Membrane-coupled immunoassay chip capable of detecting scattered light

Abstract

The present invention relates to an immunoassay chip and, more particularly, to an immunoassay chip having improved specimen detection sensitivity using a scattered-light signal of a label material.

Description

Immunoassay chip capable of detecting scattered light combined with a membrane

[0001] The present invention relates to an immunoassay chip, and more specifically, to an immunoassay chip that improves the detection sensitivity of a sample by reducing background scattered light signals and effectively detecting the scattered light signal of a labeling substance.

[0002] A biosensor is an analytical device configured to selectively measure and analyze specific substances at the molecular level by combining bioreceptors and signal transduction technology. Bioreceptors, such as antibodies, enzymes, aptamers, and cells, play the role of selectively recognizing the substance to be analyzed, while signal transduction technology detects the changes and their magnitudes that occur when the bioreceptor reacts with or binds to the target substance, converting them into a signal perceptible to humans.

[0003] Biosensors are being utilized in a wide variety of fields, including medicine, food, environment, and military; among these, they are most widely used in the diagnosis of diseases and infections in hospitals and in self-monitoring of blood sugar levels at home.

[0004] (Prior Art Literature) Korean Published Patent No. 2013-0037648

[0005] The present invention provides an immunoassay chip that enhances the signal intensity of a marker binding to a detection target within a sample, and relates to an immunoassay chip capable of measuring the scattered light signal of a marker substance by minimizing background signals.

[0006] Furthermore, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0007] To achieve the above objective, an immunoassay chip according to one embodiment of the present invention comprises: a light-absorbing pad that absorbs irradiated light; a transparent substrate disposed on the light-absorbing pad; a membrane structure disposed on the transparent substrate; and a capture antibody formed on the transparent substrate.

[0008] The above membrane structure comprises a sample pad; a conjugate pad connected to the sample pad; a first membrane pad in contact with the conjugate pad; a second membrane pad spaced apart from the first membrane pad; and an absorption pad connected to the second membrane pad, wherein the conjugate pad may include plasmonic nanoparticles as a labeling material.

[0009] The above-mentioned immobilized antibody may be formed on the transparent substrate between the first membrane pad and the second membrane pad.

[0010] The above-mentioned immobilized antibody may specifically bind to an analyte of the sample in the reaction solution.

[0011] The above reaction solution may further include NaCl and a surfactant.

[0012] The above-mentioned immobilized antibody binds to the above-mentioned sample, and the above-mentioned sample may bind to the above-mentioned detection antibody.

[0013] The above plasmonic nanoparticles may have scattering characteristics for the irradiated light.

[0014] The light-absorbing pad above may have a light diffuse reflectance lower than that of barium sulfate (BaSO4).

[0015] The light-absorbing pad may have a light diffuse reflectance of 15% or less of the light diffuse reflectance of barium sulfate (BaSO4).

[0016] The present invention has the effect of reducing background signals by limiting reflected light for an immunoassay chip and enhancing the intensity of the detection signal of a sample by utilizing scattered light from a labeling substance, thereby improving detection sensitivity even at low concentrations of the sample.

[0017] FIGS. 1a and FIGS. 1b illustrate the configuration of an immunoassay chip according to one embodiment of the present invention.

[0018] FIG. 2 illustrates a schematic diagram of plasmonic nanoparticles and detection antibodies according to one embodiment of the present invention.

[0019] FIG. 3 illustrates the principle of light signal detection of an immunoassay chip according to one embodiment of the present invention.

[0020] FIG. 4 illustrates an example of operation of an immunoassay chip according to one embodiment of the present invention.

[0021] FIGS. 5a to 5e illustrate detection signals according to the reflectance of a light-absorbing pad of an immunoassay chip according to one embodiment of the present invention.

[0022] An immunoassay chip according to one embodiment of the present invention comprises: a light-absorbing pad that absorbs irradiated light; a transparent substrate disposed on the light-absorbing pad; a membrane structure disposed on the transparent substrate; and a capture antibody formed on the transparent substrate.

[0023] The above membrane structure comprises a first membrane pad; a sample pad; a conjugate pad connected to the sample pad; a first membrane pad in contact with the conjugate pad; a second membrane pad spaced apart from the first membrane pad; and an absorption pad connected to the second membrane pad, wherein the conjugate pad may include plasmonic nanoparticles as a labeling material.

[0024] The above-mentioned immobilized antibody may be formed on the transparent substrate between the first membrane pad and the second reaction membrane pad.

[0025] The above-mentioned immobilized antibody may specifically bind to an analyte of the sample in the reaction solution.

[0026] The above reaction solution may further include NaCl and a surfactant.

[0027] The above-mentioned immobilized antibody binds to the above-mentioned sample, and the above-mentioned sample may bind to the above-mentioned detection antibody.

[0028] The above plasmonic nanoparticles may have scattering characteristics for the irradiated light.

[0029] The light-absorbing pad above may have a light diffuse reflectance lower than that of barium sulfate (BaSO4).

[0030] The light-absorbing pad may have a light diffuse reflectance of 15% or less of the light diffuse reflectance of barium sulfate (BaSO4).

[0031] Hereinafter, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0032]

[0033] Hereinafter, with reference to the drawings, an immunoassay chip according to one embodiment of the present invention will be described in detail.

[0034]

[0035] An immunoassay chip according to one embodiment of the present invention may be a device for detecting a sample (analyte), which is a substance to be detected, by injecting a reaction solution containing a sample to be detected.

[0036] The above sample is not particularly limited as long as it contains the specimen to be detected. For example, the sample may be a biological sample, such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood (whole blood), plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, etc. Biological tissue is an aggregate of cells, which may include intracellular materials that generally form one of the structural materials of human, animal, plant, bacterial, fungal, or viral structures, and specific types of aggregates such as connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Additionally, examples of biological tissue may include organs, tumors, lymph nodes, arteries, and individual cell(s).

[0037] The above-mentioned analyte may be a molecule or other substance within the sample to be detected. For example, the analyte may include antigenic substances, ligands (mono- or polyepitopes), haptens, antibodies, and combinations thereof. Specifically, the analyte may include, for example, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs, drug intermediates or byproducts, bacteria, virus particles, yeast, fungi, protozoa, and metabolites of said substances or antibodies against them, but is not necessarily limited thereto. However, it may typically be an antigen or an antibody.

[0038]

[0039] Referring to FIG. 1a, an immunoassay chip (10) according to one embodiment of the present invention comprises a light-absorbing pad (100); a transparent substrate (200); and a membrane (300). More specifically, an immunoassay chip (10) according to one embodiment of the present invention comprises a light-absorbing pad (100) that absorbs irradiated light; a transparent substrate (200) disposed on the light-absorbing pad (100); and a membrane structure (300) disposed on the transparent substrate (200).

[0040]

[0041] Additionally, an immunoassay chip (10) according to one embodiment of the present invention further includes a capture antibody (400) formed on a transparent substrate (200). More specifically, a microflow element (10) according to one embodiment of the present invention includes a light-absorbing pad (100) that absorbs irradiated light; a transparent substrate (200) disposed on the light-absorbing pad (100); a membrane structure (300) formed on the transparent substrate (200); and a capture antibody (400) formed on the transparent substrate (200).

[0042] The light-absorbing pad (100) absorbs light irradiated onto the immunoassay chip (10), and the light-absorbing pad (100) may comprise a support member having a predetermined length; and a light-absorbing material applied on the support member. The support member may be a PVC (Polyvinyl Chloride) substrate, and the light-absorbing material may be a CNT (Carbon Nanotube). The light-absorbing pad (100) may have a light reflectance lower than that of barium sulfate (BaSO4), and more specifically, may be 15% or less of the light reflectance of barium sulfate (BaSO4).

[0043]

[0044] The transparent substrate (200) transmits all light irradiated onto the immunoassay chip (10) to the light absorption pad (100), and the transparent substrate (200) may be made of glass.

[0045]

[0046] Referring to FIGS. 1a and 1b, the membrane structure (300) includes a sample pad (310), a conjugate pad (320); a first membrane pad (331); a second membrane pad (332); and an absorption pad (340). Additionally, the membrane structure (300) further includes a cover portion (350), and more specifically, the membrane structure (300) includes a sample pad (310), a conjugate pad (320), a first membrane pad (331), a second membrane pad (332); an absorption pad (340); and a cover portion (350).

[0047]

[0048] Referring to FIG. 1a and FIG. 1b, more specifically, the membrane structure (300) comprises a sample pad (310) according to the flow direction (Flow) of the reaction solution (Solution); a conjugate pad (320) connected to the sample pad (310); a first membrane pad (331) in contact with the conjugate pad (320); a second membrane pad (332) spaced apart from the first membrane pad (331); an absorption pad (340) in contact with the second membrane pad (332); and a cover portion (350) in contact with both the first membrane pad (331) and the second membrane pad (332).

[0049]

[0050] The sample pad (310) is an area where the reaction solution is loaded, and performs the function of uniformly distributing the reaction solution and spreading the reaction solution onto the membrane pad (330) (first membrane pad (331) and second membrane pad (332)).

[0051] The sample pad (310) is in contact with the conjugate pad (320), and the sample pad (310) is not limited to any type of material as long as it is made of a material capable of absorbing a liquid reaction solution, and preferably can be cellulose, polyester, polypropylene, or glass fiber.

[0052]

[0053] The conjugate pad (320) may be placed between the sample pad (310) and the membrane pad (330).

[0054] The conjugate pad (320) may include a labeling material that generates a signal detectable by visual inspection or by using a sensor. The labeling material may be a nanoparticle-detection antibody conjugate in which a detection antibody that binds to the nanoparticle and the antigen is connected.

[0055] The labeling material above refers to a nanoparticle that acts as a detectable label. Referring to FIG. 2, the labeling material may be a plasmonic nanoparticle (500), and the plasmonic nanoparticle (500) may scatter light when exposed to incident light. The plasmonic nanoparticle (500) may make the scattered signal measurable by scattering light.

[0056] The plasmonic nanoparticles (500) may have scattering properties for incident light. The nanoparticles may most preferably be gold (Au) nanoparticles. The detection antibody (510) may be formed on the plasmonic nanoparticles (500). The detection antibody (510) may bind to the sample, and the sample may be the same sample that binds to the fixed antibody (400). The detection antibody (510) formed on the plasmonic nanoparticles (500) may bind to the sample that binds to the fixed antibody (400), and accordingly, the fixed antibody (400) binds to the sample, and the sample binds to the detection antibody (510), thereby forming a sandwich structure (fixed antibody (400)-sample-detection antibody (510)-plasmonic nanoparticles (500)).

[0057] The detection antibody of the above conjugate refers to an antibody that specifically binds to the antigen to be analyzed, and includes fragments of the antibody if they possess binding specificity. The detection antibody may be a monoclonal antibody or a polyclonal antibody, and most preferably, a monoclonal antibody is used. The binding of the nanoparticle and the detection antibody includes, but is not limited to, ionic bonds, covalent bonds, metallic bonds, coordinate bonds, hydrogen bonds, and van der Waals bonds.

[0058]

[0059] More specifically, FIG. 3 shows that in the immunoanalysis chip (10) of the present invention, the plasmonic nanoparticle (500) is positioned on the fixed antibody (400), so that the light-absorbing pad (100) absorbs all the light incident on it and removes the background signal, thereby allowing only the scattered light by the plasmonic nanoparticle (500) bound in a sandwich form to the fixed antibody (400) to be measured, and as a result, more sensitive detection with a high signal-to-noise ratio can be achieved along with the measurement of the intensity of the light with high intensity.

[0060]

[0061] The membrane pad (330) has a porous structure and allows the sample to move by capillary action, and the membrane pad (330) may be a membrane, and the membrane pad (330) includes a first membrane pad (331) arranged in the direction of development of the reaction solution; and a second membrane pad (332). The first membrane pad (331) and the second membrane pad (332) may be arranged spaced apart from each other by a predetermined distance.

[0062] The first membrane pad (331) may be loaded with a reaction solution and may be configured to spread the reaction solution toward a spaced-apart second membrane pad (332).

[0063] The second membrane pad (332) may be spaced apart from the first membrane pad (331) and may spread the reaction solution loaded from the first membrane pad (331) to the absorption pad (340).

[0064] The first membrane pad (331) and the second membrane pad (332) may be spaced apart from each other so that the transparent substrate (200) is exposed between the first membrane pad (331) and the second membrane pad (332).

[0065]

[0066] For example, the membrane pad (330) (first membrane pad (331) and / or second membrane pad (332)) may be made of one or more selected from nitrocellulose, nylon, polysulfone, polyethersulfone and PVDF (Polyvinylidene fluoride).

[0067] The absorption pad (340) performs the role of absorbing the reaction solution developed in the membrane pad (330) (the first membrane pad (331) and the second membrane pad (332)), specifically, absorbs the reaction solution that has moved through the first membrane pad (331) and the second membrane pad (332), and performs the role of providing the power to move the reaction solution through capillary action.

[0068] The absorbent pad (340) is not limited to any type of material as long as it is capable of absorbing a liquid reaction solution, and preferably can be cellulose, polyester, polypropylene, or glass fiber.

[0069]

[0070] The cover portion (350) may be in contact with both the first membrane pad (331) and the second membrane pad (332), and may be positioned to cover the upper portion of the transparent substrate (200) where the first membrane pad (331) and the second membrane pad (332) are spaced apart from each other. More specifically, the cover portion (350) may protect the subsequently discharged fixed antibody (400).

[0071] The cover portion (350) may be made of a material that transmits light irradiated onto the immunoassay chip (10), and the cover portion (350) may be made of glass. Accordingly, the light transmitted through the cover portion (350) may pass through the transparent substrate (200) and then reach the light-absorbing pad (100).

[0072] More specifically, the cover portion (350) transmits all light in the visible light region, thereby allowing scattered light from the plasmonic nanoparticles (500) bound in a sandwich form to the immobilized antibody (400) to pass through the cover portion (350) again and be detected, and the light that has passed through the plasmonic nanoparticles (500) may be transmitted to and absorbed by the light absorption pad (100).

[0073] Additionally, the cover portion (350) may connect the space between the first membrane pad (331) and the second membrane pad (332), and may enable the reaction solution to move from the first membrane pad (331) to the second membrane pad (332) due to the hydrophilic properties of the glass material and the surface tension of water.

[0074]

[0075] The immobilized antibody (400) may be formed and immobilized on a transparent substrate (200). The immobilized antibody (400) may bind to an analyte contained in a sample to be detected in a reaction solution.

[0076]

[0077] The above reaction solution includes the above sample. In addition, the above reaction solution may further include NaCl and a surfactant.

[0078]

[0079] For example, referring to FIG. 4, when a reaction solution is loaded onto a sample pad (310) of a microfluidic device (10) according to one embodiment of the present invention (Fig. 4(a)), the reaction solution is spread onto a conjugate pad (320), and then the reaction solution is spread onto a first membrane pad (331) (Fig. 4(b)). Subsequently, the reaction solution is spread to a position where the immobilized antibody (400) is formed, and the surface of the reaction solution rises to a cover portion (350) so that the immobilized antibody (400) is submerged (Fig. 4(c)), and the reaction solution is spread to a second membrane pad (332), at which time a portion of the upper part of the immobilized antibody (400) is exposed from the reaction solution (Fig. 4(d)). Afterward, when the reaction solution is fully spread over the second membrane pad (332), the immobilized antibody (400) is again completely submerged in the reaction solution so that the reaction solution and the immobilized antibody (400) react sufficiently (Fig. 4 (e)).

[0080]

[0081] The present invention will be explained in more detail below through examples. These examples are merely illustrative for understanding the invention and do not limit the scope of the invention.

[0082]

[0083] Preparation Example 1. Preparation of an immunoassay chip

[0084] Referring to FIGS. 1a and 1b, a light-absorbing pad (100) is prepared by bar-coating CNT paste onto the upper surface of a PVC substrate having dimensions of 7.6 cm in length and 2.6 cm in width as a support. Subsequently, a transparent substrate (glass material) (200) having the same dimensions (7.6 cm in length and 2.6 cm in width) is placed on the prepared light-absorbing pad (100). Subsequently, a sample pad (glass fiber; Ahlstrom) (310), a conjugate pad (glass fiber; Ahlstrom) (320), a first membrane pad (nitrocellulose membrane; millipore) (331), and a second membrane pad (nitrocellulose membrane; millipore) (332) are placed on the transparent substrate (200); An absorption pad (ap22; ahlstrom) (340) is sequentially arranged, and a first reaction pad (331) and a second reaction pad (332) are spaced apart at a predetermined interval to form a membrane structure (300) (the width of the membrane structure is 3.8 to 4.0 mm). Afterwards, a fixation antibody (400) is fixed on a transparent substrate (200) between the first reaction pad (331) and the second reaction pad (332), and a cover part (glass material) (350) is positioned to cover the fixation antibody (400) by connecting the first membrane pad (331) and the second reaction pad (332) to produce an immunoassay chip (10).

[0085] The conjugate pad (320) is a plasmonic nanoparticle (500) in which gold nanoparticles (AuNP, 100 nm) are immobilized by binding them to a detection antibody (510) that specifically binds to the Influenza A nucleocapsid protein.

[0086] As a fixed antibody (400), an antibody that specifically binds to the Influenza A nucleocapsid protein was used.

[0087] The reaction solution contains Influenza A nucleocapsid protein (sample), NaCl, and a surfactant.

[0088]

[0089] Analysis Example.

[0090] The reflectance analysis in the experimental example described below was performed after the reaction solution was injected onto the sample pad (310), and after a sufficient amount of time had elapsed for the reaction solution to spread through the first membrane pad (331) and the second membrane pad (332) to the absorption pad (340) and react with the immobilization antibody (400).

[0091]

[0092] Experimental Example 1. Analysis of detection signal based on light absorption versus light reflectance

[0093] Figures 5a and 5e show photographic images of the light detection signal when the light-absorbing pad is exposed to light (exposure times: 0.1s, 1.0s, 5.0s, 20.0s, 60.0s) according to the dilution ratio of the concentration of gold nanoparticles (500) bound to the detection antibody (510), which is a labeling substance, in the immunoassay chip of Preparation Example 1 (concentration adjusted by adding water as a diluent), according to the reflectance relative to BaSO4 (barium sulfate) (light reflectance of the light-absorbing pad relative to the light reflectance of barium sulfate). The reflectance relative to BaSO4 (barium sulfate) in Figures 5a and 5e is determined by the concentration control of the CNT paste in Preparation Example 1 (addition of white paint).

[0094]

[0095] Referring to FIGS. 5a to 5e, it can be seen that as the reflectance relative to BaSO4 of the light-absorbing pad is lowered, the reflected signal generated from the reaction pad can be reduced, and accordingly, the scattered signal is measured more strongly. A low reflectance relative to BaSO4 can increase the exposure time for measuring the scattered signal of the labeling material, and since the reflected signal is reduced due to the low reflectance relative to BaSO4, only the scattered signal can be amplified by increasing the measurement exposure time, which confirms that the sensitivity is superior.

[0096]

[0097] Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims also fall within the scope of the present invention.

Claims

1. A light-absorbing pad that absorbs irradiated light; A transparent substrate disposed on the light-absorbing pad above; A membrane structure disposed on the above transparent substrate; and A capture antibody formed on the above-mentioned transparent substrate, comprising Immunoassay chip.

2. In Paragraph 1, The above membrane structure is, It includes a sample pad; a conjugate pad connected to the sample pad; a first membrane pad in contact with the conjugate pad; a second membrane pad spaced apart from the first membrane pad; and an absorbent pad connected to the second membrane pad. The above conjugate pad comprises plasmonic nanoparticles as a labeling material, Immunoassay chip.

3. In Paragraph 2, The above-mentioned immobilized antibody is formed on the transparent substrate between the first membrane pad and the second reaction membrane pad, Immunoassay chip.

4. In Paragraph 1, The above-mentioned fixed antibody specifically binds to the analyte of the sample in the reaction solution, Immunoassay chip.

5. In Paragraph 4, The above reaction solution is, A substance further containing NaCl and surfactant, Immunoassay chip.

6. In Paragraph 5, The above-mentioned fixed antibody binds to the above-mentioned sample, and The above sample is one that binds to the detection antibody, Immunoassay chip.

7. In Paragraph 5, The above plasmonic nanoparticles have scattering characteristics with respect to irradiated light, Immunoassay chip.

8. In Paragraph 1, The above light-absorbing pad has a light diffuse reflectance lower than the light reflectance of barium sulfate (BaSO4). Immunoassay chip.

9. In Paragraph 8, The light-absorbing pad above has a light diffuse reflectance of 15% or less of the light diffuse reflectance of barium sulfate (BaSO4), Immunoassay chip.

Images