KR20040047343A - The detection method using local differential of complex reflection coefficient rate for protein coupled in solid face - Google Patents

Abstract

PURPOSE: A method for detecting protein coupled in solid face using local differential of complex reflection coefficient rate is provided, thereby reducing the detection time, simplifying the detection process, requiring no skilled person, and reducing the detection cost by removing a protein marker. CONSTITUTION: The method for detecting protein coupled in solid face using local differential of complex reflection coefficient rate comprises the steps of: passing light(1) through a polarizer(2) and a compensator(3) to polarize the light; irradiating the polarized light to the sample surface of the protein coupled thin layer in solid face; detecting optical intensity of the incident and reflected light from the sample surface using an analyzer(7) and a light sensor; and calculating the complex reflection coefficient rate from the detected optical intensity using equation 1 and 2, wherein N is complex refraction rate; n is refraction rate; k is extinction coefficient; rho(ρ) is complex reflection coefficient ratio; rp is reflection coefficient of p wave; rs is reflection coefficient of s wave; δp(deltap) is phase change of p wave before and after reflection; δs(deltas) is phase change of s wave before and after reflection; and the CCD camera may be used for detecting protein coupled in solid face.

Description

The detection method using local differential of complex reflection coefficient rate for protein coupled in solid face}

The present invention is a method of detecting the binding of the antibody to the antigen by using the characteristic that the complex refractive index of the protein thin film is changed by the binding of the antibody and antigen, a kind of protein on the solid surface, and thus the complex reflection coefficient ratio of the thin film is changed. In particular, it is possible to simultaneously detect a plurality of antibody-antigen bonds occurring on a solid surface by capturing an image of a local complex reflection coefficient ratio of a sample surface area to which incident light is irradiated by an image through a CD camera. It relates to a protein binding detection method that can be.

In general, thanks to the progress of the genome project, which started in 1990, the structure of human genes has recently been revealed, and interest in functional studies of the genes that make up the human body is increasing rapidly.

Since genes express proteins and perform their functions in cells, the functional study of the gene results in the study of the protein's function.

Thus, a new field of research called proteomics, which studies the function of proteins, has recently emerged.

Proteomics is the study of the proteome, which is the sum of all the proteins expressed by a gene. Its purpose is to identify which proteins, in what amounts, and in what environments.

A powerful core technology for studying proteomics is the protein chip, which is a system that can simultaneously fix dozens-hundreds of proteins on a small chip to analyze the binding between proteins simultaneously.

In addition, the protein chip can be applied not only to the proteomics field, but also to medicine, pharmacy, and life science fields such as new drug development, disease diagnosis, and microbial detection.

In order to develop such a protein chip, detailed technologies such as protein probe development, protein immobilization, and protein-to-protein binding detection must be developed, and interdisciplinary research such as biology, chemistry, and electronic engineering is essential.

Among them, the present invention relates to a technique capable of detecting the binding between proteins occurring on the solid surface using the optical technology of physics.

In protein chip technology, the detection technology of protein-to-protein binding is an essential part, and the development of rapid and accurate detection technology is required for the development of an automatic diagnostic device.

As a method for detecting the binding between proteins on a solid surface, there is a method using a protein with a label such as a fluorescent substance and a method through instrumental analysis without using a label protein.

In the former method, the most representative example is a method of determining whether an antibody-antigen is reacted by using a secondary antibody to which a fluorescent substance is attached.

In this way, to determine the presence or absence of the target substance, a secondary antibody suitable for each antigen is required.

This is because the fluorescent substance is attached to the same antibody immobilized on the chip. When the antigen in the object is attached in response to a specific antibody suitable for itself, the fluorescent antibody is attached to the antibody, and the antibody reacts at the position. To detect this.

However, this method has the disadvantage of requiring the same number of secondary antibodies as the antibodies attached to the chip, which increases cost and complicated operations in production and measurement methods.

The latter method includes two technologies, Surface plasmon resonance (SPR) and Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF), which can analyze unknown proteins without the use of labeled proteins. There is an advantage, but it is difficult to measure a large number of antigens simultaneously.

Protein chip technology is a technology for detecting the binding between the protein array immobilized on a solid surface and the protein introduced from the outside, it is highly applicable to the development of new drugs, diagnosis of diseases, detection of pathogenic microorganisms.

To develop protein chip technology, detailed techniques such as development of protein probe, immobilization of protein probe on solid surface, and detection of binding between proteins on solid surface should be developed.

The present invention provides a new method for detecting binding between proteins.

As described above, the conventional technique for detecting the binding between proteins has a problem that it is difficult to simultaneously detect each binding of the protein in instrumental analysis without using the labeled protein or using the labeled protein.

Therefore, the present inventors have developed a technique for simultaneously detecting each bond occurring in the protein sequence without using a labeled protein.

To this end, the binding of the protein is detected by measuring the change in the complex reflection coefficient ratio according to the complex refractive index change of the protein thin film according to the protein binding on the solid surface.

In particular, by photographing the difference in the local radiant reflection coefficient ratio of the sample surface to which incident light is irradiated with a CD camera, the binding between the plurality of proteins occurring on the solid surface can be detected simultaneously.

1 is a schematic diagram showing the configuration of a polarizing optics device for detecting a local change in the complex reflection coefficient ratio of a protein thin film

2 is an image of a local difference in the complex reflection coefficient ratio generated in the cross section of light incident on a solid surface.

3A to 3D are images of binding of an antibody, which is a type of protein, to an antigen present on the surface of E. coli.

FIG. 4 is a graph showing the correlation between the average light intensity value and the E. coli concentration in the protein thin film forming region in the image of FIG.

<Explanation of symbols for main parts of drawing>

1: light source 2: polarizer

3: compensator 4: protein thin film

5: solid substrate 6: objective lens

7: analyzer (analyzer) 8: CD camera

9: light path 10: incident surface

11: incident angle 12: direction of the p-wave component of the incident wave

13: direction of the s-wave component of the incident wave

14: Shaped image of local difference of complex reflection coefficient ratio on solid surface photographed by CD camera

The present invention relates to a method for detecting binding of an antibody to an antigen by using a property in which the complex reflection coefficient ratio changes according to the change in the complex refractive index of the protein thin film according to the binding of the antibody and the antigen, which is a protein, on a solid surface. will be.

In particular, by imaging the difference in the complex reflection coefficient ratio of the sample surface area to which the incident light is irradiated with an image camera, it is possible to simultaneously detect a plurality of antibody-antigen bonds occurring on the solid surface. do.

The complex refractive index and the complex reflection coefficient ratio are the main physical properties defined in the optical field of physics and are represented by equation 1 (complex refractive index) and equation 2 (complex reflection coefficient ratio).

The p-wave component and the time when the direction of the electric field of the electromagnetic wave lies on the incident surface are called the s-wave component.

Physical meanings such as the p wave and the s wave, the plane of incidence and the angle of incidence are shown in FIG. 1.

The method for measuring the complex reflection coefficient ratio of an organic / organic thin film and the polarization optical configuration for implementing the organic thin film have various forms and are known as ellipsometry in the optical field.

The present inventors construct polarization optical equipment that can detect the change in the complex reflection coefficient ratio to measure the change in the complex reflection coefficient ratio due to the binding between proteins, and by using the same, the protein pattern and immobilized protein on the solid surface and The present invention has been completed by revealing that the binding between the target proteins can be imaged.

Hereinafter, the present invention will be described in detail.

The reflective properties of the thin film present on the solid surface depend on the complex refractive index of the thin film present, and the complex refractive index is generally calculated by measuring the complex reflection coefficient ratio.

In view of the fact that the binding between proteins on a solid surface changes the complex refractive index of a protein thin film, the present invention configures a polarizing optical device capable of measuring a change in the complex reflection coefficient ratio of a thin film to provide a complex reflection coefficient ratio of a thin film. Changes were taken by image, which confirmed the binding of proteins.

1 shows a configuration of a polarizing optical device used in the present invention, and is composed of general optical components such as a light source 1, a polarizer 2, a compensator 3, an analyzer 7, and the like.

As mentioned above, the configuration and driving principle of the polarizing optical device are presented in the existing literature.

The incident wave irradiated from the light source is properly polarized through the polarizer 2 and the compensator 3, then enters and reflects on the sample surface in which the protein thin film is present, passes through the analyzer 7, and finally the intensity of light in the light detector. Is measured.

The complex reflection coefficient ratio shown in Equation 2 is the ratio of the reflection coefficients of the p-wave component and the s-wave component of the irradiated light. In the configuration of the complex reflection coefficient ratio measuring apparatus used in the present invention, the protein thin film on the solid surface As the complex refractive index changes, the optical intensity of the reflected wave varies.

In addition, the cross section of the reflected wave is enlarged through the objective lens inserted between the sample and the analyzer, and by using the CD camera as the light detector, it is possible to capture the local difference in the light intensity of the enlarged reflected wave cross section as an image.

Therefore, through the configuration of this polarizing optical device, it is possible to image the difference in the local complex reflection coefficient ratio of the protein thin film on the surface of the sample on which the incident wave is incident.

As mentioned above, since the change in the complex reflection coefficient ratio is due to the binding of the protein, this principle makes it possible to detect the binding of the protein on the solid surface.

In Example 1, the pattern of the several nanometer-thick protein thin film formed on the solid surface was imaged by the method demonstrated above.

The difference between the present and nonexistent regions of the protein thin film array was confirmed.

Example 2 is an example applied to protein chip analysis using the technique proposed in the present invention.

The protein thin film formed on the solid surface binds to the protein present on the surface of E. coli, and thus the change in the complex refractive index of the protein thin film could be detected by the method proposed in the present invention.

The embodiment has been described in detail below. The following examples illustrate the application of the present invention, it is obvious that the scope of the present invention is not limited thereto.

(Example 1)

In this embodiment, a protein thin film pattern was formed on a solid surface, and the pattern of the protein thin film was photographed by the method suggested by the present invention.

A solid substrate on which a thin film of chromium 50 nm and gold 100 nm was deposited on a silicon wafer was fabricated.

A thin substrate of 11-MUA was formed on the solid surface by soaking the solid substrate in ethanol solution (150 mM) of 11-MUA (11-mercaptoundecanoic acid) for 12 hours or more.

The 11-MUA thin film was reacted with EDAC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) solution to allow subsequent binding to proteins.

The activated 11-MUA membrane was loaded with an aqueous solution of anti-BSA (Bovine Serum Albumine) (3.4 mg / ml).

At this time, the amount of the injected protein aqueous solution was about 0.7nl for each region, the resulting protein thin film was a circular pattern of about 250㎛ diameter.

It was refrigerated for at least 24 hours for binding of the injected protein aqueous solution and 11-MUA membrane.

After the binding was completed, the protein-bound substrate was washed with water and dried with nitrogen, and then the formation of the protein thin film was confirmed by the technique proposed in the present invention.

The light source used is a helium-neon laser of 632.8 nm, the cross section of which is approximately 600 mu m in diameter.

Figure 2 is a pattern of a protein thin film taken by the image proposed by the present invention, the thickness of the protein thin film is usually about a few nanometers can not be visually confirmed.

It is known that the refractive index component of the complex refractive index of the gold substrate is 0.15 and the extinction coefficient component is 3.5-3.6 in the wavelength range of 632.8 nm, and the complex refractive index of the protein thin film is 1.4-1.6 and the extinction coefficient component is 0.

Due to the difference in the complex refractive index between the substrate and the protein thin film, it is confirmed that the difference in the complex reflection coefficient ratio in the irradiation region of the incident wave occurs, and thus the local difference in the light intensity can be photographed.

It was also confirmed that differences in complex reflection coefficient ratios in various regions of the protein thin film can be detected simultaneously.

(Example 2)

In the present Example, by applying the technique of the present invention to the binding between the protein thin film immobilized on the solid surface and the protein introduced from the outside, the detection of the binding between proteins using the measurement of the complex reflection coefficient ratio change was confirmed.

In particular, by assembling the actual protein chip, a specific application example is presented.

As in Example 1, a solid substrate on which 50 nm of chromium and 100 nm of gold was deposited was fabricated on a silicon wafer.

A thin substrate of 11-MUA was formed on the solid surface by soaking the solid substrate in ethanol solution (150 mM) of 11-MUA (11-mercaptoundecanoic acid) for 12 hours or more.

The 11-MUA thin film was reacted with EDAC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) solution to activate it for subsequent protein binding.

An aqueous solution of protein G (concentration 5 mg / ml), which can immobilize an antibody, a type of protein, was raised.

At this time, the amount of protein introduced was approximately 0.7nl, and the pattern of the formed protein thin film was a circle having a diameter of 250㎛.

It was refrigerated for more than 24 hours to bind Protein G and MUA thin film.

After washing the refrigerated substrate with water, it was immersed in a BSA aqueous solution (concentration 3%) for more than 12 hours to prevent the binding of other proteins in the region other than the protein G thin film region.

In order to immobilize an antibody, a kind of protein, on a BSA-treated solid substrate, an aqueous antibody solution (concentration 50pM) was raised and refrigerated for 24 hours.

The immobilized antibody was selected as an antibody capable of binding to a protein on the surface of E. coli.

The refrigerated solid substrate was washed with distilled water to complete the assembly of the final protein chip.

E. coli aqueous solution was added to the assembled protein chips for each concentration (10 ³ , 10 ⁵ , 10 ⁷ CFU / ml), and refrigerated for more than 3 hours.

Protein chips reacted with the E. coli aqueous solution were washed with distilled water and dried.

Finally, the protein pattern of the protein chip reacted with the E. coli aqueous solution at different concentrations was photographed by the difference of the complex reflection coefficient ratio.

The same light source as in Example 1 was used.

3A to 3D are patterns of the protein thin film due to the difference in the complex reflection coefficient ratio shown in each protein chip.

3A, 3B, 3C, and 3D show different light intensities in the protein thin film region.

Therefore, the average light intensity of the protein thin film region was calculated, and a graph of the correlation between the calculated average light intensity and the concentration of E. coli reacted is shown in FIG. 4.

There is a high correlation between the average light intensity and the concentration of E. coli (R ² = 0.97).

This demonstrated the detection of protein binding using a local measurement of the complex reflection coefficient ratio due to the binding between proteins presented in the present invention.

As described above, according to the present invention, in order to detect the binding of the protein on the solid surface or the binding between the protein and the protein, the measurement of the local change in the complex reflection coefficient ratio is used, so that the use of the label protein, which is the main method, is omitted. .

This shortens the detection time, simplifies the detection process, does not require a highly skilled technician, and there is no expensive labeling protein, thereby reducing costs.

In addition, as a technology capable of solving the difficulty of simultaneous detection in the protein array, which is a problem of instrumental analysis represented by the recent surface plasmon resonance technology, automation for clinical application of the protein chip technology may be enabled.

Claims (3)

In a thin film on which a protein such as antigen-antibody binding occurs on the surface of a solid substrate, the polarized light passes through a polarizer and a compensator. By measuring the intensity of light in the detector, the local difference in the coefficient of radiation reflection due to the difference in the complex refraction of the protein thin film is detected, and using the local detection of the complex reflection coefficient ratio characterized by detecting the binding of the protein Method for detecting protein binding on solid surface using difference. The method according to claim 1, wherein when detecting the change in the complex refractive index of the protein thin film and the corresponding change in the complex reflection coefficient ratio by the protein binding occurring on the solid surface using the polarization reflection characteristics of the protein thin film, A method for detecting protein binding on a solid surface using a local difference of a complex reflection coefficient ratio, characterized by allowing a CD camera to confirm protein binding on a solid surface due to a phosphorus difference. 2. The complex reflection coefficient ratio of claim 1, wherein protein binding can also be detected for a protein chip and a protein immunosensor array using a protein binding detection method using local difference detection of the complex reflection coefficient ratio. Method for detecting protein binding on a solid surface using a local difference of.