KR20110112177A - Method for diagnosing of alzheimer's disease using giant magneto resistance and magnetic bead-polyprotein complex for alzheimer's disease - Google Patents

Abstract

The present invention relates to a method for diagnosing Alzheimer's disease using a giant magnetoresistive sensor and a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease. Diagnosis method of Alzheimer's disease using the giant magnetoresistance sensor according to the present invention can easily diagnose Alzheimer's disease by a simple method using a giant magnetoresistance sensor instead of conventional fluorescent material or genetic analysis, as a biosensor for diagnosing Alzheimer's disease Mass production is possible, making it useful for monitoring and treating Alzheimer's disease.

Description

Method for diagnosing of Alzheimer's disease using giant magneto resistance and magnetic bead-polyprotein complex for Alzheimer's disease using magnetic resistance sensor

The present invention relates to a method for diagnosing Alzheimer's disease using a magnetoresistive sensor and to a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease.

Alzheimer's disease is the leading cause of dementia and the most common degenerative neurological disease. Indeed, progressive Alzheimer's disease is characterized by a loss of memory, speech, language judgment, and discernment. The nature of symptoms that often confuse with older physiological illness, the severity of these symptoms, and the age at which these symptoms occur varies from person to person. For this reason, the exact etiology or treatment of Alzheimer's disease is not known, and it is difficult to diagnose Alzheimer's disease at an early stage.

Pathological features of Alzheimer's disease include senile plaques, neurofibrilary tangles, and neuronal loss. Aggregated amyloid-beta protein, which accounts for most of the senile plaques, is considered to be a major cause of Alzheimer's disease by various experimental evidence.

On the other hand, there are many ways to diagnose Alzheimer's disease. Korean Patent Laid-Open Publication No. 10-2009-0048192 describes methods for diagnosing, preventing and treating dementia (Alzheimer's disease) and screening methods thereof. Specifically, FcγRIIb and its variants, FcγRIIb extracellular domain proteins, and anti-FcγRIIb antibodies , Binding inhibitors selected from the group consisting of FcγRIIb-specific peptides, and FcγRIIb, in particular siRNA, reduce signal transduction, intracellular migration, neurotoxicity, cell death, and memory loss by FcγRIIb and Aβ, thus making it possible to diagnose, prevent, and treat Alzheimer's disease. It is described.

In addition, Korean Patent Laid-Open Publication No. 10-2007-0073778 describes a method of measuring β-amyloid in a biological sample such as blood, and describes its application to diagnosis of Alzheimer's disease. By immunoassay using an antibody that recognizes the terminal portion, the total amount of the β-amyloid 1-42 fragment having the C-terminal portions of β-amyloid 1-42 and β-amyloid 1-42 in the biological sample was measured. A method for enabling the assay of Alzheimer's disease is described.

In addition, Korean Patent Publication No. 10-2009-0098941 discloses methods and compositions that can be used to detect Alzheimer's disease in mammals, particularly humans, and in particular, serum markers for Alzheimer's disease and methods used in diagnostic procedures. It is. Since the methods use optical or chemical methods, real-time diagnosis is difficult, expensive to diagnose, and commercialization is difficult because mass production of Alzheimer's disease diagnosis kits is impossible.

Accordingly, the problem to be solved by the present invention is to provide a method for detecting Alzheimer's disease in a simpler and easier manner than conventional methods by detecting amyloid-beta protein using a magnetoresistance sensor.

In addition, an object of the present invention is to provide a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease, which is pretreated so that magnetic beads used for diagnosing Alzheimer's disease using a magnetoresistive sensor can be combined with protein biomarkers. .

In addition, the problem to be solved by the present invention, a method for producing a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease combined with a protein biomarker by combining magnetic beads used for diagnosing Alzheimer's disease using a magnetoresistance sensor with a multiprotein. To provide.

In order to solve the above problems, the present invention comprises the steps of pretreatment with magnetic beads to be able to bind with protein biomarkers that cause Alzheimer's disease; Placing the cell to be diagnosed on the magnetoresistive sensor; Placing the pretreated magnetic beads on the cell to be diagnosed and applying an external magnetic field to magnetize them; And detecting a change in the magnetic field according to whether the pretreated magnetic beads and the protein biomarker are coupled to the magnetoresistive sensor to determine whether Alzheimer's disease is present in the cell to be diagnosed. Provide diagnostic methods.

The magnetic field change may be caused by a stray field generated in the magnetic beads when the protein biomarker is present and the magnetic beads are bound.

The protein biomarker may be an amyloid-beta protein.

The magnetic beads may range in diameter from 50 nm to 5 μm.

The pretreatment may be performed by coating magnetic beads with streptavidin, binding to biotin, and then combining polyethylene glycol (poly (ethylene glycol)).

The magnetoresistive sensor may be an anisotropic magnetoresistive thin film, a giant magnetoresistive thin film, a tunnel-type magnetoresistive thin film, or the like.

The magnetoresistive sensor may include a free layer, a spacer, a pinned layer, and a pinning layer.

The magnetoresistive sensor may have a cross shape or a rod shape.

The magnetoresistive sensor may be protected by an oxide or nitride thin film layer.

The cells may be brain cells, olfactory cells, taste cells or visual cells in which amyloid-beta proteins may accumulate.

The magnetization may be performed by applying an external magnetic field in a vertical or horizontal direction with respect to the magnetoresistive sensor surface.

In addition, the present invention Alzheimer's disease, characterized in that the magnetic bead is coated with streptavidin (streptavidin), the streptavidin binds to biotin (biotin) and the linker is coupled to the protein biomarker Diagnostic magnetic bead-polyprotein complexes are provided.

The protein biomarker is an amyloid-beta protein.

The linker may be used polyethylene glycol (poly (ethylene glycol)) and the like.

In addition, the present invention comprises the steps of coating around the magnetic beads with streptavidin (streptavidin); Binding the magnetic beads coated with streptavidin with biotin; And combining the biotin with a linker, wherein the linker provides a method for preparing a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease, wherein the linker is combined with a protein biomarker.

In the diagnosis method of Alzheimer's disease using a magnetoresistance sensor according to the present invention, Alzheimer's disease can be easily diagnosed by a simple method using a giant magnetoresistance sensor instead of a conventional fluorescent substance or genetic analysis.

In addition, the method for diagnosing Alzheimer's disease using a magnetoresistive sensor according to the present invention can produce a large amount of biosensor for diagnosing Alzheimer's disease, which can be usefully used for monitoring and treating Alzheimer's disease.

1 is a process flowchart showing a method for diagnosing Alzheimer's disease using a magnetoresistive sensor according to the present invention.
Figure 2 is a process flow diagram illustrating a method for producing a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease according to the present invention.
Figure 3 is a schematic diagram showing a giant magnetoresistance sensor for diagnosing Alzheimer's disease according to the present invention.
Figure 4 is a schematic diagram showing the structure of a large magnetoresistive thin film for diagnosing Alzheimer's disease according to the present invention.
5 is a schematic diagram showing the magnetic field direction of the magnetic beads when the magnetic beads are magnetized in a direction perpendicular to the magnetoresistive sensor surface.
6 is a schematic diagram showing the magnetic field direction of the magnetic beads when the magnetic beads are magnetized in the horizontal direction with respect to the magnetoresistive sensor surface.
Figure 7 is a schematic diagram showing a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease according to the present invention.
8 is a schematic diagram showing a method for diagnosing Alzheimer's disease using a giant magnetoresistive sensor according to the present invention.

Hereinafter, the present invention will be described in detail.

In the method for diagnosing Alzheimer's disease using a magnetoresistive sensor according to the present invention, a process of pretreating magnetic beads is required to be combined with a protein biomarker that causes Alzheimer's disease.

In this case, the protein biomarker is an amyloid-beta protein, and the protein biomarker may use cells existing on the surface.

In addition, the magnetic beads are coated with streptavidin to selectively bind to amyloid-beta protein, which is known to cause Alzheimer's disease, and then selectively binds to biotin, such as poly (ethylene glycol). The pretreatment process is performed to allow binding to amyloid-beta protein using a linker. The magnetic beads preferably have a diameter in the range of 50 nm to 5 μm. If the diameter of the magnetic beads is less than 50 nm, it is difficult to sense stray fields generated in the magnetic beads. If the diameter of the magnetic beads exceeds 5 µm, the magnetic beads bind to amyloid-beta present on the cell surface. There is a difficult problem.

Through the pretreatment, magnetic beads can selectively bind with amyloid-beta protein. In other words, Alzheimer's disease contains amyloid-beta protein, so that it binds to magnetic beads, and normal cells do not bind to magnetic beads.

In the method for diagnosing Alzheimer's disease using a magnetoresistive sensor according to the present invention, a step of placing a cell to be diagnosed on the magnetoresistive sensor is performed.

The magnetoresistive sensor may be an anisotropic magnetoresistive thin film, a giant magnetoresistive thin film, a tunnel-type magnetoresistive thin film, and the like. Can be used.

The magnetoresistive sensor includes a free layer, a spacer, a pinned layer, and a pinning layer, and may have a cross or bar shape.

In addition, the magnetoresistive sensor may be protected by an oxide or nitride thin film layer, and the cells disposed on the magnetoresistive sensor are preferably brain cells, olfactory cells, taste cells, or visual cells in which amyloid-beta proteins may accumulate. .

In the method for diagnosing Alzheimer's disease using a magnetoresistive sensor according to the present invention, the pre-treated magnetic beads are placed on the cell to be diagnosed and an external magnetic field is applied to magnetize the same.

The pretreated magnetic beads are magnetized by applying an externally applied magnetic field in a vertical or horizontal direction with respect to the magnetoresistive sensor surface, and a fine stray field is generated from the magnetic beads due to the magnetization.

In the method of diagnosing Alzheimer's disease using a magnetoresistive sensor according to the present invention, magnetic field change depending on whether the pretreated magnetic beads and the protein biomarker are combined is detected by the magnetoresistive sensor to detect Alzheimer's disease of the cell to be diagnosed. It is possible to diagnose Alzheimer's disease by determining the presence or absence.

Magnetic beads combined with amyloid-beta protein can detect the change of the magnetic field caused by stray magnetic field caused by magnetization by an external magnetic field using a magnetoresistance sensor, and detect the presence of amyloid-beta protein. Therefore, the presence or absence of Alzheimer's disease can be determined by the magnetoresistance value of the magnetoresistance sensor caused by the change of the applied voltage and the output voltage supplied to the magnetoresistance sensor. In this case, as the number of amyloid-beta proteins increases, the number of bound magnetic beads increases so that the magnetoresistance value changes linearly.

In addition, the present invention Alzheimer's disease, characterized in that the magnetic bead is coated with streptavidin (streptavidin), the streptavidin is combined with biotin (biotin) is a linker to bind the protein biomarker Diagnostic magnetic bead-polyprotein complexes are provided.

The protein biomarker is preferably amyloid-beta protein, and the linker is preferably polyethylene glycol (poly (ethylene glycol)).

In addition, the present invention comprises the steps of coating around the magnetic beads with streptavidin (streptavidin); Binding the magnetic beads coated with streptavidin with biotin; And combining the biotin with a linker, wherein the linker provides a method for preparing a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease, wherein the linker is combined with a protein biomarker.

≪ Example 1 >

Magnetic beads  Preprocessing step ( S100 )

Magnetic beads about 100 nm in diameter were pretreated to detect protein biomarkers causing Alzheimer's disease. The pretreatment was coated with streptavidin around magnetic beads followed by binding the streptavidin with biotin and the biotin bound with polyethylene glycol, a linker, to bind amyloid-beta protein biomarkers. Through the pretreatment, the magnetic beads are selectively bound to amyloid-beta protein. In the case of Alzheimer's disease, the magnetic beads bind to the magnetic beads and not to the magnetic beads in the case of normal cells.

Stage of cell placement for diagnosis ( S120 )

A brain cell capable of accumulating amyloid-beta protein is disposed on the upper surface of the giant magnetoresistance sensor of the spin valve structure which is protected by the oxide thin film layer. The giant magnetoresistance sensor includes a free layer and a spacer. ), A rod type including a pinned layer and a pinning layer was used.

Magnetic beads  Magnetization stage ( S130 )

The pretreated magnetic beads were placed on the giant magnetoresistive sensor and an external magnetic field was applied. The pretreated magnetic beads applied an external magnetic field in a direction perpendicular to the magnetoresistive sensor surface, and generated a fine stray magnetic field in the magnetic beads magnetized by the external magnetic field.

Alzheimer's disease determination stage ( S140 )

Magnetic beads combined with amyloid-beta protein can detect changes in magnetic field caused by stray magnetic field caused by magnetization by external magnetic field, and can detect magnetic resistance and Alzheimer's disease when normal cells exist. Alzheimer's disease was diagnosed by comparing magnetoresistance when present. At this time, as the number of amyloid-beta proteins increases, the number of bound magnetic beads increases so that the magnetoresistance value changes linearly.

<Example 2>

After coating with streptavidin around 100 nm diameter magnetic beads (S200), the streptavidin binds to biotin (S210) and polyethylene glycol to bind the biotin with amyloid-beta protein. In combination with (S220) to prepare a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. Here, the repeated description, well-known functions and configurations that may unnecessarily obscure the subject matter of the present invention, and detailed description of the configuration will be omitted. Embodiments of the present invention are provided to more completely describe the present invention to those skilled in the art. Accordingly, the shape and size of elements in the drawings may be exaggerated for clarity.

1 is a process flowchart showing a method for diagnosing Alzheimer's disease using a magnetoresistive sensor according to the present invention.

Figure 2 is a process flow diagram illustrating a method for producing a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease according to the present invention.

Figure 3 is a schematic diagram showing a giant magnetoresistance sensor for diagnosing Alzheimer's disease according to the present invention.

Referring to FIG. 3, a GMR sensor 120 layer is deposited on a substrate 130, and a passivation layer 110 formed of an oxide is formed on the GMR sensor. Electrodes 100 made of Ta / Au are formed on both sides of the resistance sensor 120.

Figure 4 is a schematic diagram showing the structure of a large magnetoresistive thin film for diagnosing Alzheimer's disease according to the present invention.

Referring to FIG. 4, the giant magnetoresistive sensor structure used in the present invention is a spin valve composed of a free layer, a spacer, a pinned layer, and a pinning layer. Structure.

Since the spin valve structure has the advantage of obtaining a large resistance change of 5 to 10% even in a small magnetic field, it is suitable for measuring the magnetoresistance of magnetic particles bound to amyloid-beta protein in a giant magnetoresistance sensor.

The giant magnetoresistive sensor used in the present invention can measure the change in the externally applied magnetic field due to the fine stray field generated in the spherical magnetic particles having a size of several tens of nanometers to several micrometers. The resistance change characteristic of the magnetoresistive sensor can be represented as an electrical output signal.

5 and 6 are schematic diagrams showing the magnetic field direction of the magnetic beads when the magnetic beads are magnetized in the vertical direction or the horizontal direction with respect to the magnetoresistive sensor surface.

5 and 6, the direction of the magnetic field applied from the outside may be applied to the surface of the giant magnetoresistive sensor vertically or horizontally. As shown in FIG. 2, magnetic beads may be applied to the giant magnetoresistive sensor. It can be magnetized in the direction perpendicular to the surface or in the horizontal direction, and the stray field generated in the magnetic beads generated at this time affects the magnetization of the giant magnetoresistive sensor, thereby changing the resistance characteristics of the giant magnetoresistive sensor. The characteristic of the output voltage is changed.

Figure 7 is a schematic diagram showing a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease according to the present invention.

Referring to FIG. 7, magnetic beads (or magnetic particles) 300 used in the present invention are coated with streptavidin 310 to selectively bind to amyloid-beta protein 340. The magnetic beads 300 coated with the streptavidin 310 selectively bind to biotin (biotin, 320), and the biotin 320 and amyloid-beta protein 340 are polyethylene glycol (ploy (ethylene glycol), 330). Use a linker such as) to bind. Through this process, the magnetic beads 300 may be combined with the amyloid-beta protein 340.

Through this binding method, the magnetic bead is selectively bound to amyloid-beta protein, and in the case of cells containing amyloid-beta protein causing Alzheimer's disease, the magnetic bead is bound to magnetic bead, and does not contain amyloid-beta protein. Normal cells do not bind to magnetic beads.

8 is a schematic diagram showing a method for diagnosing Alzheimer's disease using a giant magnetoresistive sensor according to the present invention.

Referring to FIG. 8, the magneto-resistance sensor detects a change in the magnetic field due to stray current generated in the magnetic beads after placing the diagnosis target cell on the magnetoresistance sensor and applying an external magnetic field to magnetize the magnetic beads. The presence of beta protein can be detected. Therefore, Alzheimer's disease can be determined by comparing the magnetoresistance in the presence of normal cells with the magnetoresistance in the presence of Alzheimer's disease cells. In addition, as the number of amyloid-beta proteins increases, the number of bound magnetic beads increases so that the magnetoresistance value changes linearly. As a result, by measuring the changed magnetoresistance using the magnetic beads combined with amyloid-beta protein, it is possible to diagnose the progression of Alzheimer's disease as well as the early diagnosis of Alzheimer's disease.

The present invention has the advantage that it can be easily and simply diagnosed using conventional fluorescent materials or genetic analysis and comparison methods, and commercialized as a biosensor for diagnosing Alzheimer's disease.

As described above, the best embodiment has been disclosed in the drawings and the specification. Although specific terms have been used herein, they are used only for the purpose of describing the present invention and are not used to limit the scope of the present invention as defined in the meaning or claims. Therefore, those skilled in the art will understand that various modifications and equivalent other embodiments are possible from this. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

100: electrode (Ta / Au)
110: passivation (oxide)
120: giant magnetoresistive sensor (GMR sensor)
130: substrate
300: magnetic bead
310: Streptavidin
320: Biotin
330: Polyethylene glycol
340: Amyloid-beta protein

Claims (15)

Pretreatment of the magnetic beads to bind to the protein biomarker causing Alzheimer's disease;
Placing the cell to be diagnosed on the magnetoresistive sensor;
Placing the pretreated magnetic beads on the cell to be diagnosed and applying an external magnetic field to magnetize them; And
Diagnosis of Alzheimer's disease using a magnetoresistance sensor comprising detecting a change in the magnetic field according to whether the pre-treated magnetic beads and the protein biomarker is combined with the magnetoresistance sensor Way. The method according to claim 1,
The magnetic field change is caused by a stray field generated in magnetic beads when a protein biomarker is present and magnetic beads are combined, and the Alzheimer's disease diagnosis method using a magnetoresistive sensor. The method according to claim 1,
The protein biomarker is a method for diagnosing Alzheimer's disease using a magnetoresistive sensor, characterized in that the amyloid-beta protein. The method according to claim 1,
The magnetic bead has a diameter of 50 nm to 5 ㎛ range Alzheimer's disease diagnostic method using a magnetoresistive sensor. The method according to claim 1,
The pretreatment is a diagnosis of Alzheimer's disease using a magnetoresistance sensor, characterized in that the magnetic beads are coated with streptavidin (streptavidin) and combined with biotin and then combined with polyethylene glycol (poly (ethylene glycol)) Way. The method according to claim 1,
The magnetoresistive sensor is an anisotropic magnetoresistive thin film, a giant magnetoresistive thin film and tunnel-type magnetoresistive thin film of any one of the Alzheimer's disease diagnosis method using a magnetoresistive sensor. The method according to claim 1,
The magnetoresistive sensor is a diagnostic method of Alzheimer's disease using a magnetoresistive sensor, characterized in that it comprises a free layer, a spacer, a pinned layer and a pinning layer. The method according to claim 1,
The magnetoresistive sensor of the Alzheimer's disease diagnosis method using a magnetoresistive sensor, characterized in that the cross-shaped or rod-shaped. The method according to claim 1,
The magnetoresistive sensor is a diagnostic method of Alzheimer's disease using a magnetoresistive sensor, characterized in that the oxide or nitride thin film layer is protected. The method according to claim 1,
Said cell is a brain cell, olfactory cell, taste cell or visual cell which can accumulate amyloid-beta protein, The diagnosis method of Alzheimer's disease using a magnetoresistance sensor. The method according to claim 1,
The magnetization is a diagnostic method of Alzheimer's disease using a magnetoresistance sensor, characterized in that performed by applying an external magnetic field in the vertical or horizontal direction with respect to the magnetoresistance sensor surface. Magnetic bead-multiple diagnostics for Alzheimer's disease, characterized in that the magnetic beads are coated with streptavidin, the streptavidin is bound to biotin, and the biotin is linked to a protein biomarker. Protein complexes. The method of claim 12,
The protein biomarker is a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease, characterized in that the amyloid-beta protein. The method of claim 12,
The linker is polyethylene glycol (poly (ethylene glycol)) characterized in that the magnetic bead-polyprotein complex for diagnosing Alzheimer's disease. Coating around magnetic beads with streptavidin;
Binding the magnetic beads coated with streptavidin with biotin; And
Coupling the biotin with a linker,
The linker is a method for producing a magnetic bead-polyprotein complex for diagnosing Alzheimer's disease, characterized in that coupled to a protein biomarker.

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