KR20180031612A - A Method of Rapid Diagnosis With High Sensitivity By Using Single Diagnosis Chip Comprising Reaction and Analysis Process - Google Patents

Abstract

The present invention relates to a method for detecting a detection target material by using a diagnosis chip including reaction and analysis, and more particularly, to a diagnosis chip including a biological detection material interacting with a material to be detected and a detection part to which a sol-gel spot is fixed, and a method for detecting a material to be detected or a method for analyzing a sample.

Description

[0001] The present invention relates to a method for rapidly diagnosing a single diagnostic chip including a reaction and an assay,

The present invention relates to a method for detecting a substance to be detected using a diagnostic chip including a reaction and an analysis, and more particularly, to a method for detecting a substance to be detected including a biological detection substance interacting with a substance to be detected, The present invention relates to a chip and a method for detecting a substance to be detected based on the chip, or a method for analyzing a sample.

In the field of clinical examination, the field of point-of-care testing (hereinafter also referred to as POCT) has received attention. It is possible to shorten the time required for obtaining a test result after collecting a specimen by separating a detection target substance from a specimen and then performing a POCT analysis through a simple device.

POCT analysis can be performed using enzyme assay, immunoassay, chemical colorimetric assay, electrochemical assay, fluorescence labeling and measurement, or chemiluminescent labeling. And various techniques such as measurement are used.

In vitro diagnostic methods using conventional biochemical methods require expensive analytical equipment and have been developed as reagents for large hospitals and laboratories. As a result, consumption patterns of diagnostic products are concentrated in some hospitals and laboratories, and should be combined with expensive analytical equipments. Since the 1980s, the concept of point-of-care diagnostic testing (POCT), which is an on-site diagnostic kit, has been developed. This on-site diagnostic kit is then used for pregnancy diagnosis, drug abuse diagnosis, infectious disease diagnosis, It has been widely used and led the growth of diagnostic market since 1990. More people are interested in in vitro diagnostics due to the growing population using global in vitro diagnostic therapies and the cost of in vitro diagnostic tests that are cheaper with the introduction of POCT. The market size of immunochemical diagnosis during IVD is expected to grow at a CAGR of 5.4% from USD 14 billion in 2009, and to generate more than USD 20 billion in sales by 2016.

On the other hand, influenza is an acute respiratory virus infection that is prevalent in the winter season, and approximately 10% of the population is infected every year. It is often classified as a serious disease due to the complication of pneumonia or aggravation of underlying disease in elderly, infant and chronic disease patients. While seasonal influenza every year is due to a small number of influenza viruses, the pandemic influenza, which occurs every 10 to 40 years, is caused by an antigen-to-host mutation. It is characterized by global trends and 30-50% of the population is infected. Socioeconomic damage is enormous.

Rapid tests based on antigen-antibody reaction, which is the most frequently used for the detection of influenza virus, are relatively short in time and have a simple advantage, but have a low sensitivity and accuracy. The Real-Time PCR method used for confirmation of influenza is recognized as the most effective technology for the diagnosis of infectious diseases through high sensitivity and accuracy. However, since the inspection time is comparatively long and a request is made to a specialized institution, It is impossible. In addition, testing costs are high and it is very difficult to apply it as a large-scale initial countermeasure when infectious diseases are pandemic.

Therefore, it is necessary to develop a POCT concept product that can be applied to a relatively small scale field, and it is necessary to develop a technology having high sensitivity and accuracy.

Under these technical backgrounds, the inventors of the present application have proposed a sensitive sensitivity technology based on a three-dimensionally immobilized sol-gel. This technology is able to massively immobilize various biomarkers based on the sol-gel structure of porous three-dimensional structure. It can build a system for detecting distress in comparison with conventional biosensors and provides technology based on miniaturization diagnosis chip by fusion of micro array manufacturing technology And the sensitivity and specificity of existing Rapid kit and detection time limit of molecular diagnosis were overcome by the development of 3 - D sol - gel bio - fusion technology. In addition, by multiplexing multiple markers independently in the same space using a sol-gel microarray fabrication technique in one reaction part, it is possible to implement a multiplex technique, thereby establishing a platform base of multiple diagnosis technology.

Thus, a sol-gel-based diagnostic chip for analyzing a sample with sol-gel spots containing a biological detection material that interacts with the detection target material, rather than the well-known well or plate based solids- By providing analytical performance and multi-diagnosis platform technology and quick and accurate results based on field diagnosis, it is possible to improve the accuracy of analysis, as well as the integrated reagent storage and waste storage, And the present invention has been completed.

In order to solve the above problems, the present invention provides a method of detecting a detection target substance using a diagnostic chip.

In order to achieve the above object, the present invention provides a method for detecting a detection target material using a diagnostic chip including the following components in an integrated manner, comprising: a container mounting part containing a sample containing a detection target material; A detection unit fixed with a sol-gel spot including a biological detection substance interacting with a substance to be detected; A reaction reagent storage section containing a biological marker for the biological detection substance to confirm whether the biological substance to be detected reacts with the biological substance to be detected; A cleaning solution storage unit including a cleaning solution for removing a substance not reacting with a detection target material; A waste storage unit for removing remaining or unreacted materials after the reaction in the detection unit; And a hole equipped with a transporting means in the form of a pipette tip, a sample containing a detection target substance contained in the container mounting portion is brought into contact with a biological detection substance interacting with a detection target substance of the detection portion, Lt; RTI ID = 0.0 > imaging. ≪ / RTI >

The present invention also provides a method for analyzing a detection target material using a diagnostic chip comprising the steps of: inserting a container containing a sample containing a substance to be detected in a diagnostic chip into a container mounting part, mounting a tip-shaped carrier means in a hole; Injecting a sample containing the substance to be detected into a delivery means and injecting a sol-gel spot containing a biological detection substance interacting with the substance to be detected into a fixed detection section; A step of sucking the remaining material after the reaction in the detection unit and discharging the remaining material to the waste storage unit; Washing the unreacted material in the detection part by injecting the cleaning solution into the detection part in the cleaning solution storage part including the cleaning solution for removing the substance not reacting with the detection target substance; Withdrawing the unreacted material into the waste storage unit; And imaging the reaction result of the detection unit.

According to the present invention, on the basis of the sol-gel-based technology, which has been presented as a multi-diagnosis platform with a conventional high sensitivity / high specificity performance, the analytical process is carried out in a straight line Using the diagnostic chip for field diagnosis together with the inspection equipment, it is possible to save the diagnosis time due to the analysis progress by the quick and simple operation including the high analytical performance compared with the conventional one, and to store the sample and the reagent and / , Cross contamination between them can be prevented, and the analysis accuracy can be improved.

1 shows a front view of a diagnostic chip.
FIG. 2 is a schematic view of the detection part of the diagnostic chip and a reaction scheme for a three-dimensional sol-gel spot
3 is a detailed view of the reagent storage part of the diagnostic chip.
4 is a detailed view of the cleaning solution storage portion of the diagnostic chip.
5 is a detailed view of a waste storage portion in the diagnostic chip.
Fig. 6 is a detailed view of a hole in which the carrying means is mounted in the diagnostic chip.
7 is a detailed view of the container mounting portion of the diagnostic chip.
8 is a schematic diagram of a diagnostic device used in implementing the method according to the present invention.
FIGS. 9 and 10 show diagnostic imaging results through a conventional Rapid product (S company).
FIGS. 11 and 12 show diagnostic results by the method according to the present invention.
13 and 14 show fluorescence intensities and fluorescence image results for a three-dimensional antibody-immobilized 3D-antibody-immobilized assay using Sol-gel.
Fig. 15 shows the fluorescence image obtained by testing the negative specimen sample.
Fig. 16 shows the result of analysis of fluorescence data obtained by testing the negative specimen samples.
Fig. 17 shows fluorescence images of a positive specimen sample tested against Influenza A. Fig.
18 shows the result of analyzing fluorescence data of a positive specimen sample against Influenza A. FIG.
Fig. 19 shows a fluorescent image of a positive specimen sample tested against Influenza B. Fig.
Fig. 20 shows the result of analyzing fluorescence data of a positive specimen sample with Influenza B. Fig.
21 is a configuration diagram of a three-dimensional sol-gel antibody immobilized chip for detecting tumor markers.
Fig. 22 shows a fluorescence image of a tumor marker antigen-responsive sample.
Figures 23 to 26 show fluorescent intensity distributions by tumor marker antigen concentration.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a method for detecting a substance to be detected by using a diagnostic chip comprising the following components integrally, comprising: a container mounting portion containing a sample containing a substance to be detected; A detection unit fixed with a sol-gel spot including a biological detection substance interacting with a substance to be detected; A reaction reagent storage section containing a biological marker for the biological detection substance to confirm whether the biological substance to be detected reacts with the biological substance to be detected; A cleaning solution storage unit including a cleaning solution for removing a substance not reacting with a detection target material; A waste storage unit for removing remaining or unreacted materials after the reaction in the detection unit; And a hole equipped with a transporting means in the form of a pipette tip, a sample containing a detection target substance contained in the container mounting portion is brought into contact with a biological detection substance interacting with a detection target substance of the detection portion, Lt; RTI ID = 0.0 > imaging. ≪ / RTI >

The method according to the present invention can be realized as an optimum rapid reaction step (overcoming quantitative error error of reagent and reaction step, optimization of reaction part of measurement part, rapid design of interlocking equipment, etc.) The diagnostic chip can solve the problems caused by contamination and misuse of the reagents and reagents used by separating the reagent storage and the waste storage for each reaction step.

In one embodiment, the substance to be detected may be a biological substance and may be, for example, a nucleic acid, a peptide, a protein, a low molecular substance, an antigen, a virus or a cell. The biomaterial may be derived from body fluids. The body fluids may be, for example, feces, urine, tears, saliva, external secretions of skin, external secretions of the respiratory tract, external secretions of the intestinal tract, external secretions of the digestive tract, plasma, serum, blood, spinal fluid, Homogenates, portions of tissues, cells, cell extracts, or in vitro cell cultures.

The virus may be, for example, an influenza virus. According to one embodiment of the present invention, antibodies specific for influenza subtypes A and B can be immobilized on a dedicated diagnostic chip by sol gel immobilization technique, and the specific diagnostic potential of each subtype can be confirmed. The complex process part of the existing inspection method can be shortened to the automated measurement process from the sampling to the calculation of the result value by using the dedicated diagnostic chip of Lab on a chip type so that the presence of influenza virus can be measured easily . In addition, the miniaturized diagnostic chip configuration has the advantage of being easy to store and applicable to on-site inspection (POCT) system.

The container containing the sample can be fitted externally in detachable form to the diagnostic chip. The container containing the sample may be mounted on the diagnostic chip by mechanical, adhesive, or other means in the hole included in the diagnostic chip. By using such vessels, the sample can be stored or processed freely in the spatial and / or temporal constraints that arise from storing the sample in one piece together with other reagents.

The shape of the container can be, for example, a bottle, a tube, a tube or an ampoule, and the material is not limited. For example, the container may be partially or wholly made of plastic, glass, paper, foil or wax .

The biological detection substance that interacts with the substance to be detected may be, for example, a protein, or a nucleic acid and / or an oligonucleotide. The protein may be, for example, an antibody or a fragment thereof that specifically binds to a substance to be detected. At this time, detection of the substance to be detected can be performed by an immunoassay method by an antigen-antibody reaction.

The immunoassay may be performed according to various quantitative or qualitative immunoassay protocols. The immunoassay format may include, but is not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay (ELISA), capture-ELISA, sandwich assay, flow cytometry, But are not limited to, chemical purification.

In one embodiment, the method includes reacting a sample containing the substance to be detected with a primary biological detection substance interacting with the substance to be detected; And reacting a secondary biological detection substance that binds to the primary biological detection substance that is interacted with the detection target substance. When the detection substance that interacts with the detection target substance is an antibody, the first biological detection substance can specifically bind to the detection target substance and can bind to the antibody specifically binding to the detection target substance The reaction can be carried out as a secondary biological detection substance.

The nucleic acid and / or oligonucleotide may be, for example, an aptamer. The aptamer means a nucleic acid and / or an oligonucleotide molecule having a binding activity to a predetermined target molecule, and may bind to a target molecule to inhibit activity. The aptamer may be RNA, DNA, modified oligonucleotides or a mixture thereof. The aptamer may also be in a linear or cyclic form. The length of the aptamer is not particularly limited and may be generally about 15 to about 200 nucleotides.

At this time, the biological marker for the biological detection substance may be, for example, a radioisotope, a fluorescent dye, a dye, a protein, or an antibody or an aptamer labeled with a luminescent substance, but is not limited thereto. As a result of the reaction included in the method of the present invention, imaging can be performed by measuring and analyzing the fluorescence, the amount of radiation, the luminescence intensity, and the like represented by the biological marker.

The sol-gel spot is for immobilizing a biological detection substance interacting with a detection target substance. The sol-gel spot can be fixed while the sol solution is gelled, and can be fixed so that the spot does not fall off during reaction with a sample containing the detection target substance.

The size of the sol-gel spot may be, for example, 100-1000 m, specifically 200-400 m, and more particularly 250-350 m. If the size of the sol-gel spot is too small within the desired range, there is a problem of desorption of the sol-gel spot. If the size of the sol-gel spot is large, there is a problem of forming a uniform porous structure.

The sol-gel spots may be arranged in microarray form so that at least one sol-gel spot in the direction of one of the sol-gel spots has at least one row. In this way, various biological detection substances interacting with various detection target substances can be immobilized, so that diagnosis of diseases can be performed at one time.

Hereinafter, the diagnostic chip according to the present invention will be described in detail with reference to the drawings. The same elements are denoted by the same reference numerals as possible even if they are shown on different drawings.

Referring to FIG. 1, a diagnostic chip 100 according to the present invention includes a detection unit 110, a reagent and / or waste storage unit 120, and a sample and carrier storage unit 130. The diagnostic chip 100 according to the present invention includes a detection chip 110, a reagent and / or waste storage unit 120, a sample and a carrier storage unit 130, Height (12.5 mm).

The sample and carrier storage part 130 is constituted of a hole 131 equipped with a transporting means in the form of a pipette tip and a container mounting part 132 containing a sample containing a substance to be detected. The container secured to the container mounting portion 132 may be, for example, a diagnostic chip and a detachable bottle, tub, tube or ampoule. Individual pipette tips and containers are included for each diagnostic chip, and a tag, for example, a bar code or a QR code, can be displayed on the sample to distinguish the plurality of samples.

In some cases, the handle 133 may be attached to one side of the diagnostic chip 100 for the convenience of the user.

A sol-gel spot including a nucleic acid, a peptide, a protein, a low molecular substance, a virus or a cell-interacting biological detection substance such as an antigen, an antibody or an aptamer is immobilized on the detection unit 110 And the sol-gel spot may have a size of, for example, 100-1000 μm , specifically 200-400 μm , more specifically 250-350 μm .

In the detection unit 110, one or more sol-gel spots may be arranged in one direction and may be arranged in a microarray form so as to have at least one or more rows. Through such sol-gel spots, it is possible to immobilize a variety of biological detection substances, for example, one or more biological detection substances, which interact with the various detection target substances, so that diagnosis can be made in multiple ways at once.

The reagent and / or waste storage part 120 includes a reaction reagent storage part 121 including a biological marker for the biological detection substance, a reaction reagent storage part 121 not reactive with the detection target substance A cleaning solution storage part 122 including a cleaning solution for removing the material, and a waste storage part 123 for removing the remaining or unreacted material after the reaction in the detection part. The reaction reagent storage part 121, the washing solution storage part 122 and the waste storage part 123 are specifically shown in FIGS. 3 to 5, respectively.

The biological marker included in the reaction reagent storage section 121 may be, for example, a radioisotope capable of detecting a biological detection substance, a fluorescent dye, a dye, a protein, or an antibody or an aptamer labeled with a luminescent substance.

The solution for washing the substance not reacted with the detection target substance contained in the cleaning solution storage part 122 may be, for example, a solution containing a small amount of a surfactant, a tarnish agent or an ionized salt. The waste storage part 123 for removing the remaining or unreacted material after the reaction in the detection part may be in the form of a pad for absorbing the residual material or unreacted material after the reaction in the detection part, for example. The waste storage part 123 is not particularly limited as long as it is a porous material capable of absorbing the remaining or unreacted material after the reaction, but may be, for example, a sponge, a nonwoven fabric, a cloth or a cotton. It is possible to solve the problem that the accuracy of the inspection result is lowered due to the contamination caused by mixing the residual material or unreacted material after the reaction with the reagent by treating the waste in the waste storage part 123.

A sticker may be attached to the detection unit 110 and the reaction reagent storage unit 121, the washing solution storage unit 122 and the waste storage unit 123 may be sealed by sealing the cover member.

The single diagnostic chip 100 included in an integrated form according to the present invention may be a polyvinylidene fluoride (PVdF), a nylon, a nitrocellulose, a polyurethane, a polycarbonate, a polystyrene (PS), a polylic polyacrylic acid, polyacrylonitrile (PAN), polylactic-co-glycolic acid, polyethyleneimine, polypropyleneimine, polymethyl methacrylate, polyvinylcholide, polyvinylacetate and polystyrene divinylbenzene A polymer injected with at least one selected from the group consisting of a polystyrene divinylbenzene copolymer and a plastic injected with polystyrene and / or polymethyl methacrylate, It can be included as a material.

In another aspect, the present invention relates to a method for analyzing a detection target material using a diagnostic chip comprising the steps of: inserting a container containing a sample containing a detection target material in a diagnostic chip into a container mounting portion, mounting a pipet tip-shaped carrier means in a hole; Injecting a sample containing the substance to be detected into a delivery means and injecting a sol-gel spot containing a biological detection substance interacting with the substance to be detected into a fixed detection section; A step of sucking the remaining material after the reaction in the detection unit and discharging the remaining material to the waste storage unit; Washing the unreacted material in the detection part by injecting the cleaning solution into the detection part in the cleaning solution storage part including the cleaning solution for removing the substance not reacting with the detection target substance; Withdrawing the unreacted material into the waste storage unit; And imaging the reaction result of the detection unit.

The concrete method of analysis is as follows.

1. Preparation for analysis

A container containing a sample containing a substance to be detected, for example, a tube is inserted into the container mounting portion 132, and a transportation means in the form of a pipette tip is mounted on the hole 131. [ The mounting cover of the analytical apparatus having completed preheating is raised and the diagnostic chip 100 is mounted. The diagnostic chip 100 is mounted on a diagnostic device, and further includes a driving part, a syringe pump, and an optical part as shown in FIG.

The driving unit automatically recognizes the diagnostic chip, moves the X-axis to the device reaction unit equipped with the constant temperature system, and operates the liquid handler Y-axis driving module and the designated position. The dynamic error rate can be precisely designed to be 0.1mm. A controlled shaking function of several hundred to several thousand rpm is added to the sol-gel reaction amplification of the diagnostic chip (100) measurement part, and finally the optical part and the structure are measured at a precise position. By channeling these structures, up to four diagnostic chips can be interlocked simultaneously.

The syringe pump is operated in conjunction with the moving X-axis of the diagnostic chip 100 and injects and discharges samples of each stage at a designated position. It is possible to add the function of automatically removing and attaching the consumable tip and to add the function of punching the storage container foil to the diagnostic chip 100 by the calculated pressure result value so as to minimize the volume by fusing the multi- have. The main function, suction-ejection of the sample, can recognize the specific position fixed by the touch sensor and adjust the exact volume amount. It is possible to control the error range of the suction and discharge to a very small amount of 1 microliter.

When the fluorescence reaction is completed in the sol-gel immobilized spot of the diagnostic chip measuring unit, the optical unit moves to the optical module unit and senses an optical image. Applying a spatial algorithm between the reaction spots from a reference spot to recognize the position of the correct spot and including an operation system in pixel units of the reaction spot. The light emitting part is composed of a high sensitivity LED and the light receiving part can be composed of a high resolution CCD module. The fluorescence wavelength filter used can be constructed and installed at 550 ~ 680nm. By optimizing the light wave guide between the measurement window of the diagnostic chip and the LED-CCD, it can be sensed with high sensitivity. To prevent distortion of the resultant value due to the risk of temperature variation of the LED light, it is possible to maintain the temperature around the measuring part as a constant temperature system.

2. Analyze progress

The process can be performed while pressing the start button of the analyzer and moving into the reaction chamber while recognizing the barcode tagged to the sample of the diagnostic chip. The sealed pouch is sealed with the cover member by punching to open the sealed reagent storage unit 121, the washing solution storage unit 122 and the waste storage unit 123.

A pipette tip is fixed to the hole 131 and the pipette tip is moved so that the sample contained in the container fixed to the container mounting part 132 is sucked and injected into the detecting part 110 so that the reaction proceeds do. The biological marker of the biological detection substance in the reaction reagent storage unit 121 is sucked into the detection unit 110, and the reaction proceeds by stirring.

After the reaction of the detection unit 110 that has completed the reaction, the remaining substance is sucked and discharged to the waste storage unit 123, and the cleaning solution for removing the substance not reacting with the detection target material contained in the cleaning solution storage unit 122 The unreacted material of the detection unit 110 is sucked and discharged to the waste storage unit 123. In some cases, the process of washing with the wash solution to remove unreacted material may be repeated three times.

The results of the reaction in the detection unit 110 are as shown in Fig. 2, the selective first-order immune reaction between the biological detection substance immobilized on the sol-gel spot of the detection unit 110 and the detection target substance in the sample progresses, and a biological A selective secondary immunoreaction with the biological marker for the detection substance can proceed.

Thereafter, the diagnostic chip 100 that has completed the reaction moves to the optical section, processes the data by calculating a standard signal (Raw Signal) value, a bar code value and a machine code value measured from the diagnostic chip 100, .

Thereafter, the diagnostic chip 100 is automatically detached from the tray, and the diagnostic chip 100 having completed the reaction is removed.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Comparison of reaction results of existing Rapid product influenza and diagnostic chip sol-gel spot sensor

The purpose of this study is to investigate the difference in analytical specificity and sensitivity performance between the preparation of sol - gel spots and the analysis of a dedicated analytical instrument. The domestic influenza panel (Zeptomatrix influenza panel: 8 viral / Influenza A positive: 7 viral / Influenza B positive: 5 vials) was used to immerse domestic Rapid products (panel immersed in sample strips for 10 to 15 minutes) The test was carried out through a diagnostic chip sol-gel spot diagnostic method according to the invention.

Diagnostic imaging results through domestic Rapid products are shown in Figures 9 and 10, respectively. On the contrary, the results obtained by the method according to the present invention are shown in Figs. 11 and 12, respectively.

Specifically, the clinical sensitivity according to Table 1 was measured according to the specificity measurement standard.

[Table 1]

Sensitivity: TP / TP + FN, Specificity: TN / FP + TN

Positive predictive value: TP / TP + FP, negative predictive value: TN / TN + FN

False positive rate: FP / FP + TN = 1-specificity, false negative rate: FN / TP + FN = 1-Sensitivity

As a result, according to Tables 2 and 3, the sensitivity and specificity of Influenza A were 100% (100%, 100%, and 100%, respectively) 2), whereas the known rapid kit is low in sensitivity: 3/7 = 43% and specificity: 13/13 = 100% (Table 3), indicating that the method according to the present invention exhibits excellent sensitivity.

[Table 2]

[Table 3]

According to Tables 4 and 5, the sensitivity to Influenza B was 100% (100%) in both the sensitivity and the specificity of 5/5 = 100% and the specificity of 15/15 = 100% In contrast, the known rapid kit has a sensitivity of 3/5 = 60% and a specificity of 13/13 = 100% (Table 5), indicating that the method according to the present invention exhibits excellent sensitivity.

[Table 4]

[Table 5]

Example  2. Influenza two-dimensional antibody surface Immobilized chip  Comparison of reaction results of 3D sol-gel antibody immobilized sensor

The performance of the two-dimensionally immobilized antibody on the surface-treated substrate and the influenza antigen assay of the antibody immobilized three-dimensionally by the sol-gel of the detection method according to the present invention was evaluated.

The three-dimensional fixation is to immobilize a biological detection substance such as a protein and an antibody three-dimensionally in a porous sol-gel matrix, thereby exposing the site or position to be reacted to the pore region so that the biochemical reaction is performed at various positions.

In order to immobilize the antibody on the surface, the antibody was surface - immobilized two - dimensionally by polyelectrolyte polymer, and immobilized with 3 - dimensional antibody by sol - gel technique to induce the reaction with influenza antigen. The signal results were analyzed for the antigen - antibody reaction.

[Table 6]

According to Table 6, FIG. 13 and FIG. 14, it can be confirmed that the antibody-antigen reaction fluorescence intensity of the present invention immobilizing the three-dimensional antibody by the sol-gel technique is 4 to 9 times as sensitive as the concentration.

Example 3 Clinical Sample Test Results Using an Influenza Sol-Gel Diagnostic Sensor

The sensitivity / specificity performance for clinical sample testing was compared using influenza sol-gel diagnostic chip. Based on the clinical samples (negative sample: 200ea / positive specimen A / B each 50ea) supplied from Korea University Guro Hospital (professor Chae Chae Seung), Influenza is detected and diagnosed within 30 minutes by using Sol-gel diagnostic chip and special equipment. Respectively. (Measured by bio-RAD CFX96 ™ Real-time PCR System using Seegene's Anyplex ™ II RV16 Detection kit)

A fluorescence image obtained by testing a sample of a negative sample is shown in Fig. 15, and Fig. 16 shows a result of analysis of fluorescence data. The detection specificity for Influenza A is shown in Table 7, and the detection specificity for Influenza B is shown in Table 8 below.

[Table 7] Detection specificity for Influenza A

[Table 8] Detection specificity for Influenza B

A fluorescence image of the positive specimen sample tested against Influenza A is shown in Fig. 17, and Fig. 18 shows the result of analysis of fluorescence data. The detection specificity for Influenza A is shown in Table 9 below. According to Table 9, the sensitivity is 48/50 = 96%.

 [Table 9] Sensitivity analysis for Influenza A

A fluorescence image of the positive specimen sample tested against Influenza B is shown in Fig. 19, and Fig. 20 shows the result of analysis of fluorescence data. In this regard, the detection specificity for Influenza B is shown in Table 10 below. According to Table 10, the sensitivity is 49/50 = 98%.

[Table 10] Sensitivity analysis for Influenza B

Example  4: Tumor markers test using sol-gel diagnostic sensor Example )

Tumor markers of hepatocellular carcinoma, lung cancer, pancreatic cancer, biliary tract cancer, and prostate cancer were prepared as protein immunodiagnostic sensors fixed in the form of one sol-gel microspot, respectively. Normal reference values and body organ involvement distribution by tumor markers are shown in Table 11.

[Table 11]

A three-dimensional sol-gel diagnostic chip for tumor marker detection is constructed as shown in FIG. The tumor marker detection method is the same as the influenza method described above, and the sol-gel spot is immobilized on a diagnostic chip so that multiple tumor markers can be detected at one time.

(CA19-9: 30 U / ml) were diluted in a normal sample to a certain concentration (Meridian's Tumor Marker protein, Lot information CA19-9: 10D11915 / AFP: 3G19214 / CEA: 5H21615 / PSA: 8B04915) 22, AFP: 10 ng / ml, CEA: 5 ng / ml, PSA: 5 ng / ml). Sol-gel diagnostic chip and sample reaction fluorescence images are shown in FIG. There is no mutual nonspecific reaction and sufficient fluorescence intensity detection is possible even at the boundary value concentration interval.

Based on this, fluorescence intensity distribution by tumor marker antigen concentration is shown in FIG. 23 to FIG. 26. It is based on the fact that the intensity of fluorescence increases linearly with increasing antigen concentration and that the fluorescence intensity can be reconstructed to the clinical concentration results of the tumor markers.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

A method for detecting a detection target material by using a single diagnostic chip including the following components integrally,
A container mounting portion containing a sample containing a substance to be detected;
A detection unit fixed with a sol-gel spot including a biological detection substance interacting with a substance to be detected;
A reaction reagent storage section containing a biological marker for the biological detection substance, for confirming whether the biological substance to be detected reacts with the biological substance to be detected;
A cleaning solution storage unit including a cleaning solution for removing a substance not reacting with a detection target material;
A waste storage unit for removing remaining or unreacted materials after the reaction in the detection unit; And
A hole equipped with a pipette tip type transporting means,
Contacting a sample containing the detection target substance contained in the container mounting section with a biological detection substance interacting with the detection target substance of the detection section, and imaging the reaction result. The method according to claim 1, wherein the substance to be detected is a biomaterial. The method of claim 1, wherein the container containing the sample is a detachable bottle, tub, tube or ampoule with a diagnostic chip. The method according to claim 1, wherein the substance to be detected is a nucleic acid, a peptide, a protein, a low molecular substance, a virus or a cell. 5. The method of claim 4, wherein the virus is an influenza virus. The method according to claim 1, wherein the biological detection substance interacting with the substance to be detected is an antigen, an antibody or an aptamer. The method of claim 1, wherein the sol-gel spots have a size of 100-1000 m. The method of claim 1, wherein the sol-gel spot comprises at least one biological detection material. 2. The method of claim 1, wherein the detector is arranged in a microarray configuration such that at least one sol-gel spot in one direction has at least one row. The method of claim 1, wherein the biological marker is an antibody or aptamer labeled with a radioactive isotope, fluorescent dye, dye, protein, or luminescent substance capable of detecting a biological detection substance. The method according to claim 1, wherein the waste storage part is a pad that absorbs residual material or unreacted material after reaction in the detection part. The method of claim 1, wherein the reaction reagent storage portion, the washing solution storage portion, and the waste storage portion are sealed by sealing the cover member. The method according to claim 1,
Reacting a sample containing the substance to be detected with a primary biological detection substance interacting with the substance to be detected; And
Reacting a secondary biological detection substance that binds to the primary biological detection substance that is interacted with the detection target substance. [2] The method according to claim 1, wherein the single diagnostic chip is integrally formed of plastic, which is made of polystyrene or polymethyl methacrylate A method for analyzing a detection target substance using a diagnostic chip comprising the steps of:
Inserting a container containing a sample containing a substance to be detected in a diagnostic chip into a container mounting portion and mounting a transportation means in the form of a pipette tip in the hole;
Injecting a sample containing the substance to be detected into a delivery means and injecting a sol-gel spot containing a biological detection substance interacting with the substance to be detected into a fixed detection section;
A step of sucking the remaining material after the reaction in the detection unit and discharging the remaining material to the waste storage unit;
Washing the unreacted material in the detection part by injecting the cleaning solution into the detection part in the cleaning solution storage part including the cleaning solution for removing the substance not reacting with the detection target substance;
Withdrawing the unreacted material into the waste storage unit; And
And imaging the reaction result of the detection unit.

Images