KR20160021803A - Complex comprising bead including quantum dot-layer and method for diagnosing myocardial infarction-related disease using the same - Google Patents

Abstract

Provided is a composite containing a formulation for detecting or analyzing a target material or a bead particle having a quantum dot layer; a composition for diagnosing myocardial infarction or for detecting the target material comprising the composite; and a method for diagnosing myocardial infarction-related diseases using the composite. According to the present invention, the composite and the composition can offer a fast and highly precise analysis result in terms of the detection or the analysis for the target material.

Description

{Complex comprising bead including quantum dot-layer and method for diagnosing myocardial infarction-related disease using the same}

A complex comprising a bead particle including a quantum dot layer and an agent for detecting or analyzing a target substance, a composition for analyzing a target protein comprising the same, a composition for diagnosing a disease related to myocardial infarction using the same, and a method for diagnosing a disease related to a myocardial infarction using the same About.

Currently, acute myocardial infarction is diagnosed by observing changes in the concentration of hormones (CK-MB, Myoglobin, Cardiac Troponin) through chest pain, electrocardiography, and blood tests. It is only possible through it. Since most of the patients go to the hospital after the onset of myocardial infarction, the cost of diagnosis or treatment is very high. Although cardiac troponin is the gold standard for diagnosis of acute myocardial infarction, its specificity is less than 80%, and it is difficult to diagnose early because it is released into the blood after a heart attack. Therefore, even if it is actually an acute myocardial infarction, the possibility of negative appearance is quite high, and physical diagnostic tests such as electrocardiogram, MRI, and X-ray are still mainly performed. In addition, once an acute myocardial infarction occurs, the prognosis is fatal, so the importance of early diagnosis is increasing. Accordingly, the discovery of peptidic diagnostic markers has been accelerated, and among them, a marker well known for its relevance to cardiovascular disease is NT-proBNP (N-terminal proBNP). Although it is known to increase over time in patients with acute myocardial infarction, plasma NT-proBNP concentration is more commonly known as a marker of chronic congestive heart failure (CHF). NT-ProBNP is also released into the blood due to the death of heart cells like Troponin, so it is difficult to use for early diagnosis. Chest pain, electrocardiogram, and cardiovascular angiography are not suitable for early diagnosis, so it is necessary to find biomarkers in blood for early diagnosis, acute myocardial infarction diagnostic test with higher specificity and sensitivity, and diagnosis of diseases related to myocardial infarction.

One aspect is to provide a composite comprising a bead particle including a quantum dot layer, and an agent for detecting or analyzing a target substance.

Another aspect is to provide a composition for analyzing a target substance, a kit for immunological analysis of a target substance, and a composition for diagnosing a disease related to myocardial infarction, including a complex comprising a bead particle including a quantum dot layer and an agent for detecting or analyzing the target substance.

Another aspect is to provide a method for preparing a composite including bead particles including a quantum dot layer and an agent for detecting or analyzing the bead particles and target substances.

Another aspect is to provide a method for diagnosing a disease related to myocardial infarction using a bead particle including a quantum dot layer and an agent that detects or analyzes the bead particle and a target substance.

One aspect provides a composite comprising a bead particle including a quantum dot layer and an agent for detecting or analyzing a target material.

The bead particle is a particle having a spherical shape including a quantum dot layer, for example, the quantum dot layer may be located inside the bead particle. The term "Quantum Dot" may mean nanometer-sized particles having a 0-dimensional structure having unique optical and electronic properties, and may include, for example, nanometer-sized semiconductor crystals. The quantum dot may have a structure of a core body, a cell portion surrounding the core body, and a polymer coating layer coating the cell portion. In the present invention, the specific type of the quantum dot is not particularly limited, and can be used without limitation as long as it has biocompatibility to be used for biometric imaging. Components constituting the central body of the quantum dot are, for example, CdSe (cadmium-cesium), CdTe (cadmium-telluride), CdS (cadmium sulfide), ZnSe (zinc-cesium), ZnO (zinc oxide), or ZnS (zinc sulfide). ), etc. can be used. The components may have a characteristic of generating much stronger fluorescence in a narrow wavelength band than that of a general fluorescent material. The term "quantum dot layer" means that a plurality of quantum dots form a sheet or film form by electric force, magnetic force, chemical bonding, or external or internal force with each other. In this case, it may include not only a case where a complete film is formed, but also a case where a complete film is not formed but is located on a concentric sphere.

The bead particle may include quantum dots that are radially bonded by electrostatic attraction on an inner concentric sphere close to the surface of the bead particle. For example, each of the quantum dots may be radially positioned at the same distance from the center of the bead particle to form a single-layered sphere shell shape and be doped inside the bead particle. In the present invention, the single layer of the quantum dots provides a doped layer having a uniform density without oxidation of the quantum dots through doping of the quantum dots by electrostatic attraction in a state excluding the use of an organic polymer. Since the quantum dots exist as a single layer in the form of a concentric sphere, self-quenching phenomenon is minimized, and fluorescence amplified by resonance with the cavity of the bead particle can be emitted.

The concentric sphere may have a radius (r) of 0.5 times or more and less than 1 times, 0.7 times or more and less than 1 times, and 0.9 times or more and less than 1 times of the distance (radius; R) from the center of the bead particle to the surface. For example, it may be an interior close to the surface of the bead particle. If the concentric sphere has a radius (r) less than 0.5 times the radius (R), because the quantum dot layer is formed too deep inside the bead particle, the fluorescence emitted to the outside of the bead particle becomes too weak. . Less than 1 times the radius (R) means that the quantum dots are not exposed to the outside of the bead particles. Therefore, as the quantum dots are located inside the bead particles, the bead particles may form a pore layer (see FIG. 2). The pore body layer may be a material that is the same as the inside of the bead particle, or may be a different material. As the quantum dots are wrapped by the pore body layer and confined inside the bead particle, light stability and durability are improved compared to when the quantum dots are alone without the pore body layer, and at the same time, the quantum dots and the porous body layer The intensity of light emission is further amplified by resonance coupling between pupils.

The diameter of the bead particles may be, for example, 10 to 1000 nm, 50 to 500 nm, or 100 to 300 nm. When the diameter of the bead particles is 10 nm or less, it is difficult to form a uniform spherical single layer of quantum dots, and when the diameter of the bead particles is 1000 nm or more, it is difficult to synthesize the bead particles. When the diameter of the bead particle is 100 to 300 nm, the quantum dots exhibit appropriate luminescence properties. The bead may include at least one selected from the group consisting of silica, titania, zirconia, and zeolite, and may be made of silica, for example. Also, if the bead particle is made of an inorganic material having a high refractive index, there is no particular limitation.

In the complex, the agent that detects or analyzes the target substance binds to the bead particle, and may, for example, bind to the surface of the bead particle. The term "target substance" may be a protein, nucleic acid, peptide, cell, organelle or other physiologically active substance for which the complex of the present invention is for the purpose of detection, detection, or analysis. The term "agent for detecting or analyzing a target substance" may be a substance that specifically binds to a target substance or a fragment thereof, and may also be the target substance itself or a fragment thereof. The term “fragment” refers to a part of a target substance and includes physical, enzymatic or chemical cleavage. For example, in the case of a protein, it includes both protease cleaved and chemically cleaved. . The term "specifically binds to a target substance or fragment thereof" means that the agent contained in the complex of the present invention selectively binds to the target substance or fragment of the target substance, and does not substantially bind to substances other than the target substance. do. The term "a substance that specifically binds to a target substance" may be a protein, nucleic acid, peptide or derivative thereof that specifically binds to a target substance, for example, an antibody specific to a target substance, or a target substance-specific It may be a peptide comprising a domain that binds to. The term "antibody" is a term known in the art and is a specific immunoglobulin directed against an antigenic site. The form of the antibody includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, and all immunoglobulin antibodies are included. The antibody may refer to a complete form having two full-length light chains and two full-length heavy chains. In addition, the antibody may also include special antibodies such as chimeric antibodies, humanized antibodies, and human antibodies. The term "partial peptide having a specific binding domain" refers to a polypeptide that does not have the structure of an intact antibody, but has a specific antigen-binding site, ie, a binding domain, directed against an antigenic site. The partial peptides comprise functional fragments of antibody molecules that are not in full form antibody having two light chains and two heavy chains. The functional fragment of an antibody molecule refers to a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. The peptide may comprise at least 7 amino acids, for example 9 or more amino acids, or for example 12 or more amino acids.

The agent for detecting or analyzing the target substance may be bonded to the surface of the bead particle by chemical bonds such as covalent bonds, hydrogen bonds, ionic bonds, or van der Waals bonds, for example, covalent bonds. have. The covalent bond may be an amide bond, a disulfide bond, a phosphorylation bond, an oxime formation, or an ester bond, and may be, for example, an amide bond.

In order to bind the bead particle and the agent, the surface of the bead particle may contain a reactive group or the surface of the bead particle may be activated so that a reactive group may be introduced. The term "activation of the surface of the bead particle" refers to the introduction of a reactive functional group capable of forming a chemical bond, including covalent bonds, so as to bind other substances to the surface of the bead particle, and includes'surface modification'. The term "surface modification" means modifying or altering the surface to facilitate bonding with other materials without changing the basic physical properties of the material to be modified. As the surface of the bead particle is activated, the surface of the bead particle may include a reactive functional group such as a hydroxyl group, a carboxyl group, a thiol group, a sulfone group, or an amino group. In one embodiment, the bead particles made of silica may have an amino group on the surface (see FIG. 2).

The bead particles may be bonded to the agent by direct covalent bonds or covalently bonded through an appropriate linker. The term "linker" refers to a substance that connects the bead particles to an agent that specifically binds to a target substance, and may be a compound, sugar, peptide, or nucleic acid containing a reactive functional group. In one embodiment, glutaraldehyde may serve as a linker.

The target substance may be, for example, a P substance (Substance P, Sub P) or a neuropeptide Y (Neuropeptide Y, NpY). The term “Substance P, Sub P” refers to a neuropeptide, which is a mammalian tachykinin. In addition, the protein sequence may include SEQ ID NO: 1. The term "Neuropeptide Y (NpY)" refers to a peptide widely distributed in the central nervous system. The protein sequence may comprise SEQ ID NO: 2. In one embodiment, the complex of the present invention may include the P substance or the neuropeptide Y or a fragment thereof, or may include an antibody, nucleic acid, peptide or derivative thereof that specifically binds to the P substance, and these bead particles May be bound to the surface of

Peptide Sequence information Sequence number P substance RPKPQQFFGLM SEQ ID NO: 1 Neuropeptide Y YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY SEQ ID NO: 2

Another aspect provides a composition for analyzing a target substance comprising a complex including a bead particle including a quantum dot layer and an agent for detecting or analyzing the target substance.

The composite included in the composition is as described above. The term “target substance analysis” includes all actions necessary to conduct research on a target substance, such as detection, detection, and quantification of the target substance. The composition may further include a material necessary to perform an analysis such as detection and quantification of a target material. For example, the necessary substances may be antibodies or antigen-binding fragments, cell staining reagents, buffers, and the like.

Another aspect provides a kit for immunoassay of a target substance comprising a complex including a bead particle including a quantum dot layer and an agent for detecting or analyzing the target substance.

The complex included in the target substance immunoassay kit is as described above. The term "kit for immunological analysis" refers to a tool capable of analyzing a target substance using an immunological method, that is, a specific binding ability using an antibody to a target substance, for detection, detection, and quantification of a target substance. . The kit is, for example, an agent for detecting or analyzing a target substance contained in a complex, and an antibody specific for the target substance or the antibody of the target substance may be used. The kit may additionally contain one or more other constituent compositions, solutions or devices suitable for the method of analyzing the target substance. The kit according to the above aspect may include, for example, a complex containing an antibody that specifically binds to a target substance, a substrate for immunological detection of the antibody, a suitable buffer solution, or a complex to which a secondary antibody is bound. I can. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass.

Since the composition for analyzing the target substance and the kit for immunological analysis of the target substance are characterized by using quantum dots as a detection means, the presence of the target substance, quantitative analysis, and separation, etc. are better performed through fluorescence images from the quantum dots. In particular, it is excellent for analysis of small amounts of target materials due to its high sensitivity.

Another aspect provides a composition for diagnosing a disease related to myocardial infarction comprising a bead particle including a quantum dot layer and an agent that specifically binds to a target material, and the target material includes a complex in which the target material is P material or neuropeptide Y.

The bead particles including the quantum dot layer included in the composition for diagnosing myocardial infarction are as described above. The term "myocardial infarction-related disease" refers to any disease that causes pain due to an abnormality in the heart muscle due to acute or chronic stricture of the cardiovascular system, resulting in necrosis of the tissues or cells of the heart muscle, or a decrease in blood supply to the heart. , For example myocardial infarction, stable angina, unstable angina. The term "diagnosis" means to confirm the presence or characteristics of pathophysiology, and in the present invention, it is to confirm the presence or absence of a myocardial infarction disease including acute myocardial infarction, stable angina, angina and the like. The P substance may include the amino acid sequence of SEQ ID NO: 1, and the neuropeptide Y may include the amino acid sequence of SEQ ID NO: 2. The myocardial infarction-related disease composition may diagnose a myocardial infarction-related disease by including an antibody specific for the P substance or neuropeptide Y protein, or the P substance or neuropeptide Y protein itself, or a fragment thereof in the complex. The composition for diagnosing diseases related to myocardial infarction of the present invention overcomes the quantitative limit of commercial kits currently on the market by using a complex containing a quantum dot layer and has more sensitivity.In the case of myocardial infarction related diseases, a marker present in a trace amount in the blood , For example, it is more preferable for diagnosing diseases related to myocardial infarction as it enables accurate and sensitive detection and level measurement of P substance.

Another aspect provides a method for preparing a composite including bead particles including a quantum dot layer and an agent specifically binding to a target material.

First, a method of manufacturing the composite will be described as follows. First, it may include the step of activating the surface of the bead particle including the quantum dot layer. The term "activation" is as described above. For the activation, conventional methods known in the art such as the use of chemical substances, plasma treatment, and ionization treatment may be used. After activation, a reactive functional group such as an amino group, a carboxyl group, or a hydroxy group may be finally introduced to the surface of the bead particle. In one embodiment, the bead particles composed of silica were activated using a silane coupling agent to introduce an amino group to the surface. The introduced reactive functional group is used to bind the agent and the bead particle.

Next, it may include the step of covalently bonding the surface of the activated bead particle and an agent for detecting or analyzing a target substance through a linker. The linker refers to a general structure connecting at least the bead particles and the agent. The linker may be, for example, a covalently linked structure, and may be, for example, an amide bond. The linker may be a cross-linking agent, and may include, for example, polyethylene glycol, aldehyde, isocyanate, maleimide, and glutaraldehyde. In one embodiment, glutaraldehyde may be used as a linker to connect the agent and the beads by an amide bond.

Another aspect is myocardial muscle using a complex containing bead particles including a quantum dot layer and a substance that specifically binds to a target substance, and the target substance is a P substance (Substance P, Sub P) or a neuropeptide Y (Neuropeptide Y, NpY). Provides a method for diagnosing infarct-related diseases.

A method of diagnosing the disease related to myocardial infarction is as follows. First, the method for diagnosing myocardial infarction includes a bead particle including a quantum dot layer and an agent capable of detecting or analyzing a target substance, for example, a substance that specifically binds to a target substance, and the target substance is a P substance. (Substance P, Sub P) or neuropeptide Y (Neuropeptide Y, NpY), using a complex, may include the step of measuring the expression level of the protein of the P substance or neuropeptide Y in the sample of the patient. The target substance is the same as described above for the P substance or the neuropeptide Y complex. The term "patient" refers to an individual suspected of having an acute myocardial infarction, and includes any individual whose expression of substance P or neuropeptide Y is changed by an acute myocardial infarction. The term "sample" refers to a biological material derived from an individual, and may be blood, bone marrow, lymph, saliva, tear, urine, mucosal fluid, amniotic fluid, or a combination thereof derived from the individual.

The "measurement of protein expression level" of the above aspect is a process of checking the presence and expression level of P substance or neuropeptide Y protein in a sample in order to diagnose acute myocardial infarction. Measurement of the expression level of the protein is, for example, Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, rocket (rocket) Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation Assay), Complement Fixation Assay (Complement Fixation Assay), FACS, can be performed by a protein chip (protein chip), or a combination thereof. However, in the above method or apparatus, in order to exhibit fluorescence properties, the composite of the present invention is used instead of a commonly used material. For example, in the case of ELISA, a complex of the present invention is used instead of a fluorescence signal using an enzyme, and the expression level of a protein is measured using a fluorescence signal from the complex.

The protein expression level may be measured by, for example, an enzyme-linked immunosorbent assay (ELISA). ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes the capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and a solid support. Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibody and antigen, and a labeled that recognizes this antibody after reacting with another antibody that recognizes the antigen in a complex of an antibody attached to a solid support and an antigen. It includes various ELISA methods such as indirect sandwich ELISA using a secondary antibody (Fig. 1). That is, as mentioned above, the method of measuring the protein expression level can be performed by the conventional ELISA method, but instead of the fluorescent signal by the enzyme, the protein expression level can be measured by analyzing the fluorescent signal derived from the complex of the present invention. It can be.

For example, in the case of using the sandwich ELISA method, coating the surface of a solid substrate with an antibody that specifically binds to a P substance or a neuropeptide Y protein or a fragment thereof as a primary antibody; Inducing an antigen-antibody reaction by contacting the antibody with a blood sample from a normal individual and an individual suspected of acute myocardial infarction; Reacting the result of inducing the antigen-antibody reaction with an enzyme-conjugated secondary antibody; And it may include the step of detecting the activity of the enzyme (Fig. 1 (c)). Hydrocarbon polymers (eg, polystyrene and polypropylene), glass, metal or gel may be used as the solid substrate, for example, a microtiter plate.

When the protein expression level is measured by a direct ELISA method, coating an antibody specifically binding to SubP or NpY or a fragment thereof as a primary antibody on the surface of a solid substrate; Antigen-antibody reaction by competitively contacting the antibody with a blood sample from a normal individual and an individual suspected of acute myocardial infarction with a standard labeled (enzyme, fluorescent organic substance, or peptide standard conjugated with a nanofluorescent inorganic substance) Inducing a; It may include the step of directly detecting the concentration by measuring the result of the antigen-antibody reaction induction step with an ultraviolet reader (microplate reader) or a fluorescence reader (fluorescence reader) (Fig. 1 (b)).

In addition, the protein expression level can be measured using, for example, Western blot analysis using one or more antibodies against the protein. The whole protein is separated from the sample, electrophoresed to separate the protein according to its size, and then transferred to a nitrocellulose membrane to react with the antibody. By confirming the amount of the generated antigen-antibody complex using a labeled antibody, by confirming the amount of the protein, diseases related to myocardial infarction can be identified.

Next, the method for diagnosing a disease related to myocardial infarction may include comparing the measured expression level of the protein with a normal control group. The above comparison can be made by investigating the expression level of the protein in the normal control group and the expression level of the protein in a patient sample, and the protein level is absolute (eg, μg/ml) or relative ( Example: Relative intensity of the signal).

In addition, the method of diagnosing a disease related to myocardial infarction may include determining as a disease related to myocardial infarction when the expression level of the substance P or the neuropeptide Y protein is higher than that of the normal control group in the sample of the patient.

In the above method, when the expression level of the substance P is equal to or higher than the first predetermined value and the expression level of the neuropeptide Y is equal to or higher than the second predetermined value, it may be determined as an acute myocardial infarction. The method can be determined as acute myocardial infarction, stable angina, or angina when the expression level of the substance P is equal to or higher than a first predetermined value or the expression level of neuropeptide Y is equal to or higher than a third predetermined value. In the above method, when the expression of substance P is less than the first predetermined value and the amount of the expression level of neuropeptide Y is less than the third predetermined value, it can be determined that it is not acute myocardial infarction, unstable angina, stable angina, or angina. . The first predetermined value may be 3594 ± 1 pg/ml, the second predetermined value may be 59 ± 0.85 pg/ml, and the third predetermined value may be 40 ± 0.85 pg/ml. In one example, when comparing and analyzing the blood concentrations of substance P and neuropeptide Y in normal subjects and patients with acute myocardial infarction using the complex of the present invention, the result was significantly higher than that of normal subjects (P<0.0001).

According to an aspect, a composite including a bead particle including a quantum dot layer and an agent for detecting or analyzing a target material, and a composition including the same, can provide a fast and highly accurate analysis result in the detection or analysis of a target material. have.

In addition, according to the composition for myocardial infarction and the method for diagnosing acute myocardial infarction according to one aspect, it has high specificity and sensitivity in the diagnosis of diseases related to myocardial infarction, including acute myocardial infarction, from a patient suspected of cardiovascular disease. It can be usefully used in the judgment of disease.

1 is a diagram showing a schematic diagram showing an ELISA assay method using a monoclonal antibody specific for a target protein. (A) represents a competitive ELISA Assay, (B) a direct ELISA Assay, (C) a sandwich ELISA Assay, and (D) an applied competitive ELISA Assay method.
FIG. 2 is a schematic diagram showing an ELISA assay method using a monoclonal antibody specific to a target protein. (A) represents a competitive ELISA Assay, (B) a direct ELISA Assay, (C) a sandwich ELISA Assay, and (D) an applied competitive ELISA Assay method.
FIG. 2 shows the structure of _{activated (functionalized) SQS-NH 2} by modulating propylamine on the surface of silica bead particles including a quantum dot layer therein.
3 is a schematic diagram of a method of binding an antibody of a substance P to bead particles activated with _{NH 2.}
4 shows the result of quantitative analysis of the amount of antibody bound to bead particles including a quantum dot layer by a protein quantification method (Bradford Assay). It was confirmed that the amount of antibody bound to 100 nmole/L of SQS-NH ₂ was about 210 nmole/L, and that two antibodies were bound to _{one SQS-NH 2.}
Figure 5 shows the result of direct ELISA assay by binding an antibody against P material to silica bead particles (SQS) including quantum dots, (a) is 100 nm SQS-NH ₂ , and (b) is 300 nm It is for SQS-NH ₂ . The LOD is 1 pg/ml, and the dynamic range is (a): 1-300 pg/ml, (b): 1-10000 pg/ml.
6A and 6B are quantitative curves of the results of competitive ELISA Assay by attaching an antibody of P substance to SQS of 100 and 300 nm. Linearity was very good at 0.957 and 0.999. 6C is a graph in which the quantitative curve 6b is changed to a log scale.
In the case of FIG. 7, a result of analyzing the blood concentration of neuropeptide Y using a commercial kit.
In the case of FIG. 8, a result of analyzing the blood concentration of substance P using a commercial kit.
In the case of FIG. 9, the results of analyzing the blood concentration of substance P using SQS.
In the case of FIG. 10, the results of material P analyzed with a commercial kit and the results of analysis with a kit using SQS are compared.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of a complex comprising bead particles including a quantum dot layer and a fragment of an antibody or P substance or neuropeptide Y

1.1 Quantum dots Silica containing layer Bead Preparation and activation of particles

Silica bead particles including a quantum dot layer therein were prepared by the method described below (hereinafter, silica bead particles including a quantum dot layer in the examples and drawings of the present specification are referred to as'SQS' (quantum dot-layered silica sphere). Referred to as).

Specifically, first, take 5 ml of a core/shell structured CdSe/CdS-ODA quantum dot solution (2×10 ^-5 M) whose surface is protected with octadecylamine (ODA) and connect it to vacuum to remove the hexane solvent, and then chloro The foam was dispersed in 10 ml, and an excess of a methanol solution in which 0.05 M of mecaptopropionic acid (MPA) and 0.06 M of sodium hydroxide were dissolved together was added, followed by vigorously stirring for 30 minutes. When 2 to 3 mL of distilled water was added to this solution, the quantum dots came up as an aqueous layer, and the aqueous layer was separated, methanol and ethyl acetate were added, followed by centrifugation to recover the quantum dots. Carboxylic acid of the quantum dot surface by adjusting the pH to near 10 of the quantum dots dispersed in water using a diluted sodium hydroxide solution solution -CO2 ^- state of poly anionic monodisperse quantum dot _{CdSe / CdS (-SCH 2 CH 2} CO 2 ^- )ex 100 mL of aqueous solution (1×10 ^-6 M) was prepared. Next, 5 mL of a silica bead solution (DLS size 1.0±0.05 μm, 10 wt%) purchased from Polyscience was taken, centrifuged, and dispersed in 20 mL of methanol. To this, 0.025 mL of aminopropyltrimethoxysilane was added and refluxed for 10 hours. After cooling this solution, it washed with methanol 3-4 times using centrifugal separation. Finally, a polycationic monodisperse silica bead solution having ^{an amine on the surface of the silica bead in the state of -NH3 +} was prepared by dispersing in 10 mL of ethanol and adding a few drops of diluted hydrochloric acid to adjust the pH of the solution to about 4. The prepared polyanionic silica bead solution was slowly added to the prepared polyanionic quantum dot solution and shaken to be uniformly mixed. The precipitate was stopped at the point of formation, and the solution was vortexed for 1 minute and then centrifuged. Since fluorescence was not detected in the filtrate, it was discarded, and the precipitate was dispersed in 400 mL ethanol to prepare a silica bead solution doped with a quantum dot layer on the surface. 12 ml of distilled water and 4 ml of concentrated ammonia solution were added to the silica bead solution doped with the quantum dot layer on the prepared surface and stirred. Then, 2 ml of tetraethoxysilane (TEOS) was added and stirred for 3 hours. By repeating again, a total of 6 mL of tetraethoxysilane was grown as a silica layer on the silica beads doped with the quantum dot layer on the surface, thereby preparing silica beads doped with the quantum dot layer inside close to the surface. The solution was centrifuged, the precipitate was washed with ethanol, and then centrifuged again and dispersed in 20 mL of ethanol to finally prepare SQS.

To activate SQS, 2 mL of the above SQS solution was taken, diluted with 20 mL, and 0.4 mL of concentrated ammonia water was added and stirred. Subsequently, 0.93 mL of aminopropyltrimethoxysilane was added, followed by stirring at 20°C for 14 hours. After washing with ethanol twice using centrifugation, the concentration was finally dispersed in 10 mL of ethanol. At this time, the concentration of the embedded quantum dots was set to 1 mM.

1.2 antibody through linker and SQS Combination of

Using glutaraldehyde as a cross-linker that connects NH ₂ activated SQS with the amine group of the antibody, target peptide (P substance or fragment of neuropeptide Y) (100 nM, monoclonal, biorbyt) or Antibodies (50 nM, monoclonal, PHOENIX PHARMACEUTICALS, INC.) were ligated. In a carbonate buffer (50 mM, pH 9.6) in which glutaraldehyde (2.5%) was dissolved, 100 nm and 300 nm of SQS-NH ₂ (contained quantum dot concentration 25 nM) separated from ethanol were dissolved in 2 at 37°C. After reacting for a period of time, unbound glutaraldehyde was removed through centrifugation (10000g, 15 minutes, 4°C).

Table 2 below shows the results of measuring the size of the complex of 100 nm SQS-NH ₂ and the antibody conjugated using Zeta PAL (Brookhaven Instrument Co., USA). Since the size of the antibody is about 15-20 nm, it is determined that two or more antibodies are bound in either of the binding ratios of 1:2 or 1:4.

Coupling ratio (SQS:Ab) SQS only 1:2 1:4 Mean diameter (nm) 167.8 ± 26.5 211.4 ± 27 243.1 ± 18.5 Relative variance 1.107 1.315 0.659

Table 3 below shows the results of measuring the size of the complex in which _{the SQS-NH 2 of 300 nm and the antibody are bound.} Both of the 1:1 or 1:2 binding ratios appear to have bound two or more antibodies.

Coupling ratio (SQS:Ab) SQS only 1:1 1:2 Mean diameter (nm) 207 ± 25 219.7 ± 27 236.3 ± 18.5 Relative variance 1.107 1.315 0.659

Example 2. Immunological for blood samples using complex Assay

2.1 Preparation of the sample and Assay Way

In order to measure the concentration of P substance or neuropeptide Y in a blood sample, an experiment was performed by a sandwich ELISA assay using a commercial kit (PHOENIX PHARMACEUTICALS, INC.) and the SQS-antibody complex prepared in Example 1.

Sandwich ELISA assay experiments using commercial kits were performed on a total of three groups of samples. Specifically, serum samples of myocardial infarction and unstable angina (40 patients), stable angina (40 patients), and a normal control group (80 patients) were prepared.

Sandwich ELISA assay experiment using SQS-antibody complex was performed in a total of 4 groups, normal subjects (29 patients), acute myocardial infarction patients (30 patients), unstable angina patients (28 patients), and angina patients (30 patients). The serum samples of were tested.

After diluting 500 µl of a blood sample from a healthy person from Korea University Medical School and a blood sample from a patient with cardiovascular disease twice with 10% formic acid buffer, peptides were extracted with Oasis HLB 1cc (30mg) Extraction Cartridges Solid Phase Extraction Kit (Waters, Ireland). ). On the other hand, a 30 kDa Molecular cut-off filter (Millipore, USA) was used to separate and extract endogenous peptides in serum and plasma from the protein. The solution containing the extracted peptide is dried using an N2 Evaporator or Freeze-Dryer, and then analyzed by dissolving in 50 µl of assay buffer (provided by each ELISA kit company) for immunological analysis, or 25 µl Serum and plasma were diluted 2 times in PBS buffer and analyzed directly. For LC-MS/MS analysis, it was analyzed by dissolving in 0.2% formic acid in 100 µl of 50% methanol.

2.2 Commercial Kit Used Neuropeptide Y ( Neuropeptide Y, NpY ) Concentration measurement

The standard solution for drawing the adjustment curve was 1000 ng/ml stock diluted in steps with 1x assay buffer, and the concentration was adjusted to 100, 10, 1, 0.1, 0.01 ng/ml, respectively, and 50 µl of the positive control group as a control group. Prepared. In each well of the microplate coated with the secondary antibody, the control and standard solutions were dispensed in duplicates, and the sample diluted by 2 times was dispensed in triplets. Subsequently, 25 µl of the primary antibody (rabbit anti-peptide IgG) (PHOENIX PHARMACEUTICALS, INC.) obtained by injecting artificial neuropeptide Y into the rabbit and 25 µl of biotinylated peptide were dispensed, and then at room temperature under a speed of 300-400 rpm. Incubated for 2 hours. After washing four times with a washing buffer, 100 μl of streptavidin-horseradish peroxidase was dispensed and stirred at room temperature (300-400 rpm) for one hour. Thereafter, after washing four times with a washing buffer in the same manner as the above washing, 100 μl of a TMB substrate solution was dispensed and reacted at room temperature for one hour. Finally, after the reaction was terminated with 100 µl of a stop solution (2N HCl), the O.D. value was measured using a microplate reader at a wavelength of 450 nm.

2.3 Commercial Kit P material used ( Substance P, SubP ) Concentration measurement

For the ELISA experiment, a commercial kit of the R&D system was used, and the experiment was conducted with an ELISA assay based on the principle of competitive binding. The standard solution for drawing the calibration curve is 50 ng/ml of P material standard solution 50 µl diluted in 1 ml Calibrator Diluent RD5-45, and then 300 µl in 1 ml Calibrator Diluent RD5-45. Serial dilution was performed to prepare a concentration of 2500, 1250, 625, 312, 156, 78, 39, 0 pg/ml. Control and zero standard wells of 213-540, 517-982, and 1224-2217 pg/ml were set as control groups, and the standard solution, control, and zero standard well were all tested in duplicate. Serum samples were diluted twice and tested in triplets. Dispense 50 μl of standard and control solutions and diluted serum samples into ELISA microplates coated with goat anti-mouse polyclonal antibody included in the kit, and then 50 μl of primary antibody solution (mouse monoclonal antibody against substance P) into each well. , 50 µl of Substance P Conjugated HRP was sequentially dispensed, covered with an adhesive strip, and incubated for 3 hours at a speed of 500 ± 50 rpm in a microplate shaker. After washing each well 4 times using a washing buffer, 200 μl of Substrate Solution (TMB) was dispensed and incubated for 30 minutes. Finally, after dispensing 50µl of Stop Solution (2N sulfuric acid), it was confirmed that the blue solution turned yellow, and the O.D. value was measured using a microplate reader at a wavelength of 450 nm.

2.4 SQS -Measurement of concentration of P substance using antibody complex

After activating paralin A with ethanol in which 10% glutaraldehyde is dissolved in a 96-well microplate coated with paralin A, the P material was incubated for 2 hours to coat it, and then a microplate blocked with BSA was prepared. did. A patient serum sample diluted in PBS and a patient serum sample diluted in PBS was incubated with a molecule in which the SQS-P-substance antibody-conjugated molecule was dissolved, and the P substance or standard P substance present in the serum was reacted to bind to the antibody of the SQS-P substance. After separating the conjugate of the SQS-P material antibody and the SQS-P material antibody and P material by centrifugation, they were put together in a microplate well prepared in advance and incubated for 3 hours. If the concentration of the P substance in the serum is high, the antibody of the SQS-P substance conjugate is already saturated with the P substance and does not bind to the P substance in the plate. Therefore, the higher the blood concentration, the lower the fluorescence intensity of the ELISA assay. Is derived as.

Thereafter, after lightly washing with PBS to which 0.1% Tween was added, fluorescence was measured at wavelengths of excitation 486 nm and emission 630 nm with a multi-label plate reader. The results are shown in FIGS. 5 and 6.

It was found that the quantitative and reproducible immunological assay using the complex was very high. When using 100 nm SQS, as can be seen in Table 4 below, the accuracy is 120%, the precision is less than 10%, the quantification and reproducibility are very high, and the dynamic range is also compared to other commercial immunological assay methods. It was wide (~10 ⁵ ).

Intraday assay Interday assay Parameter accuracy
(Accuracy) (%) Precision
(Reproducibility) (%) accuracy
(Accuracy) (%) Precision
(Reproducibility) (%) QC conc.
(0.1 ng/ml) 120 ± 86 9.9 ± 4.2 100 ± 11.7 10.1 ± 5.7

Example 3. P substance and Neuropeptide Diagnosis of patients with myocardial infarction using Y

3.1 Commercial Kit In the sample used Neuropeptide Y protein concentration analysis

First, the concentrations of P substance and neuropeptide Y were analyzed from blood samples from normal and cardiovascular patients through the above ELISA method using a commercial kit.

7 a, b, c, and d are diagrams comparing the concentration of neuropeptide Y from blood samples from normal and cardiovascular patients. 7A is a case of comparing a patient with an acute myocardial infarction (AMI) for neuropeptide Y and a normal person, where * P value <0.0001, Cut-off value> 40 pg/ml. b is a comparison between a normal person and a patient with cardiovascular disease (Unstable Angina, UA, Stable Angina, SA, acute myocardial infarction) * P value <0.0001, Cut-off value> 59 pg/ml to be. c is the comparison between AMI and nonAMI, and d is the comparison of each disease (AMI, UA, SA).

Based on this result, AUC (area under curve) obtained by calculating the Receiver operating characteristic (ROC) curve, statistical significance (p, %) calculated through t-test, positive prediction rate (PPR, %), negative The prediction rate (NPR, %) is shown in Table 5 below.

Sensitivity (%) Specificity (%) cut-off (pg/mL) p value PPR (%) NPR (%) control vs. disease 100 59 ＞40 <0.0001 70 100 control vs. AMI 100 64 ＞59 <0.0001 71 88 AMI vs. non AMI 100 41 59 0.0036 20 100

3.2 Commercial Kit Concentration analysis of the P substance used

The results for the P substance concentration analyzed using a commercial kit are shown in FIG. 8. In FIG. 8, a is a comparison between a normal person and acute myocardial infarction, and a cut-off value> 364 pg/ml, but was not statistically significant. b is a comparison between a normal person and a patient with cardiovascular disease (unstable angina, stable angina, acute myocardial infarction), and * P value <0.0001, Cut-off value> 400 pg/ml. c is the comparison of AMI and nonAMI, and d is the comparison of each disease. What is unique here is that the concentration of neuropeptide Y in patients with stable angina was very high compared to those of normal and other cardiovascular diseases (p<0.0001). Table 6 shows the AUC obtained by calculating the ROC curve, the statistical significance calculated through the t-test, and the PPR and NPR.

Sensitivity
(%) Specificity
(%) cut-off
(pg/mL) p value PPR (%) NPR (%) control vs. disease 40 94 >547 <0.0001 76 69 control vs. AMI 40 97 >524 <0.0001 29 88 AMI vs. non AMI 40 63 ≤240 0.7387 87 8

3.3 Concentration analysis of P substance using complex

The results of analyzing the blood concentration of P substance using SQS are shown in FIG. 10, and in the case of acute myocardial infarction patients, it was significantly higher than that of the normal subjects (control) (p<0.0001). The AUC obtained by calculating the ROC curve, the significance calculated through the t-test statistically, and the PPR and NPR are shown in Table 7 below.

Sensitivity
(%) Specificity
(%) cut-off
(pg/mL) p value PPR (%) NPR (%) control vs. disease 87.5 100 >3594 <0.0001 100 79 control vs. AMI 100 100 >3594 <0.0001 100 100 AMI vs. non AMI 100 57.7 >2887 0.0005 100 61.5

The P substance and neuropeptide Y had an ROC of 0.8 or higher, a sensitivity of 100%, and a specificity of 64% or higher (see Table 8). From the above results, it was found that the two markers SubP and NpY are very sensitive and can be very useful as a diagnostic marker for acute myocardial infarction.

parameter
P substance ( SubP )
Complex use
P substance ( SubP ) Use of commercial kit Neuropeptide Y ( NpY )
Cut-off value
3594 pg/ml
524 pg/ml 59 pg/ml
AUC
1.00±0.000 ^a,b
0.681±0.045 ^a,b 0.799±0.047 ^a,b
Specificity
100%
97% 64%
responsiveness
100%
40% 100%

AUC, area under the curve

^a AUC ± standard error

When comparing AUC of SubP and NpY, ^b All p <0.05

The sensitivity and specificity of each assay were calculated using the cut-off value.

<110> KIST <120> Complex comprising bead including quantum dot-layer and method for diagnosing myocardial infarction-related disease using the same <130> PX113015 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 11 <212> PRT <213> Unknown <220> <223> Substance P <400> 1 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 <210> 2 <211> 36 <212> PRT <213> Unknown <220> <223> Neuropeptide Y <400> 2 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 20 25 30 Arg Gln Arg Tyr 35

Claims (18)

A composite comprising a bead particle comprising a quantum dot layer and an agent for detecting or analyzing a target substance. The complex according to claim 1, wherein the agent for detecting or analyzing the target substance is bound to the surface of the bead particle. The composite according to claim 1, wherein the quantum dot layer is located inside the bead particle. The composite according to claim 3, wherein the quantum dot layer has a concentric spherical shape having a radius of 0.5 times or more and less than 1 time of a distance from the center of the bead particle to the surface. The composite according to claim 1, wherein the diameter of the bead particles is 100 to 300 nm. The composite according to claim 1, wherein the bead is at least one selected from the group consisting of silica, titania, zirconia, and zeolite. The complex according to claim 1, wherein the preparation is a target substance or a fragment thereof, or an antibody, peptide, nucleic acid or derivative thereof that specifically binds to a target substance. The complex according to claim 1, wherein the target substance is P substance (Substance P, Sub P) or Neuropeptide Y (NpY). 9. The complex of claim 8, wherein said P material comprises the amino acid sequence of SEQ ID NO: 1 and said neuropeptide Y comprises the amino acid sequence of SEQ ID NO: A composition for analyzing a target substance comprising the complex of any one of claims 1 to 9. A kit for immunoassaying a target substance comprising the complex of any one of claims 1 to 9. A composition for diagnosing myocardial infarction related diseases comprising the complex of claim 8. Measuring the expression level of the P substance or the protein of the neuropeptide Y in the sample of the patient using the complex of claim 8;
Comparing the expression level of the measured protein with a normal control; and
Determining the acute myocardial infarction in the sample of the patient when the level of expression of the protein is higher than that of the normal control group. [Claim 14] The method according to claim 13, wherein the expression level of the P substance protein is measured using the complex of claim 8, and the expression level of the neuropeptide Y is measured using an experimental immunological assay kit of Phoenix Pharmaceuticals Inc. Wherein the fluorescence of the complex is detected and measured. [Claim 15] The method according to claim 14, wherein the expression of the P substance is at least a first predetermined value and the expression of the neuropeptide Y is at a second predetermined value or more. [Claim 15] The method of claim 14, wherein the expression of the P substance is greater than or equal to a first predetermined value or an expression level of the neuropeptide Y is equal to or greater than a third predetermined value, a cardiovascular disease including acute myocardial infarction, Lt; / RTI &gt; 15. The method of claim 14, wherein the expression of the P substance is less than a first predetermined value and the amount of expression level of the neuropeptide Y is less than a third predetermined value. The method of any one of claims 15 to 17, wherein the first predetermined value is 3594 + - 1 pg / ml, the second predetermined value is 59 + 0.85 pg / ml, and the third predetermined value is 40 + 0.85 pg / ml .

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