Multi-detection solution, fabrication method of the same, and multi-detection device utilizing the same

Abstract

A method for preparing a multi-detection solution according to the present invention may include the steps of: preparing a first metal solution containing a first metal nanoparticle; preparing a second metal solution containing a second metal nanoparticle; preparing a third metal solution containing a third metal nanoparticle; preparing a first detection solution containing a first detection substance in which a first antibody and albumin are bound to the surface of the first metal nanoparticle of the first metal solution; preparing a second detection solution containing a second detection substance in which a second antibody and albumin are bound to the surface of the second metal nanoparticle of the second metal solution; preparing a third detection solution containing a third detection substance in which a third antibody and albumin are bound to the surface of the third metal nanoparticle of the third metal solution; and mixing the first detection solution, the second detection solution, and the third detection solution to prepare the multi-detection solution.

Description

Technology Field

[0001] The present invention relates to a multi-detection solution, a method for manufacturing the same, and a multi-detection device using the same. More specifically, the invention relates to a multi-detection solution comprising a first detection substance having a first antibody bound to the surface of a first metal nanoparticle, a second detection solution having a second detection substance having a second antibody bound to the surface of a second metal nanoparticle, and a third detection solution having a third detection substance having a third antibody bound to the surface of a third metal nanoparticle, mixed together, a method for manufacturing the same, and a multi-detection device using the same. Background Technology

[0002] Lateral flow immunoassay (LFIA) devices, which are representative examples of detection devices, are mostly based on hydrophilic cellulose or nitrocellulose paper, and are detection devices that determine the presence or absence of antigens in an analyte by immobilizing a substance that detects antigens in the test area and providing a liquid analyte to check for color changes or fluorescence expression in the test line.

[0003] While currently commercialized lateral flow immunoassay devices are capable of detecting a single antigen, various detection devices capable of simultaneously detecting multiple antigens are being actively researched and developed to allow users to frequently monitor lesions or check for infections at home.

[0004] For example, Korean Published Patent Application 10-2019-0056279 discloses a test strip for lateral flow immunoassay comprising a sample pad to which a biological sample is supplied, a probe pad coated with a detection reaction agent to enable binding with a target analyte in the biological sample, and a membrane having a reaction area formed to capture a detection biological complex formed by combining the target analyte and the detection reaction agent, wherein the detection reaction agent comprises a first reaction agent and a second reaction agent, wherein a first probe material that generates fluorescence upon irradiation with test light is fixedly bound to the first reaction agent, and a second probe material that generates reflected light upon irradiation with test light is fixedly bound to the second reaction agent, and wherein both fluorescence detection by the first reaction agent and reflected light detection by the second reaction agent are possible. The problem to be solved

[0005] The technical problem that the present invention aims to solve is to provide a multi-detection device with improved accuracy of colors displayed on a test line.

[0006] Another technical problem that the present invention aims to solve is to provide a multi-detection device with improved sensitivity to colors displayed on a test line.

[0007] Another technical problem that the present invention aims to solve is to provide a multi-detection device in which a total of seven different colors are displayed on the test line depending on the mixed detection material provided to the sample section.

[0008] Another technical problem that the present invention aims to solve is to provide a method for manufacturing a multi-detection solution with reduced manufacturing process costs.

[0009] Another technical problem that the present invention aims to solve is to provide a method for manufacturing a multi-detection solution with reduced manufacturing time.

[0010] Another technical problem that the present invention aims to solve is to provide a method for manufacturing a multi-detection solution that is easy to mass-produce.

[0011] The technical problems that the present invention aims to solve are not limited to those described above. means of solving the problem

[0012] To solve the above technical problem, the present invention provides a method for manufacturing a multi-detection solution.

[0013] According to one embodiment, the method for preparing the multi-detection solution may include the steps of: preparing a first metal solution containing a first metal nanoparticle; preparing a second metal solution containing a second metal nanoparticle; preparing a third metal solution containing a third metal nanoparticle; preparing a first detection solution containing a first detection substance in which a first antibody and albumin are bound to the surface of the first metal nanoparticle of the first metal solution; preparing a second detection solution containing a second detection substance in which a second antibody and albumin are bound to the surface of the second metal nanoparticle of the second metal solution; preparing a third detection solution containing a third detection substance in which a third antibody and albumin are bound to the surface of the third metal nanoparticle of the third metal solution; and mixing the first detection solution, the second detection solution, and the third detection solution to prepare the multi-detection solution.

[0014] According to one embodiment, the step of preparing the first metal solution comprises: preparing a first base metal solution comprising a solution containing the first metal nanoparticles and citric acid; and preparing the first metal solution by substituting the solution containing citric acid within the first base solution with a potassium carbonate solution, wherein in the step of preparing the first metal solution, the citrate functional group on the surface of the first metal particle may be substituted with a carbonate functional group.

[0015] According to one embodiment, the step of preparing the second metal solution comprises: preparing a second base metal solution comprising the second metal nanoparticles and a hexadecyltrimethylammonium bromine solution; preparing a second preliminary metal solution by substituting the hexadecyltrimethylammonium bromine solution in the second base metal solution with a solution containing citric acid; and preparing the second metal solution by substituting the solution containing citric acid in the second preliminary metal solution with a potassium carbonate solution, wherein in the step of preparing the second metal solution, the citrate functional group on the surface of the second metal particles is substituted with a carbonate functional group.

[0016] According to one embodiment, the step of preparing the third metal solution comprises: preparing a third base metal solution comprising a solution containing the third metal nanoparticles and citric acid; and preparing the third metal solution by substituting the solution containing citric acid within the third base solution with a potassium carbonate solution, wherein in the step of preparing the third metal solution, the citrate functional group on the surface of the third metal particle may be substituted with a carbonate functional group.

[0017] According to one embodiment, the first metal nanoparticle and the second metal nanoparticle comprise the same metal, and the third metal nanoparticle comprises a metal different from the first metal nanoparticle and the second metal nanoparticle, and the first metal nanoparticle and the second metal nanoparticle comprise gold, and the third metal nanoparticle comprises silver, and the first metal nanoparticle and the third metal nanoparticle comprise a spherical shape, and the second metal nanoparticle comprises a rod shape, wherein the first metal nanoparticle comprises a red color, the second metal nanoparticle comprises a blue color, and the third metal nanoparticle comprises a yellow color, and the particle drag coefficients of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle may be the same.

[0018] According to one embodiment, the first antibody may be bound to alpha-fetoprotein (AFP), the second antibody may be bound to neuron-specific enolase (NSE), and the third antibody may be bound to carcino-embryonic antigen (CEA).

[0019] To solve the above technical problem, the present invention provides a multi-detection device using the multi-detection solution prepared by the manufacturing method described above.

[0020] According to one embodiment, the multi-detection device comprises: a sample portion provided with a mixed substance to be detected including a plurality of different substances to be detected; a conjugate portion provided with a multi-detection solution that receives the mixed substance to be detected from the sample portion and includes a plurality of different detection substances that correspond to and are combined with the substances to be detected within the mixed substance to be detected; and a test portion provided with an analysis material in which the substances to be detected and the detection substances are combined are provided, wherein the mixed substance to be detected includes at least one of a first substance to be detected, a second substance to be detected, and a third substance to be detected; the multi-detection solution includes a first detection substance combined with the first substance to be detected, a second detection substance combined with the second substance to be detected, and a third detection substance combined with the third substance to be detected; and the analysis material includes a first analysis material combined with the first substance to be detected and the first detection substance, a second analysis material combined with the second substance to be detected and the second detection substance, and It may include at least one of the third analysis substances in which the third detection substance and the third detection substance are combined.

[0021] According to one embodiment, the first detection substance comprises alpha-fetoprotein (AFP), the second detection substance comprises neuro-specific enolase (NSE), and the third detection substance comprises carcino-embryonic antigen (CEA); the first detection substance comprises a first metal nanoparticle and a first antibody and albumin provided on the surface of the first metal nanoparticle; the second detection substance comprises a second metal nanoparticle and a second antibody and albumin provided on the surface of the second metal nanoparticle; the third detection substance comprises a third metal nanoparticle and a third antibody and albumin provided on the surface of the third metal nanoparticle; the first analysis substance comprises the first detection substance and the first antibody of the first detection substance bound together; and the second analysis substance comprises the second detection substance and the second antibody of the second detection substance bound together. The third analysis substance comprises a third antibody of the third detection substance bound to the third detection substance, and the test line of the test section comprises the first antibody, the second antibody, and the third antibody, which are respectively bound to the first detection substance of the first analysis substance, the second detection substance of the second analysis substance, and the third detection substance of the third analysis substance, and the test section may further include a control line.

[0022] According to one embodiment, the first metal nanoparticle and the second metal nanoparticle may comprise the same metal, and the third metal nanoparticle may comprise a metal different from the first metal nanoparticle and the second metal nanoparticle, and the first metal nanoparticle and the second metal nanoparticle may comprise gold, and the third metal nanoparticle may comprise silver, and the first metal nanoparticle and the third metal nanoparticle may comprise a spherical shape, and the second metal nanoparticle may comprise a rod shape.

[0023] According to one embodiment, a multi-detection device comprising a sample portion provided with a mixed detection material including a plurality of different detection materials, a conjugate portion provided with a multi-detection solution including a plurality of different detection materials, and a test portion including a test line, wherein the multi-detection device comprises, depending on the detection material in the mixed detection material provided in the sample portion and the detection material in the multi-detection solution provided in the conjugate portion, different colors distinguishable by the naked eye are displayed on the test line, and when the mixed detection material includes at least one of a first detection material, a second detection material, and a third detection material and the multi-detection solution includes a first detection material, a second detection material, and a third detection material, a total of seven different colors may be displayed on the test line.

[0024] According to one embodiment, the first detection substance comprises alpha-fetoprotein (AFP), the second detection substance comprises neuro-specific enolase (NSE), and the third detection substance comprises carcino-embryonic antigen (CEA); the first detection substance comprises a first metal nanoparticle and a first antibody and albumin provided on the surface of the first metal nanoparticle; the second detection substance comprises a second metal nanoparticle and a second antibody and albumin provided on the surface of the second metal nanoparticle; and the third detection substance comprises a third metal nanoparticle and a third antibody and albumin provided on the surface of the third metal nanoparticle; and a first analysis substance to which the first detection substance and the first detection substance are bound, a second analysis substance to which the second detection substance and the second detection substance are bound, and the third detection substance The method comprises providing at least one of the third analysis material to which the third detection material is combined, wherein the first analysis material comprises the first detection material and the first antibody of the first detection material are combined, the second analysis material comprises the second detection material and the second antibody of the second detection material are combined, and the third analysis material comprises the third detection material and the third antibody of the third detection material are combined, and the test line of the test section may include the first antibody, the second antibody, and the third antibody, which are respectively combined to the first detection material of the first analysis material, the second detection material of the second analysis material, and the third detection material of the third analysis material.

[0025] According to one embodiment, the first metal nanoparticle and the second metal nanoparticle comprise the same metal, the third metal nanoparticle comprises a metal different from the first metal nanoparticle and the second metal nanoparticle, the first metal nanoparticle and the second metal nanoparticle comprise gold, the third metal nanoparticle comprises silver, the first metal nanoparticle and the third metal nanoparticle comprise a spherical shape, the second metal nanoparticle comprises a rod shape, the first metal nanoparticle comprises a red color, the second metal nanoparticle comprises a blue color, and the third metal nanoparticle comprises a yellow color.

[0026] According to one embodiment, in a multi-detection device comprising a sample portion provided with a mixed substance to be detected including a plurality of different substances to be detected, a conjugate portion provided with a multi-detection solution including a plurality of different detection substances, and a test portion including a test line, the multi-detection device may include the fact that the movement speed of the analyte combined with the substance to be detected and the detection substance moving from the conjugate portion to the test portion is the same.

[0027] According to one embodiment, the mixed detection substance comprises at least one of a first detection substance, a second detection substance, and a third detection substance, wherein the first detection substance comprises alpha-fetoprotein (AFP), the second detection substance comprises neuron-specific enolase (NSE), and the third detection substance comprises carcino-embryonic antigen (CEA), and the multiple detection solution comprises a first detection substance, a second detection substance, and a third detection substance, wherein the first detection substance comprises a first metal nanoparticle, and a first antibody and albumin provided on the surface of the first metal nanoparticle, the second detection substance comprises a second metal nanoparticle, and a second antibody and albumin provided on the surface of the second metal nanoparticle, and the third detection substance comprises a third metal nanoparticle, and a third antibody provided on the surface of the third metal nanoparticle. The analyte may include at least one of a first analyte in which the first antibody of the first detection substance is bound to the first detection substance, a second analyte in which the second antibody of the second detection substance is bound to the second detection substance, and a third analyte in which the third antibody of the third detection substance is bound to the third detection substance.

[0028] According to one embodiment, the first metal nanoparticle and the second metal nanoparticle comprise the same metal, the third metal nanoparticle comprises a metal different from the first metal nanoparticle and the second metal nanoparticle, the first metal nanoparticle and the second metal nanoparticle comprise gold, the third metal nanoparticle comprises silver, the first metal nanoparticle and the third metal nanoparticle comprise a spherical shape, the second metal nanoparticle comprises a rod shape, and the particle drag coefficients of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle may be the same. Effects of the invention

[0029] A method for preparing a multi-detection solution according to the present invention may include the steps of: preparing a first metal solution containing a first metal nanoparticle; preparing a second metal solution containing a second metal nanoparticle; preparing a third metal solution containing a third metal nanoparticle; preparing a first detection solution containing a first detection substance in which a first antibody and albumin are bound to the surface of the first metal nanoparticle of the first metal solution; preparing a second detection solution containing a second detection substance in which a second antibody and albumin are bound to the surface of the second metal nanoparticle of the second metal solution; preparing a third detection solution containing a third detection substance in which a third antibody and albumin are bound to the surface of the third metal nanoparticle of the third metal solution; and mixing the first detection solution, the second detection solution, and the third detection solution to prepare the multi-detection solution.

[0030] In the steps of preparing the first metal solution, preparing the second metal solution, and preparing the third metal solution, the particle drag coefficients of the first metal nanoparticles, the second metal nanoparticles, and the third metal nanoparticles can be controlled equally. Accordingly, the fluidity (movement speed) of the first detection solution, the second detection solution, and the third detection solution can be controlled equally. Accordingly, when the multi-detection solution, which is a mixture of the first detection solution, the second detection solution, and the third detection solution with equally controlled fluidity, is applied to a multi-detection device, the accuracy of the color displayed on the test line of the multi-detection device can be improved depending on the mixed substance to be detected provided to the multi-detection device.

[0031] In addition, in the steps of preparing the first metal solution, preparing the second metal solution, and preparing the third metal solution, the citrate functional group provided on the surface of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle may be substituted with a carbonate functional group. Accordingly, the ionicity of the surface of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle is reduced, so that the first antibody, the second antibody, and the third antibody can be stably bound to the carbonate functional group on the surface of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle, respectively. Accordingly, the reproducibility of the method for manufacturing the first detection material, the second detection material, and the third detection material may be improved. In addition, when the multi-detection solution comprising the first detection substance, the second detection substance, and the third detection substance is applied to the multi-detection device, the sensitivity of the color displayed on the test line of the multi-detection device may be improved depending on the mixed detection substance provided to the multi-detection device.

[0032] Accordingly, the multi-detection device using the multi-detection solution may include a sample portion provided with the mixed substance to be detected, comprising a plurality of different substances to be detected; a conjugate portion provided with the multi-detection solution, which receives the mixed substance to be detected from the sample portion and comprises a plurality of different detection substances that correspond to and are combined with the substance to be detected within the mixed substance to be detected; and a test portion provided with the test line, which comprises an analysis substance in which the substance to be detected and the detection substances are combined.

[0033] When the above mixed detection substance includes at least one of the first detection substance, the second detection substance, and the third detection substance, and the multi-detection solution includes the first detection substance, the second detection substance, and the third detection substance, a total of seven different colors that are visually distinguishable may be displayed on the test line of the multi-detection device.

[0034] In addition, the particle drag coefficients of the first metal nanoparticle of the first detection material, the second metal nanoparticle of the second detection material, and the third nanoparticle of the third detection material within the multi-detection solution provided to the junction of the multi-detection device may be the same. Consequently, when the first detection material, the second detection material, and the third detection material are provided to the sample portion of the multi-detection device, the speed at which the first analysis material (conjugated with the first antibody of the first detection material and the first detection material), the second analysis material (conjugated with the second antibody of the second detection material and the second detection material) and the third analysis material (conjugated with the third antibody of the third detection material and the third detection material) move from the junction of the multi-detection device to the test portion is controlled to be the same, thereby improving the accuracy of the color displayed on the test line of the multi-detection device. Brief explanation of the drawing

[0035] FIG. 1 is a flowchart illustrating the steps for preparing a multi-detection solution according to an embodiment of the present invention. FIG. 2 is a drawing for explaining a method for manufacturing a first detection solution, a second detection solution, and a third detection solution according to an embodiment of the present invention. FIG. 3 is a schematic diagram and image for explaining the colors displayed on the test line of the multi-detection device according to Experimental Example 1 according to the multi-detection device according to Experimental Example 1 of the present invention, depending on the substance to be detected provided to the multi-detection device according to Experimental Example 1 of the present invention. Figure 4 is a photograph and graph for comparing the flowability of the first detection solution, the second detection solution, and the third detection solution within the multi-detection solution provided at the joint of the multi-detection device according to Experimental Example 1 of the present invention. Figure 5 is a photograph and graph for comparing the selectivity and sensitivity of the multi-detection device according to Experimental Example 1 according to the concentration of antigen provided to the multi-detection device according to Experimental Example 1 of the present invention. Figure 6 is a photograph for comparing the colors displayed on the test line of the multi-detection device according to Experimental Example 1 according to the mixed antigen provided to the multi-detection device according to Experimental Example 1 of the present invention. Specific details for implementing the invention

[0036] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. However, the technical concept of the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art.

[0037] In this specification, when a component is described as being on another component, it means that it may be formed directly on the other component or that a third component may be interposed between them. Additionally, in the drawings, the thicknesses of the films and regions are exaggerated for the effective description of the technical content.

[0038] Additionally, although terms such as first, second, third, etc., have been used to describe various components in the various embodiments of this specification, these components should not be limited by such terms. These terms are used merely to distinguish one component from another. Accordingly, what is referred to as the first component in one embodiment may be referred to as the second component in another embodiment. Each embodiment described and illustrated herein also includes its complementary embodiment. Furthermore, in this specification, "and / or" is used to mean including at least one of the components listed before and after it.

[0039] In the specification, singular expressions include plural expressions unless the context clearly indicates otherwise. Furthermore, terms such as "include" or "have" are intended to specify the existence of the features, numbers, steps, components, or combinations thereof described in the specification, and should not be understood as excluding the existence or addition of one or more other features, numbers, steps, components, or combinations thereof. Additionally, in this specification, "connection" is used to include both indirectly connecting multiple components and directly connecting them.

[0040] In addition, in describing the present invention below, if it is determined that a detailed description of related known functions or configurations could unnecessarily obscure the essence of the invention, such detailed description will be omitted.

[0042] FIG. 1 is a flowchart for explaining the steps of preparing a multi-detection solution according to an embodiment of the present invention, and FIG. 2 is a diagram for explaining the method of preparing a first detection solution, a second detection solution, and a third detection solution according to an embodiment of the present invention.

[0043] Referring to FIG. 1 and FIG. 2, a first metal solution containing a first metal nanoparticle (100) is prepared (S110), a second metal solution containing a second metal nanoparticle (200) is prepared (S120), and a third metal solution containing a third metal nanoparticle (300) is prepared (S130).

[0044] The step of preparing the first metal solution may include the step of preparing a first base metal solution comprising the first metal nanoparticles (100) and a solution comprising citric acid, and the step of preparing the first metal solution by replacing the solution containing citric acid in the first base solution with a potassium carbonate solution.

[0045] In the step of preparing the first base metal solution, the first metal nanoparticle (100) may be spherical in shape and may contain gold. For example, the first metal nanoparticle (100) may be red.

[0046] And, the solution containing the citric acid can provide a citrate functional group (10) on the surface of the first metal nanoparticle (100). For example, the solution containing the citric acid may be a trisodium citrate dihydrate solution.

[0047] In addition, in the step of preparing the first metal solution by replacing the solution containing citric acid in the first base solution with the potassium carbonate solution, the solution containing citric acid in the first base solution can be easily replaced with the potassium carbonate solution by centrifuging the first base solution. For example, the centrifugation can be performed at 9,000 rf for 10 minutes. Subsequently, the first metal solution containing the first metal nanoparticle (100) and the potassium carbonate solution can be easily prepared by applying ultrasound to the first metal nanoparticle (100) and the potassium carbonate solution. For example, the density of the first metal nanoparticle (100) in the first metal solution can be 2 OD based on optical density. Accordingly, the citrate functional group (10) provided on the surface of the first metal nanoparticle (100) can be replaced with a carbonate functional group (20). Accordingly, the ionicity of the surface of the first metal nanoparticle (100) is reduced, so that the first antibody (1), described later, can be stably bound to the surface of the first metal nanoparticle (100). Accordingly, the reproducibility of the manufacturing process of the first detection material (110), described later, can be improved.

[0048] The step of preparing the second metal solution may include: preparing a second base metal solution comprising the second metal nanoparticles (200) and a hexadecyltrimethylammonium bromine solution; preparing a second preliminary metal solution by substituting the hexadecyltrimethylammonium bromine solution in the second base metal solution with a solution containing citric acid; and preparing the second metal solution by substituting the solution containing citric acid in the second preliminary metal solution with a potassium carbonate solution.

[0049] In the step of preparing the second base metal solution, the second metal nanoparticle (200) may be rod-shaped and may contain gold. For example, the second metal nanoparticle (200) may be blue. And, the hexatecyltrimethylammonium bromine solution may provide hexatecyltrimethylammonium bromine functional groups on the surface of the second metal nanoparticle (200).

[0050] And, in the step of preparing the second preliminary metal solution by replacing the hexatecyltrimethylammonium bromine solution in the second base metal solution with the solution containing citric acid, the hexatecyltrimethylammonium bromine solution in the second base metal solution can be easily replaced with the solution containing citric acid after being replaced with triple distilled water and PBS buffer solution by centrifuging the second base metal solution. For example, the centrifugation can be performed at 8,000 rf for 10 minutes. Subsequently, the second preliminary metal solution containing the second metal nanoparticles (200) and the solution containing citric acid can be easily prepared by applying ultrasound to the second metal nanoparticles (200) and the solution containing citric acid. As a result, the hexatecyltrimethylammonium bromine functional group provided on the surface of the second metal nanoparticle (200) can be substituted with a citrate functional group (10). For example, the solution containing the citric acid may be a trisodium citrate dihydrate solution.

[0051] In addition, in the step of preparing the second metal solution by replacing the solution containing citric acid in the second preliminary metal solution with the potassium carbonate solution, the solution containing citric acid in the second preliminary metal solution can be easily replaced with the potassium carbonate solution by centrifuging the second preliminary metal solution. For example, the centrifugation can be performed at 7,000 rf for 10 minutes. Subsequently, the second metal solution containing the second metal nanoparticles (200) and the potassium carbonate solution can be easily prepared by applying ultrasound to the second metal nanoparticles (200) and the potassium carbonate solution. For example, the density of the second metal nanoparticles (200) in the second metal solution can be 3 OD based on optical density. Accordingly, the citrate functional group (10) provided on the surface of the second metal nanoparticles (200) can be replaced with a carbonate functional group (20). Accordingly, the ionicity of the surface of the second metal nanoparticle (200) is reduced, so that the second antibody (2), described later, can be stably bound to the surface of the second metal nanoparticle (200). Accordingly, the reproducibility of the manufacturing process of the second detection material (210), described later, can be improved.

[0052] The step of preparing the third metal solution may include the step of preparing a third base metal solution comprising the third metal nanoparticles (300) and a solution comprising citric acid, and the step of preparing the first metal solution by replacing the solution containing citric acid in the third base solution with a potassium carbonate solution.

[0053] In the step of preparing the third base metal solution, the third metal nanoparticle (300) may be spherical in shape and may contain silver. For example, the third metal nanoparticle (300) may have a yellow color. And, the solution containing citric acid may provide citrate functional groups (10) on the surface of the third metal nanoparticle (300). For example, the solution containing citric acid may be a trisodium citrate dihydrate solution.

[0054] And, the particle drag coefficients of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300) can be controlled substantially equally.

[0055] When the first metal nanoparticle (100) and the third metal nanoparticle (300) are spherical in shape, the particle drag coefficient for the first metal nanoparticle (100) and the third metal nanoparticle (300) can be calculated using the following <Equation 1>. And when the second metal nanoparticle (200) is rod-shaped, the particle drag coefficient for the second metal nanoparticle (200) can be calculated using the following <Equation 2>. Accordingly, by using the following <Mathematical Formula 1> and the following <Mathematical Formula 2>, the diameters of the first metal nanoparticle (100) and the third metal nanoparticle (300) are controlled, and the length and width of the third metal nanoparticle (300) are controlled, so that the particle drag coefficients of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300) can be controlled to be substantially the same.

[0056] <Mathematical Formula 1>

[0057] f=6πμR

[0058] <Mathematical Formula 2>

[0059] f≒4πμL / (ln(L / a) + 0.193)

[0060] In the above <Mathematical Formula 1>, f is the particle drag coefficient, and μ is the viscosity of water (1 x 10⁻⁶ 3 Pa·s), and R is the radius of the first metal nanoparticle (100) and the third metal nanoparticle (300). And, in the above <Equation 2>, f is the particle drag coefficient, and μ is the viscosity of water (1x10 3 Pa·s), where L is the length of the second metal nanoparticle (200) and a is half the width of the second metal nanoparticle (200). For example, if the radius of the first metal nanoparticle (100) and the third metal nanoparticle (300) is controlled to 12.5 nm, the length of the second metal nanoparticle (200) can be controlled to 30 nm and the width of the second metal nanoparticle (200) can be controlled to 15 nm. Accordingly, the particle drag coefficients of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300) can be substantially the same. As a result, the fluidity (movement speed) of the first detection solution (120), the second detection solution (220), and the third detection solution (230), which are described later and each contain the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300), respectively, can be controlled equally.

[0061] In addition, in the step of preparing the third metal solution by replacing the solution containing citric acid in the third base solution with the potassium carbonate solution, the solution containing citric acid in the third base solution can be easily replaced with the potassium carbonate solution by centrifuging the third base solution. For example, the centrifugation can be performed at 14,000 rf for 10 minutes. Subsequently, the third metal solution containing the third metal nanoparticles (300) and the potassium carbonate solution can be easily prepared by applying ultrasound to the third metal nanoparticles (300) and the potassium carbonate solution. For example, the density of the third metal nanoparticles (300) in the third metal solution can be 2 OD based on optical density. Accordingly, the citrate functional group (10) provided on the surface of the third metal nanoparticles (300) can be replaced with a carbonate functional group (20). Accordingly, the ionicity of the surface of the third metal nanoparticle (300) is reduced, so that it can be stably bound to the surface of the third metal nanoparticle (300) with the third antibody (3) described later. Accordingly, the reproducibility of the manufacturing process of the third detection material (310) described later can be improved.

[0062] Referring to FIG. 1 and FIG. 2, a first detection solution (120) is prepared (S140) comprising a first detection substance (110) in which the first antibody (1) and albumin (4) are bound to the surface of the first metal nanoparticle (100) of the first metal solution, a second detection solution (220) is prepared (S150) comprising a second detection substance (210) in which the second antibody (2) and albumin (4) are bound to the surface of the second metal nanoparticle (200) of the second metal solution, and a third detection solution (320) is prepared (S160) comprising a third detection substance (310) in which the third antibody (3) and albumin (4) are bound to the surface of the third metal nanoparticle (300) of the third metal solution.

[0063] The step of preparing the first detection solution (120) may include: preparing the first metal solution, the first antibody solution containing the first antibody (1), and the albumin solution containing the albumin (4); mixing the first metal solution, the first antibody solution, and the albumin solution to prepare a first preliminary detection solution containing the first detection substance (110); and preparing the first detection solution (120) by replacing the supernatant with a running buffer (5) by centrifuging the first preliminary detection solution.

[0064] In the step of preparing the first metal solution, the first antibody solution containing the first antibody (1), and the albumin solution containing the albumin (4), the first antibody (1) in the first antibody solution may be bound, for example, to alpha-fetoprotein (AFP), which is the first substance to be detected. And, the albumin (4) in the albumin solution may be bound to the surface of the first metal nanoparticle (100) to prevent aggregation between adjacent first metal nanoparticles (100). For example, the albumin (4) may be bovine serum albumin (BSA).

[0065] In addition, in the step of preparing the first preliminary detection solution containing the first detection substance (110) by mixing the first metal solution, the first antibody solution, and the albumin solution, the first antibody (1) of the first antibody solution can be stably bound to the carbonate functional group (20) provided on the surface of the first metal nanoparticle (100) of the first metal solution by a method of providing the first antibody solution to the first metal solution and mixing and aging. Furthermore, by additionally providing the albumin solution and mixing and aging, the albumin (4) can be stably bound to the carbonate functional group (20) provided on the surface of the first metal nanoparticle (100), so that the first detection substance (110) in which the first antibody (1) and the albumin (4) are bound can be easily formed on the surface of the first metal nanoparticle (100). For example, the aging can be performed for 30 minutes at room temperature.

[0066] And, in the step of preparing the first detection solution (120) by replacing the supernatant with the running buffer (5) by centrifuging the first preliminary detection solution, the first supernatant generated by centrifuging the first preliminary detection solution may be replaced with a second albumin solution having a lower concentration of albumin (4) than that of the albumin solution. Accordingly, the first antibody (1) that is not bound to the surface of the first metal nanoparticle (100) within the first preliminary detection solution may be removed. Subsequently, the second supernatant generated by further centrifuging under the same conditions may be replaced with the running buffer (5) to prepare the first detection solution (120) containing the first detection substance (110) and the running buffer (5). For example, centrifugation may be performed at 5,000 rf for 10 minutes. For example, the running buffer (5) may include Tween 20, sucrose, and the second albumin solution. Accordingly, the density of the first metal nanoparticle (100) in the first detection solution (120) may be increased. For example, the density of the first metal nanoparticle (100) in the first detection solution (120) may be 10 OD based on optical density.

[0067] The step of preparing the second detection solution (220) may include: preparing the second metal solution, the second antibody solution containing the second antibody (2), and the albumin solution containing the albumin (4); mixing the second metal solution, the second antibody solution, and the albumin solution to prepare a second preliminary detection solution containing the second detection substance (210); and preparing the second detection solution (220) by replacing the supernatant with a running buffer (5) by centrifuging the second preliminary detection solution.

[0068] In the step of preparing the first metal solution, the second antibody solution containing the second antibody (2), and the albumin solution containing the albumin (4), the second antibody (2) in the second antibody solution may be bound, for example, to neuro-specific enolase (NSE), which is the second substance to be detected. And, the albumin (4) in the albumin solution may be bound to the surface of the second metal nanoparticle (200) to prevent aggregation between adjacent second metal nanoparticles (200). For example, the albumin (4) may be bovine serum albumin (BSA).

[0069] In addition, in the step of preparing the second preliminary detection solution containing the second detection substance (210) by mixing the second metal solution, the second antibody solution, and the albumin solution, the second antibody (2) of the second antibody solution can be stably bound to the carbonate functional group (20) provided on the surface of the second metal nanoparticle (200) of the second metal solution by a method of providing the second antibody solution to the second metal solution and mixing and aging. Furthermore, by additionally providing the albumin solution and mixing and aging, the albumin (4) can be stably bound to the carbonate functional group (20) provided on the surface of the second metal nanoparticle (200), so that the second detection substance (210) with the second antibody (2) and the albumin (4) bound can be easily formed on the surface of the second metal nanoparticle (200). For example, the aging can be performed for 30 minutes at room temperature.

[0070] And, in the step of preparing the second detection solution (220) by replacing the supernatant with the running buffer (5) by centrifuging the second preliminary detection solution, the first supernatant generated by centrifuging the second preliminary detection solution may be replaced with a second albumin solution having a lower concentration of albumin (4) than that of the albumin solution. Accordingly, the second antibody (2) that is not bound to the surface of the second metal nanoparticle (200) within the second preliminary detection solution may be removed. Subsequently, the second supernatant generated by further centrifuging under the same conditions may be replaced with the running buffer (5) to prepare the second detection solution (220) containing the second detection substance (210) and the running buffer (5). For example, centrifugation may be performed at 4,000 rf for 10 minutes. For example, the running buffer (5) may include Tween 20, sucrose, and the second albumin solution. Accordingly, the density of the second metal nanoparticles (200) in the second detection solution (220) may be increased. For example, the density of the second metal nanoparticles (200) in the second detection solution (220) may be 15 OD based on optical density.

[0071] The step of preparing the third detection solution (320) may include: preparing the third metal solution, the third antibody solution containing the third antibody (3), and the albumin solution containing the albumin (4); mixing the third metal solution, the third antibody solution, and the albumin solution to prepare the third preliminary detection solution containing the third detection substance (310); and preparing the third detection solution (320) by replacing the supernatant with a running buffer (5) by centrifuging the third preliminary detection solution.

[0072] In the step of preparing the third metal solution, the third antibody solution containing the third antibody (3), and the albumin solution containing the albumin (4), the third antibody (3) in the first antibody solution may be bound, for example, to a carcino-embryonic antigen (CEA), which is the third substance to be detected. And, the albumin (4) in the albumin solution may be bound to the surface of the third metal nanoparticle (300) to prevent aggregation between adjacent third metal nanoparticles (300). For example, the albumin (4) may be bovine serum albumin (BSA).

[0073] In addition, in the step of preparing the third preliminary detection solution containing the third detection substance (300) by mixing the third metal solution, the third antibody solution, and the albumin solution, the third antibody (3) of the third antibody solution can be stably bound to the carbonate functional group (20) provided on the surface of the third metal nanoparticle (300) of the third metal solution by a method of providing the third antibody solution to the third metal solution and mixing and aging. Furthermore, by additionally providing the albumin solution and mixing and aging, the albumin (4) can be stably bound to the carbonate functional group (20) provided on the surface of the third metal nanoparticle (300), thereby easily forming the third detection substance (310) in which the third antibody (3) and the albumin (4) are bound to the surface of the third metal nanoparticle (300). For example, the aging can be performed for 30 minutes at room temperature.

[0074] And, in the step of preparing the third detection solution (320) by replacing the supernatant with the running buffer (5) by centrifuging the first preliminary detection solution, the first supernatant generated by centrifuging the third preliminary detection solution may be replaced with a second albumin solution having a lower concentration of albumin (4) than that of the albumin solution. Accordingly, the third antibody (3) that is not bound to the surface of the third metal nanoparticle (300) within the third preliminary detection solution may be removed. Subsequently, the second supernatant generated by further centrifuging under the same conditions may be replaced with the running buffer (5) to prepare the third detection solution (320) containing the third detection substance (310) and the running buffer (5). For example, centrifugation may be performed at 12,000 rf for 10 minutes. For example, the running buffer (5) may include Tween 20, sucrose, and the second albumin solution. Accordingly, the density of the third metal nanoparticle (300) in the third detection solution (320) may be improved. For example, the density of the third metal nanoparticle (300) in the third detection solution (320) may be 10 OD based on optical density.

[0075] Referring to FIGS. 1 and FIGS. 2, a multi-detection solution is prepared by mixing the first detection solution (120), the second detection solution (220), and the third detection solution (320) (S170). Accordingly, the multi-detection solution may include the first detection solution (120), the second detection solution (220), and the third detection solution (320).

[0076] As described above, the first detection solution (120) may include the first detection material (110) and the running buffer (5) provided on the surface of the first metal nanoparticle (100) with the first antibody (1) and the albumin (4). The second detection solution (220) may include the second detection material (210) and the running buffer (5) provided on the surface of the second metal nanoparticle (200) with the second antibody (2) and the albumin (4). The third detection solution (320) may include the third detection material (310) and the running buffer (5) provided on the surface of the third metal nanoparticle (300) with the third antibody (3) and the albumin (4).

[0077] Additionally, the particle drag coefficients of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300) within the multi-detection solution may be the same. Accordingly, the fluidity of the first detection solution (120), the second detection solution (220), and the third detection solution (320) within the multi-detection solution may be the same. Accordingly, when the multi-detection solution, which is a mixture of the first detection solution (120), the second detection solution (220), and the third detection solution (320), is provided to the junction of the multi-detection device described later, the speed at which the first detection solution (120), the second detection solution (220), and the third detection solution (320) within the multi-detection solution move from the junction of the multi-detection device to the test line of the multi-detection device may be the same. Accordingly, the accuracy of the color displayed on the test line of the multi-detection device can be improved.

[0078] Furthermore, the first detection substance (110), the second detection substance (210), and the third detection substance (310) within the multi-detection solution may each include the first antibody (1), the second antibody (2), and the third antibody (3) stably bound to the surfaces of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300), respectively. Accordingly, the mixed detection substance provided to the sample portion of the multi-detection device described later can be easily combined with at least one of the first detection substance (110), the second detection substance (210), or the third detection substance (310) within the multi-detection solution provided to the junction portion of the multi-detection device, and can be provided to the test line of the multi-detection device. Accordingly, the sensitivity of the color displayed on the test line of the multi-detection device can be improved.

[0079] In conclusion, the method for preparing the multi-detection solution according to an embodiment of the present application comprises the steps of: preparing the first metal solution containing the first metal nanoparticle (100); preparing the second metal solution containing the second metal nanoparticle (200); preparing the third metal solution containing the third metal nanoparticle (300); preparing the first detection solution (320) containing the first detection substance (310) in which the first antibody (1) and the albumin (4) are bound to the surface (100) of the first metal nanoparticle in the first metal solution; preparing the second detection solution (220) containing the second detection substance (210) in which the second antibody (2) and the albumin (4) are bound to the surface of the second metal nanoparticle (200) in the second metal solution; and the third antibody (3) and the albumin (4) on the surface of the third metal nanoparticle (300) in the third metal solution. The method may include the step of preparing the third detection solution (320) comprising the third detection substance (310) combined with albumin (4), and the step of preparing the multiple detection solution by mixing the first detection solution (120), the second detection solution (220), and the third detection solution (320).

[0080] In the steps of preparing the first metal solution, preparing the second metal solution, and preparing the third metal solution, the particle drag coefficients of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300) can be controlled equally. Accordingly, the fluidity of the first detection solution (120), the second detection solution (220), and the third detection solution (320) can be controlled equally. Accordingly, when the multi-detection solution comprising the first detection solution (120), the second detection solution (220), and the third detection solution (320) is applied to the multi-detection device described later, the accuracy of the color displayed on the test line of the multi-detection device can be improved depending on the mixed detection substance provided to the multi-detection device.

[0081] In addition, in the steps of preparing the first metal solution, preparing the second metal solution, and preparing the third metal solution, the citrate functional group (10) provided on the surface of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300) may be substituted with a carbonate functional group (20). Accordingly, the ionicity of the surface of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300) is reduced, so that the first antibody (1), the second antibody (2), and the third antibody (3) can be stably bound to the carbonate functional group (20) on the surface of the first metal nanoparticle (100), the second metal nanoparticle (200), and the third metal nanoparticle (300), respectively. Accordingly, the reproducibility of the manufacturing method of the first detection material (110), the second detection material (210), and the third detection material (310) can be improved. Furthermore, when the multi-detection solution comprising the first detection material (110), the second detection material (210), and the third detection material (310) is applied to the multi-detection device described later, the sensitivity of the color displayed on the test line of the multi-detection device can be improved depending on the mixed detection material provided to the multi-detection device.

[0083] Hereinafter, the multi-detection device described above using the multi-detection solution described above is explained.

[0084] The multi-detection device may include a sample portion provided with a mixed substance to be detected comprising a plurality of different substances to be detected, a conjugate portion provided with a multi-detection solution comprising a plurality of different detection substances that correspond to and are combined with the substances to be detected within the mixed substance to be detected, and a test portion provided with an analysis substance in which the substances to be detected and the detection substances are combined. As a specific example, the multi-detection device may include the sample portion having a paper sample pad attached on a substrate which is a polyester adhesive film, the conjugate portion having a paper bonding pad attached, and the test portion having a paper test membrane attached. More specifically, the distance between the sample portion and the test portion on the substrate may be controlled to 5 mm, the test portion and the absorption portion may overlap by 1 mm, the sample portion and the conjugate portion may overlap by 2 mm, and the conjugate portion and the test portion may overlap by 3 mm. Accordingly, the mixed substance to be detected provided in the sample section can be easily transferred to the junction section, and the substance to be analyzed can be easily transferred from the junction section to the test line of the test section.

[0085] The mixed detection substance provided to the sample portion of the multi-detection device may include at least one of a first detection substance, a second detection substance, and a third detection substance. For example, the first detection substance may be alpha-fetoprotein (AFP). For example, the second detection substance may be neuro-specific enolase (NSE). For example, the third detection substance may be carcino-embryonic antigen (CEA).

[0086] Furthermore, the multi-detection solution provided to the junction of the multi-detection device may include a first detection substance bound to the first substance to be detected, a second detection substance bound to the second substance to be detected, and a third detection substance bound to the third substance to be detected. Specifically, the first detection substance may include a first metal nanoparticle, and a first antibody and albumin provided on the surface of the first metal nanoparticle. The second detection substance may include a second metal nanoparticle, and a second antibody and albumin provided on the surface of the second metal nanoparticle. The third detection substance may include a third metal nanoparticle, and a third antibody and albumin provided on the surface of the third metal nanoparticle. For example, the first metal nanoparticle and the second metal nanoparticle may be the same metal. For example, the third metal nanoparticle may be a different metal from the first metal nanoparticle and the second metal nanoparticle. For example, the first metal nanoparticle and the second metal nanoparticle may be gold nanoparticles. For example, the third metal nanoparticle may be silver nanoparticles. For example, the first metal nanoparticle and the third metal nanoparticle may be spherical in shape. For example, the second metal nanoparticle may be rod-shaped. For example, the first metal nanoparticle may be red. For example, the second metal nanoparticle may be blue. For example, the third metal nanoparticle may be yellow. For example, the particle drag coefficients of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle may be substantially the same. For example, the diameter of the first metal nanoparticle and the third metal nanoparticle may be 25 nm. For example, the length of the second metal nanoparticle may be 30 nm and the width may be 15 nm.

[0087] Furthermore, the analytical substance provided to the test line of the test section of the multi-detection device may include at least one of a first analytical substance bound to the first antibody of the first detection substance and the first detection substance, a second analytical substance bound to the second antibody of the second detection substance and the second detection substance, and a third analytical substance bound to the third detection substance and the third antibody of the third detection substance. Additionally, the test line of the test section may include the first antibody, the second antibody, and the third antibody that are respectively bound to the first detection substance of the first analytical substance, the second detection substance of the second analytical substance, and the third detection substance of the third analytical substance. For example, the test line may be a single line, and the color of the test line may be achromatic. In addition, the test section further includes a control line, and the control line may include a secondary antibody that binds to at least one of the first substance to be detected, the second substance to be detected, and the third substance to be detected. For example, the control line may be a single line, and the color of the control line may be black.

[0088] Accordingly, depending on the detection substance (the first to third detection substance) in the mixed detection substance provided in the sample section of the multi-detection device and the detection substance (the first to third detection substance) in the multi-detection solution provided in the junction section, the color of the test line of the test section may be displayed in different colors that are visually distinguishable.

[0089] As described below with reference to FIG. 3, when the mixed detection substance includes at least one of the first detection substance, the second detection substance, and the third detection substance, and the multi-detection solution includes the first detection substance, the second detection substance, and the third detection substance, a total of seven different colors may be displayed on the test line of the multi-detection device. For example, when the first detection substance is provided to the sample portion of the multi-detection device, the test line of the multi-detection device may be displayed in red by the first detection substance (e.g., red) provided to the junction portion of the multi-detection device. And when the second detection substance is provided to the sample portion of the multi-detection device, the test line of the multi-detection device may be displayed in blue by the second detection substance (e.g., blue) provided to the junction portion of the multi-detection device. Furthermore, when the third detection substance is provided to the sample portion of the multi-detection device, the test line of the multi-detection device may be displayed in yellow by the third detection substance (e.g., yellow) provided to the junction portion of the multi-detection device. Also, when the first detection substance and the second detection substance are mixed and provided to the sample portion of the multi-detection device, the test line of the multi-detection device may be displayed in purple, which is a color combination of the first detection substance and the second detection substance, by the first detection substance (e.g., red) and the second detection substance (e.g., blue) provided to the junction portion of the multi-detection device.In addition, when the first detection substance and the third detection substance are mixed and provided to the sample portion of the multi-detection device, the test line of the multi-detection device may be displayed in orange, which is a color combination of the first detection substance and the third detection substance, by the first detection substance (e.g., red) and the third detection substance (e.g., yellow) provided to the junction portion of the multi-detection device. In addition, when the second detection substance and the third detection substance are mixed and provided to the sample portion of the multi-detection device, the test line of the multi-detection device may be displayed in green, which is a color combination of the second detection substance and the third detection substance, by the second detection substance (e.g., blue) and the third detection substance (e.g., yellow) provided to the junction portion of the multi-detection device. And, when the first detection substance, the second detection substance, and the third detection substance are mixed and provided to the multi-detection device, the test line of the multi-detection device may be displayed in black, which is a color combination of the first detection substance, the second detection substance, and the third detection substance, by the first detection substance (e.g., red), the second detection substance (e.g., blue), and the third detection substance (e.g., yellow) provided to the junction of the multi-detection device.

[0090] In addition, the particle drag coefficients of the first metal nanoparticle of the first detection material, the second metal nanoparticle of the second detection material, and the third nanoparticle of the third detection material within the multi-detection solution provided to the junction of the multi-detection device may be the same. Accordingly, when the first detection material, the second detection material, and the third detection material are provided to the sample portion of the multi-detection device, the speed at which the first analysis material, the second analysis material, and the third analysis material move from the junction of the multi-detection device to the test portion is controlled to be the same, thereby improving the accuracy of the color displayed on the test line of the multi-detection device.

[0091] In conclusion, the multi-detection device according to an embodiment of the present application may include a sample portion provided with a mixed substance to be detected comprising a plurality of different substances to be detected; a conjugate portion provided with a multi-detection solution that receives the mixed substance to be detected from the sample portion and includes a plurality of different detection substances that correspond to and are combined with the substances to be detected within the mixed substance to be detected; and a test portion provided with an analysis substance in which the substances to be detected and the detection substances are combined.

[0092] When the above mixed detection substance includes at least one of the first detection substance, the second detection substance, and the third detection substance, and the multi-detection solution includes the first detection substance, the second detection substance, and the third detection substance, a total of seven different colors may be displayed on the test line of the multi-detection device.

[0093] In addition, the particle drag coefficients of the first metal nanoparticle of the first detection material, the second metal nanoparticle of the second detection material, and the third nanoparticle of the third detection material within the multi-detection solution provided to the junction of the multi-detection device may be the same. Accordingly, when the first detection material, the second detection material, and the third detection material are provided to the sample portion of the multi-detection device, the speed at which the first analysis material, the second analysis material, and the third analysis material move from the junction of the multi-detection device to the test portion is controlled to be the same, thereby improving the accuracy of the color displayed on the test line of the multi-detection device.

[0095] Hereinafter, specific experimental examples and characteristic evaluation results of a multi-detection solution according to an embodiment of the present invention and a multi-detection device to which the multi-detection solution is applied are described.

[0097] First base metal solution according to Experimental Example 1

[0098] A solution of gold(III) chloride trihydrate (1 mM, 100 mL) was provided in a 3-neck round-bottom flask washed with aqua regia and triple distilled water and boiled. Then, a solution of trisodium citrate dihydrate (17.5 mM, 10 mL) was additionally provided and stirred to react. At this time, after the pale yellow solution turned into a deep pink / red color, the mixture was stirred for an additional 30 minutes to synthesize first metal nanoparticles (gold nanoparticles, spherical shape, red, diameter 25 nm), and a first base metal solution containing the first metal nanoparticles and the trisodium citrate dihydrate solution was prepared.

[0100] First metal solution according to Experimental Example 1

[0101] A first base metal solution was prepared according to Experimental Example 1.

[0102] The first base metal solution was centrifuged (9,000 rf, 10 min) to replace the trisodium citrate dihydrate solution in the first base metal solution with a potassium carbonate solution, and sonicated for 10 seconds to prepare a first metal solution containing first metal nanoparticles (based on optical density, 2 OD) and the potassium carbonate solution.

[0104] First detection solution according to Experimental Example 1

[0105] A first metal solution according to Experimental Example 1 was prepared, a first antibody solution containing a first antibody that binds to alpha-fetoprotein (AFP) was prepared, and a 10% BSA solution (w / v in 1 x PBS) was prepared to prevent aggregation between first metal nanoparticles within the first metal solution.

[0106] After mixing the first metal solution and the first antibody solution so that the concentration of the first antibody bound to the surface of the first metal nanoparticles of the first metal solution is 10 μg / mL, the mixture was aged at room temperature for 30 minutes, and after additionally providing and mixing the 10% BSA solution (w / v in 1 x PBS) so that the ratio of the aged mixture of the first metal solution and the first antibody solution to the 10% BSA solution (w / v in 1 x PBS) is 10:1, the mixture was aged at room temperature for 30 minutes.

[0107] Then, in order to remove the first antibody not bound to the surface of the first metal nanoparticle, a mixed solution containing the first metal solution, the first antibody solution, and the 10% BSA solution was centrifuged (5,000 rf, 10 min), and the supernatant was replaced with a 1% BSA solution (w / v in 1 x PBS). After further centrifuging under the same centrifugation conditions, the supernatant was replaced with a running buffer solution (0.25% Tween 20, 10% sucrose, 1% BSA (w / v 1 x PBS)), thereby preparing a first detection solution containing the first metal nanoparticle (10 OD based on optical density) and the running buffer solution.

[0109] Second base metal solution according to Experimental Example 1

[0110] A seed solution and a growth solution were prepared to synthesize second metal nanoparticles.

[0111] The above seed solution was prepared by mixing a hexadecyltrimethylammonium bromine solution (CTAB, 200 mM, 5 mL) and a gold(III) chloride trihydrate solution (0.5 mM, 5 mL), then adding a sodium borohydroxide solution (NaBH4, 10 mM, 0.6 mL) and stirring for 2 minutes.

[0112] The above growth solution was prepared by mixing the above hexadecyltrimethylammonium bromine solution (CTAB, 200 mM) and sodium 3-methyl salicylate solution (Na-creso, 35.35 mM) in a 1:1 ratio and stirring the mixture (125 mL) at 60°C, then adding a silver nitrate solution (4 mM, 3 mL) and stirring for 15 minutes, then adding a gold chloride (III) trihydrate solution (1 mM, 125 mL) and confirming that the color of the solution has changed, then adding an L-ascorbic acid solution (0.5 mL) and stirring for 15 minutes.

[0113] Then, the seed solution and the growth solution were mixed and stirred for 30 seconds, and then reacted in an oven at 30°C for 12 hours to synthesize the second metal nanoparticles (gold nanoparticles, rod-shaped, blue, length 30 nm, width 15 nm), thereby preparing a solution containing the second metal nanoparticles. Then, the solution containing the second metal nanoparticles was centrifuged (11,000 rf, 90 min, twice) to partially replace the unreacted hexadecyltrimethylammonium bromine solution with triple distilled water, thereby preparing a second base metal solution containing the second metal nanoparticles and the hexadecyltrimethylammonium bromine solution (2.5 mM).

[0115] Second metal solution according to Experimental Example 1

[0116] A second base metal solution was prepared according to Experimental Example 1.

[0117] The second base metal solution was centrifuged (8,000 rf, 10 min) and the process of sequentially replacing the hexadecyltrimethylammonium bromine solution in the second base metal solution with triple distilled water, PBS buffer solution (pH 8.0, SDS 0.3% w / v), and a solution in which the triple distilled water and the PBS buffer solution were mixed in a 1:1 ratio was repeated three times, and finally, the solution was replaced with trisodium citrate dihydrate to prepare a second preliminary metal solution containing the second metal nanoparticles and trisodium citrate dihydrate solution.

[0118] Then, the second pre-metal solution was centrifuged (7,000 rf, 10 min) to replace the trisodium citrate dihydrate solution in the second pre-metal solution with a potassium carbonate solution, and sonicated for 10 seconds to prepare a second metal solution containing second metal nanoparticles (based on optical density, 3 OD) and the potassium carbonate solution.

[0120] Second detection solution according to Experimental Example 1

[0121] A second metal solution was prepared according to Experimental Example 1, a second antibody solution containing a second antibody that binds to neuron-specific enolase (NSE) was prepared, and a 10% BSA solution (w / v in 1 x PBS) was prepared to prevent aggregation between second metal nanoparticles within the second metal solution.

[0122] After mixing the second metal solution and the second antibody solution so that the concentration of the second antibody bound to the surface of the second metal nanoparticles of the second metal solution is 10 μg / mL, the mixture was aged at room temperature for 30 minutes, and after additionally providing and mixing the 10% BSA solution (w / v in 1 x PBS) so that the ratio of the aged mixture of the second metal solution and the second antibody solution to the 10% BSA solution (w / v in 1 x PBS) is 10:1, the mixture was aged at room temperature for 30 minutes.

[0123] Then, in order to remove the first antibody not bound to the surface of the second metal nanoparticle, a mixed solution containing the second metal solution, the second antibody solution, and the 10% BSA solution was centrifuged (4,000 rf, 10 min), and the supernatant was replaced with a 1% BSA solution (w / v in 1 x PBS). After further centrifugation under the same centrifugation conditions, the supernatant was replaced with a running buffer solution (0.25% Tween 20, 10% sucrose, 1% BSA (w / v 1 x PBS)), thereby preparing a second detection solution containing the second metal nanoparticle (15 OD based on optical density) and the running buffer solution.

[0125] Third base metal solution according to Experimental Example 1

[0126] In a 3-neck round-bottom flask washed with aqua regia and triple distilled water A mixed solution (100 mL) of trisodium citrate dihydrate solution (5 mM) and tannic acid solution (0.025 mM) was provided and boiled, after which a silver nitrate solution (25 mM, 1 mL) was additionally provided and stirred to synthesize a third metal nanoparticle seed. Then, to grow the third metal nanoparticle seed, the temperature of the solution containing the third metal nanoparticle seed was cooled to 90°C, and then the trisodium citrate dihydrate solution (25 mM, 100 μL), the tannic acid solution (2.5 mM, 250 μL), and the silver nitrate solution (50 mM, 250 μL) were additionally provided at 1-minute intervals, and the reaction process was repeated 10 times for 15 minutes to grow the third metal nanoparticle seed into a third metal nanoparticle (gold nanoparticle, spherical shape, yellow, diameter 25 nm), thereby preparing a solution containing the third metal nanoparticle. Then, after cooling the solution containing the third metal nanoparticles to room temperature, centrifugation (18,000 rf, 10 min, twice) was performed to replace the tannic acid solution in the solution containing the third metal nanoparticles with the trisodium citrate dihydrate, thereby preparing a third base solution containing the third metal nanoparticles and the trisodium citrate dihydrate (5 mM).

[0128] Third metal solution according to Experimental Example 1

[0129] A third base metal solution was prepared according to Experimental Example 1.

[0130] The third base metal solution was centrifuged (14,000 rf, 10 min) to replace the trisodium citrate dihydrate solution in the third base metal solution with a potassium carbonate solution, and sonicated for 10 seconds to prepare a third metal solution containing the third metal nanoparticles (based on optical density, 2 OD) and the potassium carbonate solution.

[0132] Third detection solution according to Experimental Example 1

[0133] A third metal solution was prepared according to Experimental Example 1, and a third antibody solution containing a third antibody that binds to carcino-embryonic antigen (CEA) was prepared, and a 10% BSA solution (w / v in 1 x PBS) was prepared to prevent aggregation between third metal nanoparticles within the third metal solution.

[0134] After mixing the third metal solution and the third antibody solution so that the concentration of the third antibody bound to the surface of the third metal nanoparticles of the third metal solution is 10 μg / mL, the mixture was aged at room temperature for 30 minutes, and after additionally providing and mixing the 10% BSA solution (w / v in 1 x PBS) so that the ratio of the aged mixture of the third metal solution and the third antibody solution to the 10% BSA solution (w / v in 1 x PBS) is 10:1, the mixture was aged at room temperature for 30 minutes.

[0135] Then, in order to remove the third antibody not bound to the surface of the third metal nanoparticle, a mixed solution containing the third metal solution, the third antibody solution, and the 10% BSA solution was centrifuged (12,000 rf, 10 min), and the supernatant was replaced with a 1% BSA solution (w / v in 1 x PBS). After further centrifuging under the same centrifugation conditions, the supernatant was replaced with a running buffer solution (0.25% Tween 20, 10% sucrose, 1% BSA (w / v 1 x PBS)), thereby preparing a third detection solution containing the third metal nanoparticle (10 OD based on optical density) and the running buffer solution.

[0137] Multiple detection solution according to Experimental Example 1

[0138] A multi-detection solution was prepared by mixing the first detection solution according to Experimental Example 1, the second detection solution according to Experimental Example 1, and the third detection solution according to Experimental Example 1.

[0140] Multi-detection device according to Experimental Example 1

[0141] A sample pad (0.5 cm x 3 cm) made of paper material was prepared as the sample part of the multi-detection device, a bonding pad (0.5 cm x 1 cm) made of paper material was prepared as the bonding part, a test membrane (0.5 cm x 2.5 cm) made of paper material was prepared as the test part, an absorbent pad (0.5 cm x 3.8 cm) made of paper material was prepared as the absorbent part, and a polyester adhesive film was prepared as the substrate.

[0142] The sample pad, the bonding pad, the test membrane, and the absorption pad were attached to the substrate. Specifically, the sample pad and the test membrane were attached to the substrate such that the distance between them was 5 mm, the test membrane and the absorption pad were attached such that they overlapped by 1 mm, the sample pad and the bonding pad were attached such that they overlapped by 2 mm, and the bonding pad and the test membrane were attached such that they overlapped by 3 mm.

[0143] Then, a blocking solution (blocking buffer solution, pH 7.4, 1% BSA, 1% sucrose, 0.1% sodium azide, w / v 1x PBS) and a multiple detection solution according to Experimental Example 1 were sequentially supplied to the conjugation pad, and an antibody mixture solution containing a first antibody, a second antibody, and a third antibody was dispensed onto the test membrane to form a test line, and a secondary antibody solution containing a secondary antibody was dispensed onto the test membrane to form a control line spaced apart from the test line, thereby fabricating a multiple detection device.

[0145] FIG. 3 is a schematic diagram and image for explaining the colors displayed on the test line of the multi-detection device according to Experimental Example 1 according to the multi-detection device according to Experimental Example 1 of the present invention, depending on the substance to be detected provided to the multi-detection device according to Experimental Example 1 of the present invention.

[0146] Referring to FIG. 3(a), the operation of the multi-detection device according to Experimental Example 1 is schematically illustrated before and after providing an antigen to the sample pad of the multi-detection device according to Experimental Example 1. Referring to FIG. 3(b), this is an image for comparing the colors displayed on the test line of the multi-detection device according to Experimental Example 1 when no antigen is provided to the sample pad of the multi-detection device according to Experimental Example 1, and when a single antigen is provided. Specifically, antigen A is alpha-fetoprotein (AFP), antigen B is neuro-specific enolase (NSE), and antigen C is carcino-embryonic antigen (CEA). Referring to FIG. 3(c), this is an image for comparing the colors displayed on the test line of the multi-detection device according to Experimental Example 1 when multiple antigens are provided to the sample pad of the multi-detection device according to Experimental Example 1.

[0147] As can be seen in FIGS. 3 (a) to (c), when no antigen is provided to the multi-detection device according to Experimental Example 1, the test line of the multi-detection device is displayed in achromatic color. That is, since no antigen is provided to the multi-detection device, it can be seen that there is no color change in the test line of the multi-detection device.

[0148] And, when the antigen A is provided to the multi-detection device, it can be seen that the test line of the multi-detection device is marked in red by the first detection material (red) provided to the junction of the multi-detection device.

[0149] And, when the antigen B is provided to the multi-detection device, it can be seen that the test line of the multi-detection device is marked in blue by the second detection material (blue) provided to the junction of the multi-detection device.

[0150] And, when the antigen C is provided to the multi-detection device, it can be seen that the test line of the multi-detection device is marked yellow by the third detection material (yellow) provided to the junction of the multi-detection device.

[0151] And, when the antigen A and the antigen B are mixed and provided to the multi-detection device, it can be seen that the test line of the multi-detection device is displayed in purple, which is a color combination of the first detection substance and the second detection substance, by the first detection substance (red) and the second detection substance (blue) provided to the junction of the multi-detection device.

[0152] And, when the antigen A and the antigen C are mixed and provided to the multi-detection device, it can be seen that the test line of the multi-detection device is displayed in orange, which is the color combination of the first detection substance and the third detection substance, by the first detection substance (red) and the third detection substance (yellow) provided to the junction of the multi-detection device.

[0153] And, when the antigen B and the antigen C are mixed and provided to the multi-detection device, it can be seen that the test line of the multi-detection device is displayed in green, which is the color combination of the second detection material and the third detection material, by the second detection material (blue) and the third detection material (yellow) provided to the junction of the multi-detection device.

[0154] And, when the antigen A, the antigen B, and the antigen C are mixed and provided to the multi-detection device, it can be seen that the test line of the multi-detection device is displayed in black, which is a color combination of the first detection substance, the second detection substance, and the third detection substance, by the first detection substance (red), the second detection substance (blue), and the third detection substance (yellow) provided to the junction of the multi-detection device.

[0156] Figure 4 is a photograph and graph for comparing the flowability of the first detection solution, the second detection solution, and the third detection solution within the multi-detection solution provided at the joint of the multi-detection device according to Experimental Example 1 of the present invention.

[0157] Referring to FIG. 4(a), UV-vis spectroscopy analysis was performed on the first detection solution (AuNPs), the second detection solution (AuNRs), the third detection solution (AgNPs), and the multiple detection solution provided to the junction of the multiple detection device according to Experimental Example 1, and referring to FIG. 4(b), the first detection substance in the first detection solution, the second detection substance in the second detection solution, and the third detection substance in the third detection solution provided to the junction of the multiple detection device were photographed by TEM (scale bar, 50 nm). Referring to FIG. 4(c), with the test line and control line of the test section of the multi-detection device not formed, the first detection solution was provided to the joint of the multi-detection device to check the flowability of the first detection solution (i), the second detection solution was provided to the joint of the multi-detection device to check the flowability of the second detection solution (ii), and the third detection solution was provided to the joint of the multi-detection device to check the flowability of the third detection solution. Referring to FIG. 4(d), with the test line and control line of the test section of the multi-detection device not formed, a mixed detection solution in which the first detection solution and the second detection solution are mixed is provided to the joint of the multi-detection device to verify the flowability of the first detection solution and the second detection solution (i), a mixed detection solution in which the second detection solution and the third detection solution are mixed is provided to verify the flowability of the second detection solution and the third detection solution (ii), a mixed detection solution in which the first detection solution and the third detection solution are mixed is provided to verify the flowability of the first detection solution and the third detection solution (iii), and a multi-detection solution is provided to verify the flowability of the multi-detection solution (iv).

[0158] As can be seen in FIG. 4(a), the first detection solution is red due to the first detection substance in the first detection solution, the second detection solution is blue due to the second detection substance in the second detection solution, and the third detection solution is yellow due to the third detection substance in the third detection solution.

[0159] As can be seen in FIG. 4(b), the first metal nanoparticles of the first detection material and the third metal nanoparticles of the third detection material are spherical in shape, and the average diameter of the first metal nanoparticles and the third metal nanoparticles is 25 nm. In contrast, the second metal nanoparticles of the second detection material are rod-shaped, and the length of the second metal nanoparticles is 30 nm and the width is 15 nm.

[0160] As can be seen in Figures 4 (c) and (d), when at least two of the first detection solution, the second detection solution, and the third detection solution are mixed and provided to the junction of the multi-detection device, it can be seen that no color separation occurs because the movement speeds of the first detection solution, the second detection solution, and the third detection solution are the same. This factor is interpreted as being due to the fact that the drag coefficients of the first metal nanoparticles of the first detection material in the first detection solution, the second metal nanoparticles of the second detection material in the second detection solution, and the third metal nanoparticles of the third detection material in the third detection solution are the same.

[0162] Figure 5 is a photograph and graph for comparing the selectivity and sensitivity of the multi-detection device according to Experimental Example 1 according to the concentration of antigen provided to the multi-detection device according to Experimental Example 1 of the present invention.

[0163] Referring to FIG. 5(a), the concentration of antigen A (alpha-fetoprotein, AFP) provided to the sample portion of the multi-detection device according to Experimental Example 1 was controlled to 20 ng / mL to 500 ng / mL, and the color displayed on the test line of the multi-detection device was checked (scale bar, 5 mm) (i); the concentration of antigen B (neuron-specific enolase, NSE) was controlled to 20 ng / mL to 500 ng / mL, and the color displayed on the test line of the multi-detection device was checked (ii); and the concentration of antigen C (carcino-embryonic antigen, CEA) was controlled to 20 ng / mL to 500 ng / mL, and the color displayed on the test line of the multi-detection device was checked (iii). Referring to Fig. 5(b), the color intensity of each color displayed on the test line of the multi-detection device in Fig. 5(a) was measured using MATLAB (MathWorks, USA).

[0164] As can be seen in Figures 5 (a) and (b), as the concentrations of antigen A, antigen B, and antigen C provided to the multi-detection device increase, the color intensity of the color of the test line displayed on the multi-detection device increases.

[0165] In addition, when the concentrations of antigen A, antigen B, and antigen C provided to the multi-detection device are controlled to 20 ng / mL, the color intensity of the colors displayed on the test lines of the multi-detection device is at the levels of 1.52, 1.35, and 1.45, respectively, so it can be seen that it is difficult to distinguish the colors. In contrast, when the concentrations of antigen A, antigen B, and antigen C provided to the multi-detection device are controlled to 50 ng / mL or higher, it can be seen that the colors displayed on the test lines of the multi-detection device are clearly distinguishable.

[0166] Accordingly, in the multi-detection device according to the embodiment of the present application, the method of controlling the concentration of a substance to be detected provided to the sample portion of the multi-detection device to 50 ng / mL or higher is a method of improving the selectivity and sensitivity of the multi-detection device.

[0168] Figure 6 is a photograph for comparing the colors displayed on the test line of the multi-detection device according to Experimental Example 1 according to the mixed antigen provided to the multi-detection device according to Experimental Example 1 of the present invention.

[0169] Referring to FIG. 6(a), after providing a mixed antigen containing antigen A (alpha-fetoprotein; AFP, 200 ng / mL) and antigen B (neuron-specific enolase; NSE, 200 ng / mL) to the sample portion of the multi-detection device according to Experimental Example 1, the color displayed on the test line of the multi-detection device was checked; referring to FIG. 6(b), after providing a mixed antigen containing antigen B and antigen C (carcino-embryonic antigen; CEA, 200 ng / mL) to the sample portion of the multi-detection device, the color displayed on the test line of the multi-detection device was checked; referring to FIG. 6(c), after providing a mixed antigen containing antigen A and antigen C to the sample portion of the multi-detection device, the color displayed on the test line of the multi-detection device was checked; and referring to FIG. 6(d), the antigen After providing a mixed antigen comprising A, the above antigen B, and the above antigen C, the color displayed on the test line of the multi-detection device was checked.

[0170] As can be seen in FIG. 6(a), when the antigen A and the antigen B are mixed and provided to the multi-detection device, the test line of the multi-detection device is displayed in purple, which is the color combination of the first detection substance (red) and the second detection substance (blue).

[0171] As can be seen in Fig. 6(b), when the antigen B and the antigen C are mixed and provided to the multi-detection device, the test line of the multi-detection device is displayed in green, which is the color combination of the second detection substance (blue) and the third detection substance (yellow).

[0172] As can be seen in Fig. 6(c), when the antigen A and the antigen C are mixed and provided to the multi-detection device, the test line of the multi-detection device is displayed in orange, which is the color combination of the first detection substance (red) and the third detection substance (yellow).

[0173] As can be seen in Fig. 6(d), when the antigen A, antigen B, and antigen C are mixed and provided to the multi-detection device, the test line of the multi-detection device is displayed in black, which is a color combination of the first detection substance (red), the second detection substance (blue), and the third detection substance (yellow).

[0174] Accordingly, it can be seen that a total of seven different colors are displayed on the test line of the test section of the multi-detection device by means of a multi-detection solution (a first detection solution, a second detection solution, and a third detection solution) provided to the junction section of the multi-detection device according to an embodiment of the present application, depending on the mixed detection substance (at least one of a first detection substance, a second detection substance, and a third detection substance) provided to the sample section of the multi-detection device according to an embodiment of the present application.

[0176] Although the present invention has been described in detail using preferred embodiments, the scope of the invention is not limited to specific embodiments and should be interpreted by the appended claims. Furthermore, those skilled in the art will understand that many modifications and variations are possible without departing from the scope of the invention. Explanation of the symbols

[0177] 1: Antibody 1 2: Second antibody 3: Third antibody 4: Albumin 5: Running buffer 10: Citrate functional group 20: Carbonate functional group 100: First metal nanoparticles 110: First detection material 120: First detection solution 200: Second metal nanoparticles 210: Second detection material 220: Second detection solution 300: Third metal nanoparticles 310: Third detection substance 320: Third detection solution

Claims

Claim 1 A step of preparing a first metal solution containing first metal nanoparticles; a step of preparing a second metal solution containing second metal nanoparticles; a step of preparing a third metal solution containing third metal nanoparticles; a step of preparing a first detection solution comprising a first detection substance in which a first antibody and albumin are bound to the surface of the first metal nanoparticles of the first metal solution; a step of preparing a second detection solution comprising a second detection substance in which a second antibody and albumin are bound to the surface of the second metal nanoparticles of the second metal solution; a step of preparing a third detection solution comprising a third detection substance in which a third antibody and albumin are bound to the surface of the third metal nanoparticles of the third metal solution; A method for preparing a multi-detection solution comprising the step of mixing the first detection solution, the second detection solution, and the third detection solution to prepare a multi-detection solution, wherein the first metal nanoparticle and the second metal nanoparticle comprise the same metal, the third metal nanoparticle comprises a metal different from the first metal nanoparticle and the second metal nanoparticle, the first metal nanoparticle and the second metal nanoparticle comprise gold, and the third metal nanoparticle comprises silver, the first metal nanoparticle and the third metal nanoparticle are spherical in shape, the second metal nanoparticle is rod-shaped, and the particle drag coefficients of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle are the same. Claim 2 A method for preparing a multiple detection solution according to claim 1, wherein the first antibody is bound to alpha-fetoprotein (AFP), the second antibody is bound to neuro-specific enolase (NSE), and the third antibody is bound to carcino-embryonic antigen (CEA). Claim 3 A method for preparing a multi-detection solution according to claim 1, wherein the step of preparing the second metal solution comprises: preparing a second base metal solution comprising the second metal nanoparticles and a hexadecyltrimethylammonium bromine solution; preparing a second preliminary metal solution by substituting the hexadecyltrimethylammonium bromine solution in the second base metal solution with a solution containing citric acid; and preparing the second metal solution by substituting the solution containing citric acid in the second preliminary metal solution with a potassium carbonate solution, wherein, in the step of preparing the second metal solution, the citrate functional group on the surface of the second metal particles is substituted with a carbonate functional group. Claim 4 A method for preparing a multi-detection solution according to claim 1, wherein the step of preparing the second metal solution comprises: preparing a second base metal solution comprising the second metal nanoparticles and a hexadecyltrimethylammonium bromine solution; preparing a second preliminary metal solution by substituting the hexadecyltrimethylammonium bromine solution in the second base metal solution with a solution containing citric acid; and preparing the second metal solution by substituting the solution containing citric acid in the second preliminary metal solution with a potassium carbonate solution, wherein in the step of preparing the second metal solution, the citrate functional group on the surface of the second metal nanoparticles is substituted with a carbonate functional group. Claim 5 A method for preparing a multi-detection solution according to claim 1, wherein the step of preparing the third metal solution comprises: preparing a third base metal solution comprising a solution comprising the third metal nanoparticles and citric acid; and preparing the third metal solution by substituting the solution containing citric acid within the third base metal solution with a potassium carbonate solution, wherein the citrate functional group on the surface of the third metal nanoparticles is substituted with a carbonate functional group in the step of preparing the third metal solution. Claim 6 A method for preparing a multi-detection solution according to claim 1, wherein the first metal nanoparticle has a red color, the second metal nanoparticle has a blue color, and the third metal nanoparticle has a yellow color. Claim 7 A multi-detection device using a multi-detection solution manufactured by a method according to any one of claims 1 to 6, comprising: a sample portion provided with a mixed substance to be detected including a plurality of different substances to be detected; and a conjugate portion provided with a multi-detection solution comprising a plurality of different substances to be detected, each corresponding to and bonded to the substances to be detected within the mixed substance to be detected. The test section includes a test line provided with an analytical substance in which the substance to be detected and the detection substance are combined, wherein the mixed substance to be detected comprises at least one of a first substance to be detected, a second substance to be detected, and a third substance to be detected, and the multiple detection solution comprises a first detection substance combined with the first substance to be detected, a second detection substance combined with the second substance to be detected, and a third detection substance combined with the third substance to be detected, wherein the first detection substance comprises a first metal nanoparticle, the second detection substance comprises a second metal nanoparticle, and the third detection substance comprises a third metal nanoparticle, wherein the first metal nanoparticle and the second metal nanoparticle comprise gold, and the third metal nanoparticle comprises silver, wherein the first metal nanoparticle and the third metal nanoparticle are spherical in shape, and the second metal nanoparticle is rod-shaped, and the particle drag of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle A multi-detection device including identical counts. Claim 8 A multiple detection device according to claim 7, wherein the first detection substance comprises alpha-fetoprotein (AFP), the second detection substance comprises neuron-specific enolase (NSE), and the third detection substance comprises carcino-embryonic antigen (CEA). Claim 9 A multiple detection device according to claim 7, wherein the first detection material comprises the first metal nanoparticle and the first antibody and albumin provided on the surface of the first metal nanoparticle, the second detection material comprises the second metal nanoparticle and the second antibody and albumin provided on the surface of the second metal nanoparticle, and the third detection material comprises the third metal nanoparticle and the third antibody and albumin provided on the surface of the third metal nanoparticle. Claim 10 In claim 9, the analysis material comprises at least one of a first analysis material in which the first antibody of the first detection material is bound to the first detection material, a second analysis material in which the second antibody of the second detection material is bound to the second detection material, and a third analysis material in which the third antibody of the third detection material is bound to the third detection material. Claim 11 In claim 10, the test line of the test section comprises the first antibody, the second antibody, and the third antibody, which are respectively bound to the first detectable substance of the first analyte, the second detectable substance of the second analyte, and the third detectable substance of the third analyte, and the test section further comprises a control line, thereby forming a multi-detection device. Claim 12 A multi-detection device comprises: a sample portion provided with a mixed substance to be detected comprising a plurality of different substances to be detected; and a junction portion provided with a multi-detection solution comprising a plurality of different substances to be detected. The multi-detection device includes a test section including a test line, wherein different colors distinguishable by the naked eye are displayed on the test line according to the detection substance in the mixed detection substance provided to the sample section and the detection substance in the multi-detection solution provided to the junction section, wherein when the mixed detection substance includes at least one of a first detection substance, a second detection substance, and a third detection substance, and the multi-detection solution includes a first detection substance, a second detection substance, and a third detection substance, a total of seven different colors are displayed on the test line, wherein the first detection substance includes a first metal nanoparticle, the second detection substance includes a second metal nanoparticle, and the third detection substance includes a third metal nanoparticle, wherein the first metal nanoparticle and the second metal nanoparticle include gold, and the third metal nanoparticle includes silver, wherein the first metal nanoparticle and the third metal nanoparticle are spherical in shape, and the second metal nanoparticle is rod-shaped. A multi-detection device including Claim 13 A multiple detection device according to claim 12, wherein the first detection substance comprises alpha-fetoprotein (AFP), the second detection substance comprises neuron-specific enolase (NSE), and the third detection substance comprises carcino-embryonic antigen (CEA). Claim 14 A multi-detection device according to claim 12, wherein the first metal nanoparticle has a red color, the second metal nanoparticle has a blue color, and the third metal nanoparticle has a yellow color. Claim 15 A multi-detection device according to claim 12, wherein the movement speed of the analysis material combined with the detection material and the detection material moving from the junction to the test section is the same, and for this purpose, the particle drag coefficients of the first metal nanoparticle, the second metal nanoparticle, and the third metal nanoparticle are the same.

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