KR101570327B1 - Parallel line biochip for multiplex diagnosis - Google Patents

Abstract

The present invention relates to a line-type biochip for parallel diagnosis, and more particularly, to a biochip comprising a plurality of line-shaped strips arranged in parallel; And a container for fixing the strip; And a control unit. When the biochip of the present invention is used, since two or more line-shaped strips can be connected in parallel, it is possible to simultaneously measure various substances present in a biological sample.

Description

[0002] Parallel line biochips for multiplex diagnosis [

The present invention relates to a line-type biochip for parallel diagnosis that can perform analysis of various elements at one time. More particularly, the present invention relates to a line type biochip for parallel diagnosis, a diagnostic kit using the same, and a diagnosis method.

Biochips are used to immobilize DNA, proteins, antibodies and other biomaterials on solid substrates such as glass, silicon, plastic, metal, nitrocellulose, and PVDF and analyze the reaction with trace amounts of samples to determine the expression patterns, And the like. A protein chip is a protein chip that fixes a protein such as an antigen or an antibody that reacts with a specific protein to a solid substrate and measures the result of binding with a specific protein in the assay sample by an analysis method using absorption, fluorescence, SPR, And as a means to interpret quantitative or biological functions.

Bio-chips can be classified into dot type and line type. Dot type can coat many types of markers on a plate like a DNA micro array, but it is troublesome to coat each plate one by one from the beginning. In the line type, since the markers to be measured on a long membrane are drawn in a plurality of lines in the form of a plurality of lines horizontally and cut vertically and used as a strip, the production process is simple and can be mass-produced. Kits capable of detecting HIV, mycobacteria, and the like are commercially available as kits that discriminate the nucleotide sequence from the line-type multiple diagnosis kits.

Line-type strip kits that measure protein are widely used in autoimmune antibodies and allergy diagnostic reagents. Allergy is an irritable immune reaction that occurs when IgE antibody against a specific foreign substance is produced in the body. It is diagnosing allergic disease by quantifying blood concentration of IgE antibody that binds to allergen which can cause allergy. The distribution of allergens is not only large by region but also influenced by food culture so dozens of allergens must be tested at the same time. Therefore, a test kit using a protein chip capable of simultaneously diagnosing various allergens can be used as an important test method, rather than performing individual tests for each allergen. Commercially available allergy diagnostic kits are prepared by immobilizing 1 to 21 allergens on nitrocellulose, reacting with serum, and analyzing specific IgE bound using fluorescence or absorbance. In this connection, Korean Patent Laid-Open Publication No. 2003-0089530 provides a kit for diagnosing asthma or rhinitis comprising a protein of Cytokeratin 18, but there is a problem that only one Cytokeratin 18 protein can be diagnosed at a time.

However, the line-type strip kit is difficult to test due to the long length of the strip when many lines are placed on the strip, and is composed of only about 20 marker lines. There is a difficulty.

As a result of efforts to develop a diagnostic kit capable of analyzing various factors in order to solve the above problems, the inventors of the present invention developed a technique of arranging line type strips in parallel so that more kinds of materials can be measured at one time The present invention has been completed.

Accordingly, it is an object of the present invention to provide a multi-diagnostic bio-chip including a parallel line-shaped strip, a multiple diagnosis kit using the same, and a diagnostic method.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a multi-parallel line-type bio-chip in which line-shaped strips are arranged in parallel.

In one embodiment of the present invention, the biochip includes a plurality of line-shaped strips arranged in parallel; And a container for fixing the strip; And a control unit.

In another embodiment of the present invention, the strip is characterized by coating a membrane on a support.

In another embodiment of the present invention, the membrane is characterized in that it comprises a marker.

In yet another embodiment of the present invention, the membrane is characterized in that it is selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), glass, and plastic.

In another embodiment of the present invention, the marker is selected from the group consisting of a protein, an antigen, an antibody, DNA, RNA, PNA, a drug, a chemical, and an aptamer.

In addition, the present invention provides a diagnostic method using a line-type biochip for multiple diagnosis, wherein a biological sample is brought into contact with a marker and the concentration of an existing substance is measured.

In one embodiment of the present invention, the marker is selected from the group consisting of protein, antigen, antibody, DNA, RNA, PNA, drug, chemical, and aptamer.

In another embodiment of the present invention, the biological sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, and urine.

In another embodiment of the present invention, the substance is characterized in that it is selected from the group consisting of immunoglobulin E (IgE), autoantibodies, cytokines, proteins, drugs, compounds, DNA, and RNA.

The present invention also provides a reader for analyzing the line diagnostic kit for multiple diagnoses.

Conventional line-type strip biochips utilize 2 to 21 kinds of materials, each of which is fixed in a line shape. When a large number of substances are to be analyzed, a plurality of line-shaped strips have to be used in a plurality of reaction vessels. Therefore, there is an inconvenience that the amount of sample consumed is large and the process of washing and reacting each reaction solution into individual biochips is repeated.

However, in the multi-diagnosis parallel line type bio-chip provided in the present invention, the individual line type strips are miniaturized, and a plurality of line type strips are arranged and attached in parallel to form one parallel strip, There is an advantage to be able to analyze.

1 is a schematic diagram of a parallel type line-type biochip of the present invention.
2 is a two-row parallel line-type biochip prepared by attaching two line-shaped strips.
3 is a four-row parallel line-type biochip prepared by attaching four line-shaped strips.

The present invention relates to a biochip capable of simultaneously diagnosing many types of biomarkers by arranging line type strips in parallel.

The present inventors have studied a method for improving the efficiency of a conventional biochip in which a length of a strip is long and a large amount of specimens are required when many kinds of markers are included on a strip in a line-shaped strip-type biochip, When the strips are arranged in parallel, a plurality of strips can be included in one biochip, so that many kinds of markers can be measured at one time. Thus, the present invention has been completed.

Accordingly, the present invention provides a line-type biochip in which line-shaped strips are arranged in parallel.

A line-type biochip in which line-shaped strips of the present invention are arranged in parallel comprises a plurality of line-shaped strips arranged in parallel; And a container for fixing the strip; And a control unit. The line-shaped strips 10 are arranged in parallel in one biochip and include a container 20 capable of fixing the strips (see FIG. 1).

Conventional line-type strips have a problem in that many strips can not be arranged because the width of the strips is wide and it is impossible to arrange them in parallel. However, the present invention solves the above problem by making the width of the line-shaped strip thinner and attaching a plurality of strips in parallel. Therefore, the width of the strip may be 0.1 mm to 100 mm, preferably 0.2 mm to 50 mm, and most preferably 0.5 mm to 25 mm.

Therefore, by disposing the line-shaped strips in parallel, it is possible to detect a wide range of substances with only a small sample compared with a series-type long biochip and save time spent in detection .

In addition, unlike dot type biochips, which have been used for the detection of various substances, since the line type strip biochip is used, the time and manpower consumed in drawing the marker on the membrane can be significantly reduced, It is possible.

According to an embodiment of the present invention, the strip includes a support. The support may be coated with a membrane, and the membrane may be nitrocellulose, nylon, polyvinylidene fluoride (PVDF), glass, or plastic, but is not limited thereto. As shown in Fig. 1, the biochip of the present invention is characterized in that two line-shaped strips are connected in parallel.

In the present invention, the term "line-shaped strip" refers to a strip formed by attaching a marker in the form of a line on a membrane. The meaning of "drawing a line" means to attach a marker to the membrane.

Further, a diagnostic method using the biochip of the present invention can be provided. At this time, the biological sample is brought into contact with the marker 30, and the concentration of the expressed substance is measured. The marker can be detected by contacting with a biological sample and measuring the concentration level of the expressed substance by comparing with the control sample. The marker can be detected as a protein, an antigen, an antibody, DNA, RNA, PNA, drug, Or an organic material or an inorganic material composed of a plaster or the like, but is not limited thereto. In addition, the biological sample may be, but not limited to, tissue, cell, whole blood, serum, plasma, saliva, cerebrospinal fluid, or urine.

The expressed material may be immunoglobulin E (IgE), autoantibody, cytokine, protein, drug, compound, DNA, or RNA. The concentration of the substance to be expressed can be measured by a method of measuring fluorescence, absorbance, luminescence, magnetism, electric current, and the like.

The biochip provided by the present invention can be used as a protein chip for allergy diagnosis, but is not limited thereto and is not limited to a biochip arranged in parallel.

Allergy is an irritable immune response that is produced in the body by immunoglobulin E (IgE) antibodies against certain foreign substances. It is very important to simultaneously diagnose allergens that may cause allergies in the diagnosis of allergies. Therefore, when the biochip of the present invention is used, it is possible to simultaneously diagnose a large number of allergens.

The present invention also provides a reader for analyzing a parallel type line-type biochip for multiple diagnosis. The reader recognizes the degree of expression of the protein in the biochip so that it can automatically determine the protein to be expressed for a certain substance.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Preparation of a biochip using a parallel line-shaped strip

100 μl of Sulfo NHS-LC-LC-Biotin (Thermo, USA) solution dissolved in DMSO at 5 mg / ml was slowly added to 1 ml of 4 mg / ml Bovine serum albumin (BSA) solution in PBS. Cut off the light with foil and allow to react by leaving 4 O / N. 4 to O / N by dialysis against PBS twice to produce biotin labeled BSA. The diluted biotin labeled BSA solution was dispensed into 25 horizontal lines on a nitrocellulose membrane (NC-membrane) cut into 5 cm long and 16 cm wide. NC membrane coated with biotin-labeled BSA in the horizontal line was dried in a dry chamber in an RT O / N solution. The dried membrane was adhered to a plastic support, cut vertically at intervals of 1.5 mm, and fixed in a rectangular plastic reaction vessel using two double-sided tapes. A PBS solution containing 0.4 ml of 0.5% BSA was added to a plastic reaction vessel coated with the membrane, followed by stirring for 1 hour. The solution in the reaction vessel was discarded and 0.4 ml of Streptavidin-Alkaline Phosphatase (Streptavidin-AP) (Promeg, USA) was added and stirred for 30 minutes. Streptavidin-AP solution is discarded and 0.4 ml of washing solution (50 mM Tris, 0.2 M NaCl, 0.05% Tween 20) is added, stirred at room temperature for 5 minutes and then removed. This procedure was added twice to completely remove unattached streptavidin-AP. 400uL of a coloring solution containing 0.2mg / ml of bromochlorophenyl phosphate and 0.3mg / ml of nitroblue tetrazolium was added to the sample reaction container and stirred at room temperature for color development reaction. After 20 minutes, remove the solution, add 400 ul of distilled water, wash, remove the solution and dry.

As a result, it was confirmed that the strips could be made thinner in the lateral direction and arranged in parallel. As shown in FIG. 2, it was confirmed that the strips attached in parallel in the plastic reaction vessel were constantly colored.

Example 2. Biochip-attached markers

43 kinds of solutions in which different allergens and proteins were dissolved were divided into two NC-membranes (A, B) as shown in Table 1, and the lines were drawn in the same manner as in Example 1 to immobilize allergens. The allergen-immobilized NC-membrane is dried overnight in a room temperature dryer, adhered to a plastic support, and cut vertically at 1.5 mm intervals to produce a strip. One strip prepared from A membrane and one strip prepared from B 멥 brain were attached side by side in one plastic reaction vessel.

Membrane A Membrane B line Marker line Marker One Biotin-BSA One Biotin-BSA 2 anti-IgE 2 Mugwort 3 Milk 3 Ragweed, short 4 Egg White 4 Alternaria alternata 5 Crab 5 Aspergillus fumigatus 6 Shrimp 6 Cladosporium herbarum 7 Acacia 7 Penicillium notatum 8 Ash mix 8 Cat 9 Birch-alder mix 9 Dog 10 Sallow willow 10 Cockroach 11 Hazelnut 11 Housedust 12 Japanese cedar 12 D. farinae 13 Oak white 13 D. pteronyssinus 14 Poplar mix 14 Sweet vernal grass 15 Sycamore mix 15 Reed 16 Bermuda grass 16 Pine 17 Orchard grass 17 Oxeye daisy 18 Timothy grass 18 Japanese hop 19 Goldenrod 19 Mackerel 20 Rye pollens 20 Kiwi 21 Pigweed 21 Banana 22 Russian thistle 22 Apple

300ul of sample dilution (PBS, 0.5% BSA) was added to plastic reaction vessel, 100ul serum of allergic patients was added, and the mixture was reacted at room temperature for 1 hour with stirring. Discard the solution in the reaction vessel, dispense 0.4 ml of washing solution (50 mM Tris, 0.2 M NaCl, 0.05% Tween 20), stir at room temperature for 5 minutes and remove. This process was added twice.

Biotin labeled mouse anti-IgE solution (400 ul) was added to the sample reaction container and stirred at room temperature. Biotin-labeled mouse anti-IgE was prepared in the same manner as Biotin-labeled BSA of Example 1. After 30 minutes of the reaction, the solution was removed and washed with 400 ul of the washing solution at room temperature for 5 minutes. After repeating this procedure twice, 400 μl of Streptavidin-AP solution was added to the sample reaction vessel and stirred at room temperature. After 30 minutes, the solution was removed and washed with 400 ul of washing solution for 5 minutes at room temperature. This procedure was added twice to completely remove unattached streptavidin-AP. 400uL of chromogenic solution containing 0.2mg / ml of bromochlorophenyl phosphate and 0.3mg / ml of nitroblue tetrazolium was added to the sample reaction vessel and stirred at room temperature. After 20 minutes, the solution was removed, and 250 ul of distilled water was added thereto. The solution was removed and dried. As a result, as shown in Fig. 3, it was confirmed that the allergen having the specific IgE in the serum tested was developed in black color. At this time, IgE in the sample could be quantified by measuring the developed strip with a reader.

Example  3. Plastic Strip type Of the diagnostic kit  Produce

The solution in which different allergens and proteins were dissolved was divided into four NC membranes (A, B, C, D) as shown in Table 2, and the lines were drawn and immobilized by the same method as in Example 1. The allergen-immobilized NC-membrane is dried overnight in a room temperature dryer, adhered to a plastic support, and cut vertically at 1.5 mm intervals to produce a strip. A membrane, B membrane, C membrane, and D membrane were cut and attached to a plastic strip (Fig. 2).

Membrane A Membrane B Membrane C Membrane D line Marker line Marker line Marker line Marker One Biotin-BSA One Biotin-BSA One Biotin-BSA One Biotin-BSA 2 anti-IgE 2 Mugwort 2 Biotin-BSA 2 Mugwort 3 Milk 3 Ragweed, short 3 anti-IgE 3 Ragweed, short 4 Egg white 4 A. alternata 4 Milk 4 A. alternata 5 crab 5 A. fumigatus 5 Egg white 5 A. fumigatus 6 Shrimp 6 C. herbarum 6 Crab 6 C. herbarum 7 Acacia 7 P. notatum 7 Shrimp 7 Cat 8 Ash mix 8 Cat 8 Tuna 8 Dog 9 alder-Birch 9 Dog 9 Codfish 9 Cockroach 10 Sallow willow 10 Cockroach 10 Salmon 10 Housedust 11 Hazelnut 11 Housedust 11 Pork 11 D. farinae 12 Japanese cedar 12 D. farinae 12 Chicken 12 D. pteronyssinus 13 Oak white 13 D. pteronyssinus 13 Beef 13 Buck-wheat 14 Poplar mix 14 Sweet vernal grass 14 Wheat flour 14 Candida albicans 15 Sycamore mix 15 Reed 15 Rice 15 Acarus siro 16 Bermuda grass 16 Pine 16 Barely meal 16 Japanese hop 17 Orchard grass 17 Oxeye daisy 17 Garlic 17 Mackerel 18 Timothy grass 18 Japanese hop 18 Onion 18 PEA 19 Goldenrod 19 Mackerel 19 Peanut 19 Walnut 20 Rye pollens 20 Kiwi 20 Yeast, bakers 20 anti IgE 1 21 Pigweed 21 Banana 21 alder-Birch 21 anti-IgE 2 22 Russian thistle 22 Apple 22 Oak white 22 anti-IgE 3 23 Rye pollens

A 4-column parallel line-shaped strip was placed in a reaction vessel, and 500 ul of a sample dilution (PBS, 0.5% BSA) was added to a plastic reaction vessel. 100 ul of the serum of the allergic patient was added and stirred at room temperature for 1 hour. Discard the solution in the reaction container, dispense 0.6 ml of washing solution (50 mM Tris, 0.2 M NaCl, 0.05% Tween 20), stir at room temperature for 5 minutes and remove. This process was added twice. Biotin labeled mouse anti-IgE solution (600 ul) was added to the sample reaction container and stirred at room temperature. Biotin-labeled mouse anti-IgE was prepared in the same manner as Biotin-labeled BSA of Example 1. After 30 minutes of the reaction, the solution was removed and washed with 400 ul of the washing solution at room temperature for 5 minutes. 600 μl of Streptavidin-AP solution, which was repeated twice, was placed in a sample reaction vessel and stirred at room temperature. After 30 minutes, the solution was removed and washed with 600 ul of wash solution at room temperature for 5 minutes to wash and remove. This procedure was added twice to completely remove unattached streptavidin-AP. 600uL of chromogenic solution containing 0.2mg / ml of bromochlorophenyl phosphate and 0.3mg / ml of nitroblue tetrazolium was placed in a sample reaction vessel and stirred at room temperature. After 20 minutes, the solution was removed and 600 ul of distilled water was added. After washing, the strip was removed from the reaction vessel and dried (FIG. 3). Allergen with specific IgE in the serum was found to be in black color.

From the above results, it is confirmed that a plurality of markers can be detected by arranging a plurality of biochips in parallel.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

10: Line strip
20: container
30: Marker

Claims (5)

A method of measuring the concentration of a substance present in a biological sample using a parallel line-type biochip,
The bio-chip includes a plurality of line-shaped strips arranged in parallel in a reaction vessel, and a vessel for holding the strip, wherein the membrane is coated with a membrane including a marker on a support,
Contacting the biological sample with the marker, and then reacting in the reaction vessel.
A method of measuring the concentration of a substance present in a biological sample using a parallel line-type biochip.
The method according to claim 1,
Wherein the marker is selected from the group consisting of a protein, an antigen, an antibody, a DNA, an RNA, a PNA, a drug, a chemical, and an aptamer.
The method according to claim 1,
Wherein the biological sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, and urine.
The method according to claim 1,
Characterized in that the substance is selected from the group consisting of immunoglobulin E (IgE), autoantibodies, cytokines, proteins, drugs, compounds, DNA, and RNA.
A reader for analyzing a substance measured using the method of any one of claims 1 to 4.

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