KR102244375B1 - Apparatus for detecting analyte by simultaneous movement of particles and solution and detection method using the same - Google Patents

Abstract

The present invention relates to an apparatus for detecting an analyte due to simultaneous movement of particles and a solution, and a detection method using the apparatus.

Description

Apparatus for detecting analyte by simultaneous movement of particles and solution and detection method using the same}

The present invention relates to an apparatus for detecting an analyte due to simultaneous movement of particles and a solution, and a detection method using the apparatus.

Point-of-care testing (POCT) is a test on site without taking a sample to an analytical laboratory. For field testing, a simple kit that can test analytes such as animal cells, bacteria, viruses, nucleic acids, proteins, and other organic or inorganic substances without using large and complex instruments is required. It is preferable to use a low-powered kit or a non-powered kit that does not use electricity at all. In addition, it should be easy, simple, and economical to operate so that it can be easily used by the general public, not skilled analysts.

The most widely used for field examinations are lateral flow kits used for pregnancy diagnosis or electrochemical diagnostic kits used for blood glucose level testing. However, the low-power or non-powered kit, which is easy to operate and simple as described above, has a very low detection sensitivity and has a limitation that only qualitative tests are possible. Various types of lab-on-a-chips are also being developed to solve this problem, but they have problems in that the structure is complex, a separate device is required, sensitivity is low, or reliability is low.

In order to solve this problem, a method of detecting an analyte by moving magnetic particles used in a solid phase has been proposed (Korean Patent Registration Nos. 10-1398764, 10-1608598, and 10-1811134). According to the above method, since the magnetic particles are moved instead of moving the liquid phase, the structure is simpler than that of the existing lab-on-a-chip, and the apparatus for performing the analysis has a simple advantage.

Korean Patent Registration No. 10-1398764 Korean Patent Registration No. 10-1608598 Korean Patent Registration No. 10-1811134

An object of the present invention is to provide a detection device capable of detecting an analyte by simultaneously moving particles and a solution without using a separate device, and a detection method using the detection device.

In order to solve the above problem,

The present invention has a sample chamber for accommodating a sample and magnetic capture particles and a detection solution injection port, and a chamber unit including a detection chamber for detection of an analyte; A detection solution supply unit connected to the detection solution injection port; A channel unit including a first flow path connecting the sample chamber and the detection chamber to be fluidly movable, and a second flow path branched from the first flow path and extending to the outside; A cylinder unit connected to the second flow path and including a plunger disposed to be movable therein, and configured to adjust an internal pressure of the cylinder according to the movement of the plunger; And a connecting member connecting the magnet and the plunger located outside the chamber unit, and when the connecting member is operated to move the magnet and the plunger from the sample chamber side to the detection chamber side, the buffer solution in the first flow path is transferred to the second flow path. It provides an analyte detection device that is moved to the cylinder unit through.

The present invention has a sample chamber and a detection solution injection port to which a sample tube for accommodating a sample and magnetic trapping particles are coupled, and a chamber unit including a detection chamber for detection of an analyte; A detection solution supply unit connected to the detection solution injection port; A channel unit including a first flow path connecting the sample chamber and the detection chamber to be fluidly movable, and a second flow path branched from the first flow path and extending to the outside; A cylinder unit connected to the second flow path and including a plunger disposed to be movable therein, and configured to adjust an internal pressure of the cylinder according to the movement of the plunger; And a connecting member connecting the magnet and the plunger located outside the chamber unit, and when the connecting member is operated to move the magnet and the plunger from the sample chamber side to the detection chamber side, the buffer solution in the first flow path is It provides an analyte detection device that is moved to the cylinder part through the flow path.

The present invention includes a chamber unit including a detection chamber for detecting an analyte, having a sample chamber and a detection solution injection port to which a sample and a sample syringe containing magnetic trapping particles are coupled; A detection solution supply unit connected to the detection solution injection port; A channel unit including a first flow path connecting the sample chamber and the detection chamber to be fluidly movable, and a second flow path branched from the first flow path and extending to the outside; A cylinder unit connected to the second flow path and including a plunger disposed to be movable therein, and configured to adjust an internal pressure of the cylinder according to the movement of the plunger; And a connecting member connecting the magnet and the plunger located outside the chamber unit, and when the connecting member is operated to move the magnet and the plunger from the sample chamber side to the detection chamber side, the buffer solution in the first flow path is transferred to the second flow path. It provides an analyte detection device that is moved to the cylinder portion through.

The present invention comprises the steps of: filling a buffer solution into a first flow path of a detection chamber and a channel portion by using the analyte detection device; Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample chamber to produce a sample mixture including a capture particle complex; Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution in the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And measuring a signal in the detection chamber.

The present invention comprises the steps of: filling a buffer solution into a first flow path of a detection chamber and a channel portion by using the analyte detection device; Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample tube to produce a sample mixture including a capture particle complex, and coupling the sample tube to the sample chamber; Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution in the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And measuring a signal in the detection chamber.

The present invention includes the steps of moving a connecting member from the detection chamber side to the sample chamber side to move the buffer solution in the cylinder to the sample chamber and the first flow path using the analyte detection device; Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample syringe to generate a sample mixture including a capture particle complex, and coupling the sample syringe to a sample chamber; Injecting the sample mixture from the sample syringe into the sample chamber and simultaneously moving the buffer solution to the detection chamber; Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution in the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And measuring a signal in the detection chamber.

The analyte detection device and detection method according to the present invention can detect an analyte without using power or with an extremely small amount of power, and can be detected by a simple method by moving particles and liquid at the same time. Compared to this, the sensitivity is high and quantitative tests are possible. In addition, the analyte detection device according to the present invention can be used in the field due to its economical and simple usage.

1 is a view showing an analyte detection device according to the present invention.
2 is a diagram showing an analyte detection device according to the present invention.
3 is a diagram showing an analyte detection apparatus according to the present invention.
4 is a diagram showing an analyte detection device according to the present invention.
5 is a photograph of parts of a detection device manufactured in an embodiment of the present invention.
6 is a photograph of a fully assembled detection device in an embodiment of the present invention.
7 is a photograph showing the movement of the detection solution by the reaction of catalase, the labeling material of the detection device prepared in an embodiment of the present invention.
8 is a graph showing that the moving distance of the detection solution is proportional to the concentration of Salmonella in an embodiment of the present invention.

In the present invention, various modifications may be made and various embodiments may be provided, and specific embodiments will be illustrated in the drawings and described in detail in the detailed description.

However, this is not intended to limit the present invention to a specific embodiment, it should be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention.

In the present invention, terms such as "comprises" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof does not preclude in advance.

Hereinafter, the present invention will be described in detail.

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Conventional devices for detecting analytes have a problem in that the detection solution is stored in the detection space (chamber) so that all the solutions in the device can be mixed. Therefore, a means for separating the detection solution from the buffer solution required for washing is required. It was required separately.

In order to solve the above problems, the analyte detection device according to the present invention can store the detection solution in the detection solution supply unit without storing the detection solution in the detection chamber by adding a separate detection solution supply unit, and the buffer solution can also be stored in the additionally installed cylinder. I can. Accordingly, the detection solution or the buffer solution can be stored in an independent dedicated space even though it has fluidity, thereby ensuring the stability of the detection device.

In addition, the analyte can be detected by a simple method by moving the particle and the liquid at the same time.

1 is a view showing an analyte detection device according to the present invention. As shown in FIG. 1, the present invention has a sample chamber 111 and a detection solution injection port 114 for accommodating a sample and magnetic capture particles, and a detection chamber 113 for detection of an analyte. Chamber unit including a; A detection solution supply unit 120 connected to the detection solution injection port 114; A channel including a first flow path 131 that connects the sample chamber 111 and the detection chamber 113 in a fluid movement manner, and a second flow path 132 branched from the first flow path 131 and extending to the outside. )part; A cylinder part connected to the second flow path 132 and including a plunger 133 disposed to be movable therein, and provided to adjust the internal pressure of the cylinder 130 according to the movement of the plunger 133 (130); And a connecting member 150 connecting the magnet 140 and the plunger 133 located outside the chamber unit, and manipulating the connecting member 150 to transfer the magnet 140 and the plunger 133 to the sample chamber ( When moving from the 111) side to the detection chamber 113, the buffer solution 115 in the first flow path 131 is moved to the cylinder part through the second flow path 132, providing an analyte detection device 100. .

Specifically, the detection device according to the present invention includes a sample chamber 111 and a detection chamber 113, and may include a channel portion connected to enable fluid movement between the sample chamber 111 and the detection chamber 113. have.

More specifically, the sample chamber 111 may have an upper portion open so that a sample containing an analyte, a capture particle, and a sample mixture 116 of an analysis reagent can be added when the analyte detection starts, When the analyte is detected after receiving the sample mixture 116, the sample chamber 111 may be sealed by using the sample chamber cover 112a on the upper side.

In the present invention, the capture particle is a particle in which a receptor for an analyte contained in a sample is fixed to a magnetic particle, and captures the analyte and moves in the sample chamber 111 by the movement of the magnet 140. 113). The receptor is a substance capable of directly binding to an analyte, and a representative example is an antibody. For example, the magnetic particles may include Fe ₃ O ₄ or Fe ₂ O ₃ .

In the present invention, the receptor immobilized on the magnetic particle may be a receptor capable of directly binding to an analyte. For example, when the analyte is a substance capable of forming an antibody such as a protein, organic molecule, virus, bacteria, plant cell, or animal cell, an antibody against each analyte may be used as a receptor. As another example, when the analyte is a lectin, a carbohydrate can be used as a receptor, and in the case of an analyte containing a carbohydrate, the lectin can be used as a receptor. As another example, when the analyte is a nucleic acid such as DNA or RNA, DNA or peptide nucleic acid (PNA) having a complementary nucleotide sequence may be used as a receptor. In addition, any substance capable of specifically binding an analyte, such as an aptamer obtained from a nucleic acid, a peptide ligand obtained from a peptide library, and a protein ligand obtained from affibody library, can be used as receptors.

In the magnetic particles, a receptor capable of binding to a receptor that directly binds to an analyte may be immobilized. In this case, in order to distinguish between the two receptors, a receptor that directly binds to an analyte is called a primary receptor, and a receptor that binds to the primary receptor is called a secondary receptor. More specifically, when the primary receptor is an antibody, a secondary antibody or protein G may be used as the secondary receptor. As another example, when the primary receptor is DNA or peptide nucleic acid (PNA), DNA or PNA complementary to a part of the nucleotide sequence of the primary receptor may be used as the secondary receptor.

At this time, the secondary antibody means, for example, if the primary antibody is made in rabbits, anti-rabbit immunoglobulin G antibody may be used as the secondary antibody.

The magnetic particles may be immobilized with a receptor capable of binding to a material produced by an analyte, and the analyte may be an enzyme. More specifically, a receptor for a substance produced in proportion to the amount of an analyte may be used. For example, when the analyte is an enzyme, a receptor for a product corresponds to this.

As another example, receptors for molecules that dissociate or change their structure in response to an analyte may also be used.

The assay reagent is a substance that reacts by mixing with a capture particle and an analyte in the first step when an analyte is detected. Examples of assay reagents used in immunoassay include analyte-label complex, antibody-label complex, and labeling particles. particle) and the like.

The analyte-labeling complex is an assay reagent that can be used in a competitive immunoassay, which is a method of detecting low molecular weight substances, and may be a complex in which a labeling material is linked to the analyte. In competitive immunoassays, the analyte and the analyte-label complex in the sample are competitively bound to the capture particle.

The antibody-labeled complex is an assay reagent that can be used in a sandwich immunoassay, a method of detecting a polymer such as a protein, and may be a complex in which a labeling substance is linked to an antibody against an analyte. When attempting to detect a protein by this method, another antibody is immobilized on the capture particle. The antibody immobilized on the capture particle is called a capture antibody, and the antibody linked to the labeling material is called a detection antibody. In the sandwich immunoassay, the analyte is bound to the capture antibody immobilized on the capture particle, and the antibody-label complex is bonded to the analyte to form a sandwich around the analyte.

The labeling particle is a particle in which a labeling substance and a receptor for an analyte are immobilized on a nonmagnetic particle, and is an assay reagent that can be used to detect cells such as bacteria. When a label particle is mixed with a sample containing bacteria and a trapped particle, since the bacteria are large, a large number of the trapped particle and the labeled particle are simultaneously bound to a single bacteria.

The nonmagnetic particles include silica particles, polystyrene particles, polycarbonate particles, dextran particles, glass particles, alumina particles, gold particles, silver particles, palladium particles, platinum particles, titania particles, zirconium particles and core-shell ( core-shell) may be at least one selected from the group consisting of particles.

The labeling material may include a material capable of generating a measurable signal such as light absorption property, fluorescence property, light emission property, radioactivity, and catalytic activity. For example, enzymes such as catalase, peroxidase, and phosphatase react on a substrate to produce a product having absorbing or fluorescent properties, or to generate light, or to generate a gas. Since it can be generated, it can be used as a labeling material. In addition, a fluorescent substance such as an organic fluorescent molecule or europium chelate can be used as a labeling substance because it can fluoresce. In addition, any substance capable of generating a measurable signal can be used as a labeling substance.

The analyte may include one or more selected from the group consisting of cells, viruses, bacteria, proteins, nucleic acids, carbohydrates, organic molecules, and metal ions.

The detection solution 117 is a solution required to measure the signal of a labeling substance. For example, when the labeling substance is an enzyme, the substrate solution of the enzyme may be the detection solution 117, and the labeling substance is a fluorescent substance. In this case, it may be a buffer solution 115 that is easy to measure fluorescence. For example, when the labeling material is catalase, the detection solution tube 121 may be used as a tool to measure the volume of oxygen gas generated as a result of the enzymatic reaction. That is, when the hydrogen peroxide solution used as the detection solution 117 is moved to the detection chamber 113, if a small amount of hydrogen peroxide solution is left in the detection solution injection port 114, the detection solution injection port ( The hydrogen peroxide solution remaining in 114) moves toward the detection solution supply unit 120. By measuring this distance, the analyte can be quantitatively detected.

Before the detection of the present invention, when the magnet 140 is positioned under the sample chamber 111, the buffer solution 115 may be accommodated in the detection chamber 113 and the first flow path 131, During execution, the detection solution 117 and the trapped particle complex 200 may move to the detection chamber 113 to detect an analyte. In this case, the capture-particle complex 200 may be an analyte-capturing particle-analysis reagent complex generated by reacting an analyte, a capture particle, and an analysis reagent. Specifically, the capture-particle complex 200 may vary depending on the type of analysis reagent. For example, when the assay reagent is an analyte-labeled complex, it may be a capture-particle complex in which the analyte and the analyte-labeled complex are simultaneously bound to the capture particle. In addition, when the assay reagent is an antibody-labeled complex, it may be a capture particle complex in which the analyte is bound to the capture particle and the assay reagent is again bound to the analyte. Finally, when the assay reagent is a labeling particle, it may be a capture particle complex in which both the capture particle and the labeling particle are bound to the analyte.

The detection chamber 113 may have a detection solution injection hole 114 on one side of the upper end, and the detection solution injection hole 114 is a detection solution supply unit in which the detection solution 117 is stored by the detection solution tube 121 ( 120), and the side connected to the detection solution supply unit 120 of the detection solution tube 121 is installed to reach the bottom of the detection solution supply unit 120 so that the detection solution 117 is in the detection solution supply unit 120 It may be movable to the detection chamber 113. A small hole may be provided at the upper end of the detection solution supply unit 120 so that pressure is not applied to the detection solution supply unit 120 when the detection solution 117 moves in the detection solution tube 121.

The channel portion may contain the buffer solution 115, and is branched from one side of the first flow path 131 and the first flow path 131 connecting the sample chamber 111 and the detection chamber 113 and extends to the outside. The second flow path 132 may be included. The diameters of the first flow path 131 and the second flow path 132 may be smaller than the diameters of the sample chamber 111 and the detection chamber 113, which is a solution in the chamber part and the channel part when an analyte is detected. This is to prevent mixing of the samples and to prevent the sample impurities in the sample chamber 111 from moving to the detection chamber 113. The buffer solution 115 enables the trapped particle complex 200 to move from the sample chamber 111 to the detection chamber 113, and may function to wash the particles in the process of moving the particles. A cylinder unit 130 may be connected to the second flow path 132, and the cylinder unit 130 may include a plunger 133 disposed to be movable therein, and according to the movement of the plunger 133 The pressure in the cylinder unit 130 is adjusted so that when the plunger 133 moves, the buffer solution 115 and the detection solution 117 can move at the same time.

The plunger 133 of the cylinder unit 130 is movable inside and can be sealed to both inner walls so that fluid may not flow out, so that the pressure inside the cylinder 130 can be adjusted according to the position of the plunger 133. I can.

The apparatus for detecting an analyte of the present invention moves the plunger 133 from the sample chamber 111 side to the detection chamber 113 side to detect the analyte, and at this time, the buffer solution stored in the detection chamber 113 While 115 moves to the cylinder unit 130 through the second flow path 132, the detection solution 117 in the detection solution supply unit 120 moves to the detection chamber 113 through the detection solution tube 121. I can. That is, the cylinder unit 130 including the plunger 133 may serve to replace the solution inside the detection chamber 113 from the buffer solution 115 to the detection solution 117.

It may include a connection member 150 for connecting the magnet 140 and the plunger 133 located at the lower end of the outside of the chamber. By manipulating the connecting member 150, the magnet 140 and the plunger 133 are moved from the sample chamber 111 to the detection chamber 113 or from the detection chamber 113 to the sample chamber 111 in one operation. Can be moved at the same time. When the connecting member 150 is manipulated to move the connecting member 150 from the sample chamber 111 side to the detection chamber 113 side, the trapped particle complex in the sample chamber 111 according to the movement of the magnet 140 ( 200 can be collected and moved to the detection chamber 113, and as the plunger 133 moves, the internal pressure of the cylinder 130 decreases and the pressure is relatively lowered, thereby causing the negative pressure to act. The solution) can be sucked (sucked up), so that the solution in the detection chamber 113 can be replaced. That is, the buffer solution 115 stored in the detection chamber 113 can move to the cylinder 130, and at the same time, the detection solution 117 from the detection solution supply unit 120 moves into the detection chamber 113, and the detection The analyte in the trapped particle complex 200 moved to the chamber 113 may be detected.

At this time, by adjusting the distance between the sample chamber 111 and the detection chamber 113, the diameter of the cylinder unit 130, the inner volume of the detection solution tube 121, etc., the magnet 140 is moved from the sample chamber 111. The distance moving to the detection chamber 113 and the moving distance of the plunger 133 required to replace the buffer solution 115 with the detection solution 117 can be matched. The moving speed of the magnet 140 may be 1 to 5 cm/min, 1 to 4 cm/min, 1 to 2 cm/min, 2 to 5 cm/min, or 2 to 3 cm/min. For example, when the moving speed of the magnet 140 is 5 cm/min or more, particles may not be collected when the magnet 140 moves and may not move together, and the moving speed of the magnet 140 is 1 cm/min. If min or less, the total detection time may be lengthened, which may affect the detection result.

In addition, the present invention may further include a driving unit for moving the connection member 150. For example, it is possible to move the connecting member 150 by hand, but since the moving speed of the detection solution 117 may affect the detection, the mechanical movement of the spiral spring motor to obtain a constant result. You can also use If you pull the string of the spring motor and wind the spring inside the motor and release it, the string pulled by the ratio of the gear is made to move linearly at a relatively constant speed. Therefore, the connecting member 150 can be moved at a constant speed by pulling this string and connecting it to the connecting member 150. In addition, for more precise movement, the connecting member 150 may be moved using an electrically driven linear actuator.

In the present invention, when the sample mixture 116 is directly added to the sample chamber 111, a modified plunger cover 112b may be additionally installed so that the cover of the sample chamber 111 acts like a plunger, and this is shown in FIG. I got it.

As shown in FIG. 2, when the sample mixture 116 is directly added to the sample chamber 111, a plunger cover 112b may be installed so that the sample chamber cover 112a acts like a plunger. At this time, a buffer solution ( 115 may be stored in the cylinder 130 instead of stored in the detection chamber 113 and the first flow path 131. That is, the chamber part and the channel part become empty. Specifically, when installing the plunger cover 112b in the sample chamber 111, before adding the sample mixture 116 to the sample chamber 111, the plunger ( When 133 is moved from the detection chamber 113 side to the sample chamber 111 side, the buffer solution 115 stored in the cylinder 130 passes through the second flow path 132 to the sample in the first flow path 131. It moves to the space between the chamber 111 and the branch point of the second flow path 132. However, the space between the detection chamber 113 and the branching point of the second flow path 132 among the first flow path 131 is still empty. Thereafter, when the sample mixture 116 is put in the sample chamber 111 and the plunger cover 112b is covered and closed at the top of the sample chamber 111, a part of the sample mixture 116 is moved toward the first flow path 131 by pressure. As a result, a part of the buffer solution 115 in the first flow path 131 fills the space between the branch points of the detection chamber 113 and the second flow path 132 in the first flow path 131 and moves to the detection chamber 113 Thus, preparations can be made to detect the analyte. To this end, the volume of the space between the branching points of the sample chamber 111 and the second flow path 132 among the first flow path 131 may be adjusted to accommodate the buffer solution 115 flowing from the cylinder 130. If the volume of this space is small, the volume of the buffer solution 115 that can be accommodated is small, so even if the plunger cover 112b is pushed and covered, the buffer solution 115 cannot reach the detection chamber 113.

In addition, FIG. 3 is a view showing that the sample tube 300 is additionally installed in the sample chamber 111 according to the present invention. As shown in FIG. 3, the present invention has a sample chamber 111 and a detection solution injection port 114 to which a sample tube 300 for accommodating a sample and a magnetic trapping particle is coupled, and a detection for detection of an analyte A chamber unit including a chamber 113; A detection solution supply unit connected to the detection solution injection port 114; A channel including a first flow path 131 that connects the sample chamber 111 and the detection chamber 113 in a fluid movement manner, and a second flow path 132 branched from the first flow path 131 and extending to the outside. )part; A cylinder part connected to the second flow path 132 and including a plunger 133 disposed to be movable therein, and provided to adjust the internal pressure of the cylinder 130 according to the movement of the plunger 133 (130); And a connecting member 150 connecting the magnet 140 and the plunger 133 located outside the chamber unit, and manipulating the connecting member 150 to connect the magnet 140 and the plunger to the sample chamber 111 Provides an analyte detection device 100 in which the buffer solution 115 in the first flow path 131 is moved to the cylinder unit 130 through the second flow path 132 when moving from the side to the detection chamber 113 side. do.

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Specifically, instead of directly adding the sample mixture 116 to the sample chamber 111, when the sample mixture 116 is added and accommodated in a separate sample tube, the sample tube is transferred to the sample chamber 111 when an analyte is detected. Can be combined to perform analyte detection. The apparatus using the sample tube may be useful when a long time is required before the detection process or when a complicated pretreatment process is required.

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Further, FIG. 4 is a view showing that a sample syringe 400 is additionally installed in the sample chamber according to the present invention. As shown in Figure 4, the present invention has a sample chamber 111 and a detection solution injection port 114 to which the sample syringe 400 in which the sample and the magnetic trapping particles are accommodated, and a detection chamber for detecting an analyte ( A chamber unit including 113); A detection solution supply unit connected to the detection solution injection port 114; A channel including a first flow path 131 that connects the sample chamber 111 and the detection chamber 113 in a fluid movement manner, and a second flow path 132 branched from the first flow path 131 and extending to the outside. )part; A cylinder part connected to the second flow path 132 and including a plunger 133 disposed to be movable therein, and provided to adjust the internal pressure of the cylinder 130 according to the movement of the plunger 133 (130); And a connecting member 150 connecting the magnet 140 and the plunger 133 located outside the chamber unit, and manipulating the connecting member 150 to transfer the magnet 140 and the plunger 133 to the sample chamber ( When moving from the 111) side to the detection chamber 113, the buffer solution 115 in the first flow path 131 is moved to the cylinder unit 130 through the second flow path 132, the analyte detection device 100 Provides.

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Specifically, the analyte detection device receives the buffer solution 115 in the cylinder 130 when the magnet is positioned under the detection chamber before detection, and then transfers the buffer solution 115 using the connecting member 150. After moving from the branched portion of the first flow path 131 to the sample chamber 111 and the second flow path 132, the sample syringe 400 in which the sample mixture 116 is accommodated in the open portion of the sample chamber 111 After coupling ), the sample mixture 116 may be injected into the sample chamber 111 using the sample syringe plunger 133. When the sample mixture 116 is injected into the sample chamber 111, the buffer solution 115 in the sample chamber 111 is formed in the empty space of the first flow path 131 and the second flow path 132 and the detection chamber 113. ) Can be moved.

The analyte detection device 100 of the present invention allows the solutions to be stored separately by adding the detection solution supply unit 120 and the cylinder 130, but all solutions are required by the plunger 133 of the cylinder unit 130. You can go to. When the buffer solution 115 is stored in the cylinder 130, the plunger 133 is pushed to move the buffer solution 115 to a part of the first flow path 131 and the sample chamber 111, and the plunger of the sample syringe 400 again. Alternatively, by pushing the lid of the sample chamber, the buffer solution 115 may be moved to the rest of the first flow path 131 and the detection chamber 113. When the cylinder plunger 133 is pulled again, the buffer solution 115 of the detection chamber 113 exits the cylinder 130 and the detection solution 117 of the detection solution supply unit 120 moves to the detection chamber 113.

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In the analyte detection device 100 of the present invention, the detection process is performed by one operation of pulling the connecting member 150 by installing a connecting member 150 connecting the cylinder plunger 133 and the magnet 140 There are features that can be. When the connecting member 150 is pulled in the state until the sample is injected, the trapped particles move toward the detection chamber 113 along with the movement of the magnet 140, and the buffer solution 115 in the detection chamber 113 is the second Three processes are simultaneously carried out through the flow path 132 to the cylinder 130 and the detection solution 117 to enter the detection chamber 113. In addition, in this process, the particles and the buffer solution 115 move in opposite directions to each other, and even a washing process occurs in which impurities present with the trapped particles are removed.

A method of detecting an analyte using the analyte detection device 100 as described above is as follows.

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First, the present invention relates to a method for detecting an analyte using the analyte detection device 100, comprising: filling a buffer solution in a first flow path of a detection chamber and a channel portion; Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample chamber to produce a sample mixture including a capture particle complex; Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution in the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And measuring an analyte detection method including measuring a signal in the detection chamber.

For example, the analyte detection method includes a sample chamber for accommodating a sample and a magnetic capture particle as a chamber part of the above-described detection device, and a detection solution injection port, and a detection chamber for detection of an analyte. It relates to a method of detecting an analyte using a detection device including a chamber unit including. Accordingly, the details described in the analyte detection device 100 may be the same as for specific matters such as analytes, trapped particles, and analysis reagents used in the detection device and the detection device to be described later.

The trapping particle is a particle in which a receptor for an analyte is fixed to a magnetic particle, and serves to capture an analyte and move it from the sample chamber to the detection chamber by the movement of the magnet. The receptor may include one capable of directly binding to an analyte, and may include, for example, an antibody.

The assay reagent is a substance that reacts by mixing the captured particles and the analyte in the first step when the analyte is detected, and the assay reagent fixes a labeling material and a receptor for the analyte to the nonmagnetic particles. Labeled particles, and the labeling material may include an enzyme or a fluorescent material.

The analyte may include one or more selected from the group consisting of cells, viruses, bacteria, proteins, nucleic acids, carbohydrates, organic molecules, and metal ions.

The detection solution is a solution required to measure the signal of a labeling substance. For example, if the labeling substance is an enzyme, the substrate solution of the enzyme may be the detection solution, and if the labeling substance is a fluorescent substance, fluorescence is measured. It can be a buffer solution that is easy to follow.

For example, a method of detecting bacteria using the analyte detection device 100 is as follows. When detecting bacteria, a receptor capable of specifically binding to an analyte and a labeling particle to which a labeling material are immobilized together can be used as an assay reagent. When bacteria, trapped particles, and labeled particles (analytical reagents) are added to the sample chamber, the substances react to form a trapped particle complex, which is a trapped particle-bacteria-labeled particle complex. When the connecting member is moved from the sample chamber side to the detection chamber side and the plunger and the magnet are moved, the trapped particle complex in the sample chamber is collected by the movement of the magnet and moves from the sample chamber to the detection chamber. From the branched portion of the 1 channel, the buffer solution in the detection chamber moves to the cylinder through the second channel, and the detection solution (ie, the substrate solution of the enzyme) moves to the detection chamber through the detection solution tube. In the detection chamber, a reaction occurs by an enzyme, which is a labeling material of the trapped particle complex, and bacteria can be detected by measuring a signal by the reaction.

In addition, the present invention relates to a method for detecting an analyte using the analyte detection device, comprising: filling a buffer solution in a first flow path of a detection chamber and a channel portion; Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample tube to produce a sample mixture including a capture particle complex, and coupling the sample tube to a sample chamber; Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution in the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And measuring a signal in the detection chamber.

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For example, the analyte detection method includes a sample chamber in which a sample tube for accommodating a sample and a magnetic trapping particle is coupled as a chamber part of the above-described detection device, and a detection solution injection port, and a detection for detection of an analyte It relates to a method of detecting an analyte using a detection device including a chamber unit including a chamber. Accordingly, the details described in the analyte detection device 100 may be the same as for specific matters such as analytes, trapped particles, and analysis reagents used in the detection device and the detection device to be described later.

Among the analyte detection methods of the present invention, a method of detecting an analyte using a separate sample tube in the sample chamber is as follows. After adding the analyte, trapped particles, and labeling particles (analytical reagents) into the sample tube to accommodate the sample mixture, open the upper part of the sample tube and invert the upper part of the sample tube in the sample chamber to go to the lower part of the sample chamber. As the sample mixture flows down into the sample chamber, a trapped particle complex formed by reacting the sample mixture and the substances in the sample chamber may be accommodated. The trapped particle composite may be trapped by a magnet located at a lower portion outside the chamber part. Thereafter, when the connecting member is moved from the sample chamber side to the detection chamber side to move the plunger and the magnet, the trapped particle complex in the sample chamber is collected according to the movement of the magnet and moves from the sample chamber to the detection chamber. Accordingly, from the branched portion of the first flow path, the buffer solution in the detection chamber moves to the cylinder through the second flow path, and the detection solution moves to the detection chamber through the detection solution tube. In the detection chamber, the labeling reaction between the labeling substance of the trapped particle complex and the detection solution occurs, and the bacteria can be detected by measuring the signal by the reaction. For example, when an enzyme is used as a labeling material, the detection solution contains a substrate of the enzyme, so when the labeling material of the capture particle complex reacts with the substrate, a colored or fluorescent substance may be generated or light may be generated.

Further, the present invention relates to a method for detecting an analyte using the analyte detection device, comprising: moving a connecting member from the detection chamber side to the sample chamber side to move the buffer solution in the cylinder to the sample chamber and the first flow path; Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample syringe to produce a sample mixture including a capture particle complex, and coupling the sample syringe to the sample chamber; Injecting the sample mixture from the sample syringe into the sample chamber and simultaneously moving the buffer solution to the detection chamber; Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution in the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And measuring a signal of the detection chamber.

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For example, the analyte detection method includes, as a chamber part of the above-described detection device, a sample chamber to which a sample and a sample syringe containing magnetic trapping particles are coupled, and a detection solution injection port, and a detection chamber for detection of an analyte. It relates to a method of detecting an analyte using a detection device including a chamber portion including. Accordingly, the details described in the analyte detection device 100 may be the same as for specific matters such as analytes, trapped particles, and analysis reagents used in the detection device and the detection device to be described later.

Among the analyte detection methods of the present invention, a method of detecting an analyte using a separate sample syringe in the sample chamber is as follows. A sample syringe is attached to the top of the sample chamber, and an analyte, capture particle, and label particle (analysis reagent) are added to the sample syringe to accommodate the sample mixture, and then the sample mixture in the sample chamber using the plunger of the sample syringe. By injecting, the sample mixture and the trapped particle complex formed by reacting the substances may be accommodated in the sample chamber. The trapped particle composite may be trapped by a magnet located at a lower portion outside the chamber part. Thereafter, when the connecting member is moved from the sample chamber side to the detection chamber side to move the plunger and the magnet, the trapped particle complex in the sample chamber is collected according to the movement of the magnet and moves from the sample chamber to the detection chamber. Accordingly, from the branched portion of the first flow path, the buffer solution in the detection chamber moves to the cylinder through the second flow path, and the detection solution moves to the detection chamber through the detection solution tube. In the detection chamber, the labeling reaction between the labeling substance of the trapped particle complex and the detection solution occurs, and the bacteria can be detected by measuring the signal by the reaction.

Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

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Example

<Detection of Salmonella bacteria using catalase as a labeling substance>

In this example, Salmonella typhimurium was detected using catalase as a labeling material.

Captured particles were prepared by immobilizing Salmonella antibodies on magnetic particles (Amogreentech, Gimpo, Korea) having a diameter of 300 nm. Chemical method using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC] because the magnetic particles have a carboxyl group on the surface Antibody was immobilized.

Label particles were prepared by first immobilizing catalase on silica nanoparticles (Microspheres-Nanospheres, Cold Spring, NY, USA) having a diameter of 100 nm and having a carboxyl group on the surface, and then immobilizing Salmonella antibodies thereon. Since silica nanoparticles also have a carboxyl group on their surface, enzymes and antibodies were immobilized by a chemical method using EDC.

5 is a photograph of parts of the detection device manufactured according to the present embodiment, and FIG. 6 is a photograph of the detection device assembled according to the present embodiment.

5 and 6, a chip at the center of the detection device was made of polydimethylsiloxane (PDMS) (FIG. 5). The thickness of the PDMS chip is 2 mm, the sample chamber and the detection chamber are open, and a groove with a width and height of 1 mm each connects the two chambers on the bottom. A hole with a diameter of 2 mm is drilled in the middle of the channel to be connected to the second flow path. The bottom surface of the PDMS chip was blocked by attaching a 1 mm-thick glass plate. As a result, the sample chamber and the detection chamber became a chamber with the bottom blocked and the top opened, and the groove on the bottom of the PDMS chip became a channel connecting the two chambers.

The PDMS chip is placed on the PDMS chip mount on the bottom plate of the detection device manufactured by processing acrylic. The base plate is also provided with a magnetic guide through which the magnet can move under the glass plate attached to the PDMS chip, and a connection member guide through which the steel wire used as the connection member can move.

The upper plate covering the PDMS chip was fabricated by processing acrylic, and it was drilled upward to extend the sample chamber, the detection chamber, and the second flow path of the PDMS chip. At the top of the hole extending the detection chamber, a detection solution injection port is drilled on the side. The whole kit was completed by fixing the top plate and the bottom plate with screws.

Capillary piston (Gilson, Middleton, WI, USA) was used as a cylinder equipped with a plunger, and Teflon tubing with an inner diameter of 1 mm was used as the detection solution tube. As the detection solution, a hydrogen peroxide solution (0.45 MH ₂ O ₂ , 0.033 mg/mL methylene blue, 0.05 M sodium phosphate, pH 7.0) containing methylene blue, a blue dye, was used.

0.5 mL of a sample containing Salmonella bacteria, 20 μL (20 μg) of trapped particles, and 10 μL (10 μg) of label particles were placed in the detection chamber, and the top of the detection chamber was covered with a tape. The connecting member was pulled at a speed of 3 cm/min to move the magnet from the sample chamber to the detection chamber. When the hydrogen peroxide solution in the detection solution tube flows into the detection chamber, the moving distance of the magnet was adjusted so that a part of it remains at the end of the detection solution tube.

7 shows the movement of the detection solution by the catalase reaction of the detection device prepared according to the present example. As shown in Fig. 7, when oxygen is generated by catalase in the detection chamber, the hydrogen peroxide solution remaining in the detection solution injection tube moves toward the detection solution tube. In FIG. 7, S denotes a position where the hydrogen peroxide solution first remained, and F denotes a position moved by oxygen generation.

8 shows the results of analyzing samples containing different concentrations of Salmonella by the above method. As shown in Figure 8, it was confirmed that the hydrogen peroxide solution moves in proportion to the Salmonella concentration. The moving distance of such a solution can be quantitatively measured using the scale drawn on the detection device without using a separate device.

100: analyte detection device 111: sample chamber
112a: sample chamber cover 112b: plunger cover
113: detection chamber 114: detection solution injection port
115: buffer solution 116: sample mixture
117: detection solution 120: detection solution supply
121: detection solution tube 130: cylinder
131: first euro 132: second euro
133: plunger 140: magnet
150: connecting member 200: trapped particle composite

Claims (18)

A chamber unit having a sample chamber for accommodating a sample and magnetic capture particles and a detection solution injection port and including a detection chamber for detecting an analyte;
A detection solution supply part connected to the detection solution injection port;
A channel unit including a first flow path connecting the sample chamber and the detection chamber to be fluidly movable, and a second flow path branched from the first flow path and extending to the outside;
A cylinder unit connected to the second flow path and including a plunger disposed to be movable therein, and configured to adjust an internal pressure of the cylinder according to the movement of the plunger; And
A magnet located outside the chamber and a connection member connecting the plunger,
An analyte detection device in which the buffer solution in the first flow path is moved to the cylinder through the second flow path when the magnet and the plunger are moved from the sample chamber side to the detection chamber side by operating the connecting member.
A chamber portion having a sample chamber to which a sample tube for accommodating a sample and magnetic trapping particles are coupled and a detection solution injection port and including a detection chamber for detecting an analyte;
A detection solution supply unit connected to the detection solution injection port;
A channel portion including a first flow path connecting the sample chamber and the detection chamber to be fluidly movable, and a second flow path branched from the first flow path and extending to the outside;
A cylinder part connected to the second flow path and including a plunger disposed to be movable therein, and configured to adjust an internal pressure of the cylinder according to the movement of the plunger; And
A magnet located outside the chamber and a connection member connecting the plunger,
An analyte detection device in which the buffer solution in the first flow path is moved to the cylinder part through the second flow path when the magnet and the plunger are moved from the sample chamber side to the detection chamber side by manipulating the connecting member.
A chamber unit including a detection chamber for detecting an analyte, and having a sample chamber and a detection solution injection port to which a sample and a sample syringe containing magnetic trapping particles are coupled;
A detection solution supply unit connected to the detection solution injection port;
A channel portion including a first flow path connecting the sample chamber and the detection chamber to be fluidly movable, and a second flow path branched from the first flow path and extending to the outside;
A cylinder part connected to the second flow path and including a plunger disposed to be movable therein, and configured to adjust an internal pressure of the cylinder according to the movement of the plunger; And
A magnet located outside the chamber and a connection member connecting the plunger,
An analyte detection device in which the buffer solution in the first flow path is moved to the cylinder part through the second flow path when the magnet and the plunger are moved from the sample chamber side to the detection chamber side by manipulating the connecting member.
The method according to any one of claims 1 to 3,
An analyte detection device in which the detection solution is transferred into the detection chamber from the detection solution supply when the magnet is moved from the sample chamber side to the detection chamber side by manipulating the connecting member.
The method according to any one of claims 1 to 3,
The diameter of the first flow passage and the second flow passage is smaller than the diameters of the sample chamber and the detection chamber.
The method according to any one of claims 1 to 3,
An analyte detection device comprising a detection solution tube connecting the detection solution injection port and the detection solution supply unit, and a side connected to the detection solution supply unit of the detection solution tube is installed so as to touch the bottom of the detection solution supply unit.
The method of claim 1,
The sample chamber is an analyte detection device for sealing the sample chamber by using a sample chamber cover on the top.
The method according to any one of claims 1 to 3,
An analyte detection device further comprising a driving unit for moving the connecting member.
The method according to any one of claims 1 to 3,
The capture particle is a particle in which a receptor for an analyte is immobilized on a magnetic particle,
The receptor is an analyte detection device capable of directly binding to an analyte.
The method according to any one of claims 1 to 3,
The analyte detection device comprising at least one selected from the group consisting of cells, viruses, bacteria, proteins, nucleic acids, carbohydrates, organic molecules, and metal ions.
The method of claim 1,
When the magnet is positioned below the sample chamber before performing detection, a buffer solution is accommodated in the detection chamber and the first flow path.
The method of claim 3,
An analyte detection device in which a buffer solution is accommodated in a cylinder when the magnet is positioned below the detection chamber before performing detection in which the sample syringe is not coupled.
Using the analyte detection device according to claim 1,
Filling a buffer solution into the detection chamber and the first flow path of the channel;
Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample chamber to produce a sample mixture including a capture particle complex;
Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution in the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And
An analyte detection method comprising the step of measuring a signal in the detection chamber.
Using the analyte detection device according to claim 2,
Filling a buffer solution into the detection chamber and the first flow path of the channel;
Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample tube to produce a sample mixture including a capture particle complex, and coupling the sample tube to a sample chamber;
Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution in the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And
An analyte detection method comprising the step of measuring a signal in the detection chamber.
Using the analyte detection device according to claim 3,
Moving the connecting member from the detection chamber side to the sample chamber side to move the buffer solution in the cylinder to the sample chamber and the first flow path;
Mixing a sample containing an analyte, a capture particle, and an analysis reagent in a sample syringe to produce a sample mixture including a capture particle complex, and coupling the sample syringe to the sample chamber;
Injecting the sample mixture from the sample syringe into the sample chamber and simultaneously moving the buffer solution to the detection chamber;
Moving the connecting member from the sample chamber side to the detection chamber side, moving the buffer solution of the detection chamber to the cylinder, and moving the detection solution and the trapped particle complex to the detection chamber; And
An analyte detection method comprising the step of measuring a signal in the detection chamber.
The method according to any one of claims 13 to 15,
The capture particle is a particle in which a receptor for an analyte is immobilized on a magnetic particle,
The method of detecting an analyte, wherein the receptor is capable of directly binding to an analyte.
The method according to any one of claims 13 to 15,
The assay reagent is a labeling particle in which a labeling material and a receptor for the analyte are immobilized on a nonmagnetic particle,
The labeling material is an analyte detection method comprising an enzyme or a fluorescent material.
The method according to any one of claims 13 to 15,
The analyte detection method comprising at least one selected from the group consisting of cells, viruses, bacteria, proteins, nucleic acids, carbohydrates, organic molecules, and metal ions.

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