KR100691528B1 - Instrument of scanning surface plasmon microscope for the analysis of protein arrays - Google Patents

Abstract

The present invention relates to an apparatus for ultra-fast analysis of protein chips, and according to the present invention, an apparatus for rapidly analyzing a protein chip in large quantities comprises a light source for irradiating light and a polarizer for p-polarizing light emitted from the light source. And a prism for irradiating p-polarized light by the polarizer, a protein chip having a plurality of protein spots arranged in constant and horizontal direction in contact with a bottom surface of the prism so that the light irradiated by the prism is incident; And the xy stage which is installed on the prism and the protein chip thereon and is sequentially moved by a driving means using GPIB communication so that light passing through the prism is irradiated to a portion including spots of a specific range of the protein chip. And sequentially photographing light reflected from the protein chip to obtain an image. It characterized in that it comprises a CCD camera.

Protein Chip, Protein Array, SPR, CCD Camera, x-y Stage

Description

Instrument of scanning surface plasmon microscope for the analysis of protein arrays}

1 is a schematic configuration diagram of a white light surface plasmon scanning microscope device according to the present invention,

2 is a graph showing the principle of the surface plasmon resonance image obtained according to the present invention,

3 is a diagram showing the pixelation of the protein chip according to the present invention,

Figure 4a is a diagram showing the results of the qualitative analysis of protein chips loaded with various types of proteins using the analysis device according to the present invention,

4B is a graph showing a quantitative analysis result using the analysis result of FIG. 4A;

Figure 5a is a diagram showing the results of qualitatively analyzing the protein chip on one type of protein by using the analysis device according to the concentration gradient of the protein,

5B is a graph showing a quantitative analysis result using the analysis result of FIG. 5A.

6 is a flow chart of the position control of the x-y stage of the analysis device according to the present invention.

Explanation of symbols for main parts of the drawings

1: white light source 2: aperture

3: convex lens 4: polarizer

5,6 mirror 7: x-y stage

8: protein chip 9: prism

10 filter 11: CCD camera

12: spot 13: slide glass

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an ultrafast analysis device of a protein chip. In particular, light reflected from an interface between a dielectric and a metal thin film by injecting light from a white light source into a protein chip is referred to as a charge coupled device (CCD). ) A device for analyzing a large amount of interactions and biological reactions of biopolymers such as proteins, carbohydrates, nucleic acids, and the like at high speed by measuring and analyzing surface plasma resonance (SPR) images using a camera. will be.

Protein chips are used to fix dozens or hundreds of proteins on small substrates or slides, and then simultaneously analyze the binding of proteins. Protein chips can be widely used in proteomics research, drug development, and diagnostics, such as protein expression and function studies, protein interaction studies.

Analysis equipment for protein chips uses detection techniques such as fluorescence, chemiluminescence, mass spectrometry, and SPR, and uses them for diagnosis and proteome research. When performing a relatively simple analysis using a protein chip, there is a method of labeling a protein with a fluorescent material and analyzing it with a fluorescence scanner like a DNA chip, but there is a problem in that all proteins are uniformly labeled with a fluorescent material and mass spectrometry is performed. The technology has the advantage of analyzing unknown samples, but it is pointed out that it is difficult to analyze a large number of samples at high speed.

On the other hand, the protein chip using SPR phenomenon is very sensitive to the physical and chemical changes occurring near the surface of the metal thin film as the change of the resonance angle or the wavelength, so that it can be applied to various fields through the surface treatment of the metal thin film. Compared to other methods, biosensor for the study of protein interactions has the advantage of detecting the interaction of biopolymers in real time without any damage or modification to the sample and without the use of radioactive or fluorescent markers. It is evaluated as a useful technique in the field.

In general, when light travels from a high refractive index medium to a low refractive index medium, total reflection occurs when the incident angle is greater than or equal to the critical angle. The phenomenon of exciting surface plasmon on a metal surface by waves is called SPR phenomenon. Metals such as gold, silver, aluminum, and the like are mainly used. Among them, silver, which has the sharpest SPR resonance peak, and gold, which exhibits excellent surface stability against external environmental changes, are commonly used.

The existing SPR sensor for protein chip analysis has a problem in that the number of channels that can be analyzed at a time is limited. Therefore, there has been a need for a technique for analyzing a large number of proteins of several tens or thousands. In particular, when the SPR sensor adopts the concept of an array type, and provides a plurality of sensing functions, it is possible to analyze several materials at the same time.

The general method of analyzing a protein chip as described above is to make incident light generated from an incident light part into a parallel beam of uniform intensity through an optical lens, and then pass through a prism to enter the chip surface, and the incident beam is reflected from the chip surface. This is measured by the light receiver.

The light receiver is a part for measuring the intensity of light reflected from the surface of the metal thin film. In the Republic of Korea Patent No. 404071, “A Protein Chip Analysis Device Using White Light SPR,” the light receiver collects light reflected from the protein chip through an optical fiber to form a plane image. And an optical array detector for detecting the spectroscopic light. However, in order to obtain a reliable result by such a measuring method, it is necessary to measure the entire area of the spot precisely within 100 um intervals, instead of measuring the center of each spot of the protein chip, which takes a long time and a large amount of data. There is a problem that needs to be stored.

In addition, there is a method of obtaining an image of the light reflected from the protein chip directly by using imaging devices such as CCD, but the conventional CCD camera method can measure only a limited area of the protein chip. There is a problem that it is difficult to apply to a technique for rapidly mass analysis of tens or thousands of proteins distributed in a large area. In order to capture with a CCD camera so that the entire area of the protein chip is in one image, the distance between the protein chip and the CCD camera needs to be increased. In this case, a large area of parallel p-polarized light is produced to irradiate the protein chip. There is a constraint that the spot image resolution of the protein chip to be analyzed is sharp and difficult to analyze. In addition, in order to make a large area of parallel p-polarized light and irradiate the protein chip, the optical system has to be relatively complicated, which has many problems in terms of economy and practicality.

The present invention is to solve the problems of the prior art, the present invention is to obtain a protein chip image with a CCD camera, it is possible to analyze a large area of the protein chip without limiting the size of the protein chip The purpose is to provide a device.

An apparatus for analyzing protein chips by a surface plasmon scanning microscope according to the present invention for achieving the above object is a light source for irradiating light, a polarizer for p-polarized light from the light source, the polarizer A prism for irradiating p-polarized light by the light source, a protein chip having a plurality of protein spots constantly arranged in a horizontal direction in contact with a bottom surface of the prism such that the light irradiated by the prism is incident thereon; It uses GPIB (General-Purpose Interface Bus) communication so that the prism and the protein chip are installed on top of each other, and the light passing through the prism is irradiated to the part including the specific range of spots of the protein chip. The xy stage which is sequentially moved by the driving means, and the light reflected from the protein chip sequentially By imaging by the camera comprises a CCD to obtain image.

In addition, the light emitted from the light source is white light, it is preferable to provide a filter between the protein chip and the CCD camera so that the light reflected from the protein chip is incident to the CCD camera through the filter.

In addition, it is preferable to further include a device for quantitative analysis from the image obtained by the CCD camera.

Hereinafter, specific embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a conceptual diagram showing a schematic configuration of the present invention. As shown in FIG. 1, the protein chip analyzing apparatus according to the present invention includes a light source 1, an aperture 2, a convex lens 3, a polarizer 4, a mirror 5, 6, and an xy stage 7. , A protein chip 8, a prism 9, a filter 10, a CCD camera 11, and the like.

The light source 1 uses white light such as tungsten lamp, tungsten halogen lamp, and xenon lamp having a near infrared wavelength range in the ultraviolet wavelength, so that the white light has a constant light intensity to provide a stable output and obtain a large area of light. Because. The convex lens 3 is used to uniformly collect the light emitted from the light source 1 and can be used in combination of several. The aperture 2 has a function of adjusting the amount of light and a function of making a balanced beam. In order to generate surface resonance plasmon resonance, a polarizer 4 is required which produces light in a transverse magnetic (TM) mode. In addition, two mirrors 5 and 6 are used so that the polarized light is above the critical angle at which total reflection occurs (surface plasmon resonance angle; θ _sp ). Even if the polarized light is incident on the protein chip 8 above the critical angle at which total reflection occurs, surface plasmons cannot be generated without passing through the prism 9. This is because the momentum of incident light cannot be combined with the momentum of the surface plasmon occurring at the metal surface.

In order to generate surface plasmons, a Kretchmann type ATR (Attenuated Total Reflectance) coupler using a prism 9 is used. The protein chip 8, which is p-polarized through the prism 9, is located on the xy stage 7, where the protein chip 8 has a diameter of 2 mm with a diameter of 12 mm like a slide glass 13. It is a small multi-matrix array chip in which a metal thin film arranged in a plurality of matrices or more arranged on a plane of a dielectric is deposited. The material of the metal thin film in this embodiment is gold (Au). In order to reduce the refractive index difference between the prism 9 and the slide glass 13, it is sealed with distilled water or a commercially available emulsion oil. The x-y stage 7 on which the protein chip 8 is mounted can be moved back and forth or left and right by a driving device (not shown), and the driving device can be configured as a step motor driven by GPIB communication.

Since the light reflected from the protein chip 9 passes through the filter 10, white light is used as a light source, and thus, the filter 10 needs to be installed. The intensity of the reflected light is measured by the CCD camera 11 imaging the light passing through the filter 10. The image data of each spot detected by the CCD camera 11 is analyzed by imaging software in a computer and the result is displayed on a monitor.

Figure 2 shows the relationship between the SPR spectrum and the light intensity (intensity) in the vicinity of the resonance position to be measured. As the SPR spectrum moves to the right, the intensity of light to be measured increases, so that the brightness of the spot 12 is brighter. In general, the greater the amount of protein attached to the protein chip, the SPR spectrum is shifted to the right. (See FIG. 5). Figure 2 is a graph of the surface plasmon resonance signal, the concave portion in the middle of the curve is the portion where the resonance wavelength is generated, the light intensity which is the y-axis value of the point where the curve meets the vertical line across the curve when the value of the SPR resonance wavelength increases Qualitative analysis results are obtained by detecting how is changed.

In FIG. 3, a constant portion surrounded by horizontal and vertical lines forms one pixel, and all pixels are measured in order. In FIG. 3, four spots 12 are set to form one pixel. The order of measurement of the pixels is determined according to the moving direction of the xy stage 7. The process of repeating the imaging after the xy stage 7 is moved by GPIB communication so that a new pixel is irradiated with light after one pixel is imaged do. Motion control, data acquisition and analysis, and display of the x-y stage 7 is accomplished through a proprietary program based on LabVIEW software. FIG. 6 shows a position control flowchart of the x-y stage 7. When the x-y stage 7 moves, it is determined whether the target position and the current position are within an allowable error range, and the program is moved or stopped again.

Figure 4a is a graph showing the results of qualitative analysis of the image captured by the CCD camera 11, the upper row is the result for Rac1 protein, the lower row is the result for RhoA protein. In addition, the leftmost column is a result obtained by measuring glutathione (GSH) coated on the metal thin film without antibodies or antigens, and is used as a reference value for determining resonance image change. The right column is the result when the antigen is reacted on the GSH, and the right column is the result when the antibody is reacted on the antigen. The contrast between each spot when the protein chip does not react on the surface of the GSH, when the antigen is reacted, or when the antibody reacts on the antigen can be seen at a glance.

Meanwhile, FIG. 4B is a graph showing a quantitative analysis result, which is a bar graph showing quantitatively calculating the contrast of an image from FIG. 4A through data processing. The three bars on the left are the results of the antigen-antibody response of the Rac1 protein, which are the reference values for analyzing the antigen and antibody responses from the left, respectively. The mean intensity at the time of antigen-antibody reaction, and the three bars on the right are the results for RhoA protein, the mean intensity of the GSH surface image, and only the antigen on the GSH surface as reference values for analyzing the antigen and antibody response, respectively. The average intensity at the time of use and the average intensity at the time of antigen-antibody reaction are shown. Here, the y-axis value of each bar is the average value at one spot, and the length of the vertical line drawn at the top of the bar represents the standard deviation.

Figure 5a shows the analyzed image when the application of different concentrations of protein on the protein chip using the analysis device according to the present invention. In FIG. 5A, the leftmost spot is a reference spot for monitoring how much protein is attached. As the spot increases to the right, the concentration of the protein increases. As the protein concentration increases, the brightness of the spot changes brighter than that of the reference spot. Able to know.

FIG. 5B is a graph showing the quantification of the intensity of each spot from each spot image of FIG. 5A. In this example, the PAK protein antigen was attached to a protein chip, and then measured and analyzed. The results show that when the protein concentration is above a certain level, it becomes saturated and there is almost no change in the light intensity. The vertical line through the broken point of the graph also shows the standard deviation of the intensity measured at each spot as in FIG. 4B.

As mentioned above, in order to increase the number of spots of protein chips that can be acquired at one time through a CCD camera, the CCD camera should be placed at a far distance. However, the resolution of the CCD camera may limit the number of spots. In addition, the longer the CCD camera is located, the smaller the spot size will be and the uniform measurement results cannot be obtained. Therefore, an analyzer using a conventional CCD camera as a light receiver is suitable for analyzing a large area of protein chips. However, according to the surface plasmon scanning microscope apparatus for protein chip analysis according to the present invention, the CCD camera is not located far from the protein chip, and GPIB communication is performed without using a complicated optical system to examine a large area protein chip. To measure the image by moving the protein chip sequentially. Protein chips with a large area can also be analyzed in a short time, so that the technique according to the present invention is a very useful technique to be used for rapid and mass analysis of proteins.

Claims (3)

A light source for irradiating light; A polarizer for p-polarizing light emitted from the light source; A prism for irradiating p-polarized light by the polarizer; A protein chip in which a plurality of protein spots are arranged in a vertical and horizontal manner in contact with a bottom surface of the prism such that light irradiated by the prism is incident; The prism and the protein chip are installed thereon, and the xy stage sequentially moved by the driving means using GPIB communication so that the light passing through the prism is irradiated to the part including the specific range of spots of the protein chip. ; A CCD camera for sequentially photographing light reflected from the protein chip to obtain an image; Surface plasmon scanning microscope device for protein chip analysis, comprising a. The method of claim 1, The light emitted from the light source is white light, and the surface of the protein chip analysis plasmon characterized in that the filter is installed between the protein chip and the CCD camera so that the light reflected from the protein chip is incident to the CCD camera through the filter. Scanning microscope device. The method according to claim 1 or 2, Surface plasmon scanning microscope device for protein chip analysis, characterized in that it further comprises a device for quantitative analysis from the image obtained by the CCD camera.

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