High-density line-type biochip for multiplex immunoassay

Abstract

The present invention relates to: a biochip which enables simultaneously measuring substances in a biological sample for a plurality of markers; and use thereof. A high-density line-type biochip of the present invention, for measuring the concentration of substances present in a biological sample by means of a multiplex immunoassay, improves the weakness in existing biochips of the difficulty when inserting multiple markers on limited reaction strips, and has the advantage of enabling the analysis of 100 or more substances at once by having a plurality of line-type strips cut narrower than conventional strips and arranged in parallel. In addition, the present invention may provide higher accuracy than existing biochips by using a standard line and a calibration line, included in the strips, when performing the immunoassay.

Description

Highly integrated linear biochip for multi-immunoassay

[0001] This application claims priority to Korean Patent Application No. 10-2024-0198342 filed on December 27, 2024, and the entire specification is a reference to this application.

[0002]

[0003] The present invention relates to a biochip capable of simultaneously measuring substances in a biological sample for a plurality of markers and to uses thereof.

[0004]

[0005] The present invention was completed by Project No. RS-2023-00218581 (1425175810, 2420000660) with the support of the Ministry of SMEs and Startups of the Republic of Korea.

[0006]

[0007] In the field of in-vitro diagnostics, biochips are a key tool for analyzing biochemical reactions by integrating biomolecules (proteins, DNA, etc.) at high density on a solid substrate. In particular, in the diagnosis of allergic diseases, biochip technology is effectively used to confirm the presence of specific immunoglobulin antibodies that react to specific causative substances (allergens) using only a small amount of serum.

[0008] Line immunoassay, a type of biochip technology, is a representative method for the Multiple Allergen Simultaneous Test (MAST). It involves immobilizing dozens of purified natural or recombinant allergens in the form of lines at regular intervals on a support such as a nitrocellulose membrane. When a patient's serum is reacted with this, antibodies in the serum bind to the corresponding allergen lines. Subsequently, labeled secondary antibodies, enzymes, and substrates are applied to induce a color reaction. The presence and degree of sensitization to each allergen can be determined through the intensity of this color development.

[0009] Conventional line-type multi-diagnostic products utilize this principle to simultaneously provide information on dozens of antigens, such as major inhaled allergens (e.g., house dust mites, pollen, mold) and dietary allergens (e.g., milk, eggs, nuts), in a single test, thereby offering the advantage of reducing time and cost in identifying the cause of allergies.

[0010] However, due to changes in modern living environments, fluctuations in vegetation distribution caused by climate change, and the introduction of new foods, the types of potential allergens have become much more complex and diverse compared to the past. Consequently, diseases caused by rare allergens that were not previously included, or allergens limited to specific regions or dietary cultures, are on the rise, and the number of patients with allergies of unknown origin is also on the rise.

[0011] In addition, as the need to consider cross-reactivity between antigens with similar protein structures in order to identify the exact causative antigen increases, there is also a growing need to individually test various types of antigen components derived from a single allergen source.

[0012] Under these circumstances, existing panels that test dozens of antigens have limitations in clearly identifying the cause in patients with complex allergic sensitization, and there is a persistent possibility of missing important causative substances during the diagnostic process.

[0013] Therefore, there is an urgent need in clinical settings for the development of an expanded multi-diagnostic product capable of simultaneously testing hundreds of antigens and antigen components at high density, which is more than existing products, so that comprehensive profiling and accurate identification of causes can be achieved with a single test.

[0014]

[0015] [Prior Art Literature]

[0016] [Patent Literature]

[0017] (Patent Document 1) KR 10-2018-0164818 (2018-12-19)

[0018]

[0019] The inventors have made diligent efforts to provide a biochip capable of simultaneously measuring substances in a biological sample for multiple markers, and have completed the present invention by confirming that immunoassay can be performed with high accuracy when using multiple strips containing more than 100 markers, as well as standard lines and correction lines.

[0020]

[0021] The present invention provides a highly integrated linear biochip for multiple immunoassay comprising: a plurality of linear strips; and a reaction vessel for receiving the linear strips.

[0022] According to a preferred embodiment of the present invention, the plurality of line-shaped strips are included in four or more.

[0023] According to a preferred embodiment of the present invention, the plurality of line-shaped strips comprises strips including a standard line and a correction line.

[0024] According to a preferred embodiment of the present invention, the correction lines are included in 1 to 10.

[0025] According to a preferred embodiment of the present invention, the strip includes 25 or more markers per strip.

[0026] According to a preferred embodiment of the present invention, the strip is received in the reaction vessel and arranged so that the marker is exposed to the outside.

[0027] According to a preferred embodiment of the present invention, the biochip comprises 100 or more markers.

[0028] In addition, the present invention provides a multi-immunoassay method comprising the step of processing a sample separated from an individual on the highly integrated linear biochip for multi-immunoassay.

[0029] According to a preferred embodiment of the present invention, the sample is one or more selected from the group consisting of blood, whole blood, serum, plasma, cerebrospinal fluid, urine, feces, saliva, tears, nasal discharge, and sweat.

[0030] According to a preferred embodiment of the present invention, the sample is a sample containing immunoglobulin.

[0031]

[0032] The present invention will be described in more detail below.

[0033]

[0034] The 'line-type strip' of the present invention may be a strip manufactured by attaching a marker in the form of a line on a membrane.

[0035] The 'multiplexed immunoassay' of the present invention may be an immunochemical analysis method that specifically detects or quantifies two or more targets (antigens or antibodies) simultaneously in a single sample and a single reaction. While utilizing antigen-antibody binding, each target can be measured in parallel by distinguishing it into physically separated locations, carriers assigned with unique identification information, and unique physicochemical signal measurements.

[0036] The 'marker' of the present invention may refer to a biomarker (protein, antigen, antibody, DNA, RNA, PNA, drug, chemical, or aptamer, etc.) that specifically binds to a specific substance to be analyzed. For example, in the case of allergy diagnosis, the marker may be various types of allergen antigens, and in the case of hormone or cancer marker diagnosis, it may be an antibody against a specific substance.

[0037]

[0038] The present invention relates to a highly integrated linear biochip that qualitatively or quantitatively measures the concentration of a specific analyte present in a biological sample using the principle of immunoassay, and has the main purpose of minimizing deviations that may occur when testing multiple samples or multiple items simultaneously and significantly improving the accuracy and reproducibility of the analysis.

[0039] Specifically, the highly integrated linear biochip for multiple immunoassay of the present invention comprises a plurality of linear strips and a reaction vessel that accommodates the linear strips. The reaction vessel (100) has a structure that is open at the top, and a reaction vessel space (130) is formed inside by a reaction vessel side wall (110) that is formed to be inclined outward from the bottom to the top and a reaction vessel base (120) that supports the strips from the bottom. A strip (200) that performs an analysis reaction is accommodated within the reaction vessel space (130). When a light source is irradiated from the outside, the reaction vessel side wall (110) has an inverted trapezoidal slope structure rather than a straight line, so that shadows of the side wall may not be formed on the strip (200). Therefore, the reaction vessel according to the present invention has the effect of enabling the display pattern of the strip (200) to be clearly read when irradiated with external light, thereby allowing for the verification of highly reliable reaction results.

[0040]

[0041] Accordingly, the present invention can provide a highly integrated linear biochip for multiple immunoassay comprising: a plurality of linear strips; and a reaction vessel for receiving the linear strips.

[0042] The above plurality of line-shaped strips may have a structure arranged on a single plate or cassette. By integrating a plurality of strips, the present invention can maximize throughput by simultaneously reacting strips with different markers on a single sample in a single experimental process.

[0043] The above-mentioned line-shaped strip may include a marker fixed to a support. The support may be a porous membrane used in conventional immunoassays, such as nitrocellulose, acetylcellulose acetate, mixed cellulose esters, nylon, PVDF (Polyvinylidene fluoride), PTFE (Polytetrafluoroethylene), glass fiber membrane, ceramic membrane, polyester, or polycarbonate.

[0044] The horizontal length of the above-mentioned line-shaped strip may be 1.3 to 1.5 mm, and preferably 1.35 to 1.45 mm.

[0045] According to a preferred embodiment of the present invention, the plurality of line-shaped strips may be included in four or more. Preferably, the plurality of line-shaped strips may be included in 4 to 10, and more preferably, the plurality of line-shaped strips may be included in 4 to 6.

[0046] According to a preferred embodiment of the present invention, the plurality of line-shaped strips may include a strip comprising a standard line and a correction line. The strip comprising the standard line and the correction line may include only one strip among the plurality of line-shaped strips, and the remaining strips may include only the standard line. By including the standard line and the correction line, the accuracy and reliability of the immunoassay can be ensured.

[0047] The above standard line may be a line formed using a standard substance of known concentration to verify the validity of the experimental procedure. For example, the standard line can be formed by a substance that always exhibits a constant reaction regardless of the type of sample. If the standard line does not appear after testing, this may indicate an error in the experimental procedure.

[0048] The above standard lines may include 1 to 3 per strip.

[0049] The aforementioned correction line may serve to verify the validity of the experimental process and correct deviations between strips. By forming one or more correction lines with different concentrations together on the strip, the user can obtain a correction standard for each test. By normalizing the response signal intensity of each marker based on the signal intensity of the correction line obtained from the biochip, deviations in result values ​​resulting from differences in the test environment, minute variations in sample injection volume, or minute performance differences between strips can be effectively corrected, thereby providing a high level of reliability.

[0050] According to a preferred embodiment of the present invention, the correction lines may be included in 1 to 10 numbers. Preferably, the correction lines may be included in 1 to 5 numbers. More preferably, the correction lines may be included in 2 to 5 numbers.

[0051] According to a preferred embodiment of the present invention, the strip may include 25 or more markers per strip. Preferably, the strip may include 25 to 100 markers per strip, and more preferably, the strip may include 25 to 60 markers per strip.

[0052] The above reaction vessel can be designed to prevent reading errors caused by shadows that may occur when reading the reaction results of the strip with an optical scanner or camera.

[0053] During optical reading, if a shadow is cast on the edge of the strip by the wall of the reaction vessel, reading may become impossible or the signal intensity in that area may be measured lower than the actual value, which can seriously impair the accuracy of the results. To solve this, the reaction vessel of the present invention has a cross-sectional shape in which the upper opening of the well where the strip is located is wider than the lower bottom surface, thereby allowing light irradiated from a light source to reach the entire surface of the strip uniformly without being blocked by the wall. As a result, a clear, shadow-free image can be obtained from all areas of the strip, thereby maximizing the accuracy and reproducibility of the reading. The material of the reaction vessel may be a matte, opaque material that does not reflect light.

[0054] Specifically, according to a preferred embodiment of the present invention, the reaction vessel has a space formed therein for accommodating the strip and includes reaction vessel side walls extending in the height direction from each end of a reaction vessel base that supports the strip from below, and the inner surface of the reaction vessel side wall may be formed to be inclined toward the outside as it extends from the bottom to the top.

[0055] The inclination between the inner surface of the reaction vessel sidewall and the base of the reaction vessel may be 91 to 179 degrees. Preferably, the inclination between the inner surface of the reaction vessel sidewall and the base of the reaction vessel may be 95 to 110 degrees.

[0056] According to a preferred embodiment of the present invention, the strip may be received in the reaction vessel and arranged so that the marker is exposed to the outside.

[0057] According to a preferred embodiment of the present invention, the biochip may include 100 or more markers. Preferably, the biochip may include 100 to 1000 markers, and more preferably, the biochip may include 100 to 360 markers.

[0058] The biochip of the present invention may be used individually or in combination. The connection may be in the form of a parallel, series, or other possible arrangement.

[0059]

[0060] In addition, the present invention may provide a multi-immunoassay method comprising the step of processing a sample separated from an individual on the highly integrated linear biochip for multi-immunoassay.

[0061] According to a preferred embodiment of the present invention, the sample may be one or more selected from the group consisting of blood, whole blood, serum, plasma, cerebrospinal fluid, urine, feces, saliva, tears, nasal discharge, and sweat, but is not limited thereto.

[0062] The above sample may be a sample containing immunoglobulin, autoantibody, cytokine, protein, drug, compound, DNA, or RNA; however, according to a preferred embodiment of the present invention, the sample may be a sample containing immunoglobulin. This may be equally applicable to samples of mammals such as dogs, cats, cattle, and horses.

[0063]

[0064] The high-density linear biochip of the present invention, which measures the concentration of substances present in biological samples using an immunoassay method, improves upon the weakness of existing biochips that make it difficult to place multiple markers into a limited reaction strip. By cutting multiple linear strips narrower than conventional strips and arranging them in parallel, it has the advantage of being able to analyze more than 100 substances at once. In addition, by using a standard line and a correction line during immunoassay, it can provide higher accuracy than existing biochips.

[0065]

[0066] Figure 1 shows a highly integrated linear biochip for multiple immunoassay according to the present invention.

[0067] FIG. 2a shows a reaction vessel with reduced shadow interference manufactured according to the present invention.

[0068] FIG. 2b shows a cross-sectional view of the reaction vessel of the present invention.

[0069] FIG. 2c shows a plan view tilted so that it can be clearly seen that the side wall (110) of the reaction vessel of the present invention is inverted trapezoidal.

[0070] FIG. 2d shows a perspective view of the reaction vessel of the present invention.

[0071] FIG. 2e shows an enlarged drawing so that it can be clearly seen that the side wall (110) of the reaction vessel of the present invention is inverted trapezoidal.

[0072] Figure 3 shows a 4-row parallel high-density line-type biochip manufactured through the present invention.

[0073] Figure 4 is a biochip manufactured in the present invention after undergoing the reaction process according to Example 2.

[0074]

[0075] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0076]

[0077] [Example 1]

[0078] Fabrication of parallel linear biochips

[0079] 128 solutions containing individual protein markers, including standard and correction lines, were immobilized by coating them onto four nitrocellulose membranes in a line shape, divided into 32 horizontal lines (Table 1). P1 to P4 corresponded to the standard lines, and C1 and C2 corresponded to the correction lines. The standard lines utilized standard substances as markers to verify whether the experiment was performed correctly. The correction lines utilized markers of known concentrations at different concentrations to correct for deviations from the test.

[0080] The protein-immobilized membranes were dried overnight in a drying oven at room temperature. Each dried membrane was attached to a plastic support, cut into 1.42 mm intervals, and attached in four parallel rows to a reaction vessel to fabricate a parallel linear biochip (Fig. 3). The angle of inclination between the plastic support to which the membrane was attached and the sidewall of the biochip was approximately 97 degrees. The width of the biochip was 10.0 mm and the length was 85.0 mm.

[0081] Membrane Amembrane Bmembrane Cmembrane Dline marker line marker line marker line marker P1Positive ControlP2Positive ControlP3Positive ControlP4Positive ControlC1Calibration30Bent grass61CCD92SesameC2Calibration31Cultivated rye62Tomato93Corn1Total IgE32Common ragweed63Carrot94Rice2Housedust33Mugwort64Garlic95Wheat flour3Dermatophagoides pteronyssinus34Ox-eye daisy65Onion96Gluten4Dermatophagoides farinae35Dandelion66Celery97Rye5Acarus siro36Plantain67Cucumber98Barley meal6Tyrophagus putrescentiae37Goosefoot, Lamb's quarters68Mushroom99Buckwheat7Cat epithelium & dander38Russian thistle69Potato100Egg white8Dog dander39Goldenrod70Pumpkin101Egg yolk9Hamster epithelium40Cocklebur71Eggplant102Milk10Mouse epithelium / Rat epithelium41Common pigweed72Kiwi103Cheese11Guinea pig epithelium42Japanese hop73Mango104Pork12Wool,sheep43Alder74Banana105Beef13Rabbit epithelium44Birch75Peach106Lamb meat14Horse epithelium45Hazel76Orange107Chicken15Penicillium notatum46Oak77Strawberry108Codfish16Cladosporium herbarum47Elm78Apple109Tuna17Aspergillus fumigatus48Olive79Cacao110Salmon18Candida albicans49Sycamore80Coconut111Pacific squid19Alternaria alternata50Willow81Peanut112Mackerel20Rhizopus nigricans51Cottonwood82Soybean113Plaice21Bee venom52White ash83White bean114Eel22Wasp venom53White pine84Hazel nut115Anchovy23Cockroach54Japanese cedar85Brazil nut116Lobster24Sweet vernal grass55Acacia86Almond117Crab25Bermuda grass56Cypress87Cashew nut118Shrimp26Orchard grass57Hevea latex88Pine nut119Blue mussel27Ryegrass58Sunflower seed89Walnut120Clam28Timothy grass59Silkworm pupa90Sweet chestnut121Oyster29Common reed grass60Yeast, bakers91Macadamia nut122Scallop,

[0082]

[0083] [시시예 2]

[0084] Biochip is used to protect the immune system

[0085] A washing solution (50 mM Tris, 0.15 M NaCl, 0.05% Tween 20) was added to the biochip prepared in [Example 1] above, stirred for 5 minutes, and then removed. A sample dilution solution (PBS, 0.02 mg / mL Bromocresol purple) and the sample to be tested were added and stirred for 35 minutes. After removing the reaction solution, the washing solution was added and stirred for 5 minutes, and then removed. The above washing process was performed one additional time. An antibody solution containing an anti-immunoglobulin antibody labeled with biotin protein was added and stirred for 30 minutes. After removing the antibody solution, the washing solution was added and stirred for 5 minutes, and then removed. The above washing process was performed one additional time. Subsequently, an enzyme conjugation solution containing an alkaline phosphatase protein labeled with Streptavidin, which can bind to biotin protein, was added and stirred for 15 minutes. After removing the enzyme conjugation solution, a washing solution was added and stirred for 5 minutes, after which it was removed. The above washing process was performed one additional time. A color-developing solution (0.2 mg / ml 5-bromo-4-chloro-3-indolyl phosphate, 0.3 mg / ml Nitroblue Tetrazolium) that reacts with alkaline phosphatase to produce a color was added and stirred for 20 minutes. After removing the color-developing solution, the sample was washed with distilled water and dried to check for color development.

[0086] As a result, it was confirmed that a black reaction appeared on the corresponding protein marker in samples containing specific immunoglobulin (Fig. 4). It was confirmed that uniform color development was specifically observed even when the horizontal width of the marker line was thin and there were more than 100 markers in the reaction vessel.

[0087]

[0088] [Explanation of the symbol]

[0089] 100: Reaction vessel

[0090] 110: Reaction vessel sidewall

[0091] 120: Reaction vessel base

[0092] 130: Reaction vessel space

[0093] 200: Strip

[0094]

[0095] The high-density linear biochip of the present invention, which measures the concentration of substances present in biological samples using an immunoassay method, improves upon the weakness of existing biochips that make it difficult to place multiple markers into a limited reaction strip. By cutting multiple linear strips narrower than conventional strips and arranging them in parallel, it has the advantage of being able to analyze more than 100 substances at once. Furthermore, by using a standard line and a correction line during immunoassay, it can provide higher accuracy than existing biochips, thus offering industrial applicability.

Claims

1. A highly integrated linear biochip for multiple immunoassay comprising: a plurality of linear strips; and a reaction vessel for receiving the linear strips.

2. A biochip according to claim 1, characterized in that the plurality of line-shaped strips comprises four or more.

3. A biochip according to claim 1, characterized in that the plurality of line-shaped strips include strips comprising a standard line and a correction line.

4. A biochip according to paragraph 3, characterized in that the correction lines include 1 to 10 of them.

5. A biochip according to claim 1, characterized in that the strip comprises 25 or more markers per strip.

6. A biochip according to claim 1, characterized in that the strip is received in the reaction vessel and arranged so that a marker is exposed to the outside.

7. The biochip according to claim 1, characterized in that the biochip comprises 100 or more markers.

8. A multiple immunoassay method comprising the step of processing a sample separated from an individual on a highly integrated linear biochip for multiple immunoassay according to claim 1.

9. A method according to claim 8, characterized in that the sample is one or more selected from the group consisting of blood, whole blood, serum, plasma, cerebrospinal fluid, urine, feces, saliva, tears, nasal discharge, and sweat.

10. A method according to claim 8, characterized in that the sample is a sample containing immunoglobulin.

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