KR20150111015A - Biochip for the detection and determination of the concentration of cardiac biomakers, and method for the detection and determination of cardiac biomakers using the same - Google Patents

Abstract

The present invention relates to a protein analysis method for measuring a complex of an antibody-gene conjugate, an antigen and a labeling substance-secondary antibody conjugate by using a biochip of a 9G guanine DNA chip type; a method for quickly and simultaneously detecting various types of heart disease biomarker proteins (cTnI, NT proBNP and BNP) at a pictogram concentration level used to separate a normal state and an abnormal state of the heart disease biomarker proteins by using a standard concentration-sensitivity graph and for analyzing the concentration thereof; and a method for diagnosing heart diseases, monitoring heart disease progression and treatment processes of heart diseases, and determining whether the heart diseases are completedly cured or not.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for monitoring progress and treatment status of cardiovascular diseases by a standard concentration-sensitivity table of cardiovascular biomarkers, and a biochip for such monitoring and determination of cardiac biomarkers using the same}

The present invention relates to a method for diagnosing cardiovascular disease by rapidly and quantitatively monitoring the concentration of cardiovascular biomarkers using a concentration-sensitivity graph of a standard sample containing a cardiovascular biomarker, and a method for monitoring progress and treatment status of cardiovascular diseases .

More specifically, the present invention creates a concentration-sensitivity graph of a standard sample containing a heart disease biomarker using a biochip that can be measured in proportion to the sensitivity of the measurement means according to the concentration of the heart disease biomarker, Type biochip to detect heart disease biomarkers in a blood sample of a subject and to quantitatively determine their concentration by comparison with the standard sample concentration-sensitivity graph, thereby diagnosing heart disease, And more particularly to a method for monitoring status.

The heart of a man-sized fist has a very large effect on the human body when its function is deteriorated because it is easy to suffer from diseases with high mortality such as myocardial infarction, hypertension, heart failure, arrhythmia and cardiomyopathy as well as deterioration of blood circulation. According to statistics of the World Health Organization (WHO), cardiovascular disease is the world's leading cause of death with a death rate of 29.2%. In Korea, the incidence and prevalence of cardiovascular disease has increased sharply in the last 30 years, The mortality rate is high (Reference, National Statistical Office, 2006).

Cardiovascular disease accounts for one-third of deaths from domestic vascular disease and is considered to be a major cause of acute deaths in the 40-50 age group. Especially, in the case of a sudden middle-aged man who is very proud of his health, 80% is an acute myocardial infarction (AMI). In addition, it is known that if mortality is not detected early due to age increase, it is known that periodic screening is essential. Because this disease is mainly caused by clogging of cardiovascular system, if the symptoms are judged early and proper treatment is not done, it will mostly lead to death. Since most of the deaths due to this disease are socially and economically important in the 40s and 50s, the development of diagnostic technologies and products that can diagnose the disease early and take appropriate measures will enhance the vitality of the national economy, It can be said that it is necessary to improve the public health.

The American College of Cardiology guidelines (ACC / AHA) suggest that at least two of the three conditions, first of all, chest pain over 20 minutes, second, changes in ECG, and increased cardiac marker concentrations Acute myocardial infarction (AMI) is diagnosed.

Acute myocardial infarction (AMI) is important because rapid diagnosis and treatment are important because more than half of all deaths occur within one hour of symptom onset. However, basic tests such as X-ray and electrocardiogram, and echocardiography require time and equipment. Approximately 20% of AMI patients do not show electrocardiographic changes. Chest pain and electrocardiogram are the criteria for AMI diagnosis The importance of cardiac bio-markers as a major diagnostic criterion for acute myocardial infarction has been emphasized more and more, and therefore rapid development of blood diagnostic methods has been required.

Myocardial infarction diagnostic devices, which are widely used and widely used nowadays, diagnose biomarkers in blood using fluorescence resonance transfer (FRET) technique, which takes 15 minutes for each marker and only for one marker at a time And most of the detection limit is 1 ~ 10 ng / mL.

Rapid and timely diagnosis and determination of rapid changes in cardiac marker markers is highly needed for acute myocardial infarction (AMI). It is very useful not only for self-diagnosis, hospital visit, and rapid treatment when there is a slight pain in the chest but also as a method for confirming that the treatment process is completed by detecting the concentration of the heart disease marker in the diagnosis and treatment process to the cure level .

B-type natriuretic peptide (BNP) or nterminal proBNP (NT proBNP), which is a useful biomarker for differential diagnosis of cardiac troponin I (cTnI) and acute respiratory distress, and diagnosis of heart failure and prognosis, ) Is widely used.

The cTnI level was 250 pg / ml, NT proBNP was 80 pg / ml, and BNP was 100-400 pg / ml, which distinguishes between normal and abnormal values (Reference: American Heart Association Circulation, 2002; 105: 2328-2331. , en.wikipedia.org/wiki/Troponin), it is very important to quantitatively analyze biomarkers from several hundred pg / ml to several tens of pg / ml.

Techniques such as ELISA plate or NC membrane, which have been widely used for analysis of cardiovascular biomarker proteins, mainly use a detection method in which an antibody protein is immobilized on a solid substrate by chemical bonding or physical adsorption, and then protein concentration is measured. In the fixing technique by chemical bonding, since the activity of the immobilized protein decreases with time after immobilization, it is difficult to secure high sensitivity. In the immobilization technique by physical adsorption, it is difficult to maintain the protein activity after immobilization of the immobilized protein, And it is difficult to ensure reproducibility. In addition, proteins immobilized on a solid substrate will be denatured and non-specific binding to immobilized proteins as time goes by, resulting in several hundred pg / ml to several tens of pg / ml, which allows discrimination of multiple heart disease biomarkers into an abnormal state and a normal state. lt; RTI ID = 0.0 > mg / ml. < / RTI >

For this reason, known diagnostic methods and products for cardiovascular markers are mostly diagnostic of cardiogenic marker markers, and quantitative concentration analysis is possible at more than 1 ng / ml, but it has been difficult to confirm the concentration change below this level.

In the case of acute myocardial infarction (AMI), rapid diagnosis and treatment are required. In order to determine the progress of the heart disease and the treatment state based on the measurement of the concentration of the heart disease biomarker protein using the biochip, The development of a technique for simultaneously and rapidly detecting various coronary heart disease biomarker proteins contained in trace amounts of pg to several hundred pg is very important.

On the other hand, many attempts have been made to use biochips such as protein chips as a method or means for the detection and concentration measurement of cardiovascular biomarker proteins. However, protein chip-based technology for analyzing the labeled protein has mainly used a method of fixing a large number of proteins on a chip surface in a narrow space by using various molecular recognition technologies, and has been mainly used for research. In particular, analysis of the majority of the labeled proteins was carried out in a sandwich form of the antibody-antigen-antibody between the binding antibody and the secondary antibody attached to the substance required for analysis, so that reproducible analysis was difficult.

The inventors of the present invention have disclosed a gene chip capable of detecting a desired gene with a rapid and high sensitivity by using an amino-calixarene derivative and a continuous guanine-attached gene in Korean Patent Application No. 10-2005-0096322, The application 10-2008-0093800 discloses a gene chip capable of hybridization at room temperature using an amino-calixamine derivative and a continuous guanine-attached gene.

In a paper published by the inventors of the present invention (Chem. Commun., 2011, 47 , 7716-7718), a technique for detecting a biomarker at a picogram level using a genetic chip disclosed in the above-mentioned document is disclosed have. The above technique is a method in which a plurality of binding antibodies and a secondary antibody are rapidly bound to each labeled protein in solution and then each protein is immobilized using gene-gene binding at a predetermined position in the chip. Most of the problems have been solved. However, although the above-mentioned article refers to detecting not only cardiovascular biomarkers but general biomarkers existing at the picogram level, it is not possible to quantitatively analyze the concentrations of these biomarker proteins.

Therefore, by detecting a variety of heart disease biomarkers simultaneously and quantitatively determining their concentrations at the picogram level, changes in the concentration of biomarkers in the process and / , There has been a great need for effective means for monitoring progress and / or treatment status of heart disease.

Korean Patent Application No. 10-2008-0093800

Chem. Commun., 2011, 47, 7716-7718

The present inventors have developed a gene chip (hereinafter also referred to as 9G DNA chip) composed of a substrate, an amino-calixamine derivative and a 9G-gene, and used an antibody-gene conjugate, an antigen and a labeling- It has been reported that biomarker proteins such as CRP as an antigen can be detected at the level of picogram using a method of amplifying the sensitivity. However, it is difficult to distinguish the abnormal state of the heart disease biomarker from the abnormal state and several hundred pg / ml Methods for quantitatively analyzing or determining their concentrations at the level of tens of pg / ml have not yet been developed.

The present inventors have further studied the detection and concentration determination of an antigen protein using the above-described gene chip. As a result, it has been found that an antibody-gene conjugate, an antigen and a labeling substance-antibody conjugate form a complex and quantitatively bind to the gene chip Based on these findings, a concentration-sensitivity graph of a standard sample containing a cardiovascular biomarker could be generated. Using the same type of biochip, the cardiovascular biomarkers were detected in a blood sample of a subject, The present invention has been accomplished based on the discovery that quantitative determination of these concentrations at the picogram level as well as the standard sample concentration-sensitivity graphs can be performed to monitor the progression and treatment status of heart diseases.

According to the present invention, it is possible to rapidly and simultaneously detect various kinds of cardiovascular biomarker proteins at a picogram concentration level that allows discrimination between an abnormal state and a normal state of a heart disease biomarker protein, and simultaneously quantitatively analyze their concentrations. It is possible to monitor the progress and treatment process of the heart disease as well as the diagnosis of heart disease and make a cure judgment.

Figure 1 is a schematic diagram of antibody-gene and antibody-phosphor syntheses.
Figure 2 is a Nanodrop measurement for a material obtained from antibody-gene and antibody-phosphor syntheses.
Fig. 3 is a schematic view showing a form and a manufacturing method of a biochip for protein detection.
Fig. 4 shows a protein detection method and an experimental procedure.
FIG. 5 shows the results of measurement using a standard sample and a sensitivity contrast concentration graph.
FIG. 6 is a graph showing optimal internal standard spot sensitivity results and protein concentration versus internal standard spot sensitivity. FIG.
FIG. 7 shows the position of an internal standard spot (IS) and a protein detection spot (PD) with a biochip for protein detection using an internal standard spot.
Figure 8 is the actual concentration and measured concentration results of the three heart disease biomarker proteins (cTnI, NT proBNP, BNP) obtained by adjusting the gain to the internal standard spot.
Figure 9 shows the results measured at a dilution from one half to one-thirty of the three heart disease biomarker proteins (cTnI, NT proBNP, BNP) in a blood sample.

There is provided a method for quantitative analysis of cardiovascular biomarkers comprising the following steps (1) to (3):

(1) A standard concentration-sensitivity graph of anticholinergic biomarker proteins, which are antigens, is prepared using a biochip,

(2) analyzing a subject's blood sample with a biochip to obtain a measurement sensitivity of heart disease biomarker proteins contained in blood,

(3) determining the converted concentration of heart disease biomarker proteins by comparing the measured sensitivities described above with the standard concentration-sensitivity graphs described above,

(4) Determination of progression of heart disease or treatment state from the converted concentration of the obtained heart disease biomarker proteins.

According to one embodiment of the present invention, the biochip described above can be selected from biochips capable of detecting heart disease biomarkers at the picogram level as well as quantitatively analyzing the concentration at the picogram level. As this type of biochip, for example, a biochip including the following (a) to (d) can be mentioned:

(a) a solid substrate,

(b) means of attachment to a solid substrate and an amino-calixarene derivative having a recognition site for a continuous guanine base,

(c) an immobilized gene having a continuous guanine base and a desired base (sequence), and

(d) an antibody-gene conjugate comprising a complementary binding gene and an antibody bound to the immobilized gene.

In the biochip, the sample to be measured may be added to (e) a heart-healthy biomarker protein antigen, (f) a marker-antibody conjugate before or during measurement, and (d) , And may be separately added before or after the sample is added.

In the above-described biochip, the above-mentioned aminocylian arene derivative is attached to the solid substrate by chemical or physical bonding between the above-mentioned attachment means and the surface of the solid substrate, and the immobilized gene described above is bound to the solid substrate by a continuous guanine base Is attached to the recognition site of the continuous guanine base of the aforementioned immobilized gene.

According to one embodiment of the present invention, step (1) for creating a standard concentration-sensitivity graph as described above may comprise:

A solution containing a predetermined concentration of at least one kind of heart disease biomarker protein (1a) antigen and a solution (s) obtained by diluting the same at a predetermined ratio are prepared,

(1b) The standard solutions described above were analyzed by biochip to obtain a standard sensitivity,

(1c) A standard concentration-sensitivity graph is prepared from the above-mentioned standard concentration and the obtained standard sensitivity.

According to one variation of the present invention, the above-described standard density-sensitivity graph is a graph form combining numerical values of standard concentration and numerical values of standard sensitivity, and instead of this, a table in which standard concentration values and standard sensitivity images are combined Density-sensitivity spot) can be created and used.

According to one embodiment of the present invention, the component (d) may be added beforehand or separately with the sample containing the components (e) and (f), although it may be added in advance in the production of the biochip.

According to one embodiment of the present invention, the aforementioned cardiovascular biomarker protein antigen is one or more, preferably all three, of cTnI, NT proBNP and BNP, and the above-mentioned antibodies may be selected from the antibodies of the antigens .

According to one embodiment of the present invention, the continuous guanine base may be formed by continuously connecting seven or more, preferably nine, guanine (G) bases. The desired base (sequence) is not particularly limited in length, and may be selected from 13 mer or less, 14 to 17 mer, 18 to 21 mer, or 22 mer or more, as necessary.

The solid substrate may be selected from the group consisting of metals such as gold, silver, and platinum, glass, glass fiber sheets, silicon wafers, and crystal crystals, and the surface of the substrate may be modified with an amine group, a siloxane group, .

According to one embodiment of the present invention, the aminocylian arene derivative may be selected from aminocarboxylarene derivatives represented by the following general formula (1) or (2)

[Chemical Formula 1]

(2)

Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R _{'6, R' 7, R} '8 are each independently _{-H, -CH 3, -C 2 H} 5, -C 3 H 7, -OCH 3, -Cl, -C 6 H 5, -OH, - OCH ₂ CH ₃ , -Br, -CF ₃ , -OCH ₂ C ₆ H ₅ , -OC ₆ H ₅ , -OC ₆ H ₄ CH ₃ , -OC ₆ H ₄ C (CH ₃ ) ₃ , -OC ₆ H _{4 CF 3, -OC 6 H 4} Cl, -OCOCH 3, -NHCOCH 3, -CONHCH 3, -CN, and selected from the group consisting of COOH, and -COOR, in the -COOR R is -CH ₃ or -C ₂ H ₅ .

Y _1, Y _2, Y ₃ and Y ₄ are independently -H, each other _{- (CH 2) n-CH} = O, - (CH 2) n-SH, - (CH 2 CH 2 O) m-CH 2 _{CH 2 -CH = O, - (} CH 2 CH 2 O) m-CH 2 CH 2 -SH, - (CH 2) mC 6 H 4 - (CH 2) cZ, and -CO- (CH ₂₎ m- _{1-c 6 H 4 - (} CH 2) is selected from the group consisting of cZ (n = 2 ~ 15, m = 1 ~ 10, c = 0 ~ 10, Z is -SH, -CHO, -COOH and -NH ₂ , and -C ₆ H ₄ - and C ₆ H ₅ are defined as phenyl groups).

_{Preferably, R 1 = R 2 = R} 3 = R 4 = R 5 = R 6 = R 7 = R 8 and _{R '1 = R' 2 =} R '3 = R' 4 = R '5 in the formula (1) = R ' ₆ = R' ₇ = R ' _{8 where} R ₁ ≠ R' ₁ and R ₁ = R ' ₁ = R ₃ = R' ₃ = R ₅ = R ' ₅ = R ₇ = R ' ₇ and R ₂ = R' ₂ = R ₄ = R ' ₄ = R ₆ = R' ₆ = R ₈ = R ' ₈ and R ₁ ≠ R ₂ .

The substituents Y ₁ , Y ₂ , Y ₃ and Y ₄ in the general formulas (1) and (2) may be bonded to the surface of a solid substrate or a functional group attached to the surface of the solid substrate by ionic bonding, covalent bonding, 1 and < RTI ID = 0.0 > 2 < / RTI >

In one embodiment of the present invention, when the solid substrate is gold, at least one of Y ₁ , Y ₂ , Y ₃ and Y ₄ in formula (1) or (2) may be selected from the thiol group. When the solid substrate is selected from the group consisting of glass having an amine functional group, a silicon wafer and crystal crystals, at least one of Y ₁ , Y ₂ , Y ₃ and Y ₄ may be selected from aldehyde groups, The linkage of the substrate with the compound of formula (I) or (II) can be achieved through an imine group or an amine group reduced therefrom.

According to the present invention, the above-mentioned labeling substance can be selected from the group consisting of phosphors, enzymes, radioactive substances, fine particles and pigments.

Yet another object of the present invention is to provide a method for quantitatively analyzing an antigen protein using the above-described method, wherein at least one, preferably 3 to 10 species of antigen proteins are selected from the group consisting of the marker proteins shown in FIG. 10, And to provide a quantitative analysis method of an antigen protein to be quantitatively analyzed.

It is another object of the present invention to simultaneously quantitatively analyze the concentration of cardiovascular marker protein including cTnI, NT proBNP, BNP, etc. from 1,000 pg / ml to 25 pg / ml for monitoring the treatment process or for early diagnosis, And to provide a biochip that can be used for the purpose.

A further object of the present invention is to provide a method for preparing a standard concentration-sensitivity graph or a standard concentration-sensitivity table as an internal standard indispensable for the analysis of protein concentration, a novel gene immobilization technology for the same and a protein concentration analysis method using the internal standard .

According to the present invention, a standard concentration-sensitivity graph or a standard concentration-sensitivity table is prepared as an internal standard, the concentration is quantitatively determined from the measurement sensitivity of the biomarker detected using the bio-chip, The present invention can be applied to a method for monitoring the progress and treatment state of a disease using measured concentration.

Hereinafter, the present invention will be described in detail.

Specifically, the biochip of the present invention may have the following configuration:

(a) a solid substrate which can be selected from the group consisting of metals such as gold, silver, platinum, glass, silicon wafers, and crystal crystals;

(b) an aminocarixarene derivative attached to the solid substrate and having an attachment site to a solid substrate and a recognition site of a continuous guanine base, wherein the aforementioned continuous guanine base is specifically 5 or more, Means at least 7, in particular at least 9 consecutive guanine bases;

(c) a linking gene attached to the aminocylian arene derivative and having a continuous guanine base and a target base (sequence), wherein the linking gene is attached to the above-mentioned aminocalixarene derivative through a continuous guanine base has exist; And

(d) an antibody-gene conjugate complementary to the linking gene, wherein the antibody-gene conjugate described above comprises a base (sequence) complementary to the above-mentioned objective base (sequence) and the aforementioned antibody protein .

Using the biochip according to the present invention, it is possible to detect complexes formed by gene-gene binding of (d) an antibody-gene conjugate, (e) an antigen, and (f) a labeling substance-attached secondary antibody. Specifically, (e) adding an antigen and (f) a mixture of a labeling substance-attached secondary antibody to the biochip comprising (a) - (d) (D) an antibody-gene conjugate, (e) an antigen, and (f) a labeling substance-attached 2 (a) A conjugate of the secondary antibody can be formed.

Amino Calixarene derivatives recognizing a continuous guanine base in the present invention, a biochip attached to a solid substrate, is referred to as a "9G gene chip ", and a hybridization reaction of 80% or more within 10 minutes at room temperature Is a possible gene chip technology (cf. Chem. Commun., 2011, 47 / 7101-7103). Techniques related to such a room temperature hybridization chip are disclosed in Korean Patent Application No. 10-2008-0093800 by the present inventor, which is incorporated herein by reference.

The length of the guanine base and the specific base attached to the biochip prepared by immobilizing a gene having seven or more consecutive guanine bases by multimolecular recognition can be determined based on the use of a gene of 13 mer or less, -21mer gene, and a gene having more than 22mer gene.

In the present invention, the reaction of the biomarker protein with the antibody is carried out in a solution in which the activity of the antibody is maintained using the antibody-gene conjugate, and then hybridized with the antibody-gene conjugate on the 9G gene chip. Specificity binding problems that have occurred in the method of the present invention to solve the problem of heart disease biomarker protein at the level of picogram, specifically 1 to 1000 pg / ml, more specifically 5 to 800 pg / ml, preferably 10 To 600 pg / ml, particularly preferably 25 to 400 pg / ml (see Figures 8 to 9).

According to the present invention, the upper limit of the concentration range is not important, since the concentration can be determined by dilution method or by extending the standard concentration-sensitivity range even at a concentration higher than the picogram level, Gram level or more. In addition, since it is possible to control the gain of the scanner or to measure the kinds of the labeling materials even at a concentration of 25 pg / ml or less and a concentration of 5 pg / ml or less as measured in the present invention, Can be sufficiently overcome according to the method of the present invention.

To do this, 50-60 or more probe genes were designed and immobilized on a gene chip, and fluorescence expressed on the chip was analyzed using a complementary gene with fluorescence, and the probe gene with the highest specificity and sensitivity was selected A biochip in which genes were immobilized so that the concentration of cTnI, NT proBNP, and BNP in the cardiovascular biomarker protein can be detected simultaneously or simultaneously on one chip (see FIG. 3).

Then, the antibody-gene conjugate was prepared and purified as in Example 1 using a gene having a base complementary to the gene immobilized on the next gene chip, and the protein-protein complex for the preparation of the antibody- In order to confirm the number of gene binding, the optimal number of binding was selected for confirming the number of genes bound to each protein using the purified gene - protein conjugate Nanodrop and hybridizing with the immobilized gene. In order to detect a biomarker protein, a conjugate of a secondary protein having a label attached thereto was prepared and purified as in Example 2. The number of labeled substances bound to the secondary protein was determined using Nanodrop, Respectively. At this time, the display material which can be used may be a phosphor, an enzyme, a radioactive material, a fine particle and a pigment, but a phosphor is used in the present invention.

In the present invention, a standard concentration-sensitivity graph for quantitative analysis is prepared by applying an internal standard using a standard spot using a gene mix immobilization technique at a predetermined ratio to a biochip and converting the sensitivity of the detected biomarker protein to a protein amount (See Figs. 5 to 6).

The internal standard is a technique for preparing standard spots that can detect the sensitivity of the expression between 0% and 100% by mixing and immobilizing two or more genes. For example, a standard solution having a standard concentration Standard sensitivity-graph or standard concentration-sensitivity table made by digitizing and graphing or standardizing the standardized sensitivity of the biomarker protein, and standardizing various detection apparatuses used for analyzing the biomarker protein related to cardiovascular disease At the same time, it is possible to provide a system that can verify the distribution, storage, and use of biochips for diagnosis of heart disease. Also, it is possible to check the problems of equipment that may occur when using the detection equipment early, and to provide accurate analysis results.

Standard solutions having concentrations of, for example, 400 pg / ml, 200 pg / ml, 100 pg / ml, 50 pg / ml and 25 pg / ml were prepared for each of the heart disease biomarkers cTnI, NT proBNP and BNP, The measurement sensitivity was obtained by measuring with a specially prepared biochip for analysis of heart disease biomarker protein (see FIG. 7), and a standard concentration-sensitivity graph and / or table was prepared from the concentration and measurement sensitivity of the standard solution. Next, by adjusting the gain of the detector, the sensitivity of the detected biomarker protein was converted into the amount of protein, and the actual concentration and the detection concentration were compared to confirm the correspondence. In conclusion, the accuracy of the concentration analysis technique by the internal standard (standard concentration-sensitivity graph) prepared according to the present invention and the biochip for analyzing the heart disease biomarker protein used in the present invention was confirmed (see FIG. 8).

In order to confirm that cTnI, NT proBNP and BNP, which are cardiovascular biomarkers, can be detected at the same time, the three proteins cTnI, NT proBNP and BNP in the blood sample are diluted from 1/2 to 1/32 and measured according to the diluted ratio It was confirmed that the numerical values are analyzed at an accurate ratio.

This result shows that the present invention provides biochip technology that can quantitatively analyze one or three kinds of heart disease biomarker protein to pg / ml level, thereby monitoring the treatment of cardiovascular disease and informing it to a complete cure.

According to one variant of the invention, if the value of the measured sensitivity obtained from the blood sample is outside of the numerical range of the standard concentration-sensitivity graph and it is difficult to directly convert the concentration, the converted concentration can be estimated by extrapolation , And the blood sample can be diluted by a predetermined ratio, for example, by 1/2. This dilution can be performed until the sensitivity of the measurement is within the numerical range of the above-described graph, and diluted to a magnification of, for example, 1/2, 1/4, 1/8, 1/16, 1/32, The biochip can also be configured to measure simultaneously the samples. The diluted concentration can be converted from the measured sensitivity obtained in the diluted sample, and the converted concentration of the original blood sample can be obtained by multiplying the dilution ratio by the inverse.

The converted concentration of cardiovascular biomarker protein obtained according to the method of the present invention can be used to diagnose acute myocardial infarction (AMI) or to monitor its progress and treatment status. The diagnosis of acute myocardial infarction (AMI) may be made when the converted concentration of one or more of the cardiovascular biomarker proteins exceeds a predetermined value, and the determination of mitigation of heart disease by stagnation or reduction of the converted concentration Can be dropped. The above-mentioned predetermined figures can be determined according to the official opinions of professional organizations or experts.

The methodology for quantitative analysis of biomarker proteins according to the present invention is also applicable to the biomarkers of other primary organs diseases described below:

- Lung cancer: CEA, SCC, NSE, SLX

- Liver cancer: AFP, PIVKA-II

- Pancreatic cancer (gallbladder cancer, bile duct cancer): CEA, CA 19-9, CA 50, DUPAN-2, Esterase 1, Span-1 Ag, SLX, NCC-ST-439

- Gastric cancer: CEA, CA 19-9, CA 72-4

- Colorectal cancer: CEA, CA 19-9, CA 72-4

Prostate Cancer: PAP, PSA, γ-Sm

- Breast cancer: CEA, CA 15-3, TPA, NCC-ST-439, BCA 225

- Uterus cancer: SCC Antigen, CA 125, CEA, TPA

- ovarian cancer: CA 125, CEA, SCC, STN, CA 72-4, SLX

- Esophageal cancer: SCC, CEA

According to one variation of the present invention, a method for quantitative analysis of the antigen protein of the present invention is carried out by selecting one or more, specifically, 3 to 10, antigen proteins in the group consisting of the marker proteins of the primary organs or diseases Whereby the abnormality or abnormality of the primary organs can be judged, the diagnosis of the disease can be made, and the progress and / or the treatment situation thereof can be monitored.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited thereto.

Example 1: Antibody-Gene Binding and Purification

The substances used for antibody-gene binding are shown in Table 1 below, and the nucleotide sequences of the target gene (TD) used are shown in Table 2.

The material used in Example 1 Molecular Weight density cTnI Ab (capture Antibody) 160,000 g / mol 7.1mg / ml, 35ul NT proBNP Ab (capture Antibody) 160,000 g / mol 6mg / ml, 42ul BNP Ab (capture Antibody) 160,000 g / mol 6mg / ml, 41ul 2-iminothiolane 137 g / mol 4mg / ml TD06 9000 g / mol 100 pmol / ul TD01 9000 g / mol 100 pmol / ul TD10 9000 g / mol 100 pmol / ul Sulfo-SMCC 436 g / mol 80 mg / ml 10 X PBS 500 mM EDTA

Target gene Base sequence TD06 5 '- NH ₂ -TTTTTTTTTCGGGCAGGCCATAGCGA TD01 5'- NH ₂ -TTTTTTTTTGCGCCTCCCCAAGTCGT TD10 5 '- NH ₂ -TTTTTTTTTCCGCGCTCGCAAGGTAT

The target genes TD06, TD01 and TD10 were purchased from Bioneer, and amine groups (-NH ₂ ) were attached to the 5'-side of each target gene.

Example 1-1: Preparation of antibody-gene conjugate (cTnI Ab-TD06)

The synthesis of cTnI Ab-TD06 was performed in three steps as depicted in FIG. 1 and was performed as follows.

end. (Step 1) Synthesis of TD06-SMCC

1) 60 μl of the TD06 gene (concentration 900 ng / μl) and 5 μl of Sulfo-SMCC (concentration 80 mg / ml) were mixed in a 0.6 ml reaction tube.

2) 10 l of 10XPBS was added to the mixture of 1).

3) 3 ul of 500 mM EDTA was added to the mixture of 2).

4) The reaction mixture was reacted at room temperature for 2 hours.

5) After the reaction, TD06-SMCC was purified using BioWORLD Sephadex G-25 packed column (3 ml size) and mobile phase was 1 × PBS.

6) The purified TD06-SMCC was verified by measuring its purity and concentration using ND1000 UV / Vis spectrometer. The results are shown in FIG. 2 and 200ul of TD06-SMCC (concentration 202.4ng / ul) was obtained.

I. (Step 2) Synthesis of cTnI Ab-SH

1) A reaction tube of 0.6 ml was mixed with 35 ul of cTnI Ab (concentration 7.1 mg / ml) and 2 ul of 2-iminothiolane (concentration 4 mg / ml).

2) 30 [mu] l of 10XPBS was added to the mixture of 1).

3) 5 ul of 500 mM EDTA was added to the mixture of 2).

4) The reaction mixture was reacted at room temperature for 30 minutes.

5) After the reaction, cTnI Ab-SH was purified using a Sephadex G-25 packed column (3 ml size) of bioWORLD Inc. and 1 × PBS was used as the mobile phase.

6) Purified cTnI Ab-SH was verified by measuring the purity and concentration using ND1000 UV / Vis spectrometer. The results are shown in FIG. 2 and 190 ul of cTnI Ab-SH (concentration 1.10 mg / ml) was obtained.

All. (Step 3) Synthesis of cTnI Ab-TD06

1) In a reaction tube having a size of 0.6 ml, 200ul of the TD06-SMCC (concentration 202.4ng / ul) synthesized in the step 1 and 190ul of the cTnI Ab-SH (concentration 1.10mg / ml) synthesized in the step 2 were mixed.

2) The reaction mixture was reacted at 37 ° C for 30 minutes and then at 4 ° C for 1 hour.

3) After the reaction, cTnI Ab-TD06 was purified using 3000 MWCO spin filter (Millipore).

4) Purified cTnI Ab-TD06 was verified by measuring its purity and concentration using ND1000 UV / Vis spectrometer. The results are shown in FIG. 2 and 80 ul of cTnI Ab-TD06 (concentration 2.05 mg / ml) was obtained.

Example 1-2: Preparation of antibody-gene conjugate (NT proBNP Ab-TD01)

Synthesis of NT proBNP Ab-TD01 was synthesized in the same manner as in Example 1-1) using 42ul of NT proBNP Ab (concentration 6mg / ml) to obtain 80ul NT proBNP Ab-TD01 (concentration 2.08mg / ml) 2 shows the results.

Example 1-3: Preparation of antibody-gene conjugate (BNP Ab-TD10)

BNP Ab-TD10 was synthesized in the same manner as in Example 1-1) except that 41ul of BNP Ab (concentration 6mg / ml) was obtained and 60ul of BNP Ab-TD06 (concentration 2.39mg / ml) .

Example 2: Preparation of a labeling substance-secondary antibody conjugate

As the amplification substance of the antigen detection signal, a labeling substance secondary antibody conjugate was prepared. A fluorescent substance (Cy5) was used as a labeling substance, and the substances used for antibody-fluorescent binding are shown in Table 3 below.

The material used in Example 5 Molecular Weight density cTnI AB (Secondary Antibody) 160,000 g / mol 3.7mg / ml, 65ul NT proBNP AB (Secondary Antibody) 160,000 g / mol 8mg / ml, 30ul BNP AB (Secondary Antibody) 160,000 g / mol 7.6mg / ml, 33ul Cy5-bis NHS ester 792 g / mol 1 pack in DMSO 20ul 10 X PBS - - 500 mM EDTA - -

Example 2-1: Preparation of labeling substance secondary antibody conjugate (cTnI AB-Cy5)

The synthesis of cTnI AB-Cy5 is shown in FIG. 2 and the materials used are shown in Table 3. Cy5-bis NHS ester was purchased from GE Healthcare Life Sciences, Cy5-bis NHS ester 5pack.

[ Synthesis of cTnI AB-Cy5 ]

1) Add 20ul DMSO to Cy5-bis NHS ester 1pack and mix.

2) 5 ul of Cy5-bis NHS ester of 1) was added to a cTnI AB tube containing 65 ul of cTnI AB (concentration 3.7 mg / ml).

3) 3 ul of 500 mM EDTA was added to the above 2) and mixed.

4) The mixture of 3) was reacted at room temperature for 20 minutes.

5) After the reaction, cTnI AB-Cy5 was purified using BioWORLD Sephadex G-25 packed column (3 ml size) and the mobile phase was 1 × PBS.

6) Purified cTnI AB-Cy5 was verified by measuring its purity and concentration using ND1000 UV / Vis spectrometer. The results are shown in FIG. 2 and 180ul of cTnI AB-Cy5 (concentration 1.2mg / ml) was obtained.

Example 2-2: Preparation of labeling substance-secondary antibody conjugate (NT proBNP AB-Cy5)

NT proBNP AB-Cy5 was synthesized in the same manner as in Example 2-1) by using 30ul of NT proBNP AB (concentration 8mg / ml) to obtain 190ul of NT proBNP AB-Cy5 (concentration 1.1mg / ml) 2 shows the results.

Example 2-3: Preparation of labeling substance-secondary antibody binding substance (BNP AB-Cy5)

The synthesis of BNP AB-Cy5 was carried out in the same manner as in Example 2-1) using 33ul of BNP AB (concentration 7.6mg / ml) to obtain 150ul of BNP AB-Cy5 (concentration 1.4mg / ml) The results are shown.

Example 3: Production of biochip for protein detection

Oligonucleotide (9G-oligo DNA) having 9 consecutive guanine bases was immobilized on an aminocylic array monolayer according to the method described in Korean Patent No. 10 0786577, FIGS. 1 and 11, 9G-DNA chip was prepared and used as a biochip for protein detection (see FIG. 3).

The 9G-oligo gene used in this example is described in Table 4 below:

Oligo
gene The base sequence (5 ', 3') density HC GGGGGGGGG CTTTATCAT CC ACT GTT CTC GGC ACG 100 pmol / ul PCP06 GGGGGGGGG CTTTATCAT TCG CTA TGG CCT GCC CG 100 pmol / ul PCP01 GGGGGGGGG CTTTATCAT ACG ACT TGG GGA GGC GC 100 pmol / ul PCP10 GGGGGGGGG CTTTATCAT ATA CCT TGC GAG CGC GG 100 pmol / ul

Example 4 Detection of Antigen Proteins cTnI, NT proBNP and BNP

Methods and experimental procedures for detecting heart disease biomarkers as antigenic proteins are schematically illustrated in FIG. Three antigen proteins cTnI, NP proBNP and BNP were used as standard heart biomarker proteins and used as standard samples.

1) Preparation of antigen standard sample

Standard samples were prepared by purchasing cTnI Ag (0.5 mg / ml), NT proBNP Ag (0.9 mg / ml) and BNP Ag (3.5 ng / ml) from Hytest.

A standard sample 1 containing the antigen proteins cTnI, NT proBNP and BNP at a concentration of 400 pg / ml was prepared and diluted 1/2 × with 1 × PBS solution to a concentration of 200 pg / ml, 100 pg / ml, 50 pg / ml and 25 pg / ml Containing standard samples 2-5 were prepared as in Table 5 below.

Protein Standard sample 1 Standard sample 2 Standard sample 3 Standard Sample 4 Standard sample 5 cTnI 400 pg / ml 200 pg / ml 100 pg / ml 50 pg / ml 25 pg / ml NT proBNP 400 pg / ml 200 pg / ml 100 pg / ml 50 pg / ml 25 pg / ml BNP 400 pg / ml 200 pg / ml 100 pg / ml 50 pg / ml 25 pg / ml

2) Preparation of Antibody Mixture

end. 15 ul of the antibody-gene conjugate cTnI Ab-TD06 (100 ug / ml), NT proBNP Ab-TD01 (100 ug / ml) and BNP Ab-TD10 (100 ug / ml) were added to an e-tube having a capacity of 1.5 ml .

I. 60 ul of cTnI AB-Cy5 (100 ug / ml), NT proBNP AB-Cy5 (100 ug / ml) and BNP AB-Cy5 (100 ug / ml)

All. 1XPBS 375ul was added to prepare an antibody mixture of 600ul.

3) Sample mixing, loading and incubation

end. An incubation mixture was prepared by mixing 100ul of each antibody mixture and 300ul of an incubation buffer in an e-tube of 1.5ml size.

I. 20ul of the standard samples (1-5) in Table 5 and 40ul of the incubation mixture were mixed and then loaded on the chip and then incubated at room temperature for 2 hours

4) Wash, dry and scan

After the incubation, the cells were washed twice in 2X for 2 minutes in a washing solution (4XSSC), followed by drying at 3000 rpm for 1 minute using a centrifuge. The dried biochip was scanned using a commercial scanner (Scanarray Gx Scanner, Perkin Elmer).

5) Analysis of results

Figures 5 and 6 show the measurement results for a standard sample. The standard sensitivities measured for the standard concentrations are summarized in Table 6 below, and the graphs of the standard concentrations versus standard sensitivities ("Standard Sample Sensitivity vs. Concentration Graphs" are shown in FIG.

Standard sample concentration 400 pg / ml 200 pg / ml 100 pg / ml 50 pg / ml 25 pg / ml Fluorescence sensitivity
(Gain 90) cTnI 65000 45000 28000 17000 10000 NT proBNP 65000 44000 28000 16000 10000 BNP 65000 45000 27000 16000 10000

The blood sample of the subject is analyzed by biochip, and the resultant fluorescence intensity value is compared with the graph of FIG. 6 to analyze the concentration of the target protein contained in the subject's blood sample.

An internal standard spot (standard density-sensitivity table) as shown in FIG. 5 was prepared so as to obtain the standard fluorescence sensitivity at the same value in all the scanning equipments. To analyze the protein concentration, a gain 90 ) Was determined as the standard sensitivity.

Example 5: Detection method using internal standard spot

1) Manufacture of biochip for detection using internal standard spot

When analyzing the biomarker concentration using a biochip for protein detection, the internal standard spot (standard concentration-sensitivity graph or standard concentration-sensitivity graph) is set so that the measured sensitivity can be compared with the sensitivity of the internal standard spot and the converted concentration can be determined. Sensitivity tables) were prepared and the materials used for internal standard spot immobilization and hybridization reactions are listed in Table 7 below.

Oligo gene The base sequence (5 ', 3') density HC GGGGGGGGG CTTTATCAT CC ACT GTT CTC GGC ACG 100 pmol / ul NC GGGGGGGGG CTTTATTTT TAT AAA TAT AAT ATA ATT 100 pmol / ul HC TD-Cy5 Cy5-TTT TTT CGT GCC GAG AAC AGT GG 1 pmol / ul

The internal standard spot sensitivity was determined using the mixed gene as shown in Table 8 below so as to be able to show all the sensitivities of 65000 to 10000 values at the concentration of 400 pg / ml to 25 pg / ml of the standard sample of Example 4. The standard spot 1 (IS 1) Mixed with the NC gene so that the HC gene ratios were 5%, 2.5%, 1% and 0.5%, respectively, for the standard spot 2 (IS 2), the standard spot 3 (IS 3) and the standard spot 4 Respectively. Table 8 below shows mixed gene ratios (HC: NC) representing standard concentrations of internal standard spots.

Internal standard spot Mixed gene HC gene ratio density HC gene NC gene IS 1 50ul 950ul 5% 10 pmol / ul IS 2 25ul 975ul 2.5% 10 pmol / ul IS 3 10ul 990ul One% 10 pmol / ul IS 4 5ul 950ul 0.5% 10 pmol / ul

Using the PCP06, PCP01 and PCP10 genes described in Table 4 and the IS1, IS2, IS3 and IS4 mixed genes listed in Table 8, the same procedures as described in Example 11 of Korean Patent No. 10-0786577 The biochip described in 7 was prepared.

2) Determine internal standard spot sensitivity using HC TD-Cy5

HC TD-Cy5, which has a nucleotide sequence complementary to the HC gene, is used to analyze the sensitivity at the gain 90 of FIG. 6, so that fluorescence sensitivity after the hybridization reaction with the four internal standard spots can be represented at all levels ranging from 65000 to 10000 The internal standard spot sensitivity was determined as follows.

end. 15 μl of HC TD-Cy5 (1 pmol / ul) in Table 9 was mixed with 100 ml of incubation buffer to prepare an incubation mixture containing HC TD-Cy5 at a concentration of 0.15 fmol / ul.

I. 1XPBS 30ul and < RTI ID = And 30 μl of incubation buffer was added to the chip, followed by incubation at room temperature for 2 hours.

All. After incubation, the cells were washed with 2x2min of washing solution (4XSSC) and centrifuged at 3000rpm for 1 min. The dried biochip was scanned on a Perkin Elmer Scanarray Gx Scanner to obtain the sensitivity at gain 90 of FIG. 6 and the protein concentration was analyzed using the sensitivity contrast concentration graph for protein analysis.

3) Protein cTnI, NT proBNP, BNP detection using internal standard spot

end. The intensities of the cTnI, NT proBNP, and BNP standard samples were compared with the concentrations measured on the biochip for protein detection according to the present invention by adjusting the scanner gain (gain) using the internal standard spot sensitivity. The same procedure as in Example 4 was carried out using the standard samples 1 to 5 described in Table 5 above.

I. The results obtained by converting the sensitivities shown in the standard samples 1 to 5 of cTnI into the concentrations using the internal standard spot (standard concentration-sensitivity graph) are shown in FIG. 8, and the actual standard concentrations are matched with the converted concentrations measured by the biochip Lt; / RTI > For the other cardiovascular biomarkers NT proBNP and BNP, the results were also obtained in which the converted concentrations converted from the standard concentrations of the standard samples equally corresponded to the measured sensitivities.

Example 8: Detection of plasma sample (serum)

(Sample 1) having a sensitivity of 65000 or more was prepared by a biochip, although the concentration of the cardiovascular biomarker was unknown, and the sample diluted with 1XPBS from 1/2 to 1/32 as shown in Table 9 6) was further prepared.

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Plasma samples (No. 22) 1/2 dilution 1/4 dilution 1/8 dilution 1/16 dilution 1/32 dilution

1) 30 ul of plasma sample and 30 ul of 1XPBS were mixed and diluted 1/2.

2) 1) diluted sample 30ul and 1XPBS 30ul were mixed and diluted 1/4.

3) Dilution of 2) 30ul of sample and 30ul of 1XPBS were mixed and diluted 1/8.

4) 30 μl of the diluted sample of 3) and 30 μl of 1XPBS were mixed and diluted 1/16.

5) Dilution of 4) 30ul of sample and 30ul of 1XPBS were mixed and diluted 1/23.

6) Using the plasma samples in Table 9, the results were obtained in the same manner as in Example 4), and the results are shown in FIG.

Concentration analysis showed that, for plasma samples that did not know the exact concentration, the included coronary heart disease biomarker protein antigens, cTnI and NT proBNP, had sensitivities reduced by exactly 1/2 in the samples diluted by ½ concentration , And it was confirmed that quantitative concentration analysis can be performed at a concentration of a picogram level up to a low concentration of 25 pg / ml at a high concentration of 400 pg ml.

In the first plasma sample stock solution (sample 1), the sensitivity was 65000 or more, and the concentration was 400 pg / ml or more, so that it was difficult to accurately convert the concentration. The concentration converted from the sensitivity measured in the 1/2 diluted sample was 322 pg / ml, The plasma sample concentration was determined to be 644 pg / ml.

INDUSTRIAL APPLICABILITY The present invention can be industrially used in the field of protein chip manufacturing and diagnosis and treatment using the same.

Claims (17)

A method for quantitative analysis of heart disease biomarkers comprising the steps of:
(1) A standard concentration-sensitivity graph of anticholinergic biomarker proteins, which are antigens, is prepared using a biochip,
(2) analyzing a subject's blood sample with a biochip to obtain a measurement sensitivity of heart disease biomarker proteins contained in blood,
(3) determining the converted concentration of heart disease biomarker proteins by comparing the measured sensitivities described above with the standard concentration-sensitivity graphs described above,
(4) determining the state of progression of heart disease or the state of treatment from the converted concentration of the obtained heart disease biomarker proteins;
Here, the above-mentioned biochip includes the following (a) to (d)
(a) a solid substrate,
(b) means of attachment to a solid substrate and an amino-calixarene derivative having a recognition site for a continuous guanine base,
(c) an immobilized gene having a continuous guanine base and a desired base (sequence), and
(d) an antibody-gene conjugate obtained by binding a complementary binding gene and an antibody to the immobilized gene;
Here, the sample to be measured by the biochip is (e) a marker substance-antibody conjugate (f) in addition to the heart disease biomarker protein antigen, before or during the measurement, and the component (d) May be added separately before, after, or upon addition of the sample,
Here, the above-mentioned aminocylic arerene derivative is attached to the solid substrate by chemical or physical bonding between the above-mentioned attachment means and the surface of the solid substrate, and the immobilized gene described above is formed by the continuous guanine base, Attached to the recognition site of the guanine base. The method according to claim 1, wherein the step (1) described above comprises quantitatively analyzing heart disease biomarker protein.
A solution containing a predetermined concentration of at least one kind of heart disease biomarker protein (1a) antigen and a solution (s) obtained by diluting the same at a predetermined ratio are prepared,
(1b) The standard solutions described above were analyzed by biochip to obtain a standard sensitivity,
(1c) A standard concentration-sensitivity graph is prepared from the above-mentioned standard concentration and the obtained standard sensitivity. The method according to claim 2, wherein the standard density-sensitivity graph is a table (standard density-sensitivity spot) in which standard density values and standard sensitivity images are combined in place of the graph format combining standard density values and standard sensitivity values And measuring the concentration of the biomarker protein. The bio-chip according to claim 1, wherein the bio-chip comprises components (a), (b) and (c) Wherein the method comprises the steps of: 2. The method according to claim 1, wherein the heart disease biomarker protein antigen described above is at least one selected from the group consisting of cTnI, NT proBNP and BNP, and the antibody is an antibody of the antigen, Analysis method. The method according to any one of claims 1 to 5, wherein said heart disease biomarker protein includes all of cTnI, NT proBNP and BNP. The method according to claim 1, wherein the target base (sequence) has a length of 13 mer or less, 14 to 17 mer, 18 to 21 mer, or 22 mer or more. 2. The method according to claim 1, wherein the solid substrate is selected from the group consisting of gold, silver, platinum, glass, glass fiber sheet, silicon wafer, and crystal crystals. The method for quantitative analysis of cardiovascular biomarker protein according to claim 1, wherein the aminoglycos arene derivative is an aminocarboxylarene derivative represented by the following formula 1 or 2:
[Chemical Formula 1]

(2)

_{[Wherein, R 1, R 2, R} 3, R 4, R 5, R 6, R 7, R 8, R '1, R' 2, R '3, R' 4, R '5, R' ₆ , R ' ₇ and R' ₈ independently of one another are -H, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -OCH ₃ , -Cl, -C ₆ H ₅ , -OH, -OCH _{2 CH 3, -Br, -CF 3} , -OCH 2 C 6 H 5, -OC 6 H 5, -OC 6 H 4 CH 3, -OC 6 H 4 C (CH 3) 3, -OC 6 H 4 _{CF 3, -OC 6 H 4 Cl} , -OCOCH 3, -NHCOCH 3, -CONHCH 3, -CN, and selected from the group consisting of COOH, and -COOR, in the -COOR R is -CH ₃ or -C ₂ H ₅ .
Y _1, Y _2, Y ₃ and Y ₄ are independently -H, each other _{- (CH 2) n-CH} = O, - (CH 2) n-SH, - (CH 2 CH 2 O) m-CH 2 _{CH 2 -CH = O, - (} CH 2 CH 2 O) m-CH 2 CH 2 -SH, - (CH 2) mC 6 H 4 - (CH 2) cZ, and -CO- (CH ₂₎ m- _{1-c 6 H 4 - (} CH 2) is selected from the group consisting of cZ (n = 2 ~ 15, m = 1 ~ 10, c = 0 ~ 10, Z is -SH, -CHO, -COOH and -NH ₂ , and -C ₆ H ₄ - and C ₆ H ₅ are defined as phenyl groups). The method of claim 8 or 9, wherein said solid substrate is selected from gold and wherein at least one of Y ₁ , Y ₂ , Y ₃ and Y ₄ of formula (1) or (2) is selected from thiol groups as a means of attachment to a solid substrate Wherein the quantitative analysis of heart disease biomarker protein is performed. 8. The method of claim 8 or 9, wherein said solid substrate is selected from the group consisting of glass modified with amine functional groups, silicon wafers and crystal crystals, and wherein Y ₁ , Y ₂ , Y ₃ and Y ₄ is selected from aldehyde groups, whereby the linkage of (a) and (b) is selected from an imine group or an amine group reduced therefrom, the quantification of a cardiovascular biomarker protein Analysis method. The method according to claim 1, wherein the labeling substance is selected from the group consisting of phosphors, enzymes, radioactive substances, fine particles and pigments. The method according to claim 1, wherein if the measurement sensitivity of the heart disease biomarker protein of the blood sample obtained in the step (2) described above is higher than the range of the standard concentration-sensitivity graph obtained in step (1), the blood sample of the subject is diluted Obtaining a measurement sensitivity, and determining a converted concentration from the obtained measurement sensitivity and dilution ratio. A method for diagnosing acute myocardial infarction (AMI) using a concentration of a cardiovascular biomarker protein obtained by the method according to claim 1. A method for monitoring the progress and treatment status of acute myocardial infarction (AMI) using the concentration of the cardiovascular biomarker protein obtained by the method according to claim 1. A method for quantitative analysis of an antigen protein using the method according to claim 1, wherein the antigen protein is selected from the group consisting of the marker proteins of the following primary organ diseases:
- Lung cancer: CEA, SCC, NSE, SLX
- Liver cancer: AFP, PIVKA-II
- Pancreatic cancer (gallbladder cancer, bile duct cancer): CEA, CA 19-9, CA 50, DUPAN-2, Esterase 1, Span-1 Ag, SLX, NCC-ST-439
- Gastric cancer: CEA, CA 19-9, CA 72-4
- Colorectal cancer: CEA, CA 19-9, CA 72-4
Prostate Cancer: PAP, PSA, γ-Sm
- Breast cancer: CEA, CA 15-3, TPA, NCC-ST-439, BCA 225
- Uterus cancer: SCC Antigen, CA 125, CEA, TPA
- ovarian cancer: CA 125, CEA, SCC, STN, CA 72-4, SLX
- Esophageal cancer: SCC, CEA The method for quantitative analysis of antigenic proteins according to claim 16, wherein the antigen is selected from the group consisting of marker proteins of primary organs diseases.

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