KR101062316B1 - Signal Amplification Method of Surface Plasmon Resonance Sensor Using Carbon Nanotubes - Google Patents

Abstract

The present invention relates to a signal amplification method of a surface plasmon resonance sensor using carbon nanotubes, and more particularly, by amplifying a signal of the surface plasmon resonance sensor by combining carbon nanotubes with a biomolecule for detecting a target protein such as an antibody. The present invention relates to a method for amplifying a surface plasmon resonance sensor using carbon nanotubes to rapidly and accurately analyze a target protein.

Surface Plasmon Resonance, Carbon Nanotubes, Protein Chips, SPR Signal Amplification

Description

Signal amplification method of surface plasmon resonance sensor using carbon nanotubes {Method for signal enhancement of surface plasmon resonance using carbon nanotube conjugation}

The present invention relates to a signal amplification method of a surface plasmon resonance sensor using carbon nanotubes, and more particularly, by amplifying a signal of the surface plasmon resonance sensor by combining carbon nanotubes with a biomolecule for detecting a target protein such as an antibody. The present invention relates to a method for amplifying a surface plasmon resonance sensor using carbon nanotubes to rapidly and accurately analyze a target protein.

Methods for quantitative analysis of specific proteins include enzyme immunoassay, enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay. Among these, ELISA is a method of binding an enzyme to an antibody to confirm an antigen-antibody reaction. The method is simple, inexpensive, and can be analyzed in large quantities, making it one of the most widely used antigen-antibody assays. have. The ELISA method is widely used because it is very sensitive and does not use radioactivity as in radioimmunoassay. Radioimmunoassays have high sensitivity but safety is a problem in that they use radioactive materials.

Therefore, Surface Plasmon Resonance (SPR) method that can recognize the interaction of biological materials by measuring the change of refractive index by an analytical technique, which is highlighted as an alternative to overcome the disadvantages of the prior art described above. There is. The SPR method can measure interactions between molecules using optical principles without a separate label such as a fluorescent material, and can measure the progress of a reaction in real time. SPR technology is widely used as a method for measuring biosensors and biochips by using a phenomenon that changes a signal when a biomaterial such as a protein is bound to a sensor surface (Nice, EC and Catimel, B., Bioassays , 1999, 21, 339-352). Surface plasmons are quantized vibrations of free electrons that propagate along a conductor surface, such as a metal surface. The surface plasmon is excited by incident light incident on the metal thin film at an angle greater than or equal to the critical angle of the dielectric medium through a dielectric medium such as a prism and causes resonance at a constant angle. The angle of incidence, ie the resonance angle, at which this SPR occurs is sensitive to the change in refractive index of the material in proximity to the metal thin film. SPR sensor is used to measure the thickness of a sample which is close to the metal thin film, that is, the quantitative analysis, qualitative analysis and thin film of the sample from the refractive index change of the sample. However, the conventional SPR sensor has a problem that the sensitivity is low to analyze a low concentration of biomaterials (Homola, J., Yee, SS and Gauglitz, G., Sens. Actuators B , 1999, 54, 3-15).

Looking at the conventional studies for amplification of the SPR signal, Korean Patent No. 0511055 captures the protein to be analyzed with a primary antibody and binds it to a secondary antibody and then horseradish peroxidase (Horseradish Peroxidase; "HRP") A technique for amplifying the SPR signal by forming a precipitate on the surface of the chip by binding to and adding a substance that reacts with the enzyme is disclosed. International Patent Publication No. 01/09388 discloses a technique using colloidal metal nanoparticles that capture analytes such as proteins as primary antibodies and conjugated secondary antibodies. In addition, the Republic of Korea Patent No. 0737689 discloses a technique for increasing the sensitivity of the SPR signal by using a secondary amplification method through the target protein, gold nanoparticles and enzyme precipitation reaction.

Conventional SPR method has the advantage that can directly measure the binding of the biological material without a separate label, but separate signal amplification technology is required to measure the trace amount of the biological material.

It is an object of the present invention to provide a method for amplifying a signal of an SPR sensor chip using a complex of surface-modified carbon nanotubes and a biomolecule specific for a target protein.

Another object of the present invention is to provide a method for quickly and effectively quantifying proteins present in various concentrations, especially trace amounts, using the SPR sensor system for quantifying proteins.

In order to achieve the above object, the present invention

Preparing a complex consisting of carbon nanotubes and biomolecules specific for the target protein; And

It provides a method for amplifying a signal of the SPR sensor chip comprising the step of reacting the sample containing the target protein and the complex.

The invention also

i) forming a self assembled monolayer on the surface of the gold thin film of the Surface Plasmon Resonance (SPR) chip;

ii) immobilizing the biomolecule for the target protein on the self-assembled monolayer;

iii) preparing a complex consisting of carbon nanotubes and biomolecules specific for the target protein;

iv) amplifying the SPR signal by reacting the sample containing the target protein with the carbon nanotube-biomolecule complex, or

First reacting the sample containing the target protein with the SPR chip and secondly reacting the carbon nanotube-biomolecule complex; And

and v) measuring the amount of the target protein using the refractive index change relationship for the material immobilized on the gold thin film surface of the SPR chip.

The present invention can quickly and effectively quantify trace proteins using the SPR sensor system that can effectively amplify the signal of the SPR sensor using the modified carbon nanotubes, so that various concentrations of protein analysis can be conducted. .

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention

Preparing a complex consisting of carbon nanotubes and biomolecules specific for the target protein; And

A method for amplifying a signal of an SPR sensor chip comprising reacting a sample containing a target protein with the complex.

The carbon nanotubes are not particularly limited, but it is preferable to use high hydrophobic multi-wall carbon nanotubes, and more preferably, to increase dispersibility, a carboxyl group, a hydroxyl group, an aldehyde group, and an amine group. It is preferable to use a surface modified with at least one of various functional groups such as a thiol group, a carbonyl group and the like.

The method of modifying the surface of the carbon nanotubes is not particularly limited, but the carbon nanotubes are reacted by applying heat in an acidic solvent, cooled at room temperature, and a carbon nanotube solution having a neutral pH is added by adding water and filtering. It is preferable to modify the surface by sonication.

Biomolecules specific for the target protein are not particularly limited, and enzymes, proteins, peptides, nucleotides, nucleic acids, polysaccharides, fatty acids, lipids, and the like may be used alone or in combination of two or more. More preferably, an antibody specific for a target protein such as a monoclonal or polyclonal antibody of the target protein or a fusion protein in which these antibodies are combined with horseradish-derived peroxidase or the like is preferably used.

The antibody specific for the target protein is not particularly limited, but specifically with the target protein erythropoietin, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Monoclonal or polyclonal antibodies that can bind can be used.

The method for preparing the complex consisting of the carbon nanotubes and the biomolecules specific for the target protein is not particularly limited, but the surface-modified carbon nanotubes and the secondary antibody are preferably reacted at 0 to 4 ° C. for 10 to 30 hours. Do.

In addition, in the SPR sensor chip of the present invention, a self-assembled monolayer may be formed on a surface thereof.

In the present invention, the term "self-assembled monolayer" refers to a regularly ordered organic molecular film produced on the surface of the SPR chip, carboxyl group (-COOH), or carboxyl group (-COOH) and hydroxyl group (-OH) It is preferable to have either.

The self-assembled monolayer may be formed by supporting an SPR chip in a mercapto alkyl carboxylic acid solution.

Examples of the mercapto alkyl carboxylic acid include mercaptoundecanoic acid, mercapto-hexadecanoic acid (MHA), or 3-mercapto-propionic acid, 3-MPA), and the like to form a molecular film having a hydroxyl group includes mercaptoundecanol (MUOH). The compound may be used alone or in combination of two or more to form a self-assembled monolayer.

The mercapto alkyl carboxylic acid solution is preferably prepared by dissolving mercapto alkyl carboxylic acid in an aqueous solvent at a ratio of 1: 9.

Although it does not restrict | limit especially as said aqueous solvent, Alcohol, such as ethanol, is preferable.

In addition, the SPR chip having a self-assembled monolayer on the surface may be immobilized with a biomolecule for a target protein such as an antibody capable of capturing the target protein.

As the biomolecule for the target protein, enzymes, proteins, peptides, nucleotides, nucleic acids, polysaccharides, fatty acids, lipids, etc. may be used alone or in combination of two or more. More preferably, an antibody specific for a target protein, such as a monoclonal antibody or a polyclonal antibody of the target protein, or a fusion protein in which these antibodies are combined with horseradish-derived horseradish, is used. Good to do.

The antibody to the target protein is not particularly limited, but specifically binds to a target protein, erythropoietin, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Monoclonal or polyclonal antibodies can be used.

The invention also

i) forming a self assembled monolayer on the surface of the gold thin film of the Surface Plasmon Resonance (SPR) chip;

ii) immobilizing the biomolecule for the target protein on the self-assembled monolayer;

iii) preparing a complex consisting of carbon nanotubes and biomolecules specific for the target protein;

iv) amplifying the SPR signal by reacting the sample containing the target protein with the carbon nanotube-biomolecule complex, or

First reacting the sample containing the target protein with the SPR chip and secondly reacting the carbon nanotube-biomolecule complex; And

and v) measuring the amount of the target protein using the refractive index change relationship for the material immobilized on the gold thin film surface of the SPR chip.

The method for quantifying the protein of the present invention will be described in detail with reference to FIG. 1 as follows.

The first step is to form a self-assembled monolayer on the surface of the metal thin film of the SPR chip.

The self-assembled monolayer is a layer having any one of a carboxyl group (-COOH), or a carboxyl group (-COOH) and a hydroxyl group (-OH), and a SPR chip in a mercapto alkyl carboxylic acid solution. It can be formed by supporting.

Examples of the mercapto alkyl carboxylic acid include mercaptoundecanoic acid, mercapto-hexadecanoic acid (MHA), or 3-mercapto-propionic acid, 3-MPA), and the like to form a molecular film having a hydroxyl group includes mercaptoundecanol (MUOH). The compound may be used alone or in combination of two or more to form a self-assembled monolayer.

The mercapto alkyl carboxylic acid solution is preferably prepared by dissolving mercapto alkyl carboxylic acid in an aqueous solvent at a ratio of 1: 9.

Although it does not restrict | limit especially as said aqueous solvent, Alcohol, such as ethanol, is preferable.

The second step is to fix biomolecules for a target protein such as a primary antibody on the self-assembled monolayer of the SPR chip.

As the biomolecule for the target protein, enzymes, proteins, peptides, nucleotides, nucleic acids, polysaccharides, fatty acids, lipids, etc. may be used alone or in combination of two or more. More preferably, an antibody specific for a target protein, such as a monoclonal antibody or a polyclonal antibody of the target protein, or a fusion protein in which these antibodies are combined with horseradish-derived horseradish, is used. Good to do.

The antibody to the target protein is not particularly limited, but specifically binds to a target protein, erythropoietin, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Monoclonal or polyclonal antibodies can be used.

In addition, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added to the SPR chip before antibody immobilization for interaction between the carboxyl group of the self-assembled monolayer and the N-terminus of the antibody to the target protein. It is preferable to treat at least one imide compound such as 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC), or N-hydroxysuccinimide (NHS).

In addition, the step of immobilizing the antibody to the target protein may be used to control the binding of nonspecific proteins, for example, to remove unreacted activators or to block portions where non-selective reactions may occur in the SPR chip. After fixation, the method may further include treating ethanolamine, serum albumin, polyethylene glycol, aminodextran, or the like alone or two or more.

The third step is to prepare a complex consisting of a carbon nanotube and a secondary antibody specific for the target protein.

The carbon nanotubes preferably use high hydrophobic multi-wall carbon nanotubes, and have functions such as carboxyl group, hydroxyl group, aldehyde group, amine group, thiol group, or carbonyl group in order to increase dispersibility. Preference is given to using those whose surfaces have been modified with at least one of the groups.

Biomolecules specific for the target protein are not particularly limited, and enzymes, proteins, peptides, nucleotides, nucleic acids, polysaccharides, fatty acids, lipids, and the like may be used alone or in combination of two or more. More preferably, an antibody specific for a target protein such as a monoclonal or polyclonal antibody of the target protein or a fusion protein in which these antibodies are combined with horseradish-derived peroxidase or the like is preferably used.

The antibody specific for the target protein is not particularly limited, but specifically binds to a target protein, erythropoietin, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Monoclonal or polyclonal antibodies can be used.

The method for preparing the complex consisting of biomolecules specific for the carbon nanotubes and the target protein is not particularly limited, but the surface-modified carbon nanotubes and the secondary antibody may be reacted at 0 to 4 ° C. for 10 to 30 hours. Do.

In addition, the complex consisting of biomolecules specific to the carbon nanotubes and the target protein is used alone or two or more of ethanolamine, aminodextran, polyethylene glycol, or serum albumin in order to suppress nonspecific reaction with the surface of the SPR chip. It may be further treated with the material.

The fourth step is to amplify the signal of the SPR chip by reacting the sample containing the target protein with the antibody.

The target protein is not particularly limited, but may be a protein, peptide or the like, such as erythropoietin or granulocyte-macrophage colony stimulating factor. In addition to the target protein, carbohydrates, nucleic acids, fatty acids, or vitamins may be used.

The target protein is preferably contained in 0.001ng to 500㎛ per 1ml of the sample so that the change in the refractive index according to the signal amplification of the SPR sensor can be measured.

The SPR signal is further amplified by the reaction of a complex consisting of a target protein, a carbon nanotube, and a biomolecule specific to a target protein as compared with a signal generated through a reaction between a target protein and a biomolecule specific to the target protein. Can be. The amplified signal changes the SPR refractive index to allow the determination of trace proteins.

The fifth step is to quantify the target protein, which can be quantified by converting the amount of the target protein by using the refractive index change relationship for the material immobilized on the metal thin film surface of the SPR chip.

Hereinafter, the present invention will be described in more detail based on the following examples. The following examples are intended to illustrate the invention, not to limit the invention.

Example 1 Quantification of EPO Protein Using Antigen- Antibody Response and Signal Amplification in SPR Sensor Chip

One) Gold foil  Chip Fabrication and Chip Activation

Gold thin film chips were immersed at 65 ° C. for 30 minutes in a solution of 95% sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3: 1, and then the gold thin film chips were washed several times with distilled water and ethanol. The gold thin film chip was immersed in an ethanol solution in which 10 mM MUA was dissolved at room temperature for about 20 hours to form a self-assembled monolayer. The gold thin film chip was washed with ethanol and distilled water in order to complete the gold thin film chip.

2) Gold foil  Immobilization of Primary Antibodies on a Chip

In order to activate the gold thin film chip having the surface treatment, 50 µl of a solution of 0.2 M EDC and 0.05 NHS in distilled water was reacted on the chip for 15 minutes. The chip was then immobilized with 500 μl of PBS (phosphate-buffered saline, pH 7.4) containing 100 μg / ml monoclonal EPO antibody for 1 hour. In order to remove unreacted activator, 1M ethanolamine solution (pH 8.5) was then reacted with the chip for 10 minutes, and in order to prevent the non-selective reaction on the chip, 1% Bovine serum albumin (hereinafter referred to as BSA) was dissolved in PBS and reacted for 1 hour.

3) SPR  Sensor EPO  Antigen-Antibody Immune Response Measurement

The chip subjected to the above process was measured using an SPR sensor system (BiacoreX, GE Healthcare, Sweden), and then the reaction was performed at 25 ° C. at a flow rate of 2-100 μl / min.

A solution of EPO antigen protein (KFDA, Korea) dissolved in PBS buffer at a concentration of 0.1-125,000 μg / ml was injected to allow antigen-antibody binding. Blanks were set in PBS buffer in this experiment. After the SPR measurement, a buffer solution of 0.1 M Glycine-HCl (pH 2.0) was used for reuse of the sensor chip.

4) Complexation of Carbon Nanotubes and Secondary Antibodies

100 mg of multi-wall carbon nanotubes having high hydrophobicity were added to a solution mixed with sulfuric acid and nitric acid in a volume ratio of 3: 1, and then reacted at 70 ° C. for about 3 hours, and then cooled at room temperature. Thereafter, a sufficient amount of water was added and filtered to make pH 7.0 of the carbon nanotube solution. Next, ultrasonication was performed for about 6 hours. Through the above process, the carbon nanotubes were shortened in length, and the surface thereof was modified with carboxyl groups that are well dispersed in distilled water.

To prepare a carbon nanotube-secondary antibody complex, 500 μl of the carboxyl-modified carbon nanotube solution was centrifuged (12,000 rpm, 15 minutes). Carbon nanotubes thus obtained were placed in PBS buffer solution, sonicated and suspended, and then centrifuged (12,000 rpm, 15 minutes). In order to activate the carbon nanotubes, 0.4M EDC (12.5 μl) and 1 mL secondary antibody (100 μg / ml in PBS buffer, pH 7.4) were mixed and reacted at 4 ° C. for 20 hours. After the reaction, in order to prevent the non-specific reactions in the carbon nanotube-secondary antibody complex, 500 μl of a solution of 10 mg / ml amino dextran in PBS buffer solution was added thereto for 2 hours. Reacted for a while.

5) using a complex of carbon nanotubes and a secondary antibody SPR  Signal amplification

SPR values changed by antigen-antibody reaction of antigenic protein EPO and reaction of antigenic protein and carbon nanotube-secondary antibody complex according to concentration (1 to 125 µg / ml) were measured.

As shown in Figure 2, it was confirmed that the SPR signal change by the carbon nanotube complex is amplified as the concentration of EPO increases.

Example 2 Quantification of Proteins According to Signal Amplification of SPR Chip by Coupling EPO Antigen Protein with Primary Antibody-Carbon Nanotube Complex

One) Gold foil  Immobilization of Secondary Antibodies on a Chip

A protein chip was prepared in the same manner as in Example 1), and the immobilized chip was immobilized with 500 μl of PBS containing 100 μg / ml of polyclonal EPO antibody for 1 hour. In order to remove unreacted activator, 1M ethanolamine solution (pH 8.5) was reacted with the chip for 10 minutes, and 1% of BSA was dissolved in PBS to prevent the non-selective reaction on the chip. The solution was allowed to react for 1 hour.

2) EPO  Binding of Antigen Proteins to Primary Antibody-Carbon Nanotube Complexes

Signal amplification was confirmed on the SPR chip to which the secondary antibody was immobilized by a single sample injection using a complex of monoclonal primary antibody and carbon nanotube prepared in the same manner as in Example 1, 4).

To this end, 20 μl of a carbon nanotube-primary antibody complex and 20 to 1000 μl of a sample in which EPO antigen protein is dissolved in PBS buffer at a concentration of 1 to 125 μg / ml are mixed, respectively, to form a primary antigen-antibody binding. To lose. Thereafter, a mixture solution of antigen-antibody binding was injected for 15 minutes at a flow rate of 2 μl / min to enable secondary antigen-antibody binding. Blank used a sample in which a PBS solution was mixed with a carbon nanotube-primary antibody complex.

As shown in Figure 3, it can be seen that the RU value of the SPR increases according to the amount of the antigen sample, it was confirmed that the amplified value about 100 times or more than the RU value of the SPR when the antigen sample is added.

In order to reuse the chip after measuring the immune response, the chip was used for 5 minutes using a 0.1 M Glycine-HCl (pH 2.0) buffer solution on the sensor chip.

Example 3 Quantification of GM-CSF Protein Using Antigen- Antibody Response and Signal Amplification in SPR Sensor Chip

The antigen-antibody reaction and the reaction of the antigen protein with the carbon nanotube-secondary antibody complex according to the same method as in Example 1, except that GM-CSF (R & D systems, 1 to 125 µg / ml) was used as the antigenic protein. The SPR value changed by was measured.

As shown in Figure 4, it was found that the SPR signal change by the carbon nanotube complex increases as the GM-CSF concentration is increased, the signal amplified by the carbon nanotubes increased 17 times on average.

In addition, the concentration of GM-CSF antigen protein was reduced to 0.1 ng / ml, and the SPR value changed by the carbon nanotube-secondary antibody complex reaction was measured. Could confirm.

<Example 4> Quantification of protein according to signal amplification of SPR chip by binding GM-CSF antigen protein and primary antibody-carbon nanotube complex

A protein chip was prepared according to the same method as Example 1, and signal amplification was confirmed on the SPR chip to which the secondary antibody was immobilized using a complex of monoclonal primary antibody and carbon nanotubes prepared according to the same method as Example 2.

To this end, 20 μl of carbon nanotube-primary antibody complexes and 20 to 1000 μl of GM-CSF antigen protein dissolved in PBS buffer at a concentration of 1 to 10 μg / ml were mixed, respectively, to prevent primary antigen-antibody binding. It was done. Next, a mixture of antigen-antibody binding was injected for 15 minutes at a flow rate of 2 μl / min to allow secondary antigen-antibody binding.

As shown in Figure 5, it can be seen that the RU value of the SPR increases according to the amount of the antigen sample, it was confirmed that the amplified value about 40 times more than the RU value of the SPR when the antigen sample was added. It was confirmed that the sample was about 40 times higher in proportion to the antigenic protein concentration than the RU value of the SPR when the sample was added.

1 is a schematic diagram showing a method for quantifying a protein of the present invention according to amplification of SPR signals using carbon nanotubes.

Figure 2 is a protein quantification method according to an embodiment of the present invention, the primary antigen-antibody reaction of the primary antibody and EPO antigen protein immobilized on the SPR chip, the secondary of the carbon nanotube-secondary antibody complex and the immobilized antigen protein It is a graph showing the change value of the SPR refractive index by the antigen-antibody reaction.

Figure 3 is a protein quantification method according to an embodiment of the present invention, the first antigen-antibody reaction of the carbon nanotube-primary antibody complex and EPO antigen protein, the secondary antibody immobilized on the SPR chip and the primary antigen-antibody reaction It is a graph showing the change value of the SPR refractive index by the secondary antigen-antibody reaction of the target protein passed through.

Figure 4 is a protein quantification method according to an embodiment of the present invention, the primary antigen-antibody reaction of the primary antibody immobilized on the SPR chip and hGM-CSF antigen protein, the carbon nanotube-secondary antibody complex and the immobilized antigen protein It is a graph showing the change value of the SPR refractive index by the secondary antigen-antibody reaction.

Figure 5 is a protein quantification method according to an embodiment of the present invention, the first antigen-antibody reaction of the carbon nanotube-primary antibody complex and hGM-CSF antigen protein, the secondary antibody immobilized on the SPR chip and the primary antigen- It is a graph showing the change value of the SPR refractive index by the secondary antigen-antibody reaction of the target protein subjected to the antibody reaction.

Claims (5)

Preparing a complex consisting of surface-modified carbon nanotubes and biomolecules specific for the target protein; And A method of quantifying protein using signal amplification of an SPR sensor chip comprising the step of reacting the sample containing the target protein and the complex. The method of claim 1, Biomolecule is a method for quantifying proteins using signal amplification of the SPR sensor chip, characterized in that at least one selected from the group consisting of antibodies, enzymes, proteins, peptides, nucleotides, nucleic acids, carbohydrates, fatty acids and lipids. The method of claim 1, Carbon nanotubes are formed from the group consisting of carboxyl group (-COOH), hydroxyl group (-OH), amine group (-NH ₂ ), aldehyde group (-CHO), thiol group (-SH) and carbonyl group (-CO) on the surface. Method for quantifying proteins using signal amplification of the SPR sensor chip having one or more selected functional groups. The method of claim 1, Carbon nanotubes or SPR chip is characterized in that one or more selected from the group consisting of ethanolamine, serum albumin, aminodextran and polyethylene glycol is treated to prevent non-specific biomolecule binding, using the signal amplification of the SPR sensor chip Method for Quantifying Proteins. The method of claim 1, Target protein quantification method using a signal amplification of the SPR sensor chip, characterized in that it comprises 0.001 ng to 500 μg per 1 ml of the sample.

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