Biosensor based on tethered phospholipid bilayer membrane as well as preparation method and application of biosensor
By forming phase-separated hydrophilic and hydrophilic mixed monolayers and cholesterol-doped active lipid molecular layers on solid substrates, the restricted fluidity and complex labeling process in the construction of ion channel protein lipid bilayers in the prior art are solved. The problem of this paper realizes stable and adaptable biosensors to a variety of characterization technologies, demonstrating excellent electrical performance and long-term stability.
Patent Information
- Application Number
- CN202411378722.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2024-12-31
AI Technical Summary
In the prior art, the construction of ion channel proteins has problems such as limited protein fluidity, complex histidine tag labeling process and lack of ion reservoirs, making it difficult to maintain the natural activity and adaptation of ion channel proteins. Characterization technology.
Using a solid substrate with a gold film on the surface, a phase-separated hydrophilic and mixed monolayer is formed by self-assembly, and a cholesterol-doped active lipid molecular layer is spread thereon, using hydrophobic alkyl mercaptan compounds and hydrophilicity. Thiodo compounds fix the active lipid molecular layer to form a stable phospholipid bilayer membrane biosensor, adapted to a variety of characterization technologies.
It has achieved stable and adapted to a variety of characterization technologies, which can accommodate and maintain the activity of ion channel proteins, exhibit excellent electrical performance and long-term stability, and is suitable for the study of membrane proteins or ion channel proteins.
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Figure CN119224088A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biosensors, and in particular relates to a biosensor based on a tethered phospholipid bilayer membrane and a preparation method and application thereof. Background Art
[0002] As an important component of the cell membrane, ion channel proteins are essential for maintaining a variety of key biological functions of cells. They regulate the difference in ion concentration inside and outside the cell by promoting ion-specific transmembrane transport, thereby participating in a variety of physiological processes. At present, the methods for studying ion channel proteins mainly include genetic engineering technology, patch clamp technology, and in situ spectroelectrochemical technology. These methods can help scientists gain a deeper understanding of the structure, function, and regulatory mechanism of ion channel proteins. In order to maximize their original biological functions, a device needs to be designed to better simulate their natural biological environment.
[0003] One strategy in the prior art is to attach dissolved proteins to appropriately functionalized metal surfaces via histidine tags, and then use an active agent-lipid exchange strategy to reconstruct a lipid bilayer around the immobilized proteins. However, the devices constructed using this strategy have the following three major problems: (1) the mobility of proteins is severely restricted in these protein-tethered lipid bilayers; (2) the process flow for expressing and purifying proteins with histidine tags is relatively complicated and there are certain technical barriers; (3) there is a lack of ion reservoirs that can accommodate ions. Summary of the invention
[0004] The present invention aims to solve the technical problems in the prior art and provide a biosensor based on a tethered phospholipid bilayer membrane and its preparation method and application. The biosensor of the present invention has a simple construction process and stable properties, can be used to accommodate and maintain the natural activity of ion channel proteins, and is suitable for a variety of characterization techniques.
[0005] In order to solve the above technical problems, the technical solutions of the present invention are as follows:
[0006] A biosensor based on a tethered phospholipid bilayer membrane, comprising:
[0007] A solid substrate coated with a gold film;
[0008] a phase-separated hydrophilic-hydrophobic mixed monolayer self-assembled on the solid substrate;
[0009] An alternating single and double active lipid molecular layer doped with cholesterol molecules Chol spread on the hydrophilic and hydrophobic mixed monolayer;
[0010] The hydrophobic alkylthiol compound in the hydrophilic-hydrophobic mixed monolayer is an anchoring molecule for fixing the active lipid molecule layer; the hydrophilic thiol compound in the hydrophilic-hydrophobic mixed monolayer is a spacer molecule for ensuring the formation of an ion reservoir;
[0011] The active lipid molecule layer is used to provide an active biomimetic interface;
[0012] The cholesterol molecule Chol doped in the active lipid molecule layer is used to increase the stability of the biosensor.
[0013] In the above technical solution, preferably, the hydrophobic alkyl mercaptan compound is one of n-decyl mercaptan, n-dodecyl mercaptan (DT), n-tetradecyl mercaptan, n-hexadecyl mercaptan and n-octadecyl mercaptan (ODT).
[0014] In the above technical solution, preferably, the hydrophilic thiol compound is one of mercaptoethanol (ME), mercaptoethylammonium (CA), mercaptopropionic acid (MPA) and n-thio-β-D-glucose (β-Tg).
[0015] In the above technical scheme, preferably, the lipid molecules in the active lipid molecule layer are a mixture of one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylglycerol (PG), and the charge diversity of the lipid molecules can quantitatively regulate the membrane potential of the biosensor.
[0016] A method for preparing a biosensor based on a tethered phospholipid bilayer membrane comprises the following steps:
[0017] Step S1, preparing a phase-separated hydrophilic-hydrophobic mixed monolayer by substitution adsorption or co-adsorption;
[0018] The steps for preparing a phase-separated hydrophilic-hydrophobic mixed monolayer by substitution adsorption are as follows:
[0019] At room temperature, a solid substrate coated with a gold film is immersed in an ethanol solution of a hydrophilic thiol compound, and a hydrophilic monolayer is formed on the solid substrate through Au-S self-assembly; after the assembly is completed, the free hydrophilic thiol compound molecules are rinsed with ethanol and blown dry for standby use; then, the solid substrate modified with the hydrophilic thiol compound is immersed in an ethanol solution of a hydrophobic alkylthiol compound, and a phase-separated hydrophilic-hydrophobic mixed monolayer is formed on the solid substrate through replacement adsorption;
[0020] The steps for preparing a phase-separated hydrophilic-hydrophobic mixed monolayer by co-adsorption are as follows:
[0021] At room temperature, a solid substrate coated with a gold film is immersed in a mixed solution of a hydrophobic alkylthiol compound and a hydrophilic thiol compound, and a phase-separated hydrophilic-hydrophobic mixed monolayer is formed on the solid substrate through Au-S self-assembly;
[0022] Step S2, preparing active lipid molecules doped with cholesterol molecules Chol by thin film hydration ultrasound method;
[0023] Step S3, spreading the active lipid molecules doped with cholesterol molecules Chol prepared in step S2 on the hydrophilic-hydrophobic mixed monolayer formed in step S1 in an aqueous solution by means of vesicle fusion, and incubating at room temperature to form an alternating single- and double-layer active lipid molecule layer doped with cholesterol molecules Chol, thereby constructing the biosensor;
[0024] Step S4: repeatedly washing the biosensor constructed in step S3 with an aqueous solution to wash away the adsorbed and free lipid molecules.
[0025] In the above technical solution, preferably, in step S1, when the phase-separated hydrophilic-hydrophobic mixed monolayer is prepared by substitution adsorption, the concentrations of the hydrophilic thiol compound and the hydrophobic alkylthiol compound are both 0.5-2 mmol·L -1 .
[0026] In the above technical solution, it is further preferred that in step S1, when the phase-separated hydrophilic-hydrophobic mixed monolayer is prepared by replacement adsorption, the adsorption time of the hydrophilic thiol compound is 5 to 15 minutes, and the adsorption time of the hydrophobic alkyl thiol compound is 2 to 3.5 hours.
[0027] In the above technical solution, preferably, in step S1, when the phase-separated hydrophilic-hydrophobic mixed monolayer is prepared by co-adsorption, the total concentration of the mixed solution of the hydrophobic alkylthiol compound and the hydrophilic thiol compound is 0.5-2 mmol·L -1 The molar ratio of the hydrophobic alkylthiol compound to the hydrophilic thiol compound is 0.1 to 0.7; and the adsorption time of the mixed solution is 0.5 to 2 hours.
[0028] In the above technical solution, preferably, the molar ratio of cholesterol molecules Chol to lipid molecules in the active lipid molecules doped with cholesterol molecules Chol in step S2 or S3 is 0.5-1; and the incubation time in step S3 is 1.5-3 hours.
[0029] A biosensor based on a tethered phospholipid bilayer membrane is applied to membrane proteins or ion channel proteins.
[0030] The beneficial effects of the present invention are:
[0031] The biosensor based on the tethered phospholipid bilayer membrane of the present invention comprises a self-assembled amphiphilic molecular monolayer attached to a metal electrode, and a single-double-layer alternating lipid layer formed by the fusion of cholesterol Chol-doped lipid vesicles on the monolayer (the bottom layer of the biosensor is a hydrophilic and hydrophobic monolayer, and when the phospholipid vesicles fuse, a single-double-layer alternating structural lipid layer is formed according to the hydrophilic and hydrophobic interaction). The method can be used to accommodate complete ion channel proteins and has an ion conduction function, and can be adapted to a variety of characterization techniques. The biosensor is very close to the natural environment of the ion channel protein in terms of structure, and exhibits very excellent electrical properties and long-term stability.
[0032] The biosensor based on the tethered phospholipid bilayer membrane of the present invention has many advantages such as high impedance, stable properties, and mass production, and is suitable for multiple characterization techniques such as electrochemistry, patch clamp, and in situ spectroscopy.
[0033] The preparation method of the biosensor based on the tethered phospholipid bilayer membrane of the present invention has good reproducibility in the preparation process, simple operation process, and can be mass-produced. The obtained biosensor can be widely used in the research of membrane proteins or ion channel proteins. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.
[0035] Figure 1 The chemical structures of hydrophobic alkylthiol compounds and hydrophilic thiol compounds are shown in FIG.
[0036] Figure 2 The chemical structure diagram of the head group of lipid molecules (phosphatidylcholine PC, phosphatidylethanolamine PE and phosphatidylserine PS, phosphatidylglycerol PG);
[0037] Figure 3 It is a schematic structural diagram of a biosensor based on a tethered phospholipid bilayer membrane of the present invention;
[0038] Figure 4 This is the infrared characterization image of the MPA / ODT-PC (Chol) biosensor;
[0039] Figure 5 It is a phase separation proof diagram of MPA / ODT binary mixed monolayer;
[0040] Figure 6 This is the electrochemical stability characterization diagram of the MPA / ODT-PC (Chol) biosensor;
[0041] Figure 7 This is the infrared characterization image of β-Tg / ODT-PC(Chol) biosensor;
[0042] Figure 8 This is the impedance characterization diagram of β-Tg / ODT-PC (Chol) biosensor;
[0043] Fig. 9 A schematic diagram of the structure of a biosensor containing an ion channel protein;
[0044] Fig.10 This is an infrared characterization diagram of the adsorption process of thiourea-based anion transporter on MPA / ODT-PC (Chol) biosensor;
[0045] Fig.11 Transports NO3 as a thiourea-based anion transporter - Infrared characterization of . DETAILED DESCRIPTION
[0046] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0047] The solid substrate with a gold film on the surface used in the following examples was prepared with reference to the following literature:
[0048] Electrochem.Commun.2002,4(12),973-977.
[0049] The 0.5 mg / mL cholesterol molecule Chol-doped PC vesicle solution used in the following examples was prepared with reference to the following literature:
[0050] J.Phys.Chem.B 2010,114(29),9504-9509.
[0051] Example 1. Preparation of MPA / ODT-PC (Chol) biosensor
[0052] In this example, MPA / ODT-PC (Chol) biosensor was prepared by Au-S self-assembly (co-adsorption) and vesicle fusion. A freshly prepared solid substrate with a gold film on the surface was immersed in a solution with a molar ratio of 8:2 and a final concentration of 0.5 mmol·L -1An ethanol solution of a binary mixture of MPA and ODT was prepared, and the adsorption time was 1 hour. After the formation of a phase-separated amphiphilic self-assembled monolayer (hydrophilic-hydrophobic mixed monolayer), the surface was repeatedly washed with an ethanol solution to remove free thiol molecules, and the surface was dried with a nitrogen gas stream for standby use. Next, the solid substrate pre-modified with a hydrophilic-hydrophobic mixed monolayer was placed in a 0.5 mg / mL cholesterol molecule Chol-doped PC vesicle solution, and the fusion time was 3 hours. After the vesicle fusion was completed, it was carefully washed several times with an aqueous solution until all intact vesicles were washed clean, thereby constructing the MPA / ODT-PC (Chol) biosensor.
[0053] Figure 1 : is the chemical structure diagram of the hydrophobic alkylthiol compound and the hydrophilic thiol compound of the present invention. MPA and ODT are used in this example.
[0054] Figure 2 The chemical structure diagram of the head groups of lipid molecules phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, and phosphatidylglycerol PG. Phosphatidylcholine PC is used in this example.
[0055] Figure 3 It is a schematic diagram of the structure of the biosensor constructed based on the tethered lipid membrane of the present invention.
[0056] Figure 4 This is the infrared characterization of the MPA / ODT-PC (Chol) biosensor in Example 1. Figure 4 The upper middle panel shows that the MPA-ODT binary mixed monolayer has a unique peak at 1715 cm -1 The characteristic peak at 2921cm-1 is attributed to the carbonyl stretching vibration, and the characteristic peak at 2921cm-1 of the ODT molecule -1 The characteristic peak attributable to the asymmetric stretching vibration of methylene indicates the successful construction of the MPA / ODT binary hydrophilic-hydrophobic mixed monolayer. Figure 4 The lower middle panel shows that the characteristic peak of phospholipids was observed immediately after the addition of PC(Chol) vesicles, which appeared at 1092 cm -1 The characteristic peaks attributable to the phosphate groups indicate that the PC(Chol) vesicles are adsorbed and fused on the MPA / ODT mixed monolayer. At the same time, a negative band was observed in the OH stretching region, which was attributed to the adsorption and fusion of the PC(Chol) vesicles to expel the interfacial water from the self-assembled monolayer surface, indicating that the PC(Chol) vesicles were successfully unfolded on the MPA / ODT mixed monolayer.
[0057] The present invention also compares the performance of MPA / ODT-PC (Chol) biosensor and ME / ODT-PC membrane, and the results are as follows:
[0058] Figure 5 This is a diagram demonstrating the phase separation of the MPA / ODT binary mixed monolayer in Example 1. Figure 5 The electrochemical reduction analysis in A shows that the hydrophilic-hydrophobic mixed monolayer formed after MPA / ODT is adsorbed on the gold substrate in a molar ratio of 8:2 has obvious reduction analysis peaks unique to the ODT monolayer and the MPA monolayer, indicating that the formed MPA / ODT binary mixed hydrophilic-hydrophobic monolayer is in a phase separation state. Figure 5 B is the phase separation characterization of the ME / ODT binary mixed hydrophilic-hydrophobic monolayer. The results show that the phase separation trend of the MPA / ODT monolayer is significantly better than that of the ME / ODT monolayer, which may be due to the stronger hydrophilicity of the carboxyl group than the hydroxyl group. The phase-separated hydrophilic-hydrophobic mixed monolayer is the primary condition for the formation of an ion reservoir in the biosensor of the tethered phospholipid bilayer membrane, and is a necessary condition for the study of ion channel proteins. The hydrophilicity of the carboxyl group is usually stronger than that of the hydroxyl group. Therefore, the MPA / ODT-PC (Chol) sensor has a larger and more complete ion reservoir than the ME / ODT-PC membrane, and is more suitable for the study of ion channel proteins.
[0059] Figure 6 This is a characterization diagram of the electrochemical stability of the MPA / ODT-PC (Chol) biosensor in Example 1. Figure 6 A shows that the MPA / ODT-PC (Chol) biosensor exhibits high impedance, similar to the traditional ODT-PC solid-supported mixed phospholipid bilayer membrane. Especially in the positive potential region, its ion leakage current is better than that of the ME / ODT-PC membrane. The incorporation of cholesterol molecules Chol undoubtedly further increases the electrochemical stability of the sensor. Figure 6 B Results show that for the MPA / ODT-PC(Chol) sensor, the capacitance value increases from 1.65 μF cm-1 for the MPA / ODT monolayer to -2 The MPA / ODT-PC(Chol) membrane was reduced to 0.98 μF cm -2 The membrane capacitance value is close to that of natural biological membranes. The above results show that the prepared MPA / ODT-PC (Chol) sensor has the advantages of high impedance and stable properties, and can be used for the study of ion channel proteins.
[0060] Example 2. Preparation of β-Tg / ODT-PC (Chol) biosensor
[0061] In this example, the β-Tg / ODT-PC (Chol) biosensor was prepared by Au-S self-assembly (replacement adsorption) and vesicle fusion. A solid substrate coated with a gold film was immersed in 2 mmol·L -1The β-Tg-modified solid substrate was immersed in an ethanol solution of 1 mmol·L Au-S for 15 minutes to form a hydrophilic monolayer on the gold substrate through Au-S self-assembly. After the assembly was completed, the free β-Tg molecules were rinsed with ethanol and dried for later use. -1 After being placed in an ethanol solution of ODT for 1 hour, a phase-separated hydrophilic-hydrophobic mixed monolayer is formed on a gold substrate by substitution adsorption. After the formation of a phase-separated amphiphilic self-assembled monolayer, the surface is repeatedly washed with an ethanol solution to remove free thiol molecules, and the surface is blown dry with a nitrogen gas stream for standby use. Next, the modified gold film is placed in a 0.5 mg / mL cholesterol Chol-doped PC vesicle solution for 3 hours. After the vesicle fusion is completed, it is carefully washed several times with an aqueous solution until all intact vesicles are washed clean, thereby constructing the β-Tg / ODT-PC (Chol) biosensor.
[0062] Figure 7 This is the infrared characterization of the β-Tg / ODT-PC (Chol) biosensor in Example 2. Figure 7 In A, β-Tg adsorbed on a gold substrate shows a peak at 3259 cm -1 The characteristic peak at OH stretching vibration indicates the successful construction of the β-Tg hydrophilic monolayer. Then, the ethanol solution of hydrophobic ODT was added for replacement adsorption. Figure 7 The characteristic peaks of hydrophobic alkyl chain-methylene are shown in B, indicating the successful construction of β-Tg / ODT hydrophilic-hydrophobic mixed monolayer. Figure 7 C is the adsorption and fusion of PC(Chol) vesicles on the β-Tg / ODT hydrophilic and hydrophobic mixed monolayer. The negative band observed in the OH stretching region is attributed to the adsorption and fusion of PC(Chol) vesicles to expel interfacial water from the self-assembled monolayer surface, indicating that PC(Chol) vesicles are successfully unfolded on the β-Tg / ODT mixed monolayer.
[0063] Figure 8 This is the impedance characterization of the β-Tg / ODT-PC (Chol) biosensor in Example 2. The results show that the β-Tg / ODT-PC (Chol) biosensor has high impedance, and its membrane capacitance value is close to the capacitance value of the natural biological membrane, indicating that the prepared β-Tg / ODT-PC (Chol) sensor has the advantages of high impedance and stable properties, and can be used for the study of ion channel proteins.
[0064] Example 3: Verification of ion conductivity of MPA / ODT-PC (Chol) biosensor - thiourea-based anion transporter
[0065] In this example, 40 μg / mL of thiourea-based anion transport carrier was added to the MPA / ODT-PC (Chol) biosensor constructed in Example 1, and 100 mmol·L -1 NaNO3+10mmol·L -1 The adsorption was incubated in HEPPS solution for 3 hours, and the process was monitored in real time by infrared spectroscopy. After the adsorption was completed, the free and adsorbed ion transporters were carefully washed away with buffer solution. Furthermore, the anions in the solution were exchanged for SO4 by ion exchange. 2- , and the process was monitored in situ using infrared spectroscopy.
[0066] Fig. 9 Schematic diagram of the structure of the biosensor containing the ion transport carrier in Example 3.
[0067] Fig.10 Infrared characterization of the adsorption process of the thiourea-based anion transporter on the MPA / ODT-PC (Chol) biosensor in Example 3. The results show that the thiourea-based anion transporter presents a positive absorption band, while the characteristic absorption peak of the phospholipid molecule presents a negative band, indicating that the thiourea-based anion transporter is successfully inserted into the MPA / ODT-PC (Chol) biosensor to form an ion channel.
[0068] Fig.11 For the thiourea-based anion transporter in Example 3 to transport NO3 - Infrared characterization of the results. The results show that the anion transporter can specifically transport NO3 - , that is, the constructed biosensor has the ability to control the specific selection of ions entering and exiting the membrane.
[0069] The hydrophobic alkylthiol compounds, hydrophilic thiol compounds, lipid molecules used in the above embodiments, and the adsorption time, molar ratio of each substance, incubation time, etc. involved in the preparation process can be arbitrarily adjusted within the scope defined by the present invention, and can achieve technical effects similar to those of the above embodiments, which are not listed here one by one.
[0070] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A biosensor based on a tethered phospholipid bilayer membrane, characterized in that: include: A solid substrate coated with a gold film; a phase-separated hydrophilic-hydrophobic mixed monolayer self-assembled on the solid substrate; An alternating single and double active lipid molecular layer doped with cholesterol molecules Chol spread on the hydrophilic and hydrophobic mixed monolayer; The hydrophobic alkylthiol compound in the hydrophilic-hydrophobic mixed monolayer is an anchoring molecule for fixing the active lipid molecule layer; the hydrophilic thiol compound in the hydrophilic-hydrophobic mixed monolayer is a spacer molecule for ensuring the formation of an ion reservoir; The active lipid molecule layer is used to provide an active biomimetic interface; The cholesterol molecule Chol doped in the active lipid molecule layer is used to increase the stability of the biosensor.
2. The biosensor based on tethered phospholipid bilayer membrane according to claim 1, characterized in that: The hydrophobic alkyl mercaptan compound is one of n-decyl mercaptan (DT), n-dodecyl mercaptan, n-tetradecyl mercaptan, n-hexadecyl mercaptan and n-octadecyl mercaptan (ODT).
3. The biosensor based on tethered phospholipid bilayer membrane according to claim 1, characterized in that: The hydrophilic mercapto compound is one of mercaptoethanol (ME), mercaptoethylammonium (CA), mercaptopropionic acid (MPA) and normal-thio-β-D-glucose (β-Tg).
4. The biosensor based on tethered phospholipid bilayer membrane according to claim 1, characterized in that: The lipid molecules in the active lipid molecular layer are a mixture of one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylglycerol (PG), and the charge diversity of the lipid molecules can quantitatively regulate the membrane potential of the biosensor.
5. A method for preparing a biosensor based on a tethered phospholipid bilayer membrane according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step S1, preparing a phase-separated hydrophilic-hydrophobic mixed monolayer by substitution adsorption or co-adsorption; The steps for preparing a phase-separated hydrophilic-hydrophobic mixed monolayer by substitution adsorption are as follows: At room temperature, a solid substrate coated with a gold film is immersed in an ethanol solution of a hydrophilic thiol compound, and a hydrophilic monolayer is formed on the solid substrate through Au-S self-assembly; after the assembly is completed, the free hydrophilic thiol compound molecules are rinsed with ethanol and blown dry for standby use; then, the solid substrate modified with the hydrophilic thiol compound is immersed in an ethanol solution of a hydrophobic alkylthiol compound, and a phase-separated hydrophilic-hydrophobic mixed monolayer is formed on the solid substrate through replacement adsorption; The steps for preparing a phase-separated hydrophilic-hydrophobic mixed monolayer by co-adsorption are as follows: At room temperature, a solid substrate coated with a gold film is immersed in a mixed solution of a hydrophobic alkylthiol compound and a hydrophilic thiol compound, and a phase-separated hydrophilic-hydrophobic mixed monolayer is formed on the solid substrate through Au-S self-assembly; Step S2, preparing active lipid molecules doped with cholesterol molecules Chol by thin film hydration ultrasound method; Step S3, spreading the active lipid molecules doped with cholesterol molecules Chol prepared in step S2 on the hydrophilic-hydrophobic mixed monolayer formed in step S1 in an aqueous solution by means of vesicle fusion, and incubating at room temperature to form an alternating single- and double-layer active lipid molecule layer doped with cholesterol molecules Chol, thereby constructing the biosensor; Step S4: repeatedly washing the biosensor constructed in step S3 with an aqueous solution to wash away the adsorbed and free lipid molecules.
6. The preparation method according to claim 5, characterized in that: In step S1, when a phase-separated hydrophilic-hydrophobic mixed monolayer is prepared by substitution adsorption, the concentrations of the hydrophilic thiol compound and the hydrophobic alkylthiol compound are both 0.5-2 mmol·L -1 .
7. The preparation method according to claim 6, characterized in that: The adsorption time of the hydrophilic mercapto compound is 5 to 15 minutes, and the adsorption time of the hydrophobic alkylthiol compound is 2 to 3.5 hours.
8. The preparation method according to claim 5, characterized in that: In step S1, when a phase-separated hydrophilic-hydrophobic mixed monolayer is prepared by co-adsorption, the total concentration of the mixed solution of the hydrophobic alkylthiol compound and the hydrophilic thiol compound is 0.5 to 2 mmol·L -1 The molar ratio of the hydrophobic alkylthiol compound to the hydrophilic thiol compound is 0.1 to 0.7; and the adsorption time of the mixed solution is 0.5 to 2 hours.
9. The preparation method according to claim 5, characterized in that: In step S2 or S3, the molar ratio of cholesterol molecules Chol to lipid molecules in the active lipid molecules doped with cholesterol molecules Chol is 0.5-1; and the incubation time in step S3 is 1.5-3 hours.
10. Use of the biosensor based on tethered phospholipid bilayer membrane according to any one of claims 1 to 4 on membrane proteins or ion channel proteins.