KR100496939B1 - Substrate for biosensor and method for preparing the same - Google Patents

Abstract

The present invention is a solid substrate having a metal thin film on one surface; A peptide self-assembled through a terminal cysteine residue on the metal thin film of the solid substrate; A protein having specificity for the constant region of an antibody bound to the peptide; And it relates to a substrate for a biosensor comprising an antibody coupled to the protein.

Description

Substrate for biosensor and method for preparing the same}

The present invention relates to a biosensor, and more particularly, to a substrate for a biosensor using the reaction specificity of the antibody and a method of manufacturing the same.

In general, the biosensor using the reaction specificity of the antibody is composed of a detection unit that can selectively recognize the target material to be detected and a signal conversion unit for converting the detected signal into an electrical signal. In biosensors using antibodies, it is important to exclude cross-reactivity of heterologous proteins and increase detection sensitivity of a target substance. The density, uniformity, and activity of biomolecules immobilized on a solid surface determine the detection limits and nonspecific binding of individual DNA, RNA, antigens, and other protein molecules. Plays a crucial role in exclusion.

As a method of attaching biomolecules on a solid surface, various methods are known. Among them, Langmuir-Bloegett (LB) technology and Self-Assembly (SA) technology are representative. The LB technology takes advantage of the property that the amphiphilic molecules sprayed on the surface of the water exist in the form of monomolecules between the gas-liquid interface. Such monomolecular films can be deposited on the surface of solid substrates using techniques developed by Langture and Blojet (Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett To Self-Assembly , 1st). ed .; Academic Press; Boston, 1991; pp. 101-219). LB technology can control the density of monolayers stacked on the solid surface by arbitrarily adjusting the density per area of the material dispersed on the water surface, and can produce monolayer molecular film and multilayer molecular film on the solid substrate according to the cumulative frequency control. have. However, this method is time consuming and requires complex equipment.

Self-assembly (SA) technology uses a so-called bottom-up method that assembles and blocks nano-sized molecules while controlling their size and composition, and assembles them into large structures with certain properties and functions. For example, an alkyl thiol having a thiol group may be used as the self-assembled molecule. After dissolving a compound with a thiol group at the end of a long-chain alkyl group having 10 or more carbon atoms in a solution, a gold-coated substrate is placed in the solution to cause adsorption due to the affinity between the thiol and the gold surface. You lose. The wettability property of the surface changes according to the functional group of the self-assembled molecular terminal, and the properties of the substrate change according to the length of the alkyl group, so that the lamination structure and properties can be controlled according to the purpose.

There are two methods of immobilizing proteins using the self-assembly (SA) technique, immobilization by physical adsorption and immobilization by chemical covalent bonding. The immobilization method by physical adsorption utilizes the property that proteins are adsorbed by hydrophobic or electrostatic interactions between metal substrates and proteins. Protein immobilization technique by physical adsorption is easy to denature the protein, difficult to form a monomolecular membrane, poor durability of the membrane, consequently difficult to preserve protein activity (Sigal GB et al., Anal. Chem. 1996, 68, 490-). 497.). As such, physical adsorption is limited to the target of the immobilized protein, it is difficult to control the amount of protein immobilized in the unit area.

In order to improve the durability of the immobilized protein, a method of forming a stable protein thin film by inducing covalent bond with organic self-assembled thin film through surface functional group substitution of the protein is known (N. Patel et al., Langmuir. 1997, 13, 6485). -6490). The immobilization method using such a covalent bond is performed using a crosslinking material capable of binding both the substrate surface and the protein. The most well-known technique is the method of self-assembling organic alkanethiolates on gold-plated surfaces and inducing functional group substitution and covalent bonding of proteins through surface modification (Cass, T. and Ligler FS, Immobilized Biomolecules in Analysis-A Practical Approach , Oxford University Press, New York, 1998; pp. 60-72.). However, the substitution reaction using a crosslinking material can often degrade the intrinsic activity of the protein by modifying the three-dimensional structure of the protein, and when there are a large number of functional groups to be substituted on the surface, controlling the orientation of the immobilized protein becomes a problem. These immobilization methods require ancillary reactions for functional group substitution of biomolecules, and are time consuming and material consuming.

Therefore, there is a need for a biosensor capable of enhancing the immobilization reaction with the target material in order to increase the selectivity and detection sensitivity for the target material.

Accordingly, a main object of the present invention is to provide a substrate for a biosensor and a method of manufacturing the same, which inhibit nonspecific binding to heterologous proteins in a biosensor using an antibody and enable low protein concentration measurement.

In order to achieve the object of the present invention, the present invention is a solid substrate having a metal thin film on one surface; A peptide self-assembled through a terminal cysteine residue on the metal thin film of the solid substrate; A protein having specificity for the constant region of an antibody bound to the peptide; And it provides a substrate for a biosensor comprising an antibody coupled to the protein.

In the substrate of the present invention, the metal thin film is preferably formed of gold, silver, copper or platinum.

In the substrate of the present invention, the peptide preferably contains 5-25 amino acids, more preferably 15-25 amino acids.

In the substrate of the present invention, the solid substrate is preferably treated using a mercaptoethanol solution or a mercaptopropionic acid solution as a surface treating agent.

In the substrate of the present invention, the antibody is preferably a monoclonal antibody.

In the substrate of the present invention, the protein preferably has specificity with the constant region of the antibody, and has a charge distribution capable of interacting with the peptide.

In order to achieve another object of the present invention, the present invention comprises the steps of forming a metal thin film on one surface of a solid substrate; Forming a self-assembled thin film of a peptide having a cysteine residue at one end on the metal thin film; Binding a protein and a peptide on the substrate; And it provides a method for producing a substrate for a biosensor, comprising the step of binding the protein and the antibody.

In the process of the invention, the peptide preferably comprises 5-25 amino acids, more preferably 15-25 amino acids.

In the method of the present invention, before the step of combining the protein and the peptide, the substrate may further comprise the step of treating with a mercaptoethanol solution or a mercaptopropionic acid solution.

Hereinafter, with reference to the accompanying drawings will be described in more detail.

1 is a schematic view showing a substrate for a biosensor according to an embodiment of the present invention. In Fig. 1, reference numeral 1 is a substrate, 2 is a peptide having a cysteine residue at one end, 3 is a surface treatment agent film, 4 is a protein having specificity for the constant region of the antibody, and 5 is an antibody. A metal thin film is formed on the surface of the substrate 1 made of silicon or glass. The metal thin film may be formed of gold, silver, copper or platinum, with gold being particularly preferred. The substrate 1 on which the metal thin film is formed is immersed in a solution containing a peptide having a cysteine residue at one end. After 1-2 hours, a self-assembled thin film is formed on the substrate 1.

The surface treatment agent is treated to block the hydrophobic interaction by the metal on the surface of the self-assembled substrate (1). As a surface treatment agent, a mercaptoethanol solution or a mercaptopropionate solution is used, A peptide substrate is immersed in this solution, and the mercaptoethanol thin film 3 or the mercaptopropionic acid thin film 3 is formed.

In order to immobilize the antibody, a protein (4) having specificity in the constant region of the antibody and having a charge distribution capable of interacting with the peptidic is introduced into the self-assembled thin film. The substrate into which the protein is introduced is called an antibody immobilization platform. To this end, the substrate is immersed in a protein solution to fix the protein. The charged functional groups present in the peptide molecule allow the protein to be immobilized on the substrate. Therefore, the peptide molecule and the protein should be properly paired to be used. That is, a protein having a negative charge is used when the peptide molecule has a positive charge, and a protein having a positive charge is used when the peptide molecule has a negative charge.

In order to introduce the antibody (5) to the substrate, the antibody immobilization platform is immersed in the antibody solution to induce binding of the protein and the antibody. In the present invention, the antibody may be any antibody used in the art, and monoclonal antibodies are preferred, and particularly immunoglobulin G-based antibodies are more preferred. After antibody immobilization, the immobilized antibody can be washed away by dipping in phosphate buffer.

The substrate prepared in this manner can increase the selectivity and sensitivity for the target material.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Antibody Immobilization Platform

1) Substrate and Peptide Preparation

After a 2 nm thick chromium (Cr) film was formed on the cover glass with a thickness of 0.1 mm by sputtering, a 43 nm gold (Au) thin film was formed thereon. The formed gold thin film was immersed in a piranha solution (a solution made by mixing 70 parts by volume of sulfuric acid and 30 parts by volume of hydrogen peroxide solution) for 5 minutes, washed with ethanol for 30 minutes, and then deionized water for 2 hours. Soaking was completed. Solid phase synthesis was used to prepare peptides consisting of 18 amino acids with cystein residues at the ends. The amino acid sequence of the synthesized peptide consisted of CKGRGKGRGKGRGKGRGK (C: cysteine, K: lysine, G: glycine, R: arginine). The synthesized peptide has a molecular weight of 1842 and represents a water-soluble substance.

2) Formation of self-assembled thin film

The self-assembled thin film was formed by immersing the substrate on which the gold thin film was formed in an aqueous solution containing the peptide for 2 hours. The self-assembled peptide substrate was immersed in 300 mM mercaptoethanol solution for 12 hours to form a mercaptoethanol membrane to block the hydrophobic interaction between metal-proteins.

3) protein introduction

For antibody immobilization, protein A (Sigma-Aldrich), a protein having specificity to the constant region of the antibody, was introduced. To this end, the peptide substrate was immersed in an aqueous protein solution at a concentration of 0.01 mg / mL for 12 hours.

4) Determination of Protein Immobilization

The following experiments were carried out to confirm the protein immobilization on the antibody immobilization platform. A substrate (substrate 1) on which a gold thin film is formed; A substrate in which only a peptide is introduced into the substrate 1 (substrate 2); A substrate (substrate 3) having a protein A introduced thereinto; A substrate (substrate 4) in which protein G (Prozyme inc.) Is introduced into the substrate 2; For the substrate (substrate 5) into which IgG was introduced into substrate 2, protein immobilization was confirmed using surface plasmon resonance. Substrates 4 and 5 were prepared in the same manner except for using protein G and IgG instead of protein A in the method of manufacturing substrate 3, respectively.

The substrates 1 to 5 were each obtained using a surface plasmon resonance apparatus (Optrel, model name: Multiskop) as shown in FIG. 2. According to FIG. 2, it can be seen that the position of the incident angle of the light source in which the surface plasmon resonance occurs in the state in which the peptide is immobilized is increased as compared with the case of the pure gold thin film. That is, the coupling between the metal thin film and the surface plasmon resonance generation angle (incident angle at the lowest reflectance) was confirmed. In the case of protein G, the increase in the incidence angle was very small, but in the case of protein A, the incidence angle was significantly increased. This means that Protein A is immobilized on the peptide surface. In the case of protein G, there was almost no interaction due to the surface charge distribution and the incongruity of the three-dimensional structure of the protein. In the case of IgG, IgG was also immobilized by increasing the incident angle of the light source that causes surface plasmon resonance, but its effectiveness is limited in the biosensor using the antibody due to the nonspecific interaction with the peptide. Too much quantity is not adequate for immobilization.

Example 2: Preparation of Antibody Immobilized Substrate

1) Introduction of Antibodies

As an antibody for immobilization in this example, a monoclonal antibody against bovine serum albumin (BSA) was used, and the antiserum albumin antibody (anti-BSA) was prepared in Example 1 in an aqueous solution of 120 nM. The antibody immobilization platform was immersed for 6 hours to induce binding of the protein and the antibody. After antibody immobilization, the immobilized antibody was washed by soaking in phosphate buffer at pH 7.4 for 6 hours.

2) determination of the minimum detection concentration;

A bovine serum albumin that specifically reacts with the immobilized antibody was used as an antigen to prepare a signal response curve (see FIG. 3) according to the antigen concentration. Antigen-antibody reactions were induced by immersing a substrate immobilized in an antigen solution having an antigen concentration of 0M, 100pM, 1nM, 10nM, and 100nM for 120 minutes.

3 shows the position of the surface plasmon resonance generation angle according to the bovine serum albumin concentration. According to Figure 3, as the concentration of bovine albumin increases, the surface plasmon resonance generation angle also shows a tendency to increase, showing that bovine serum albumin is detected even at a very low concentration of 100pM concentration. In other words, when the concentration is increased 10 times, the surface plasmon production angle increases by about 0.1 degrees. Since the difference in the surface plasmon production angle between 0 M and 100 pM is 0.1 degrees, the estimated surface plasmon production angle by measuring 10 pM is 0. Since it is the same as the case of M, it can be seen that the low serum concentration limit of bovine albumin is 100 pM.

As described above, according to the present invention, non-specific reactions of proteins are suppressed through mixed self-assembly of functional peptide molecules and organics, and antibody immobilization with orientation is ensured through selective adsorption of protein receptors. Thus, it can be widely used in biotechnology such as diagnostic kits, biosensor development and enzyme immobilization for the production of useful materials, protein chips and the like.

1 is a schematic view showing a substrate for a biosensor according to an embodiment of the present invention.

Figure 2 compares the surface plasmon resonance generation angle according to the immobilization of peptides or proteins.

Figure 3 shows the change in the surface plasmon resonance generation angle according to the concentration of bovine serum albumin.

<Description of the symbols for the main parts of the drawings>

1. Substrate

2. Peptides with cysteine residues at one end

3. Surface treatment thin film

4. Protein

5. Antibodies

Claims (9)

A solid substrate having a metal thin film on one surface thereof; A peptide self-assembled through a terminal cysteine residue on the metal thin film of the solid substrate; A protein A having a charge distribution capable of interacting with the peptide with specificity to the constant region of the antibody, bound to the peptide; And A substrate for a biosensor comprising an antibody coupled to the protein. The substrate according to claim 1, wherein the metal thin film is formed of gold, silver, copper or platinum. The substrate of claim 1, wherein the peptide consists of 5-25 amino acids. The substrate according to claim 1, wherein the solid substrate is treated using a mercaptoethanol solution or a mercaptopropionic acid solution as a surface treating agent. The substrate of claim 1, wherein the antibody is a monoclonal antibody. delete Forming a metal thin film on one surface of the solid substrate; Forming a self-assembled thin film of a peptide having a cysteine residue at one end on the metal thin film; Binding a protein A having a charge distribution capable of interacting with the peptide with specificity to the constant region of the peptide and the antibody on the substrate; And Comprising the step of binding the protein and the antibody, a method for producing a substrate for a biosensor. 8. The method of claim 7, wherein said peptide comprises 5-25 amino acids. 8. The method of claim 7, further comprising treating the substrate with a mercaptoethanol solution or a mercaptopropionic acid solution prior to the step of combining the protein and the peptide.