stability (stable for more than 90 days), longer storage time after formation, and higher mechanical strength. Moreover, different types correspond to different interfacial interaction mechanisms and application scenarios.

[0068] (2) The anchoring molecule designed and prepared using a molecular membrane anchoring method helps to promote the embedding of bio-nanoporous proteins into the amphiphilic block copolymer biomimetic membrane. This method exhibits good membrane-protein interactions, and the self-assembled biomimetic membrane structure can embed transmembrane biomolecules such as channel proteins for gene sequencing and drug molecule detection. After the amphiphilic block copolymer is stabilized by self-assembling into a biomimetic membrane, the addition of the anchoring molecule increases the embedding rate of bio-nanoporous proteins into the amphiphilic block copolymer biomimetic membrane, saving embedding time and the amount of bio-protein used; and it also increases the stability of bio-protein nanopores on the amphiphilic block copolymer biomimetic membrane, extending the sequencing lifetime of the nanopore sequencing chip.

[0069] (3) The amphiphilic block copolymer biomimetic membrane prepared by introducing organosilicon surfactant has high membrane quality, uniformity, and good stability. It can be embedded with biological nanoporous proteins and used for nanosequencing. At the same time, the biomimetic membrane embedded with biological nanoporous proteins using this molecular membrane anchoring method was compared with known sequences. It was found that the sequencing accuracy of the biomimetic membrane embedded with nanoporous proteins using this amphiphilic block copolymer biomimetic membrane is high, reaching more than 95%. Moreover, the pore loss rate of the nanopore sequencing chip at 48h (6.33%) is much lower than that of the biomimetic membrane without anchoring molecules (47.37%). Attached Figure Description

[0070] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0071] Figure 1 This is a schematic diagram illustrating the film formation principle of the self-assembled biomimetic membrane of the amphiphilic block copolymer of the present invention.

[0072] Figure 2 The polyether-modified polydimethylsiloxane regulated block copolymer (HO-PMOXA6-PDMS) of Specific Embodiment 19 of the present invention 44 The membrane current signal results of the biomimetic membrane formed by the self-assembly of PMOXA6-OH are shown in the figure.

[0073] Figure 3 This is a synthetic route diagram of polydimethylsiloxane (4-aminobutyric acid) ester, the anchoring molecule used in the molecular membrane anchoring method of the present invention.

[0074] Figure 4 The present invention relates to an amphiphilic triblock copolymer (HO-PMOXA6-PDMS) 44 Transmembrane current diagram of biological protein nanopores embedded in a PMOXA6-OH biomimetic membrane via molecular membrane anchoring method;

[0075] Figure 5 In a specific embodiment 28 of the present invention, the amphiphilic triblock copolymer (HO-PMOXA6-PDMS) 44 The sequencing signal results of a PMOXA6-OH biomimetic membrane after adding anchoring molecules embedded in the nanopores of biological proteins via a molecular membrane anchoring method;

[0076] Figure 6 Comparative Example 4 of the present invention uses an amphiphilic triblock copolymer (HO-PMOXA6-PDMS) 44 Image of sequencing signal results after direct embedding of biological protein nanopores in a PMOXA6-OH biomimetic membrane;

[0077] Figure 7 The amphiphilic triblock copolymer (HO-PMOXA6-PDMS) of Specific Example 28 and Comparative Example 4 of this invention 44 Comparison of the trend of sequencing channel ratio with sequencing time after PMOXA6-OH biomimetic membrane is embedded in biological protein nanopores;

[0078] Figure 8 The image shows the 1H NMR spectrum of the anchoring molecule polydimethylsiloxane bis(4-aminobutyric acid) ester synthesized in this invention. Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0080] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0081] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0082] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even more typically + / -1%, even more typically + / -0.5%.

[0083] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0084] Definition of noun:

[0085] Molecular membrane anchoring refers to the technique of fixing target molecules (proteins, nucleic acids, polysaccharides, functionalized polymers, etc.) onto the surface or inside the molecular membrane carrier in a directional or non-directional manner. The core is to stabilize the target molecules and maintain their biological activity / functional properties through intermolecular interactions or chemical bonds. It is widely used in fields such as biomimetic membranes, biosensors, membrane separation, and drug delivery.

[0086] Biomolecular nanoporous proteins, also known as channel proteins, are integrated membrane proteins that span the cell membrane bilayer. They form hydrophilic channels through their conformation to mediate the flow of specific ions / small molecules (such as Na+). + K + Ca² + Passive transport of water down its concentration / electrochemical gradient (without ATP consumption); transport does not bind to the substrate and can reach rates of up to 10. 6 -10 8 The number of cells per second is much higher than that of carrier proteins.

[0087] Example: The amphiphilic block copolymer self-assembled biomimetic membrane is formed by the self-assembly of film-forming molecules; the film-forming molecules include an amphiphilic triblock copolymer, the configuration of which is hydrophilic segment A-hydrophobic segment B-hydrophilic segment A; and the amphiphilic triblock copolymer is modified by an organosilicon surfactant before self-assembling into an amphiphilic block copolymer biomimetic membrane.

[0088] In some specific embodiments, the film-forming molecules include an amphiphilic diblock copolymer, wherein the amphiphilic diblock copolymer has an AB configuration, that is, hydrophilic segment A-hydrophobic segment B;

[0089] In some specific embodiments, the concentration of the silicone surfactant in the amphiphilic triblock copolymer solution is 0.5-10%; the silicone surfactant includes one or more of the following types: polyether type, carboxyl / sulfonic acid type, amino / quaternary ammonium salt type, and multi-hydrophilic group type.

[0090] When the organosilicon surfactant is a polyether type, it includes one or a mixture of polyether-modified polydimethylsiloxane, polyether-modified silicone oil, polyether-modified heptamethyltrisiloxane, and polyethoxylated trisiloxane.

[0091] When the organosilicon surfactant is of the carboxyl / sulfonic acid type, it includes carboxypropyl polydimethylsiloxane and / or sulfonic acid-modified polysiloxane;

[0092] When the organosilicon surfactant is of the amino / quaternary ammonium salt type, it includes aminopropyl polydimethylsiloxane and / or quaternized polysiloxane;

[0093] When the organosilicon surfactant is of the multi-component hydrophilic group type, it includes polyethoxylated trisiloxane and / or 3-[methoxypoly(ethoxy)]propyl-methyl-bis(trimethylsiloxy)silane.

[0094] Organosilicon surfactants possess bifunctional hydrophilic blocks, serving as the "regulatory core" for the self-assembly of triblock polymers. One end achieves strong binding to the substrate, while the other end enhances biocompatibility. Through interface compatibility optimization, energy barrier reduction, and precise template guidance, they solve the assembly challenges of amphiphilic block copolymers on nanopore sequencing chip substrates. Furthermore, different types correspond to different interfacial interaction mechanisms and application scenarios. The organosilicon surfactants described in this invention are classified into the following categories based on the type of hydrophilic group (which determines their interfacial interaction characteristics and applicable scenarios):

[0095] 1) Polyether type

[0096] PDMS-b-PEO (polydimethylsiloxane-polyoxyethylene block copolymer) and PMPS-PEO (polymethylphenylsiloxane-polyoxyethylene) have PEO chains as their hydrophilic groups, exhibiting excellent biocompatibility and good water solubility. They also have extremely high compatibility with triblock polymers containing PEO blocks, resulting in mild interfacial interactions. Triblock polymers containing PEO hydrophilic blocks (such as PS-b-PEO-b-PS and PDMS-b-PEO-b-PDMS) are used in nanopore sequencing chips for "molecular recognition arrays" (such as antibody / antigen immobilization) and for microfluidic channel inner wall modification.

[0097] 2) Carboxyl / sulfonic acid type

[0098] Carboxypropyl polydimethylsiloxane and sulfonic acid-modified polysiloxane have negatively charged hydrophilic groups -COOH / -SO3H. They are strongly adsorbed to polar substrates (such as amino-modified silicon wafers) through electrostatic interactions, resulting in strong interfacial bonding. In addition, triblock polymers containing cationic or hydrophobic blocks (such as P2VP-b-PS-b-P2VP, where P2VP is poly-2-vinylpyridine) are also included.

[0099] 3) Amino / quaternary ammonium salt type

[0100] Aminopropyl polydimethylsiloxane and quaternized polysiloxane, with hydrophilic groups of -NH2 / -N. + (CH3)3, positively charged; readily binds to negatively charged triblock polymers (such as those containing sulfonic acid groups), and can interact with phosphate groups of DNA / RNA. Triblock polymers containing anionic blocks (such as PS-b-PSS-b-PS, where PSS is sodium polystyrene sulfonate).

[0101] 4) Multi-group hydrophilic group type

[0102] PDMS-PEO-COOH (polydimethylsiloxane-polyoxyethylene-carboxyl group) and siloxane-polyether-phosphate ester contain two or more hydrophilic groups (such as PEO+COOH, PEO+phosphate ester), combining "mild compatibility" and "strong adsorption", with high flexibility in interface regulation; in addition, there are triblock polymers with complex structures (such as PEO-b-PS-b-PDMS, containing multiple polar blocks), which can be used for multi-component nanopore sequencing chips (such as simultaneous detection of protein + nucleic acid) and high-sensitivity sensor surface assembly.

[0103] The organosilicon surfactant can be one or more of the following: polyether type, polyether-modified polydimethylsiloxane, polyoxyethylene and polypropylene copolymer, polyether-modified heptamethyltrisiloxane, polyether-modified silicone oil (polyether-siloxane), ethoxy-modified trisiloxane, 3-[methoxypoly(ethoxy)]propyl-methyl-bis(trimethylsiloxy)silane (polyethoxy-siloxane). It can also be a multi-functional hydrophilic group type, such as polyethoxy-modified trisiloxane, 3-[methoxypoly(ethoxy)]propyl-methyl-bis(trimethylsiloxy)silane; or a mixture of the above organosilicon surfactants, such as a mixture of one or more of polyoxyethylene and polypropylene copolymer (PEO).

[0104] In some specific embodiments, the organosilicon surfactant includes one or more of polyether-modified polydimethylsiloxane, polyether-modified silicone oil, and polyether-modified heptamethyltrisiloxane.

[0105] In some specific embodiments, the concentration of the organosilicon surfactant in the amphiphilic triblock copolymer solution is 1-5%.

[0106] In some specific embodiments, the concentration of the organosilicon surfactant in the amphiphilic triblock copolymer solution is 1-3%.

[0107] In some specific embodiments, the hydrophilic segment A includes one or more of polyacrylamide, polyacrylamide, polyalkylacrylamide, polyethoxyacrylate, polyethoxymethacrylate, polyethylene glycol, polymethyloxazoline, and polyhexyloxazoline.

[0108] The hydrophobic segment B includes one or more of the following: polysiloxane, polyolefin, polyalkyl acrylate, polyoxypropylene, polyacrylate, and polylactic acid.

[0109] In some specific embodiments, the amphiphilic triblock copolymer is a polyoxazoline-based triblock copolymer, comprising:

[0110] One of the following: PEOXA-PEO-PEOXA, PMOXA-PB-PMOXA, PMOXA-PE-PMOXA, PMOXA-PEO-PMOXA, PMOXA-PDMS-PMOXA, or PMOXA-PLA-PMOXA. Specifically:

[0111] (1) PEOXA-PEO-PEOXA [poly(2-ethyloxazoline)-b-poly(epoxyalkyl)-b-poly(2-ethyloxazoline)];

[0112] (2)PMOXA-PB-PMOXA [poly(2-methyloxazoline)-b-poly(1,-butadiene)-b-poly(2-methyloxazoline)];

[0113] (3) PMOXA-PE-PMOXA [poly(2-methyloxazoline)-b-poly(ethylene)-b-poly(2-methyloxazoline)];

[0114] (4)PMOXA-PEO-PMOXA [poly(2-methyloxazoline)-b-poly(cycloalkane)-b-poly(2-methyloxazoline)]; its blocks are linked by ethyl- or propyl- or propyl-ethoxy groups;

[0115] (5) PMOXA-PDMS-PMOXA [poly(2-methyl)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline)]

[0116] (6)PMOXA-PLA-PMOXA[poly(2-methyloxazoline)-b-poly(lactic acid)-b-poly(2-methyloxazoline)].

[0117] In some specific embodiments, the amphiphilic triblock copolymer is PMOXA. n -PDMS m -PMOXA n Or PMOXA n -PEO m -PMOXA n Or PMOXA n -PLA m -PMOXA n The weight-average molecular weight is 3500-10000 Da, the degree of polymerization of the hydrophilic segment A is n; the degree of polymerization of the hydrophobic segment B is m, m+n≤55 and the ratio of n:m is 1:(6-8).

[0118] In some specific embodiments, the siloxane triblock copolymer is HO-PMOXA. n -PDMS m -PMOXA n -OH or MA-PMOXAn -PDMS m -PMOXA n -MA, where n and m represent the degree of polymerization, m+n≤55 and the ratio of n:m is 1:(6-8). The hydrophilic segment A is capped with maleic anhydride or hydroxyl groups. The anhydride group generates a carboxyl group after ring opening, which can enhance the hydrophilicity, water solubility, or ionization ability of the polymer. The hydroxyl end group serves as an active site, enabling block copolymerization, graft modification, and cross-linking curing of the polymer. At the same time, it imparts a certain degree of hydrophilicity to the polymer (hydroxyl groups can form hydrogen bonds).

[0119] The preparation method of the above-mentioned amphiphilic triblock copolymer in this invention includes the following steps:

[0120] S101: Ethylene glycol, lactide, and the first catalyst are added to a reaction vessel to obtain a first mixture;

[0121] In some specific embodiments, the reaction vessel includes a pressure-resistant sealing tube.

[0122] In some specific embodiments, the molar ratio (feed ratio, the same below) of the ethylene glycol and the lactide includes 1:15. 40.

[0123] S102: React the first mixture in an oil bath at a first temperature for a first time to obtain a first product;

[0124] In some specific embodiments, the first time period includes 15-20 hours;

[0125] In some specific embodiments, the first temperature includes 100-110°C;

[0126] In some specific embodiments, the first catalyst comprises stannous octoate.

[0127] S103: The first product, pyridine and trifluoromethanesulfonic anhydride are mixed and reacted for a second time to obtain the second product;

[0128] In some specific embodiments, the molar ratio of the first product to the trifluoromethanesulfonic anhydride includes 1:5. 10;

[0129] In some specific embodiments, the second time includes 0.25 seconds. 1 hour;

[0130] S104: Mix the second product and 2 methyl 2 The oxazoline mixture was reacted at a third time to obtain the third product;

[0131] In some specific embodiments, the third time includes 0.25 seconds. 1 hour;

[0132] S105: Triethylamine is added to the third product and reacted for a fourth time to obtain the fourth product;

[0133] In some embodiments, the fourth time includes 0.5 2h;

[0134] In some embodiments, the molar ratio of the third product to the triethylamine includes 1:2. 5.

[0135] In some embodiments, the second product and the 2 methyl 2 The molar ratio of oxazoline includes 1:4. 15.

[0136] In some embodiments, the method further includes S106 mixing and reacting the fourth product with isocyanate methacrylate to obtain a fifth product, namely the amphiphilic triblock copolymer.

[0137] amphiphilic triblock copolymer PMOXA n -PDMS m -PMOXA n Or PMOXA n -PEO m -PMOXA n A preparation method comprising the following steps:

[0138] S101: Mix hydroxyalkyl-terminated polydimethylsiloxane (PDMS) or hydroxyalkyl-terminated polyepoxide (PEO), pyridine and trifluoromethanesulfonic anhydride and react for a first time to obtain the first product;

[0139] In some specific embodiments, the molar ratio of the hydroxyl-terminated double-ended polydimethylsiloxane (PDMS) to the trifluoromethanesulfonic anhydride is 1:5. 10;

[0140] In some specific embodiments, the first time includes 0.25 seconds. 1 hour;

[0141] S102: Combine the first product and 2 methyl 2 The oxazoline mixture is reacted for a second time to obtain the second product;

[0142] In some specific embodiments, the second time includes 0.25 seconds. 1 hour.

[0143] S103: Triethylamine is added to the second product and reacted for a third time to obtain the third product;

[0144] In some embodiments, the third time includes 0.5 2h.

[0145] In some embodiments, the molar ratio of the second product to the triethylamine includes 1:2. 5.

[0146] In some embodiments, the first product and the 2 methyl 2 The molar ratio of oxazoline includes 1:4. 15.

[0147] In some embodiments, the method further includes S104 mixing and reacting the third product with isocyanate methacrylate to obtain a fourth product, namely the amphiphilic triblock copolymer.

[0148] The reagents used in the above polymer synthesis process (including intermediate chain segments such as PEO and PDMS) are basically commercially available reagents and are not synthesized in-house.

[0149] Application of this amphiphilic block copolymer self-assembled biomimetic membrane in the preparation of biosensors.

[0150] In some specific embodiments, bio-nanoporin is embedded in the biomimetic membrane; specifically, a molecular membrane anchoring method is used to promote the embedding of bio-nanoporin into the biomimetic membrane by anchoring molecules; wherein, the anchoring molecule is an aptamer-long siloxane chain structure.

[0151] In some specific embodiments, the anchoring molecule is polydimethylsiloxane bis(4-aminobutyric acid) ester, whose general structural formula is:

[0152] H2N-(CH2)3-C(=O)-O-(CH2)3-[Si(CH3)2-O] n -Si(CH3)2-(CH2)3-OC(=O)-(CH2)3-NH2, with a polymer range n of 12-44.

[0153] Preferably, the specific structural formula of polydimethylsiloxane bis(4-aminobutyric acid) ester is as follows:

[0154] m represents the degree of polymerization.

[0155] In some specific embodiments, the bio-nanoporin is α-hemolysin, or Mycobacterium smegmatis pore protein A (MspA), or Escherichia coli curvature fimbriae protein G (CsgG); the biosensor is a nanopore sequencing chip.

[0156] The specific steps of this molecular membrane anchoring method are as follows:

[0157] S1 Preparation of Anchored Molecules: Hydroxyl-terminated polydimethylsiloxane (HO-PDMS-OH) and 4-aminobutyric acid or its active ester derivatives are used as raw materials, and di-tert-butyl dicarbonate (Boc2O) is used as an amino protecting agent; a condensing agent is added, and an esterification reaction is carried out in an organic solvent to generate polydimethylsiloxane bis(4-aminobutyric acid) ester; during the reaction, it is necessary to strictly control the reaction temperature, time, raw material molar ratio, and other conditions to ensure the full progress of the esterification reaction and the purity of the product;

[0158] S2 is embedded in bio-nanoporin:

[0159] S21: First, a nanopore sequencing chip is used to self-assemble into an amphiphilic block copolymer biomimetic membrane;

[0160] S22: After the amphiphilic block copolymer biomimetic membrane assembled in step S21 has stabilized, anchoring molecules and biological nanoporous protein solution are added; then, the nanoporous protein is embedded into the amphiphilic block copolymer self-assembled biomimetic membrane by voltage driving, autonomous fusion or protein vesicle fusion.

[0161] Among them, the stability of the amphiphilic block copolymer biomimetic membrane refers to the time when the transmembrane current is stable and basically without fluctuation after 10-15 minutes of self-assembly into the biomimetic membrane.

[0162] In some specific embodiments, step S21 specifically includes the following steps:

[0163] S211: Dissolve the triblock polymer in a nonpolar solvent to obtain a polymer membrane solution, and add an organosilicon surfactant at a concentration of 0.5-10% of the polymer membrane solution to obtain a nonpolar polymer membrane solution; the mass concentration of the triblock copolymer in the nonpolar polymer membrane solution is 10-20 mg / mL;

[0164] S212: Prepare polar solutions according to the specified proportions;

[0165] S213: After pretreatment of the nanopore sequencing chip, the polar solution prepared in step S212 is first introduced into the nanopore sequencing chip. Then, the nonpolar polymer membrane solution prepared in step S211 is added to the microporous channels of the nanopore sequencing chip. After waiting for 15-20 minutes, the polar solution is added, thereby forming a polar-nonpolar-polar system in the microporous channels of the nanopore sequencing chip. This allows the amphiphilic polymer to self-assemble into an amphiphilic block copolymer biomimetic membrane, thus completing the membrane formation process. Specifically, a polar-nonpolar-polar system is formed in the gear structure region of the microporous channels of the nanopore sequencing chip. The membrane formation principle is as follows: Figure 1 As shown, the self-assembly process of amphiphilic triblock copolymers in nanopore sequencing chips includes five stages: The first stage involves pre-wetting the microchannels with a polar solution to form a uniform substrate using the hydrophilicity of the channel walls; the second stage introduces a non-polar solution to construct a stable liquid-liquid interface with a "polar layer attached to the wall and a non-polar phase in the center"; the third stage involves the enrichment and initial adsorption of the amphiphilic polymer at the interface, with the polar segments anchoring the polar layer and the non-polar segments extending into the non-polar phase; the fourth stage triggers interfacial polymerization or self-assembly by adjusting conditions to form an initial membrane with both strength and semi-permeability; finally, the fifth stage introduces a polar solution to flush out impurities and induce phase separation and solidification, ultimately resulting in a continuous, uniform, and tightly bound functional separation membrane of polar-non-polar-polar phases within the channels—the amphiphilic block copolymer biomimetic membrane.

[0166] The concentration of the amphiphilic triblock copolymer, film-forming temperature, solvent concentration, and microfluidic flow rate (the speed at which the polymer membrane solution flows through the nanopore sequencing chip during film-forming operations) need to be adjusted adaptively according to the specific experimental conditions.

[0167] In some specific embodiments, the pretreatment of the nanopore sequencing chip is as follows: 2-3 μL of nonpolar polymer film solution is pre-coated on the surface of the micropores of the nanopore sequencing chip, and then baked on a hot plate for 3-5 min.

[0168] In some specific embodiments, the non-polar solvent in step S211 is silicone oil, which includes at least one of methylphenyl silicone oil, PDMS, n-decane, and hexadecane;

[0169] The triblock polymer is one or more of the siloxane triblock copolymers HO-PMOXA-PDMS-PMOXA-OH and / or MA-PMOXA-PDMS-PMOXA-MA;

[0170] The organosilicon surfactant is added to the prepared amphiphilic triblock copolymer membrane solution.

[0171] In some specific embodiments, the polar solution in step S212 is an aqueous buffer solution, including one or more of the following: phosphate buffer solution, HEPES buffer solution containing KCl or NaCl, CAPS buffer solution containing KCl or NaCl, and CAPS buffer solution containing KCl, H3PO4, K2[Fe(CN)6], and K3[Fe(CN)6].

[0172] In some specific embodiments, after the amphiphilic block copolymer biomimetic membrane is stabilized in step S22, anchoring molecules are added, and biological protein nanopores are inserted into the biomimetic membrane through voltage-driven, autonomous fusion, or protein vesicle fusion, thereby enabling biosensing applications such as nanopore gene sequencing, drug molecule detection, and protein detection. The nanopore sequencing chip involved is a 256-channel chip.

[0173] The core principle of nanopore sequencing devices is to immobilize a nanopore protein on an amphiphilic molecular membrane, then unwind the DNA double helix into single strands, and use motor proteins to pull these single DNA strands through the nanopore. Different bases on the DNA single strands have different chemical structures and carry different charges. As they pass through the nanopore, they cause changes in the current across the amphiphilic molecular membrane, generating an electrical signal. This change in current is used to identify the bases. Therefore, the stability of the amphiphilic molecular membrane is crucial for nanopore sequencing.

[0174] In some specific embodiments, the pore-embedding voltage applied to both ends of the amphiphilic polylactic acid block copolymer biomimetic membrane in step S22 is 350-400mV, which can embed nanoscale protein pores into the amphiphilic polylactic acid block copolymer biomimetic membrane.

[0175] In some specific embodiments, the nanopore sequencing chip typically has ≥2 micropore structures.

[0176] In some specific embodiments, the nanopore sequencing chip has a structure of 256 micropores.

[0177] In some specific embodiments, the final concentration of the bio-nanoporous protein solution at the biomimetic membrane with a single microporous structure is 1-2 × 10⁻⁶. 6 mg / mL.

[0178] In some specific embodiments, the added volume of the nanoporous protein solution at the biomimetic membrane of a single microporous structure includes 1 5×10 3 μL.

[0179] In some specific embodiments, the total volume of the added nanoporous protein solution includes 0.6-0.8 μL.

[0180] In some specific embodiments, the via voltage includes 300-400mV.

[0181] In some specific embodiments, the membrane capacitance of the biomimetic membrane is 15-60pF.

[0182] This invention introduces an organosilicon surfactant into an amphiphilic triblock copolymer solution, achieving strong binding to the nanopore sequencing chip substrate on one end and enhancing biocompatibility with the amphiphilic block copolymer biomimetic membrane on the other. The concentration of the organosilicon surfactant mainly affects the membrane's microstructure and macroscopic properties. The microstructure includes the degree of phase separation, membrane porosity, and thickness, while the macroscopic properties include surface wettability and mechanical stability.

[0183] Generally, organosilicon surfactants have low surface energy properties. The higher the concentration, the stronger the hydrophobicity of the membrane surface and the significantly increased water contact angle. This is crucial for the self-assembly of block polymers in the flow channels of nanopore sequencing chips.

[0184] Impact on mechanical stability: A suitable concentration (typically 0.5%-10% of the block copolymer mass) can fill the gaps in the copolymer phase region and improve the tensile strength of the membrane; however, excessively high concentrations will form a weak layer within the membrane, making it prone to rupture under microfluidic pressure. The concentration of the organosilicon surfactant in the amphiphilic block copolymer solution is preferably 0.5-10%, more preferably 1-5%, and most preferably 1-3%.

[0185] The following 18 specific examples explore the effects of different concentrations and types of silicone surfactants on the film formation rate of biomimetic films. The specific film formation methods are as follows:

[0186] 1) Prepare a nonpolar polymer film solution by adding the triblock polymer PMOXA6-PDMS. 44 PMOXA6 was dissolved in three different silicone surfactants to obtain polymer film solutions of 10 mg / mL. The three silicone surfactants were: polyether-modified polydimethylsiloxane, polyether-modified polydimethylsiloxane, and polyether-modified silicone oil; the concentrations of the silicone surfactants added were 0.1%, 1%, 3%, 5%, 10%, and 20%, respectively.

[0187] 2) Preparation of polar solution: Use a buffer solution with a pH of 7.5 containing 625 mM KCl, 10 mM HEPES ((4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)), and 5 mM EDTA (ethylenediaminetetraacetic acid);

[0188] 3) Nanopore sequencing chip preparation: The chip is pretreated by coating 2 μL of nonpolar polymer film solution onto the surface of the micropores of the nanopore sequencing chip and baking it on a hot plate for 3 min.

[0189] 4) Pass the polar solution prepared in step 2) into the nanopore sequencing chip;

[0190] 5) Biomimetic Membrane Formation: The nonpolar amphiphilic triblock copolymer membrane solution prepared in step 1) and regulated by the organosilicon surfactant was added to the microporous channels of the nanopore sequencing chip. After waiting for 15 minutes until it stabilized (i.e., the transmembrane current was stable with minimal fluctuations), a polar solution was added. A polar-nonpolar-polar system was formed in the gear structure region of the microporous channels of the nanopore sequencing chip. The amphiphilic triblock copolymer self-assembled into an amphiphilic triblock copolymer biomimetic membrane, thus completing the membrane formation process. The results of the concentration comparison experiment of the organosilicon surfactant and the membrane formation test of the block copolymer are shown in Table 1.

[0191] Table 1. Concentration comparison experiment of organosilicon surfactants and film formation test results of block copolymers

[0192]

[0193] As can be seen from Table 1, the film formation rate gradually increases with the increase of the concentration of organosilicon surfactant, reaching its maximum at 3%. After that, the film formation rate gradually decreases with further increases in the concentration of organosilicon surfactant.

[0194] When the concentration of the organosilicon surfactant reached 20%, film formation rate data was missing. This is presumably because the excessively high concentration caused the film to rupture under microfluidic pressure, preventing the formation of a complete and effective biomimetic film, thus hindering accurate measurement of the film formation rate. Therefore, for the siloxane triblock copolymer PMOXA6-PDMS... 44 -PMOXA6, the film-forming effect is best when the concentration of organosilicon surfactant is 3%.

[0195] Besides the concentration of silicone surfactants affecting film formation rate, different types of silicone surfactants also have a certain impact on film formation rate. For example, polyether-modified polydimethylsiloxane has a film formation rate of 80.47% at a concentration of 3%, while polyether-modified heptamethyltrisiloxane has a film formation rate of 68.36% at the same concentration, and polyether-modified silicone oil has a film formation rate of 71.09% at a concentration of 3%. This indicates that the interaction between silicone surfactants with different structures and triblock copolymers varies, thus affecting the film formation effect.

[0196] In practical applications, it is necessary to select the appropriate type of organosilicon surfactant and determine its optimal concentration based on the specific type of triblock copolymer and the required biomimetic membrane properties. This not only improves the film formation rate but also ensures that the prepared amphiphilic block copolymer self-assembled biomimetic membrane has good microstructure and macroscopic properties, meeting the subsequent application requirements in fields such as biosensing, such as achieving efficient nanopore gene sequencing, accurate drug molecule detection, and sensitive protein detection.

[0197] Organosilicon surfactants were introduced to regulate the self-assembly of amphiphilic triblock copolymers into biomimetic membranes. All solutions were commercially available reagents. Polar solvents included phosphate buffer solutions, HEPES buffer solutions containing KCl or NaCl, CAPS buffer solutions containing KCl or NaCl, or CAPS buffer solutions containing KCl, H3PO4, K2[Fe(CN)6], and K3[Fe(CN)6]. Non-polar solvents included polymer membrane solutions containing amphiphilic triblock polymers such as silicone oil, methylphenyl silicone oil, n-hexane, decane, or hexadecane. Non-polar solvents could be silicone oils, such as methylphenyl silicone oil or PDMS, and the polymer could be the triblock polymer HO-PMOXA6-PDMS. 44 -PMOXA6-OH or / and MA-PMOXA6-PDMS 44 The mass concentration of PMOXA6-MA triblock copolymer is 10-20 mg / mL;

[0198] The preferred organosilicon surfactants are polyether-modified polydimethylsiloxane, polyoxyethylene, polypropylene copolymer (Silwet L-77), polyether-modified heptamethyltrisiloxane, polyether-modified silicone oil, ethoxy-modified trisiloxane, polyether-modified heptamethyltrisiloxane, 3-[methoxypoly(ethoxy)]propyl-methyl-bis(trimethylsiloxy)silane, etc., and their concentration in the amphiphilic triblock copolymer solution is preferably 1-5%.

[0199] The film-forming quality of the biomimetic membrane of the present invention will be described in detail below through several specific embodiments.

[0200] Specific Example 19: This specific example involves introducing polyether-modified polydimethylsiloxane to regulate the self-assembly of amphiphilic block copolymers into a biomimetic membrane, including the following steps:

[0201] 1) Prepare a nonpolar polymer film solution by adding the triblock polymer HO-PMOXA6-PDMS. 44 -PMOXA6-OH was dissolved in silicone oil AR20 to obtain a polymer film solution of 10 mg / mL, and 2% polyether-modified polydimethylsiloxane was added;

[0202] 2) Preparation of polar solution: Use a buffer solution with a pH of 7.5 containing 625mM KCl, 10mM HEPES, and 5mM EDTA;

[0203] 3) Nanopore sequencing chip preparation: The nanopore sequencing chip is pretreated by coating 2 μL of nonpolar polymer film solution onto the surface of the micropores of the nanopore sequencing chip and baking it on a hot plate for 3 min.

[0204] 4) Pass the polar solution prepared in step 2) into the microfluidic chip;

[0205] 5) Biomimetic membrane formation: The nonpolar amphiphilic triblock copolymer membrane solution prepared in step 1) and regulated by organosilicon surfactant is added to the microporous channel of the nanopore sequencing chip. After stabilization for 15 minutes (when the transmembrane current is stable and basically without fluctuation), a polar solution is added. A polar-nonpolar-polar system is formed in the gear structure region of the microporous channel of the nanopore sequencing chip. The amphiphilic triblock copolymer self-assembles into an amphiphilic triblock copolymer biomimetic membrane, and the membrane formation process is completed.

[0206] The film-forming effect of the prepared amphiphilic triblock copolymer biomimetic membrane was detected by electrical characterization of the membrane capacitance. Generally, a suitable membrane thickness is more suitable for the embedding of biological protein pores and the stability testing of biological protein pores, with a corresponding membrane capacitance value of 15pF-60pF, and an even better value of 20pF-50pF.

[0207] Polyether-modified polydimethylsiloxane regulated block copolymer (HO-PMOXA6-PDMS) 44 The membrane current signal diagram of the biomimetic membrane formed by the self-assembly of PMOXA6-OH is shown in the figure below. Figure 2 As shown. Figure 2 The current signal graph of this biomimetic membrane shows that, within a monitoring period of 0–500 s, the current signal of the biomimetic membrane exhibits slight fluctuations near the 0 nA baseline, with the overall amplitude remaining stable within ±20 pA, without significant drift or step changes, reflecting the characteristics of the block copolymer (HO-PMOXA6-PDMS). 44 The membrane structure of the biomimetic membrane formed by the self-assembly of PMOXA6-OH has good stability and anti-interference ability.

[0208] The film formation effect is detected by electrical characterization of the membrane capacitance. Each structural unit has a first electrode at its bottom, which can contact a polar solvent (polar solvent-nonpolar solvent-polar solvent) and a second electrode at the other end of the nanopore sequencing chip structure, away from the bottom of the structural unit. This second electrode can contact another polar solvent. Therefore, each structural unit is actually a membrane capacitor, and the electrical characterization will differ depending on the thickness of the film layer. The capacitance value can characterize the state of different micropores, such as whether a film has formed and the thickness of the formed film layer.

[0209] The results showed that the amphiphilic block copolymer PMOXA6-PDMS, which was self-assembled by organosilicon surfactant polyether modified polydimethylsiloxane, was effective. 44 The PMOXA6 biomimetic membrane has a membrane capacitance range of 15-60 pF, a membrane number of 206, high membrane quality, consistent membrane uniformity, good stability, and can embed biological nanoporous proteins for sequencing.

[0210] The structural formula of polyether-modified polydimethylsiloxane is as follows:

[0211] .

[0212] Specific Example 20: This specific example introduces polyether-modified heptamethyltrisiloxane to regulate the self-assembly of amphiphilic block copolymers into a biomimetic membrane. The difference between Specific Example 20 and Specific Example 19 is the addition of a different organosilicon surfactant to the amphiphilic block copolymer membrane solution; in this Specific Example 20, the organosilicon surfactant is polyether-modified heptamethyltrisiloxane. The membrane formation steps for self-assembling the amphiphilic triblock copolymer biomimetic membrane onto the nanopore sequencing chip are the same as in Specific Example 19.

[0213] The results showed that PMOXA6-PDMS, an amphiphilic triblock copolymer whose self-assembly was regulated by organosilicon surfactant polyether-modified polydimethylsiloxane, was effective. 44 The PMOXA6 biomimetic membrane has a membrane capacitance range of 15-60 pF, a membrane number of 175, high membrane quality, consistent membrane uniformity, good stability, and can embed biological nanoporous proteins for sequencing.

[0214] The chemical structural formula of the polyether-modified heptamethyltrisiloxane is as follows:

[0215] .

[0216] Specific Example 21: This specific example introduces polyether-modified silicone oil to regulate the self-assembly of amphiphilic block copolymers into a biomimetic membrane. The difference between Specific Example 21 and Specific Example 19 is the addition of a different organosilicon surfactant to the amphiphilic triblock copolymer membrane solution; in this Specific Example 21, the organosilicon surfactant is polyether-modified silicone oil. The membrane formation steps for the self-assembly of the amphiphilic block copolymer biomimetic membrane on the nanopore sequencing chip are the same as in Specific Example 19.

[0217] The results showed that PMOXA6-PDMS, an amphiphilic triblock copolymer whose self-assembly was regulated by organosilicon surfactant polyether-modified polydimethylsiloxane, was effective. 44 The PMOXA6 biomimetic membrane has a membrane capacitance range of 15-60 pF, a membrane number of 183, high membrane quality, consistent membrane uniformity, good stability, and can embed biological nanoporous proteins for sequencing.

[0218] The structural formula of polyether-modified silicone oil is as follows:

[0219] .

[0220] Comparative Example 1:

[0221] The difference between Comparative Example 1 and Specific Examples 19, 20, and 21 is that Comparative Example 1 did not add an organosilicon surfactant to the amphiphilic block copolymer membrane solution, while the membrane formation steps for self-assembling an amphiphilic block copolymer biomimetic membrane on a nanopore sequencing chip were the same as in Specific Example 19.

[0222] 1) Prepare a nonpolar polymer film solution by adding the triblock polymer HO-PMOXA6-PDMS. 44 -PMOXA6-OH dissolved in silicone oil AR20 to obtain a polymer membrane solution of 10 mg / mL;

[0223] 2) Preparation of polar solution: Use a buffer solution with a pH of 7.5 containing 625mM KCl, 10mM HEPES, and 5mM EDTA;

[0224] 3) Nanopore sequencing chip preparation: The nanopore sequencing chip is pretreated by coating 2 μL of nonpolar polymer film solution onto the surface of the micropores of the nanopore sequencing chip and baking it on a hot plate for 3 min.

[0225] 4) Pass the polar solution prepared in step 2 into the nanopore sequencing chip.

[0226] 5) Biomimetic membrane formation: The nonpolar amphiphilic block copolymer membrane solution prepared in step 1 and regulated by organosilicon surfactant is added to the microporous channel of the nanopore sequencing chip. After stabilization for 15 minutes (when the transmembrane current is stable and basically without fluctuation), a polar solution is added. A polar-nonpolar-polar system is formed in the gear structure region of the microporous channel of the nanopore sequencing chip. The amphiphilic polymer self-assembles into an amphiphilic block copolymer biomimetic membrane, and the membrane formation process is completed.

[0227] Then, the stability of the biomimetic membrane formed by the self-assembly of amphiphilic block copolymers was tested on the nanopore sequencing chip.

[0228] Specific embodiments 19, 20, and 21 of the present invention, and comparative embodiment 1, utilize organosilicon surfactants to regulate PMOXA6-PDMS in a nanopore sequencing chip. 44 The test results of the biomimetic membrane formed by the PMOXA6 amphiphilic block copolymer and the membrane stability test are shown in Table 2.

[0229] Table 2 Comparative test results of specific embodiments 19-21 and comparative embodiment 1

[0230]

[0231] The test results in Table 2 show that, compared with Example 1, the amphiphilic block copolymer PMOXA6-PDMS 44The PMOXA6 biomimetic membrane has a low number of membrane elements (only 92) and is relatively thick, with a membrane capacitance ranging from 10-40 pF (generally, membrane thickness is inversely proportional to membrane capacitance). Furthermore, this biomimetic membrane exhibits poor density and significant leakage, with a 90-day membrane shedding rate as high as 45.65%. In contrast, the amphiphilic triblock copolymer modified with organosilicon surfactants exhibits a high membrane formation rate that remains essentially constant over time, even at 90 days, and still allows for the embedding of biological nanoporous proteins. Simultaneously, the membrane capacitance is between 15-60 pF, and the 90-day membrane shedding rate is less than 7%, while the 90-day membrane shedding rate of polyether-modified polydimethylsiloxane-modified membrane is less than 2.5%.

[0232] A comparison of specific embodiments 19, 20, and 21 shows that the added organosilicone surfactant can effectively promote the amphiphilic triblock copolymer PMOXA6-PDMS. 44 PMOXA6 self-assembles into a biomimetic membrane on the micropores of a nanopore sequencing chip. The biomimetic membrane exhibits high quality, uniformity, stability, and mechanical strength, with a membrane capacitance range of 15-60 pF. Specifically, the biomimetic membranes prepared in Examples 19, 20, and 21 (membrane capacitance range 15-60 pF) are thinner than the biomimetic membrane in Comparative Example 1 (membrane capacitance range 10-40 pF), making them more suitable for embedding biological protein nanopores. Furthermore, among all organosilicon surfactant-regulated amphiphilic block copolymer biomimetic membranes, the triblock copolymer (HO-PMOXA6-PDMS) regulated by polyether-modified polydimethylsiloxane stands out. 44 The self-assembly of PMOXA6-OH into a biomimetic membrane exhibits superior film-forming properties.

[0233] Furthermore, this invention utilizes organosilicon surfactants to regulate the amphiphilic block copolymer PMOXA6-PEO in nanopore sequencing chips. 33 -PMOXA6 and PMOXA4-PLA 21 Examples and comparative examples of biomimetic films formed from PMOXA4, with specific film formation methods the same as those for the amphiphilic block copolymer PMOXA6-PDMS. 44 -PMOXA6 (specific example 19), the biomimetic membrane test results and membrane stability test results are shown in Tables 3 and 4.

[0234] Table 3 Comparative test results of specific embodiments 22-24 and comparative embodiment 2

[0235]

[0236] Table 4 Comparative test results of specific embodiments 25-27 and comparative embodiment 3

[0237]

[0238] As can be seen from Tables 3 and 2, the addition of organosilicon surfactants can also effectively promote the amphiphilic triblock copolymer PMOXA6-PEO. 33 -PMOXA6 and PMOXA4-PLA 21 PMOXA4 self-assembles into a biomimetic membrane on the micropores of a nanopore sequencing chip. Amphiphilic triblock copolymer PMOXA6-PDMS 44 The intermediate segments of PMOXA6 are structurally similar to those of organosilicon surfactants, thus enabling superior biomimetic film formation and resulting in a higher film-forming rate than other amphiphilic triblock copolymers such as PMOXA6-PEO. 33 -PMOXA6 and PMOXA4-PLA 21 -PMOXA4, and the film-forming effect of the triblock copolymer self-assembly into a biomimetic film by organosilicon surfactant polyether modified polydimethylsiloxane is relatively excellent.

[0239] Stability testing of biomimetic membranes formed by the self-assembly of amphiphilic block copolymers (PMOXA6-PDMS) on nanopore sequencing chips was performed. 44 The PMOXA6 self-assembled membrane exhibits high quality, uniformity, and stability. The triblock copolymer membrane stabilizes for approximately 90 days, significantly longer than in Comparative Examples 2 and 3 where no silicone surfactant was added to the amphiphilic block copolymer membrane solution. Even after 90 days, the triblock copolymer membrane, regulated by the silicone surfactant, remains suitable for embedding biological proteins into nanopores, making it suitable for biosensing applications (such as nanopore gene sequencing, drug molecule detection, and protein detection). In contrast, the membrane shedding rate in Comparative Examples 2 and 3 increased to approximately 60% by day 90 with increasing storage time.

[0240] To verify that the silicone surfactant was indeed introduced into the amphiphilic block copolymer self-assembled biomimetic membrane, the water contact angle (WCA) of the biomimetic membranes prepared in the specific examples 19-27 and comparative examples 1-3 was used for characterization. Specifically, droplets with a volume of approximately 1-5 nL were directly dropped into the micropores of the nanopore sequencing chip for testing. The test results are shown in Table 5. When the silicone surfactant was introduced, the membrane surface energy decreased, and the water contact angle increased significantly. The larger the contact angle, the stronger the hydrophobicity.

[0241] Table 5. Contact angle test results of biomimetic films formed by introducing organosilicon surfactants into block copolymers.

[0242]

[0243] As shown in Table 5, the contact angle values ​​of the biomimetic membranes prepared in Examples 19-27 after the introduction of the organosilicon surfactant are significantly greater than those in Comparative Examples 1-3 without the addition of the organosilicon surfactant. This indicates that the organosilicon surfactant was successfully introduced into the self-assembled biomimetic membrane of the amphiphilic block copolymer and changed the surface properties of the biomimetic membrane, enhancing its hydrophobicity. Specifically, for the same triblock copolymer, the contact angle values ​​of the biomimetic membranes prepared with different organosilicon surfactants differed. Among them, the biomimetic membrane prepared by polyether-modified polydimethylsiloxane had a relatively large contact angle value (116°), further demonstrating its excellent film-forming control effect, which is consistent with the film formation rate and stability test results. This indicates that appropriate hydrophobicity is conducive to the embedding of biological proteins and improves the sensitivity and stability of detection.

[0244] In summary, the organosilicon surfactant introduced in this invention can effectively promote the self-assembly of amphiphilic triblock copolymers into biomimetic membranes on the micropores of nanopore sequencing chips. The membrane formation rate and quality are high, the biomimetic membrane has good uniformity and stability, and the membrane shedding rate is extremely low. It can be maintained for 90 days, and even after 90 days of membrane formation, it can still maintain a state suitable for the embedding of biological nanopore proteins.

[0245] Specific Example 28: An amphiphilic triblock copolymer biomimetic membrane is used to anchor molecules into biological protein nanopores via molecular membrane anchoring, and sequencing is performed on a nanopore sequencing chip (256 micropore structures) (pore embedding test). The specific steps are as follows:

[0246] S1: First, prepare and synthesize the anchoring molecule polydimethylsiloxane bis(4-aminobutyric acid) ester. The synthetic route of the anchoring molecule is as follows: Figure 3 As shown, the specific steps are as follows:

[0247] 1) Add di-tert-butyl dicarbonate (Boc2O), add 0.15 g (1.50 mmol) of amino-Bocated 4-aminobutyric acid, 4.30 g (1.00 mmol) of hydroxyl-terminated polydimethylsiloxane (HO-PDMS-OH) (Mw4300) and 15 mL of dichloromethane to a three-necked flask, and stir until completely dissolved; then add 0.045 g (0.368 mmol) of condensing agent N,N'-dicyclohexylcarbodiimide (DCC) and 0.045 g (0.218 mmol) of catalyst 4-dimethylaminopyridine (DMAP) and react for 15 min;

[0248] 2) The amino-Boc-modified 4-aminobutyric acid obtained in step 1) was slowly added dropwise, generating a large amount of white solid. Then, the mixture was stirred at 40°C for 4 hours.

[0249] 3) Add trifluoroacetic acid (concentration 50%-100%) to the solution in step 2) to remove the Boc group of polydimethylsiloxane bis(4-aminobutyric acid) ester;

[0250] 4) The solution obtained in step 3) was filtered under reduced pressure in a glass funnel, and the filtrate was collected. Vacuum rotary evaporation yielded a white, viscous product, which is polydimethylsiloxane bis(4-aminobutyric acid) ester. The product from this stage was purified as follows: the crude product was dissolved in dichloromethane, and subjected to silica gel column chromatography (eluent: dichloromethane / methanol = 10:1). The target fraction was collected, the solvent was removed by rotary evaporation, and the product was dried under vacuum to obtain the target product. NMR was performed at room temperature using a 600MHz NMR spectrometer, with tetramethylsilane (TMS) as an internal standard, and the sample dissolved in deuterated chloroform. Figure 8 The image shows the 1H NMR spectrum of the anchoring molecule, polydimethylsiloxane bis(4-aminobutyric acid) ester. Analysis 1 The H-NMR test results showed that the silane hydrogen corresponded to the chemical shift at position a (0 ppm) with an integral of 379, the amino hydrogen corresponded to the chemical shift at position e (1.5 ppm) with an integral of 4, and the other alkyl hydrogen protons b, c, and d also corresponded, indicating that the target product structure was correct. After confirming that the target product, polydimethylsiloxane bis(4-aminobutyric acid) ester, had been obtained, the next step of the experiment was carried out.

[0251] The structural formula of the anchoring molecule polydimethylsiloxane bis(4-aminobutyric acid) ester is:

[0252] ;

[0253] Where m is the degree of polymerization;

[0254] S2: The steps for adding anchoring molecules into the biomimetic membrane of an amphiphilic block copolymer via a molecular membrane anchoring method, embedding them into the biological protein nanopores, and then sequencing are as follows:

[0255] 1) Following the method in Specific Example 19, an amphiphilic triblock copolymer (HO-PMOXA6-PDMS) was self-assembled on a nanopore sequencing chip. 44 -PMOXA6-OH) biomimetic film, biomimetic film capacitance 15-60pF, biomimetic film is relatively thick;

[0256] 2) After the amphiphilic block copolymer has self-assembled into a stable biomimetic membrane (after 15 minutes of self-assembly into a biomimetic membrane, the transmembrane current is stable with almost no fluctuations), add the anchoring molecule polydimethylsiloxane bis(4-aminobutyric acid) ester, followed by adding 0.4 μL of a nanoporous protein (e.g., α-Hemolysin, MspA, or CsgG) solution diluted 100-200 W times with buffer. The protein concentration is usually lower than that of the block copolymer membrane.

[0257] 3) By applying an embedding voltage of 280mV to both ends of the amphiphilic polylactic acid block copolymer biomimetic membrane, nanoscale protein pores (referred to as nanopores, such as...) can be embedded in the amphiphilic polylactic acid block copolymer biomimetic membrane. Figure 4 (As shown). After embedding MspA nanopores, the membrane state fluctuated slightly but generally tended to stabilize. After 30 minutes, the applied voltage was stopped, and the number of embedded protein nanopores (embedding number) was recorded. The opening current was below 400 pA. The data are recorded in Table 6.

[0258] 4) Change the sequencing buffer (500mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0), add 4uL of library and start sequencing. The sequencing signal is as follows. Figure 5 As shown, after a stable sequencing signal is output, the trend of stable sequencing signal output and the proportion of sequencing channels with sequencing time is recorded. Figure 7 As shown.

[0259] Figure 5 This study demonstrates that after the biomimetic nanoporous protein is embedded in the biomimetic membrane, the nucleic acid molecules in the library undergo current changes as they pass through the nanopores, exhibiting baseline fluctuations and characteristic current jumps, enabling high-throughput sequencing at the single-molecule level.

[0260] After multiple membrane capacitance and sieve pore tests, the MspA nanopores remained stable on the amphiphilic block copolymer biomimetic membrane for more than 60-90 days, with high membrane pore matching. Furthermore, the biomimetic membrane is dense, uniform, has high mechanical strength, and is non-leakage, allowing the nanopore sequencing chip to perform continuous and stable sequencing for more than 2 days.

[0261] Regarding the accuracy of the sequencing results, after comparison with known sequences, it was found that sequencing using the amphiphilic block copolymer biomimetic membrane embedded with nanoporous proteins has a high accuracy rate, reaching over 95%.

[0262] Regarding reproducibility experiments, the steps of Specific Example 28 were performed multiple times, and the biomimetic membranes prepared each time showed similar performance in the pore-filling test and sequencing experiment, indicating that the preparation method and sequencing process have good reproducibility.

[0263] Meanwhile, experiments were conducted on anchoring molecules polydimethylsiloxane bis(4-aminobutyric acid) ester with different degrees of polymerization. It was found that when the polymerization degree was too low (m < 12), the end groups were exposed more and the initial anchoring efficiency of biomimetic membrane bio-nanoporin was high, but the binding with the membrane was weak and the stability was poor. Medium degree of polymerization (m 12-44) has good flexibility and the best balance between anchoring efficiency and stability. High degree of polymerization (m > 44) is prone to entanglement and end group blockage, resulting in low anchoring efficiency of biomimetic membrane bio-nanoporin and easy damage to the membrane structure.

[0264] This invention designs and prepares biomolecular nanoporous protein anchoring molecules on amphiphilic block copolymer biomimetic membranes. The PDMS backbone provides strong hydrophobicity, making it suitable for embedding in hydrophobic surfaces (such as lipid membranes or solid nanopores). PDMS achieves surface functionalization through chemical grafting, such as aldehyde modification, for use in biochips. The bis(4-aminobutyric acid) ester side chain -amino group can be coupled to the target molecule via EDC / NHS coupling or electrochemical bonding. Amino silicone oil is anchored through hydrogen bonding and electrostatic interactions. The PDMS surface is coupled with antibodies and bioprotein molecules via amino groups.

[0265] When the amphiphilic block copolymer biomimetic membrane provided by this invention is used for nanopore sequencing, the size of its core hydrophobic block can match the size of the nanopore protein, thereby enabling the nanopore to be embedded in it and ensuring that the nanopore can exist stably for a long time, extending the sequencing time and enabling stable sequencing.

[0266] Comparative Example 4: Biological protein nanopores were directly embedded in an amphiphilic block copolymer biomimetic membrane without anchoring molecules and sequenced. The difference between Comparative Example 2 and Specific Example 28 is that in Comparative Example 2, after self-assembling into an amphiphilic block copolymer biomimetic membrane, no anchoring molecules were added; that is, no molecular membrane anchoring method was used. Instead, nanoporous proteins (e.g., α-Hemolysin, MspA, or CsgG solution) were directly embedded. The specific steps are as follows:

[0267] 1) Following the method in Specific Example 19, an amphiphilic block copolymer (HO-PMOXA6-PDMS) was self-assembled into a nanopore sequencing chip. 44 -PMOXA6-OH) biomimetic film, biomimetic film capacitance 15-60pF, biomimetic film is relatively thick.

[0268] 2) After the amphiphilic block copolymer self-assembles into a biomimetic membrane and stabilizes (after 15 minutes of self-assembly into a biomimetic membrane, the transmembrane current is stable with almost no fluctuation), add 0.9 μL of nanoporous protein (a-Hemolysin or MspA or CsgG solution) diluted 100-200W times with buffer. Usually, the protein concentration should be lower than that of the block copolymer membrane.

[0269] 3) An embedding voltage of 380 mV was applied to both ends of the amphiphilic polylactic acid block copolymer biomimetic membrane, which allowed for the embedding of nanoscale protein pores (referred to as nanopores) into the membrane. After embedding the MspA nanopores, the membrane state fluctuated slightly but generally stabilized. The voltage was stopped after 30 minutes, and the number of embedded protein nanopores (embedding number) was recorded. The opening current was approximately 400 pA. The data are recorded in Table 6.

[0270] 4) Change the sequencing buffer (500mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0), add 4uL of library and start sequencing. The real-time current signal during sequencing is as follows: Figure 6 As shown, there is a stable sequencing signal output, and the trend of the sequencing channel ratio changing with sequencing time is statistically analyzed. Figure 7 As shown. Figure 6 This is a real-time monitoring graph of the current signal of the nanopore sequencing chip. The baseline current fluctuates steadily in the first half, while the signal in the second half shows a significant increase accompanied by a stepwise decrease. This indicates that the characteristic change in current caused by the blockage of the pore after the nucleic acid chain fragments of the library enter the nanopore reflects the real-time current fluctuations during the sequencing process.

[0271] Table 6 Comparison results between specific embodiment 28 and comparative embodiment 4

[0272]

[0273] After multiple membrane capacitance and sieve pore tests, the MspA nanopores remained stable on the amphiphilic block copolymer biomimetic membrane for more than 200 days, with high membrane pore matching. Furthermore, the biomimetic membrane is dense, uniform, has high mechanical strength, and is non-leakage. The nanopore sequencing chip can continuously and stably sequence for more than 2 days (206 membranes).

[0274] like Figure 7 As shown, the comparison between Specific Example 28 and Comparative Example 4 demonstrates that adding anchoring molecules via molecular membrane anchoring to the biomimetic membrane self-assembled from amphiphilic block copolymers improves the embedding rate of biomimetic nanoporous proteins into the amphiphilic triblock copolymer biomimetic membrane, saving embedding time and the amount of biomimetic protein used. Furthermore, it increases the stability of the biomimetic protein nanopores on the amphiphilic block copolymer biomimetic membrane and extends the sequencing lifetime of the nanopore sequencing chip. Simultaneously, the addition of anchoring molecules significantly reduces the pore loss rate. Within 48 hours, the pore loss rate of Specific Example 28 was only 6.33%, while the pore loss rate of Comparative Example 4 was as high as 47.37%. This fully demonstrates the importance of anchoring molecules in enhancing the interaction between biomimetic nanoporous proteins and the biomimetic membrane. This interaction not only improves the stability of the nanopores but also reduces sequencing interruptions caused by nanopore detachment, thereby improving the continuity and reliability of sequencing.

[0275] In summary, the addition of anchoring molecules to amphiphilic block copolymer biomimetic membranes via molecular membrane anchoring not only optimizes the embedding process of biological nanoporous proteins but also significantly improves the performance and stability of nanopore sequencing.

[0276] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0277] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. An amphiphilic block copolymer self-assembled biomimetic membrane, characterized in that, The biomimetic membrane is formed by the self-assembly of film-forming molecules; the film-forming molecules include an amphiphilic triblock copolymer, the configuration of which is hydrophilic segment A-hydrophobic segment B-hydrophilic segment A; and the amphiphilic triblock copolymer is modified by an organosilicon surfactant and then self-assembled into an amphiphilic triblock copolymer biomimetic membrane. The concentration of the organosilicon surfactant in the amphiphilic triblock copolymer solution is 1-5%; The silicone surfactant includes one or more of the following types: polyether type, carboxyl / sulfonic acid type, and amino / quaternary ammonium salt type; The amphiphilic structure of the organosilicon surfactant acts as a "bridge" to eliminate the polar repulsion between the substrate and the polymer, enabling the triblock polymer to adhere stably to the substrate surface. The amphiphilic triblock copolymer is PMOXA. n -PDMS m -PMOXA n Or PMOXA n -PEO m -PMOXA n Or PMOXA n -PLA m -PMOXA n The weight-average molecular weight is 3500-10000 Da, the degree of polymerization of the hydrophilic segment A is n; the degree of polymerization of the hydrophobic segment B is m, m+n≤55 and the ratio of n:m is 1:(6-8).

2. The amphiphilic block copolymer self-assembled biomimetic membrane of claim 1, wherein, When the organosilicon surfactant is a polyether type, it includes polyether-modified polydimethylsiloxane and / or polyether-modified heptamethyltrisiloxane; When the organosilicon surfactant is of the carboxyl / sulfonic acid type, it includes carboxypropyl polydimethylsiloxane and / or sulfonic acid-modified polysiloxane; When the organosilicon surfactant is of the amino / quaternary ammonium salt type, it includes aminopropyl polydimethylsiloxane and / or quaternized polysiloxane.

3. Use of the amphiphilic block copolymer self-assembled biomimetic membrane according to claim 1 or 2 for the preparation of a biosensor, characterized in that, Embedding bio-nanoporin into a biomimetic membrane; specifically, using a molecular membrane anchoring method to promote the embedding of bio-nanoporin into the biomimetic membrane by anchoring molecules; wherein, the anchoring molecule is an aptamer-long siloxane chain structure; The anchoring molecule is polydimethylsiloxane bis(4-aminobutyric acid) ester, whose general structural formula is: H2N-(CH2)3-C(=0)-0-(CH2)3-[Si(CH3)2-0] n -Si(CH3)2-(CH2)3-O-C(=0)-(CH2)3-NH2, n is 12-44.

4. The use according to claim 3, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The specific steps of the molecular membrane anchoring method are as follows: S1 Preparation of anchoring molecules: Hydroxyl-terminated polydimethylsiloxane and 4-aminobutyric acid or its active ester derivatives are used as raw materials, and di-tert-butyl dicarbonate is used as an amino protecting agent; a condensing agent is added, and an esterification reaction is carried out in an organic solvent to generate polydimethylsiloxane bis(4-aminobutyric acid) ester. S2 embedded in bio-nanoporin: S21: First, a nanopore sequencing chip is used to self-assemble into an amphiphilic triblock copolymer biomimetic membrane; S22: Then, 10-15 minutes after the amphiphilic triblock copolymer in step S21 has self-assembled into a biomimetic membrane, add anchoring molecules and a biological nanoporous protein solution; then embed the nanoporous protein into the amphiphilic triblock copolymer self-assembled biomimetic membrane by voltage driving, autonomous fusion or protein vesicle fusion.

5. The use according to claim 4, wherein the compound is ###0002### The specific steps of step S21 are as follows: S211: Dissolve the triblock polymer in a non-polar solvent to obtain a polymer membrane solution, and add an organosilicon surfactant at a concentration of 1-5% of the polymer membrane solution to obtain a non-polar polymer membrane solution. S212: Prepare a polar solution according to the specified proportion; S213: After pretreatment of the nanopore sequencing chip, the polar solution prepared in step S212 is first introduced into the nanopore sequencing chip, and then the non-polar polymer membrane solution prepared in step S211 is added into the micropore channels of the nanopore sequencing chip. After waiting for 15-20 minutes, the polar solution is added, thereby forming a polar-nonpolar-polar system in the micropore channels of the nanopore sequencing chip, so that the amphiphilic polymer self-assembles into an amphiphilic triblock copolymer biomimetic membrane, and the membrane formation process is completed.

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