KR20090070379A - A preparation method of biosensor or gas-sensor using liposome - Google Patents

Abstract

A method for manufacturing a biosensor or gas sensor using a liposome is provided to combining lipid monolayer containing a functional group for the fixation of receptor and have high fixation efficiency and reproductivity. A method for manufacturing a bio sensor in which a receptor is fixed comprises: a step of adding hydrocarbon compound containing thiol group on the surface of sensor chip and coating self-assembled monolayer; a step of preparing a liposome comprising a functional group for fixation of receptor and lipid; and a step of reacting the liposome on the coated sensor chip and re-coating on the self-assembled monolayer. A material for fixing the receptor comprises a lipid and hydrocarbon containing the functional group for fixation of receptor. The step of preparing the liposome is performed by mixing MBP-PE[1,2-Dipalmitoyl-sn-Glycero-Phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] lipid and lipid.

Description

A preparation method of biosensor or gas-sensor using liposome}

The present invention relates to a method for manufacturing a biosensor or a gas sensor using liposomes, and more particularly, by using a liposome made of a lipid containing a functional group necessary for fixing a receptor, a receptor molecule is fixed to a surface of a sensor chip. The present invention relates to a method for detecting a gaseous organic compound by coating a surface of a sensor chip with lipids having different functional groups.

In order to fix receptor molecules on the surface of a biosensor, a chemical method of preparing a self-assembled monolayer (SAM) with a compound containing a thiol group and then introducing an appropriate functional group on the surface of the self-assembled monolayer This is the most commonly used.

Among the functional groups in the receptor molecule, an amino group is most used for receptor fixation, and a thiol group is also used. When immobilizing an antibody, an aldehyde group formed by partially oxidizing a carbohydrate contained in the antibody may be used.

Looking at the prior art, after the self-assembled monomolecular layer made of a compound such as 11-mercapto-1-undecanoic acid (MUA) to fix the receptor having an amino group, the self-assembled monolayer N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide [N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide to a carboxyl group on the surface; EDC] and N-hydroxysuccinimide (NHS) were sequentially reacted. As a result, the NHS group was introduced in the position where the carboxyl group was, and the receptor molecule which has an amino group was made to react here.

In addition, when the receptor molecule has a thiol group, a self-assembled monolayer was made of a compound such as 11-amino-1-undecanethiol. Then, N- [γ-maleimidobutyryloxy] succinimide {N-[γ-Maleimidobutyrtloxy] succinimide; GMBS} was reacted to introduce a maleimide group in place of the amino group. As a result, the receptor molecule having a thiol group was reacted to fix the receptor.

On the other hand, when immobilizing the antibody, the antibody was reacted with sodium m-periodate (or sodium iodide) to form an aldehyde group on the carbohydrate in the antibody. An azide group was introduced by introducing NHS groups and reacting carbohydrazide with carbohydrazide in the same manner as when fixing an amino group on the surface of the sensor chip. When an antibody containing an aldehyde group is reacted, a Schiff's base is formed, which is then reacted with cyanoborohydride to stabilize the binding.

As described above, in the case of immobilizing the antibody by using a hydrize functional group, starting from the sensor chip on which the SAM is formed, the EDC / NHS reaction, the carbohydrazide reaction, the ethanolamine reaction, and the antibody immobilization Reaction, cyanoborohydride reaction has to go through five stages and the total time is about 5 hours.

As such, introducing a proper functional group through a conventional chemical reaction may be a very difficult process for users who do not major in chemistry. In addition, when several steps of chemical reactions are required, the yield of the reaction may be low, and thus the receptor fixation efficiency may be lowered, which may make it difficult to obtain the same result when the experiment is repeated.

In order to solve these disadvantages of the chemical fixation method, a sensor chip in which an avidin protein is immobilized has been conventionally used. Avidin has strong and selective binding properties to biotin. It is relatively simple to chemically bind biotin to receptor molecules, and in the case of antibodies, it is possible to purchase antibodies that have biotin previously bound to them.

Therefore, by adding a biotin-coupled receptor to an avidin-fixed sensor chip, the receptor could be easily fixed in a short time. However, since avidin is relatively easily denatured, sensor chips containing avidin are difficult to store and are very expensive.

To overcome the drawbacks of these prior arts, the inventors have applied liposomes. As shown in FIG. 1, the lipid is composed of a polar head portion and a nonpolar tail portion, and may be represented by a structure having two nonpolar tails on the polar head. Due to the structural properties of the lipids, when the lipids are placed in an aqueous environment, the lipid bilayers are formed to hide the hydrophobic tails, as shown in FIG. 2, resulting in liposomes. Will be made.

The self-assembled monolayer made of octadecanethiol has a hydrophobic surface, and when the liposomes forming the lipid bilayer are reacted thereto, the hydrophobic tails of the lipids in the liposomes are hydrophobic. It is in contact with the surface and can form a lipid monolayer. By mixing lipids having functional groups capable of fixing receptors in these lipids, it is possible to effectively manufacture receptor-immobilized biosensors.

As a result of the efforts made by the present inventors, the lipid containing the functional group for immobilizing the receptor is mixed with the matrix and prepared as a liposome, thereby binding the SAM to the present invention to easily introduce the functional group for immobilizing the receptor. In addition, the present invention can be applied to the manufacture of gas sensors as follows.

The existing gas sensor mainly uses the property that gas is adsorbed on the metal oxide. However, metal oxides do not provide a variety of surfaces, making it difficult to distinguish between different gases, such as volatile organic compounds. Therefore, in order to increase the diversity of the gas sensor surface, the sensor surface is added with polymers (McGil, 2000, Sensors and Actuators B, vol. 65. p5-9; Seyama, 2005, Analytical Chemistry, vol. 77, p4228-4234). Coating with one thiol compound (Pasquinet, 2004, Journal of Colloid and Interface Science, vol. 272, p21-27), an ionic liquid (Jin, 2006, Analytical Chemistry, vol. 78, p6980-6989) Although studies have been conducted, no satisfactory effect has been achieved.

The present inventors have led to the present invention to manufacture a gas sensor that can distinguish the various volatile organic compounds by applying the results of the biosensor research.

Accordingly, an object of the present invention is to provide a biosensor manufacturing method for simply fixing a receptor without undergoing a chemical reaction of several stages in introducing an appropriate functional group on the surface of the biosensor, and also applying the same to select various volatile organic compounds. It is to provide a method of manufacturing a gas sensor that can be detected by.

An object of the present invention was achieved by preparing a liposome including a lipid containing a functional group for immobilizing a receptor and establishing a simple method of manufacturing a biosensor in which it is bound to a SAM of a biosensor, and confirming the application of the manufactured sensor.

The present invention comprises the steps of coating a self-assembled monolayer by adding a hydrocarbon compound comprising a thiol group on the surface of the sensor chip connected to the microcrystalline scale; Preparing a liposome consisting of a substance comprising a matrix and a receptor-fixing functional group, and reacting the prepared liposome with a sensor chip coated with a self-assembled monolayer to recoat the self-assembled monolayer to fix the receptor. It provides a method for producing a biosensor.

In the present invention, the substance having the functional group for immobilizing the receptor is preferably any one of a group consisting of a lipid and a hydrocarbon compound including the functional group for immobilizing the receptor.

Application of the present invention, liposomes are preferably prepared by mixing MBP-PE lipids and matrix lipids to prepare a biosensor having a thiol group-immobilized, and a palmitic hydrizide hydrocarbon compound and matrix lipids. It is also possible to produce a biosensor in which a receptor having an aldehyde group is immobilized by mixing liposomes by mixing. In addition, the liposome may be composed of biotin-PE lipids and matrix lipids to prepare a biosensor in which avidin protein is immobilized.

In preparing the liposomes of the present invention, the material containing the substrate and the receptor-fixing functional group is preferably dried and stored in the form of a film in such a quantity that one sensor chip can be coated in one vial, and the liposomes are used. Can be prepared.

In the present invention, it is preferable to use a substrate having a higher phase transition temperature than experimental conditions. For example, the dilipitoyl phosphatadyl cholin (DPPC) having a phase transition temperature of 41 ° C. may be used.

The present invention provides a receptor-fixed biosensor manufactured by the above method and composed of a lipid monolayer comprising a sensor chip, a self-assembled monolayer and a functional group.

On the other hand, the present invention is characterized in that it provides a method for producing a gas sensor that can react and detect organic compounds in the same manner as described above.

In addition, it is characterized by providing a gas sensor which is manufactured by the above method and comprises a lipid monolayer comprising a sensor chip, a self-assembled monolayer and a functional group.

The present invention relates to a method for manufacturing a biosensor or a gas sensor using liposomes, using the preparation method of the present invention is not only simple and shorter than the conventional method, but also has high efficiency and reproducibility in receptor fixation. In addition, it is a very excellent invention because it does not need to use a number of reagents and can be easily prepared by non-chemical majors.

Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these examples.

Biosensor of the present invention comprises a step of forming a self-assembled monolayer, liposome manufacturing step, the step of binding the prepared liposomes on the self-assembled monolayer. Hereinafter, a biosensor having a thiol group, a biosensor having an aldehyde group, a biosensor having avidin protein, and a gas sensor using the same will be described in detail. Their structural formula is shown in FIG. 4.

Example  One : Thiol groups  Preparation of Biosensors with Fixed Receptors

Measuring instrument preparation for biosensor

A quartz crystal microbalance (QCM) was purchased from Japan's Crystal Sunlife. The size of the square quartz crystal is 8 × 8mm, the diameter of the circular gold electrode is 5mm, and the natural frequency of the crystal microbalance is about 10MHz. Since the frequency of the crystal microbalance decreases in proportion to the increase in mass, if the receptor to which the analyte is bound is fixed on the surface of the microcrystal scale, the frequency changes as the analyte binds to be used as a biosensor. Can be. The crystal microbalance biosensor system was manufactured in this laboratory and is equipped with a cell equipped with a crystal microbalance, an oscillator circuit that supplies electricity to the crystal microbalance and sends vibration information of the crystal microbalance to a frequency meter, and a syringe pump that transfers the solvent ( KDS, Holliston, USA), injection valve (Upchurch, Oak Harbor, USA), frequency meter (Dagatron, Korea), and computer. In the present invention, a batch cell having a well to which a solution can be added on the crystal microbalance electrode and a flow cell in which the solution flows through the tubing and make contact with the crystal microbalance electrode were used together. First, a receptor protein that specifically binds to a detection substance in a batch cell is fixed and then replaced with a flow cell to inject a buffer solution through a pump, and a sample to be detected is injected through an injection valve. When the analyte binds to the antibody while passing through the surface of the crystal mounted on the flow cell, the mass changes, causing a change in the vibration frequency. The frequency can be measured by a frequency meter and sent to a computer for analysis.

Self-assembled monolayer ( SAM ) formation

To clean the electrode surface of the crystal microbalance, the crystal microbalance was immersed in 60 ° C. piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 7: 3) for 1 minute, rinsed with distilled water and ethanol, and dried with nitrogen gas. Next, add a microcrystalline scale to a solution of 1-octadecanethiol (1-octadecanethiol, Sigma-Aldrich, USA) in 2mM in ethanol and stir for 12 to 15 hours to prepare a self-assembled monolayer (self-assembled monolayer, SAM) was formed. The sensor chip formed with SAM was taken out, rinsed with ethanol, dried with nitrogen gas, and mounted in a cell.

As described above, when a hydrocarbon compound including a thiol group such as octadecanethiol, that is, an alkane thiol molecule is added, sulfur atoms chemisorb onto the metal surface and an attractive force acts between hydrophobic hydrocarbon chains. As shown, a self-assembled monolayer (SAM) is made.

Liposome manufacturing

Liposomes were prepared to bind with the SAM. In preparing liposomes, a lipid containing a functional group necessary for receptor fixation may be mixed with a background lipid to easily introduce a functional group capable of immobilizing a receptor on the surface of the sensor chip. In this case, by using a lipid having a phase transition temperature higher than the experimental conditions as a background lipid, a harder lipid monolayer can be made. If the surface viscoelasticity is important, such as quartz crystal microbalance (QCM), it is advantageous to make such a hard surface. In most experimental conditions, it is preferable to use dipamitoyl phosphatadyl cholin (DPPC) having a phase transition temperature of 41 ° C. as a background lipid.

In this embodiment, two lipids of dipalmitoyl phosphatidyl cholin (DPPC) and 1,2-Dipalmitoyl-sn-Glycero-Phosphoethanolamine-N- [4- (p-maleimidophenyl) butyramide (MBP-PE) are mixed. Liposomes were made. DPPC was used here as a substrate for forming liposomes and MBP-PE was used to allow maleimide groups to appear in the liposomes. Chloroform: A 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) solution was prepared at a concentration of 12 mg / mL using methanol (1: 2) solvent. 1,2-Dioleoyl-sn- (Glycero-3-Phosphoethanolamine-N- [4- (p-maleimidophenyl) butyramide] (MBP-PE) was dissolved in chloroform at a concentration of 8.2 mg / ml, MBP-PE in 200 μL of DPPC solution. 200 μL of the solution was mixed and 40 μL of the solution was evenly spread on the bottom of the glass vial, nitrogen gas was blown gently, and the lipid solution was dried to form a film.The vial which was not used immediately was filled with nitrogen gas and then -20 or -70 ° C. Store at low temperature.

In this way, the user can fix the receptor very easily by providing a vial in which the lipid is pre-cured in the form of a film to make a liposome capable of fixing the receptor to one sensor chip. If liposomes are made in advance and not provided in vials, they are more convenient to use, but there is a disadvantage in that organ storage is difficult. Therefore, the lipids are pre-dried in the form of a film and stored for a long time at low temperature.

 Add 120 μL of 0.1M 4- (2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffer solution with pH 7.0 to the vial. After filling with nitrogen gas, the stomach is blocked and sonicated to become small unilamella vesicle (SUV).

There are two ways to apply ultrasonic waves. The first method is to leave the vial for an hour in an ultrasonic cleaner with the water temperature set to 50-60 ° C. In the second method, a 500 mL beaker is filled with two-thirds of water and the vial is floated for 20 minutes with an ultrasonic homogenizer. Being an SUV can be seen by turning the opaque white into a slightly cloudy clear color. When buffer solution is added to a lipid film vial, multilamella vesicles (MLVs) are initially formed, which have multiple layers of lipids, resulting in an opaque white color. When ultrasonic energy is applied to it, the attraction between the lipid molecules is broken down and rearrangement occurs to reach equilibrium. The result is a small liposome, small unilamella vesicle (SUV), that is slightly cloudy.

With the liposome Self-assembled monolayer  Combination

The self-assembled monolayer made of octadecanethiol has a hydrophobic surface, and when the liposomes forming the lipid bilayer are reacted thereto, the hydrophobic tails of the lipids in the liposomes are hydrophobic. In contact with the surface can form a lipid layer (monolayer). In addition, when preparing liposomes, a lipid including a functional group necessary for immobilizing a receptor is mixed with a background lipid, and as shown in FIG. 7, a functional group capable of immobilizing a receptor on a surface of a sensor chip can be easily introduced. By applying this, the prepared liposomes were reacted with the sensor chip having the self-assembled monolayer formed in the following manner. The QCM that formed SAM with 1-octadecanethiol was mounted in a batch cell. Impurities adhere well to the hydrophobic surface of SAM formed of 1-octadecanethiol, and then first washed three times with 100 μL of 40 mM octyl glucoside, followed immediately by 100 μL of liposome solution and placed at 50 ° C. for 1 hour and 30 minutes. Put it. The batch cell was connected to a frequency meter and the liposome solution was removed, and 100 μL of 0.1 M sodium hydroxide (NaOH) was added and left for 30 seconds. Then, washed three times with phosphate buffered saline (PBS). It was confirmed whether the receptor of the prepared thiol-group receptor fixing biosensor.

Thiol groups  Having receptors Fixed  Confirm

Thiol groups can be introduced simply by adding cysteine residues to the peptide. In the case of proteins, thiol groups can be introduced by reacting with Traut's reagent (2-iminothiolane-HCl). After introducing a thiol group into the antibody as a Traut's reagent, 100 μL of the antibody at a concentration of 50 μg / mL was added and reacted for 1 hour. The antibody solution was then removed and washed three times with PBS. Add 100μL of bovine serum albumin (BSA) at 50μg / mL and let it react for 30 minutes. In this way, after immobilizing an anti-goat IgG antibody in a modified microbalance (anti-goat IgG antibody) and sequentially injecting different concentrations of antigens (goat IgG), frequency changes in proportion to the concentration are observed as shown in FIG. It became. At this time, after observing the frequency change by injecting a concentration of antigen, Dissociation solution (0.2M Glycine-HCl, pH2.3 + 1% DMSO) was injected to completely remove the bound antigen and then injected with the next concentration of antigen. As described above, the frequency of the modified microbalance changes not only in proportion to the concentration but also for repeated use, which means that the receptor antibody is stably fixed through chemical bonding.

Example 2 Preparation of a Biosensor Immobilized with an Oxidized Antibody Having an Aldehyde Group

Self-assembled monolayer (SAM) formation

The self-assembled monolayer was formed in the same manner as in Example 1.

Liposome manufacturing

The preparation of liposomes was carried out in the same manner as in Example 1 except for using 2.16 mg / mL of palmitic hydrazide dissolved in chloroform instead of MBP-PE solution. Palmitic hydrazide is not a lipid but has a long hydrophobic chain and can be inserted between lipids. In addition, the same result can be obtained by using a long chain hydrocarbon having the same functional group rather than a lipid as described above, and in general, a long chain hydrocarbon is more economical than a lipid because of its long synthesis, stability, and low cost. The reaction produced DPPC and palmitic hydrazide liposomes.

Coupling the Liposomes and Self-assembled Molecular Layers

It was carried out in the same manner as in Example 1. A manufactured biosensor coupled to liposomes mixed with palmitic hydrazide is illustrated in FIG. 9.

Confirmation of fixation of aldehyde-containing receptor

A self-assembled monolayer was formed on the metal surface of the sensor chip. After binding a single layer of liposome lipid, the QCM surface was washed three times with 100 mM sodium acetate buffer solution at pH 5.5 and oxidized with sodium m-pyriodate. In addition, it was reacted for 1 hour. Finally, after washing three times with ultrapure water, 100μL of 0.1M cyanoborohydride was added for reduction of the double bond and reacted for 1 hour to stabilize fixation of the receptor having an aldehyde group. In this way, the immobilized aldehyde group (anti-goat IgG antibody) was immobilized in the crystal microbalance and injected with different concentrations of antigens (goat IgG) in order to change the frequency in proportion to the concentration as shown in FIG. Was observed. At this time, after observing the frequency change by injecting a concentration of antigen, Dissociation solution (0.2M Glycine-HCl, pH2.3 + 1% DMSO) was injected to completely remove the bound antigen and then injected with the next concentration of antigen. As described above, the frequency of the modified microbalance changes not only in proportion to the concentration but also for repeated use, which means that the receptor antibody is stably fixed through chemical bonding.

In the present embodiment, the conventional method takes about 5 hours, whereas the present invention takes about 2 hours and 30 minutes in three steps of formation of a lipid layer, an antibody immobilization reaction, and a cyanoborohydride reaction. It can be greatly shortened. In addition, conventional methods require four steps to immobilize the antibody, whereas our method requires only one step reaction, resulting in higher fixation efficiency and reproducibility. It also eliminates the need for multiple reagents, making it easy for users who are not familiar with chemistry.

Example  3: Avidin  Preparation of Protein-Fixed Biosensors

Self-assembled monolayer ( SAM ) formation

Formation of the self-assembled monolayer was carried out in the same manner as in Example 1.

Liposome manufacturing

Preparation of liposomes was carried out in Example 1 except that 0.5 mg / ml biotin-PE (biotin-PE; 1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine-n-Biotinyl) dissolved in chloroform instead of MBP-PE solution was used. It was done in the same way.

With the liposome Self-assembled monolayer  Combination

It was carried out in the same manner as in Example 1.

Avidin  Check it pinned

A self-assembled monolayer was formed on the metal surface of the sensor chip, and a single layer of liposome lipid was bound thereon, followed by reaction with a 50 μg / mL avidin protein solution for 10 minutes. In this way, the avidin was immobilized on the microbalance and then the anti-rabbit IgG antibody was added to the avidin again. Since four biotins can bind to the avidin protein, the avidin bound to the biotin on the surface of the modified microbalance can be immobilized on the surface of the modified microbalance by again binding to the biotin bound to the antibody. As a result of injecting different concentrations of antigen (rabbit IgG) into the modified microbalance in which the antibody was immobilized, a frequency change in proportion to the concentration was observed as shown in FIG. 11. At this time, after observing the frequency change by injecting a concentration of antigen, Dissociation solution (0.2M Glycine-HCl, pH2.3 + 1% DMSO) was injected to completely remove the bound antigen and then injected with the next concentration of antigen. In this way, the modified microbalance does not only change in frequency in proportion to the concentration, but also can be used repeatedly, which means that the receptor avidin is stably fixed through chemical bonds.

Example  4: manufacturing of gas sensor

Self-assembled monolayer ( SAM ) formation

The self-assembled monolayer was formed in the same manner as in Example 1.

Liposome manufacturing

Four types of liposomes for gas sensors based on DPPC were prepared. One type of liposome was made of only base lipoprotein, and the other three were biotin-PE, MBP-PE, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N- (7-nitro-2-1,3). -benzoxadiazol-4-yl) (NBD-PE) were each mixed at a mass ratio of 24: 1. Liposomes were prepared in the same manner as in Example 1.

With the liposome Self-assembled monolayer  Combination

It was carried out in the same manner as in Example 1.

e- nose Manufacture

The e-nose was prepared by arranging sensors having various functional groups prepared by the above method in an array.

In addition to the method performed in this embodiment, only one gas sensor may be used from the viewpoint of a person skilled in the art, and another several sensors may be arranged to prepare an e-nose for analyzing various compounds. Since several gas sensors respond to different organic compounds in different patterns, it is possible to determine the type of organic compounds by analyzing these patterns. 12 to 14 show the respective gas sensors.

In this experiment, four fertilized microbalances were used: DPPC, Biotin-PE, MBP-PE and NBD-PE [1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N- (7-nitro-2-1,3- benzoxadiazol-4-yl)] four types of gas sensors were prepared by coating with four lipids, respectively, and the response to the gaseous organic compounds was measured. As a result, as shown in Figure 15, it was confirmed that the reaction differently depending on the type of gas.

Example  5: Review the efficiency of the sensor according to the background geology

In order to examine the efficiency of the sensor according to the background lipids, biosensors were manufactured with different background lipids. The base lipids were compared with phosphatidyl cholin, a lipid present in the liquid state at room temperature, mixed lipids of cholesterol (PC-CH), and two DPPC solids at room temperature.

Liposomes were mixed with biotin-PE in each matrix and a liposome was used to form a lipid layer on the self-assembled monolayer of octadecanethiol. The avidin protein was bound to this, and the avidin was bound to an antibody against rabbit immunoglobulin G having biotin bound thereto. Frequency changes were measured while injecting different concentrations of rabbit immunoglobulin G.

As a result, as shown in FIG. 16, the reactivity was nearly doubled when using DPPC compared to using PC-CH as the base lipid. In addition, as shown in FIG. 17, even in the experiment of repeating the process of injecting the same concentration of sample and dissociating the bound sample into the acidic buffer solution, a nearly constant value can be obtained to form a very stable surface. there was.

1 illustrates the structure of a lipid.

2 is a schematic of the structure of a lipid bilayer and a liposome.

3 is a schematic diagram showing a structure in which a lipid monolayer is formed on a hydrophobic surface.

4 shows some lipid or hydrocarbon structures that can be used for each application.

5 is a schematic diagram of a self-assembled monolayer structure formed when an alkane thiol compound is added to a metal surface of a sensor.

FIG. 6 is a schematic of the lipid monolayer structure formed when liposomes are added onto a hydrophobic self-assembled monolayer on the surface of a sensor.

7 is a schematic diagram of a lipid layer formed when liposomes are mixed with lipids having functional groups necessary for immobilizing receptors on a matrix, and the liposomes are added on a hydrophobic self-assembled monolayer.

8 is a graph showing a frequency change generated by injecting an antigen into a biosensor in which an antibody having a thiol group is immobilized.

FIG. 9 is a schematic diagram of a lipid monolayer structure formed by mixing liposomes by mixing long-chain hydrocarbons having functional groups necessary for immobilizing receptors on a matrix and adding the liposomes onto a hydrophobic self-assembled monolayer.

10 is a graph showing a frequency change generated by injecting an antigen into a biosensor in which an antibody having an aldehyde group is immobilized.

Figure 11 is a graph showing the frequency change resulting from the injection of the antigen (rabbit IgG) in the fixed crystal balance fixed antibody.

FIG. 12 shows the gas sensor surface structure coated with liposomes composed only of lipids with specific functional groups.

FIG. 13 shows a gas sensor surface structure in which liposomes are mixed with lipids having a specific functional group and then coated with liposomes.

FIG. 14 shows a gas sensor surface structure in which a long chain hydrocarbon compound having a specific functional group is mixed with a base lipid to form a liposome, and then coated using the liposome.

FIG. 15 is a graph illustrating a reaction comparison of gaseous organic compounds of four gas sensors made by coating with four lipids, respectively, DPPC, biotin-PE, MBP-PE, and NBD-PE.

FIG. 16 is a graph showing a comparison of reactivity when using a mixed lipid of phosphatidyl cholin and cholesterol (PC-CH) as a base lipid and when using DPPC as a base lipid.

FIG. 17 is a graph showing responsiveness when phosphatidyl cholin and cholesterol mixed lipids (PC-CH) are used as the base lipid and when DPPC is used as the base lipid.

Claims (10)

Coating a self-assembled monolayer by adding a hydrocarbon compound including a thiol group to the surface of the sensor chip connected to the crystal microbalance; Preparing a liposome consisting of a substance comprising a matrix and a receptor-fixing functional group; And Reacting the prepared liposomes with a sensor chip coated with a self-assembled monolayer and recoating the self-assembled monolayer; Consisting of, the manufacturing method of the biosensor fixed. The method of manufacturing a biosensor according to claim 1, wherein the substance having a functional group for immobilizing a receptor is any one of a group consisting of a lipid and a hydrocarbon compound including a functional group for immobilizing a receptor. The method of claim 1, wherein the preparing of the liposome is performed by mixing MBP-PE lipids and matrix lipids. The method of claim 1, wherein the preparing of the liposome is performed by mixing a palmitic hydrizide hydrocarbon compound and a matrix. The method of claim 1, wherein the step of preparing the liposome is prepared by mixing the biotin-PE lipids and matrix lipids. According to claim 1, wherein the step of preparing a liposome is stored in the form of a film containing the substrate and the functional groups for receptor fixation rolled in an amount in such a way that one sensor chip can be coated in one vial and used Method for producing a biosensor, characterized in that for preparing liposomes. The method of claim 1, wherein the background lipid is DPPC. A receptor-immobilized biosensor manufactured by the method of claims 1 to 7 comprising a lipid monolayer comprising a sensor chip, a self-assembled monolayer and a functional group. A method of manufacturing a gas sensor capable of reacting and detecting organic compounds in the same manner as in claim 1. A gas sensor manufactured by the method of claim 9, comprising a lipid monolayer comprising a sensor chip, a self-assembled monolayer, and a functional group.

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