KR102236276B1 - A Method of Rapid Diagnosis With High Sensitivity By Using Single Diagnosis Chip Comprising Reaction and Analysis Process - Google Patents

Abstract

The present invention is a method of detecting a detection target material using a diagnostic chip including reaction and analysis, specifically, a diagnosis including a biological detection material that interacts with the detection target material and a detection unit having a fixed sol-gel spot The present invention relates to a chip and a method of detecting a detection target substance or a method of analyzing a sample based on the chip.

Description

{A Method of Rapid Diagnosis With High Sensitivity By Using Single Diagnosis Chip Comprising Reaction and Analysis Process}

The present invention is a method of detecting a substance to be detected using a diagnostic chip including reaction and analysis, specifically, a diagnosis including a biological detection substance that interacts with the substance to be detected and a detection unit having a fixed sol-gel spot The present invention relates to a chip and a method of detecting a detection target substance or a method of analyzing a sample based on the chip.

In the clinical examination field, the field of point, care, and testing (hereinafter, also referred to as POCT) is attracting attention. By separating the substance to be detected from the sample and analyzing the POCT through a simple device, there is an advantage in that the time from collecting the sample to obtaining the test result can be shortened and measurement can be performed simply.

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

Previously, in vitro diagnostic forms using biochemical methods require expensive analysis equipment and have been developed as reagents mainly for large hospitals and laboratories. Accordingly, the consumption of diagnostic drugs is concentrated in some of the general hospitals and laboratories, and because it must be combined with expensive analysis equipment, it acts as a risk factor for domestic companies, resulting in a market that is mostly dependent on imports. Since the 1980s, the concept of point??of??care??testing (POCT), a point-of-care diagnostic kit, was born, and these spot-type diagnostic kits have since been used for pregnancy diagnosis, substance abuse diagnosis, infectious disease diagnosis, cancer diagnosis, heart disease, etc. With wide application, it has led the growth of the diagnostic market since 1990. The global population using in vitro diagnostic therapy is on the rise, and more people are showing interest in in vitro diagnostic testing due to the in vitro diagnostic test cost that becomes cheaper with the introduction of POCT. The market size of immunochemical diagnostics among in vitro diagnostics is expected to grow from $14 billion in 2009 to an average annual growth rate of 5.4%, forming sales of more than $20 billion in 2016.

On the other hand, influenza is an acute respiratory viral infection that is prevalent in winter, affecting about 10% of the population every year, and is often classified as a serious disease due to the complication of pneumonia or exacerbation of the underlying disease in the elderly, infants, and patients with chronic diseases. Seasonal influenza, which is popular every year, is caused by mutation of the antigen of the influenza virus, whereas pandemic influenza, which occurs every 10 to 40 years, is caused by antigen-versus mutation, and it is characterized by a global epidemic and infects 30-50% of the population. The socio-economic damage is enormous.

The rapid antigen test based on antigen-antibody reaction, which is most commonly used for detecting influenza viruses, takes relatively little time and has the advantage of being convenient, but has a disadvantage of low sensitivity and accuracy. The real-time PCR method used for confirmatory testing of influenza is recognized as the most effective technology for diagnosing infectious diseases through its high sensitivity and accuracy. It is impossible. In addition, due to the high cost of testing, it is very difficult to apply it as a large-scale initial countermeasure in case of a pandemic of infectious diseases.

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

Under this technical background, the inventors of the present application proposed a high-sensitivity technology based on a three-dimensional immobilized sol-gel. This technology is a technology that can immobilize a variety of biomarkers in large quantities based on a porous three-dimensional sol-gel structure.It can build a high-sensitivity detection system compared to existing biosensors, and provides a miniaturized diagnostic chip-based technology by fusion of microarray manufacturing technology. It was possible to overcome the low sensitivity/specificity performance of the existing Rapid kit and the limitation of detection time of molecular diagnosis with the development of 3D sol-gel bio-fusion technology. In addition, the sol-gel microarray fabrication technology in one reaction part enables the implementation of multiplex technology by independently fixing multiple markers in the same space, thereby building a platform foundation for multiple diagnostic technology.

Therefore, the sol-gel-based high sensitivity/high specificity through the diagnostic chip for sample analysis in which the sol-gel spot containing the biological detection material interacting with the detection target material is fixed, not the conventional well or plate-based. Analytical performance and multi-diagnosis platform technology and on-site diagnosis-based rapid and accurate results can be obtained, thereby improving the accuracy of analysis and including an integrated reagent storage unit and waste storage unit, It was confirmed that the problem caused by misuse can be solved, and the present invention was completed.

In order to solve the above problems, the present invention provides a method of detecting a substance to be detected 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 that includes the following configuration integrally, comprising: a container mounting part containing a sample containing the detection target material; A detection unit in which a sol-gel spot including a biological detection material interacting with the detection target material is fixed; A reaction reagent storage unit including a biological marker for the biological detection substance to check whether the detection target substance and the biological detection substance react; A washing solution storage unit including a washing solution for removing a substance that has not reacted with the substance to be detected; A waste storage unit for removing residual substances or unreacted substances after the reaction in the detection unit; And a hole in which a pipet tip-type transport means is mounted, and a sample including a detection target material included in the container mounting part is brought into contact with a biological detection material that interacts with the detection target material of the detection unit, and a reaction result is obtained. It provides a method comprising imaging.

The present invention also provides a method for analyzing a substance to be detected using a diagnostic chip comprising the following steps: Inserting a container containing a sample containing the substance to be detected among the diagnostic chips into the container mounting portion, and inserting a pipette tip mounting a tip) type of conveying means in the hole; Inhaling the sample containing the detection target substance by means of a transport means, and injecting the sol-gel spot containing the biological detection substance interacting with the detection target substance into a fixed detection unit; Suctioning the residual material after the reaction in the detection unit and discharging it to a waste storage unit; Washing unreacted substances in the detection unit by injecting a cleaning solution into the detection unit in a cleaning solution storage unit including a cleaning solution for removing a substance that has not reacted with the detection target substance; Suctioning the unreacted material and discharging it to a waste storage unit; And imaging the reaction result of the detection unit.

According to the present invention, based on the sol-gel-based technology presented as a conventional high sensitivity/high specificity multi-diagnosis platform, the analysis process is integrated into a sol-gel immobilization detection unit, a reagent storage unit, and a straight line including a waste storage unit. By using the diagnostic chip for on-site diagnosis together with the test equipment, it includes higher analytical performance compared to the existing ones, the ability to shorten the diagnosis time according to the analysis process with only a quick and simple operation, and a structural function that enables quantitative storage of samples, reagents and/or reactants. , By being able to prevent cross-contamination between them, analysis accuracy can be improved.

1 shows a front view of a diagnostic chip.
2 is a detailed diagram of a detection unit among diagnostic chips and a schematic diagram of a reaction for a three-dimensional sol-gel spot
3 is a detailed diagram of a reaction reagent storage unit among diagnostic chips.
4 is a detailed view of a cleaning solution storage unit among diagnostic chips.
5 is a detailed view of a waste storage unit among diagnostic chips.
6 is a detailed view of a hole in which a transportation means is mounted among diagnostic chips.
7 is a detailed view of a container mounting part among diagnostic chips.
8 is a schematic diagram of a diagnostic device used to implement a method according to the present invention.
9 and 10 show diagnostic imaging results through a conventional domestic Rapid product (S company).
11 and 12 show diagnosis results by the method according to the present invention.
13 and 14 show fluorescence intensity and fluorescence image results for a three-dimensional Sol-gel antibody-immobilization assay in which Sol-gel is three-dimensionally fixed with an antibody.
15 is a result showing a fluorescence image of a negative specimen test.
16 shows the results of analyzing fluorescence data for testing a negative specimen sample.
17 is a result showing a fluorescence image of a positive specimen sample tested against Influenza A.
18 shows the results of analyzing fluorescence data of testing positive specimen samples for Influenza A.
19 shows a fluorescence image of a positive specimen sample tested against Influenza B.
FIG. 20 shows the results of analyzing fluorescence data of a positive specimen sample tested for Influenza B.
21 is a block diagram of a three-dimensional sol-gel antibody immobilization chip for detecting tumor markers.
22 shows a fluorescence image of a tumor marker antigen sample response.
23 to 26 show the distribution of fluorescence intensity for each tumor marker antigen concentration.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In one aspect, the present invention provides a method for detecting a detection target material using a diagnostic chip integrally including the following configuration, comprising: a container mounting part containing a sample containing the detection target material; A detection unit in which a sol-gel spot including a biological detection material interacting with the detection target material is fixed; A reaction reagent storage unit including a biological marker for the biological detection substance to check whether the detection target substance and the biological detection substance react; A washing solution storage unit including a washing solution for removing a substance that has not reacted with the substance to be detected; A waste storage unit for removing residual substances or unreacted substances after the reaction in the detection unit; And a hole in which a pipet tip-type transport means is mounted, and a sample including a detection target material included in the container mounting part is brought into contact with a biological detection material that interacts with the detection target material of the detection unit, and a reaction result is obtained. It relates to a method comprising imaging.

The method according to the present invention is composed of an optimal rapid reaction step (overcoming the quantitative error error of reagent and reaction step, optimization of the reaction of the measuring unit, design for speeding up the linkage of equipment, etc.), and can implement a sensitivity of 90% or more compared to the existing one, and is used in the present invention. By dividing the reagent storage unit and the waste storage unit according to each reaction step, the diagnostic chip can solve problems caused by contamination and misuse of samples and reagents used.

In one embodiment, the detection target material may be a biological material, for example, a nucleic acid, a peptide, a protein, a small molecule material, an antigen, a virus, or a cell. The biological material may be derived from body fluids. The body fluids are, for example, feces, urine, tears, saliva, external secretions of the skin, external secretions of the respiratory tract, external secretions of the intestine, external secretions of the digestive tract, plasma, serum, blood, spinal fluid, lymph fluid, body fluids, tissues, tissues. It may be a homogenate, a portion of a tissue, a cell, a cell extract, or an in vitro cell culture.

The virus may be, for example, an influenza virus. According to an embodiment of the present invention, antibodies specific for influenza subtypes A and B are immobilized on a dedicated diagnostic chip by sol-gel immobilization technology, and specific diagnostic possibilities can be confirmed in a sample for each subtype. Since the complex process part of the existing test method can be shortened to an automated measurement process from sample collection to result calculation using a dedicated diagnostic chip in the form of Lab on a chip, the presence of influenza virus can be easily measured. . In addition, there is an advantage in that it is easy to store and can be applied to a field inspection (POCT) system with a miniaturized diagnostic chip configuration.

The container containing the sample may be inserted from the outside in a form that is detachable from 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 a container, it is possible to store or process the sample freely from the spatial and/or temporal constraints caused by storing the sample integrally with other reagents.

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

The biological detection substance interacting with the detection target substance may be, for example, a protein, or a nucleic acid and/or an oligonucleotide. The protein may be, for example, an antibody or fragment thereof that specifically binds to a substance to be detected. In this case, detection of the substance to be detected may be performed by an immunoassay method based on an antigen-antibody reaction.

The immunoassay can be performed according to various quantitative or qualitative immunoassay protocols. The immunoassay format is radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbant assay), capture-ELISA, sandwich analysis, flow cytometry, immunofluorescence staining and immunohistochemistry. Including, but not limited to, chemical tablets.

In one embodiment, the step of reacting a sample containing the detection target material and a primary biological detection material that interacts with the detection target material; And reacting a second biological detection substance that binds to the first biological detection substance interacting with the detection target substance. When the detection substance interacting with the detection target substance is an antibody, the primary biological detection substance can specifically bind to the detection target substance, and an antibody that specifically binds to the antibody that specifically binds to the detection target substance The reaction can be carried out with a secondary biological detection material.

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

In this case, the biological marker for the biological detection material may be, for example, an antibody or aptamer labeled with a radioactive isotope, a fluorescent dye, a dye, a protein, or a luminescent material, but is not limited thereto. The reaction result imaging included in the method of the present invention may be performed by measuring and analyzing fluorescence, radiation dose, and luminescence intensity, etc., which are expressed by biological markers.

The sol-gel spot is for fixing a biological detection material that interacts with the detection target material, and may be fixed while the sol solution is gelled, and the spot may not fall off when reacting with a sample containing the detection target material.

The size of the sol-gel spot may be, for example, 100-1000 μm, specifically 200-400 μm, and more specifically 250-350 μm. 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, and 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 the form of a micro array such that one or more sol-gel spots in one direction from the detection unit have at least one row. Through this, since various biological detection substances that interact with various detection target substances can be fixed, it is possible to diagnose multiple diseases at once.

Hereinafter, it will be described in detail through the drawings of the diagnostic chip according to the present invention. The same components have the same reference numerals as possible even though they are indicated on different drawings.

Referring to FIG. 1, the 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 transportation means storage unit 130. The 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 transportation means storage unit 130, including a width (91.0 mm) X length (25 mm) X It can have a size of height (12.5mm).

The sample and transport means storage unit 130 includes a hole 131 in which a transport means in the form of a pipet tip is mounted and a container mounting unit 132 containing a sample containing a substance to be detected. The container fixed to the container mounting part 132 may be, for example, a bottle, a tub, a tube, or an ampoule detachable from a diagnostic chip. Individual pipette tips and containers are included for each diagnostic chip, and tags, for example, barcodes or QR codes, may be displayed on samples to distinguish a plurality of samples.

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

In the detection unit 110, a sol-gel spot including a detection target material, for example, a nucleic acid, a peptide, a protein, a small molecule material, a virus or a biological detection material that interacts with cells, for example, an antigen, an antibody, or an aptamer, is fixed. In addition, the sol-gel spot may have a size of, for example, 100-1000 μm , specifically 200-400 μm, and more specifically 250-350 μm .

In the detection unit 110, one or more sol-gel spots are arranged in one direction, and may be arranged in a micro-array shape to have at least one row. Through such a sol-gel spot, various biological detection substances, for example, one or more or two or more biological detection substances interacting with various detection target substances can be fixed, so that multiple diseases can be diagnosed at once.

The reagent and/or waste storage unit 120 includes a reaction reagent storage unit 121 including a biological marker for the biological detection substance and a reaction reagent storage unit 121 that does not react with the detection target substance in order to check whether the detection target substance and the biological detection substance react. It includes a washing solution storage unit 122 including a washing solution for removing substances and a waste storage unit 123 for removing residual substances or unreacted substances after reaction in the detection unit. In each of FIGS. 3 to 5, a reaction reagent storage unit 121, a washing solution storage unit 122, and a waste storage unit 123 are shown in detail.

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

A solution for washing a substance that has not reacted with a substance to be detected included in the washing solution storage unit 122 may be, for example, a solution containing a small amount of a surfactant, an anti-protein agent, or an ionizing salt substance. The waste storage unit 123 for removing residual or unreacted substances after the reaction by the detection unit may be, for example, in the form of a pad for absorbing residual substances or unreacted substances after the reaction by the detection unit. The waste storage unit 123 is not particularly limited as long as it is a porous material capable of absorbing residual material or unreacted material after the reaction, but may be, for example, a sponge, nonwoven fabric, cloth, or cotton. By treating the waste in the waste storage unit 123, it is possible to solve the problem that the accuracy of the test result is deteriorated due to contamination caused by mixing of the residual material after the reaction or the unreacted material with the reagent.

In some cases, a sticker is 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 a cover member.

The single diagnostic chip 100 included as an integral type according to the present invention is PVdF (polyvinylidene fluoride), nylon, nitrocellulose, PU (polyurethane), PC (polycarbonate), PS (polystyrene), PLA (polylatic). acid), PAN (polyacrylonitrile), PLGA, (polylactic-co-glycolic acid), PEI (polyethyleneimine), PPI (polypropyleneimine), PMMA (polymethyl methacrylate), PVC (polyvinylcholride), PVAc (polyvinylacetate), and polystyrene divinylbenzene A plastic injected with one or more types selected from the group consisting of a copolymer (polystylene divinylbenzene copolymer) may be included as a material, and specifically, a plastic injected with polystyrene and/or polymethyl methacrylate may be used. It can be included as a material.

In another aspect, the present invention relates to a method for analyzing a substance to be detected using a diagnostic chip comprising the following steps: a container containing a sample containing the substance to be detected among the diagnostic chips is inserted into the container mounting portion, and a pipette tip mounting a conveying means in the form of a (pipet tip) in the hole; Inhaling the sample containing the detection target substance by means of a transport means, and injecting the sol-gel spot containing the biological detection substance interacting with the detection target substance into a fixed detection unit; Suctioning the residual material after the reaction in the detection unit and discharging it to a waste storage unit; Washing unreacted substances in the detection unit by injecting a cleaning solution into the detection unit in a cleaning solution storage unit including a cleaning solution for removing a substance that has not reacted with the detection target substance; Suctioning the unreacted material and discharging it to a waste storage unit; And imaging the reaction result of the detection unit.

The detailed analysis method 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 part 132, and a conveying means in the form of a pipet tip is mounted in the hole 131. Raise the mounting cover of the analysis device that has been preheated and mount the diagnostic chip 100. The diagnostic chip 100 is mounted on a diagnostic device, and further includes a driving part, a syringe pump, and an optical portion, as shown in FIG. 8.

The drive unit automatically recognizes the diagnostic chip, moves the X-axis to the device reaction unit equipped with a constant temperature system, and has a liquid handler Y-axis drive module and a system that operates at a designated position. The error rate for copper can be precisely designed as 0.1mm. In order to amplify the sol-gel reaction of the measurement unit of the diagnostic chip 100, a controlled shaking function of hundreds to thousands of rpm is added, and it is finally composed of an optical unit and a structure that is measured at a precise position. By channelizing this structure, up to 4 diagnostic chips can be linked at the same time.

The syringe pump is operated in conjunction with the moving X-axis of the diagnostic chip 100 and injects-discharges samples of each step at a designated position. By adding the function of automatically attaching and detaching consumable tips and punching the storage container foil on the diagnostic chip 100 by the calculated pressure result, the volume can be minimized by fusing the functions of the operating equipment. have. The main function, the suction-discharge of the sample, can control the exact volume by recognizing a specific position fixed by the touch sensor. It can be possible to control the margin of error of the suction and discharge to a very small amount of 1 microliter.

When the fluorescence reaction is completed at the sol-gel immobilization spot of the diagnostic chip measurement unit, the optical unit moves to the optical module unit and senses the optical image. In order to recognize the exact location of the spot, a spatial algorithm between reaction spots from a reference spot is applied, and a calculation system in units of pixels of the reaction spot may be included. The light-emitting part is composed of a high-sensitivity LED, and the light-receiving part may be composed of a high-resolution CCD module. The fluorescent wavelength filter used may be configured and mounted at 550 to 680 nm. By optimizing the light wave guide between the measurement window of the diagnostic chip and the LED-CCD, high-sensitivity sensing is possible. In order to prevent distortion of the result value due to the risk of temperature variation of the LED light, a constant temperature system can be used around the measurement unit.

2. Analysis progress

The step may proceed while pressing the start button of the analysis device and moving into the reaction chamber while recognizing the barcode tagged on the sample of the diagnostic chip. The pouch sealed and sealed with a cover member by punching is opened so that the reaction reagent storage unit 121, the washing solution storage unit 122, and the waste storage unit 123 are opened.

A pipet tip is fixed in the hole 131, the pipet tip is moved to suck the sample contained in the container fixed to the container mounting part 132, inject it into the detection unit 110, and stir to proceed with the reaction. do. The biological marker for the biological detection material of the reaction reagent storage unit 121 is sucked, injected into the detection unit 110, and stirred to allow the reaction to proceed.

After the reaction of the detection unit 110 in which the reaction is completed, the residual material is sucked and discharged to the waste storage unit 123, and is used as a cleaning solution for removing substances that have not reacted 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 a washing solution to remove unreacted substances may be repeated three times.

The reaction result in the detection unit 110 is as shown in FIG. 2. According to FIG. 2, a selective primary immune reaction between the biological detection material fixed to the sol-gel spot of the detection unit 110 and the detection target material among the samples proceeds, and to check whether the detection target material and the biological detection material react. A biological marker and a selective secondary immune response to the detection substance can proceed.

Thereafter, the diagnostic chip 100, which has completed the reaction, is moved to the optical unit, and processes the data by calculating the standard signal value, the barcode value measured from the diagnostic chip 100, and the device code value, and the data is displayed on the display. Is sent out.

Thereafter, the diagnostic chip 100 is automatically separated from the tray, and the diagnostic chip 100 on which the reaction is completed is removed.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Comparison of the reaction results of the conventional rapid product of influenza and the diagnostic chip sol-gel spot sensor

The purpose of this study is to investigate the results of the difference in analytical specificity and sensitivity performance by making a sol-gel spot sensor and assaying a dedicated analysis device compared to the existing product Rapid. International influenza panel Zeptomatrix influenza panel (negative: 8 / Influenza A positive: 7 / Influenza B positive: 5) using domestic rapid products (submerged the panel in the sample injection part of the strip for 10 to 15 minutes) and this The test was conducted through the diagnostic chip sol-gel spot diagnosis method according to the present invention.

The diagnostic imaging results through domestic Rapid products are shown in Figs. 9 and 10, respectively. On the other hand, the results of the method according to the present invention are shown in Figs. 11 and 12, respectively.

Specifically, it was measured according to the criteria for measuring clinical sensitivity and specificity according to Table 1.

[Table 1]

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

Positive predictive degree: TP/TP+FP, negative predictive degree: 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 to Influenza A was 7/7 = 100%, specificity: 13/13 = 100%, and both sensitivity and specificity were 100% (Table 2) On the other hand, the known Rapid kit has low sensitivity: 3/7 = 43%, specificity: 13/13 = 100% (Table 3), and it can be seen 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 is 5/5 = 100%, specificity: 15/15 = 100% in the method according to the present invention, and both sensitivity and specificity are 100% (Table 4). On the other hand, the known Rapid kit is low in sensitivity: 3/5 = 60%, specificity: 13/13 = 100% (Table 5), and it can be seen that the method according to the present invention exhibits excellent sensitivity.

[Table 4]

[Table 5]

Example 2. Influenza two-dimensional antibody surface With immobilized chips Comparison of reaction results of 3D sol-gel antibody-immobilized diagnostic sensors

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

The three-dimensional fixation is to three-dimensionally fix a biological detection substance such as a protein and an antibody on a porous sol-gel matrix, thereby exposing a site or location to be reacted to a void portion so that a biochemical reaction occurs at various locations.

In order to immobilize the antibody on the surface, a surface treatment (polyelectrolyte polymer) was performed to immobilize the antibody in two dimensions, and a three-dimensional antibody immobilization method was performed using a sol-gel technique to induce a reaction with an influenza antigen. The signal result of the antigen-antibody reaction was analyzed.

[Table 6]

According to Table 6, FIGS. 13 and 14, it can be seen that the sensitivity of the antibody-antigen reaction fluorescence intensity is 4 to 9 times for each concentration with the present invention in which the three-dimensional antibody is immobilized using the sol-gel technique.

Example 3: Clinical sample test results using influenza sol-gel diagnostic sensor

The sensitivity/specificity performance for the clinical sample test was compared using an influenza sol-gel diagnostic chip. Based on clinical samples (negative samples: 200ea/positive samples A/B each 50ea) supplied from Korea University Guro Hospital (Professor Chae-Seung Lim), influenza was detected and diagnosed within 30 minutes using the influenza Sol-gel diagnosis chip and dedicated equipment. I did. (Reference equipment for disease diagnosis at Korea University Guro Hospital: Measured by Bio-RAD's CFX96™ Real-time PCR System using Seegene's Anyplex™ II RV16 Detection kit)

Fig. 15 shows a fluorescence image of the negative specimen sample, and Fig. 16 shows the result of analyzing the fluorescence data. In this regard, the detection specificity for Influenza A is shown in Table 7 below, 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 a positive specimen sample tested against Influenza A is shown in FIG. 17, and FIG. 18 shows the results of analyzing fluorescence data. In this regard, 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 a positive specimen sample tested against Influenza B is shown in FIG. 19, and FIG. 20 shows a result of analyzing 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 marker test using a sol-gel diagnostic sensor (enlarged application Example )

The tumor markers of liver cancer, lung cancer, pancreatic cancer, biliary tract cancer, and prostate cancer were each fixed in the form of a single sol-gel micro-spot, and were fabricated with a protein immunodiagnostic sensor. Table 11 shows the normal reference values and distribution of body organ involvement by tumor marker.

[Table 11]

The three-dimensional sol-gel diagnostic chip for detecting tumor markers is configured as shown in FIG. 21. The tumor marker detection method was performed in the same manner as the influenza test method, and a sol-gel spot is immobilized on one diagnostic chip so that multiple tumor markers can be detected at once.

Commercially available tumor marker material (Meridian's Tumor Marker protein, Lot information CA19-9: 10D11915/ AFP: 3G19214/ CEA: 5H21615/ PSA: 8B04915) and diluted to a certain concentration in a normal sample (CA19-9: 30U/ ml, AFP: 10 ng/ml, CEA: 5 ng/ml, PSA: 5 ng/ml) sol-gel diagnostic chip and sample reaction fluorescence images are as shown in FIG. 22. There is no mutually non-specific reaction, and sufficient fluorescence intensity can be detected even in the threshold concentration range.

Based on this, the distribution of fluorescence intensity for each tumor marker antigen concentration is as shown in FIGS. 23 to 26. As the antigen concentration increases, it is possible to confirm a linear increase in fluorescence intensity, and it has the basis for reconstructing the corresponding fluorescence intensity into the clinical concentration result value of the tumor marker.

As described above, a specific part of the content of the present invention has been described in detail. For those of ordinary skill in the art, this specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereby. It will be obvious. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (15)

A container mounting part containing a sample containing a substance to be detected;
A detection unit in which the biological detection material interacting with the detection target material is three-dimensionally fixed to the sol-gel spot;
A reaction reagent storage unit including a biological marker for the biological detection substance to check whether the detection target substance and the biological detection substance react;
A washing solution storage unit including a washing solution for removing a substance that has not reacted with the substance to be detected;
A waste storage unit for removing residual substances or unreacted substances after the reaction in the detection unit; And
A method of detecting a substance to be detected using a single diagnostic chip integrally included with a hole equipped with a pipet tip type transport means,
And contacting a sample containing a detection target material included in the container mounting part with a biological detection material that interacts with the detection target material of the detection part, and imaging a reaction result. The method of claim 1, wherein the detection target material is a biological material. The method of claim 1, wherein the container containing the sample is a bottle, tub, tube, or ampoule that can be separated from the diagnostic chip. The method of claim 1, wherein the detection target substance is a nucleic acid, a peptide, a protein, a small molecule substance, a virus, or a cell. The method of claim 4, wherein the virus is an influenza virus. The method of claim 1, wherein the biological detection substance interacting with the detection target substance is an antigen, an antibody, or an aptamer. The method of claim 1, wherein the sol-gel spot has a size of 100-1000 μm. The method of claim 1, wherein the sol-gel spot comprises one or more biological detection substances. The method of claim 1, wherein the detection unit is arranged in the form of a micro array such that one or more sol-gel spots have at least one row in one direction. The method of claim 1, wherein the biological marker is an antibody or aptamer labeled with a radioactive isotope, a fluorescent dye, a dye, a protein, or a luminescent material capable of detecting a biological detection material. The method of claim 1, wherein the waste storage unit is a pad for absorbing residual substances or unreacted substances after reaction in the detection unit. The method of claim 1, wherein the reaction reagent storage unit, the washing solution storage unit, and the waste storage unit are sealed by sealing a cover member. The method of claim 1,
Reacting a sample containing the detection target material with a primary biological detection material that interacts with the detection target material; And
And reacting a second biological detection substance that binds to the first biological detection substance interacting with the detection target substance. The method of claim 1, wherein the single diagnostic chip included as an integral type is made of plastic injected with polystyrene or polymethyl methacrylate. Inserting a container containing a sample containing a substance to be detected among the diagnostic chips into the container mounting portion, and mounting a pipet tip-type transport means in the hole;
Inhaling a sample containing the detection target substance by means of a transport means, and injecting a biological detection substance interacting with the detection target substance into a detection unit including a three-dimensionally fixed sol-gel spot;
Suctioning the residual material after the reaction in the detection unit and discharging it to a waste storage unit;
Washing unreacted substances in the detection unit by injecting a cleaning solution into the detection unit in a cleaning solution storage unit including a cleaning solution for removing a substance that has not reacted with the detection target substance;
Suctioning the unreacted material and discharging it to a waste storage unit; And imaging the reaction result of the detection unit,
The diagnostic chip includes a container mounting part, a detection part, and a storage part integrally. A method of analyzing a substance to be detected using a diagnostic chip.

Images