JP2011080999A - Antigen detection method, antigen detector using the same, and microfluid chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antigen detection method and an antigen detector using the same, and also to provide a microfluid chip. <P>SOLUTION: The antigen detection method includes the steps of: producing an antibody nanobead by bonding a nanobead to a first antibody; producing an antigen antibody nanobead by bonding the resulting antibody nanobead to an antigen; bonding the resulting antigen antibody nanobead to a second antibody by applying at least either an electric field or a magnetic field to the resulting antigen antibody nanobead; and detecting the antigen antibody nanobead bonded to the second antibody. This enables active mixing by using a spatially uneven and temporary variable electromagnetic field, thus achieving a reduction in reaction time and enabling quick detection of an antigen from a small amount of blood by controlling the flow so that the nanobead moves to a particular site of the capture antibody. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an antigen detection method, an antigen detection apparatus using the same, and a microfluidic chip. More specifically, the present invention can efficiently detect by forming an electromagnetic field that is spatially nonuniform and temporally changes. The present invention relates to an antigen detection method, an antigen detection apparatus using the same, and a microfluidic chip.

  In a general method for detecting the presence or absence of a specific antigen from a sample, first, an antibody that selectively binds to the specific antigen is attached to a nanobead containing a fluorescent component. The antibody fluorescent nanobeads thus formed are prepared. When a sample to be tested for the presence of a specific antigen is mixed with antibody fluorescent nanobeads at a certain ratio, when a specific antigen-antibody reaction occurs, antigen-antibody fluorescent nanobeads are formed by combining the antibody and antigen. Is done. Eventually, when antigen-antibody fluorescent nanobeads are detected, it can be seen that a specific antigen is present in the sample.

  In general, in a biosensor using microfluidics, a small amount of sample is passed through a mixing chamber through a portion containing fluorescent antibody nanobeads.

  The antigen-antibody fluorescent nanobeads that have passed through the mixed region pass through a detection chamber on the fluid flow path. The detection region includes a region in which an antibody is attached to the wall surface of the fluid channel.

  The antigen-antibody fluorescent nanobeads, in which the antigen is bound to the antibody attached on the surface of the fluorescent nanobeads, pass through the detection region, and the antibody immobilized on the fluid path wall binds to the antigen on the surface of the antigen-antibody fluorescent nanobeads Then, antigen-antibody fluorescent nanobeads adhere to the surface of the detection region.

  In the detection region, after a sufficient reaction time has elapsed, antibody nanobeads that have not reacted (not attached to the antibody immobilized on the surface of the pathway) are washed away. When the detection region is irradiated with light from the outside (illuminate), fluorescence is generated from the immobilized antibody fluorescent nanobeads by a selective antigen-antibody reaction.

  That is, the biosensor can detect an antigen using a small amount of sample by detecting such fluorescence, and can measure the concentration of the antigen in the sample.

  On the other hand, the antigen-antibody fluorescent nanobeads are colloidal and flow while performing Brownian movement in the fluid, so that natural binding can be performed. However, in order to bind the antigen and the antibody, it is necessary to locate the antigen-antibody fluorescent nanobead and the antibody at a sufficiently close distance. That is, sufficient time is required so that the antigen-antibody fluorescent nanobeads to which the antigen is bound can react with the antibody immobilized on the wall surface.

  Also, when the fluid flows in the channel, the flow velocity of the fluid becomes zero on the channel wall surface, and a large colloidal particle is mainly in the center of the channel due to a laminar flow phenomenon having a maximum velocity at the center of the channel. Gather together and move with the fluid. That is, the antigen-antibody fluorescent nanobeads that flow in a concentrated manner in the center of the channel must be brought close to the channel wall where the immobilized antibody is present so that the binding between the antigen and the antibody is performed.

  Eventually, it takes a long time to bind the antibody on the channel wall and the antigen-antibody fluorescent nanobeads depending only on the Brownian motion with the fluid stopped. Furthermore, when a fluid flows, a laminar flow is formed from the characteristics of the fluid flow, and the antigen-antibody fluorescent nanobeads tend to flow in a concentrated manner at the center of the channel. That is, since the probability that the antigen-antibody fluorescent nanobeads and the immobilized antibody react with each other decreases, a long time is required and a larger amount of sample is required.

  That is, the conventional technique has the disadvantage that a sufficient amount of sample and a sufficient amount of sample must be provided in order for the necessary binding reaction between the antigen in the sample and the antibody fluorescent nanobead to be performed. There is the disadvantage that a sufficient amount of time and a sufficient amount of sample must be provided so that the necessary binding reaction takes place between the prepared antibody and the antigen-antibody nanobeads.

US Patent Publication No. 2008-0135101

  An object of the present invention is to provide an antigen detection method capable of efficiently detecting an antigen by forming a spatially nonuniform and temporally changing electromagnetic field.

  Another object of the present invention is to provide an antigen detection apparatus that uses an antigen detection method that can efficiently detect an antigen by forming an electromagnetic field that is spatially inhomogeneous and changes with time.

  Still another object of the present invention is to provide a microfluidic chip using an antigen detection method capable of efficiently detecting an antigen by forming a spatially nonuniform and temporally changing electromagnetic field.

  An antigen detection method for achieving the above-described object of the present invention includes a step of binding a first antibody and nanobeads to generate antibody nanobeads, and binding the generated antibody nanobeads to an antigen to generate an antigen antibody. Generating a nanobead; applying at least one of an electric field and a magnetic field to the generated antigen-antibody nanobead to bind the generated antigen-antibody nanobead and the second antibody; and binding to the second antibody Detecting the produced antigen-antibody nanobeads.

  Here, the nanobead may include at least one of a dielectric and a metal.

  Here, the dielectric may have a dipole moment.

  Here, the nanobead may include a fluorescence component.

  Here, in the step of generating antigen-antibody nanobeads, at least one of an electric field and a magnetic field may be applied to the antibody nanobeads.

  Here, in the step of binding the generated antigen-antibody nanobead and the second antibody, at least one of the applied electric field and magnetic field may be inhomogeneous.

  Here, the second antibody may be attached to a fixed position in the step of binding the generated antigen-antibody nanobead and the second antibody.

  An antigen detection apparatus using an antigen detection method for achieving the object of the present invention described above is a mixed region for generating an antibody-antibody nanobead by binding an antibody nanobead formed of a first antibody and a nanobead to an antigen. And a detection region for binding the antigen-antibody nanobead and the second antibody, and an electromagnetic field generation unit for forming at least one of an electric field and a magnetic field in at least one of the mixed region and the detection region .

  Here, the nanobead may include at least one of a dielectric and a metal.

  Here, the dielectric may have a dipole moment.

  Here, the nanobead may include a fluorescent component.

  Here, at least one of the electric field and the magnetic field of the electromagnetic field generator may be non-uniform.

  Here, the second antibody may be attached to the detection region.

  The microfluidic chip using the antigen detection method for achieving the above-described object of the present invention is a mixed region for producing an antigen-antibody nanobead by binding an antibody nanobead formed of a first antibody and a nanobead to an antigen. And a detection region for binding the antigen-antibody nanobead and the second antibody, and an electromagnetic field generation unit for forming at least one of an electric field and a magnetic field in at least one of the mixed region and the detection region .

  Here, the nanobead may include at least one of a dielectric and a metal.

  Here, the dielectric may have a dipole moment.

  Here, the nanobead may include a fluorescent component.

  Here, the second antibody may be attached to the detection region.

  Here, at least one of the electric field and the magnetic field of the electromagnetic field generator may be non-uniform.

  Here, the electromagnetic field generator may be formed of at least one of an electrode array and a micro coil array.

  According to the antigen detection method as described above, the antigen detection apparatus using the same, and the microfluidic chip, if nanobeads affected by the electromagnetic field exist inside the electromagnetic field that changes temporally and spatially, the nonuniformity of the electromagnetic field Come to exercise. In particular, active mixing can be performed using a spatially inhomogeneous and time-varying electromagnetic field, reducing reaction time and moving the nanobeads to a portion of the immobilized antibody. The flow can be controlled so that the antigen can be detected from a small amount of blood in a short time.

It is a flowchart for demonstrating the antigen detection method by one Embodiment of this invention. It is a block diagram for demonstrating the antigen detection apparatus using the antigen detection method by one Embodiment of this invention. It is a conceptual diagram for demonstrating the microfluidic chip | tip using the antigen detection method by one Embodiment of this invention.

  The present invention can be embodied as various embodiments with various modifications. Hereinafter, specific embodiments will be illustrated in the drawings and described in detail.

  However, this should not be construed as limiting the invention to the particular embodiments, but should be understood to include all modifications, equivalents and alternatives within the spirit and scope of the invention.

  Terms such as “first, second” can be used to describe various components, but the above components should not be limited by the above terms. The above terms are only used to distinguish one component from another. For example, the first component can also be referred to as the second component, and similarly, the second component can be referred to as the first component without departing from the scope of rights of the present invention. The term “and / or” includes any item of a combination of a plurality of related items described or a plurality of related items described.

  When a component is referred to as being “coupled” or “connected” to another component, it can also be directly coupled or connected to the other component, It should be understood that other components may also be present. On the other hand, when a component is referred to as being “directly connected” or “directly connected” to another component, it should be understood that there are no other components in between. is there.

  The terminology used in the present application is used to describe particular embodiments and is not intended to limit the present invention. The singular form includes the plural form unless the context clearly dictates otherwise. In this application, a term such as “comprising” or “having” means that a feature, number, step, operation, component, part, or combination thereof described in the specification is present, and It should be understood that it does not exclude the presence or addition of one or more other features or numbers, steps, actions, components, parts or combinations thereof.

  Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Has meaning. Terms such as those defined in commonly used dictionaries should be construed as having a meaning consistent with the meaning contained within the context of the related art, and are ideal unless explicitly defined in this application. Or overly formal meaning.

  Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In order to facilitate overall understanding in describing the present invention, the same reference numerals are used for the same components in the drawings, and duplicate descriptions for the same components are omitted.

  FIG. 1 is a flowchart for explaining an antigen detection method according to an embodiment of the present invention.

  Referring to FIG. 1, the method for detecting an antigen according to an embodiment of the present invention includes a step S110 of combining a first antibody and a nanobead to generate an antibody nanobead, and binding the generated antibody nanobead and the antigen. Generating antigen antibody nanobeads S120, applying at least one of an electric field and a magnetic field to the generated antigen antibody nanobeads to bind the generated antigen antibody nanobeads to the second antibody S130, And S140 for detecting antigen-antibody nanobeads bound to the second antibody.

  First, the nanobead may include at least one of a dielectric and a metal, and the dielectric may have a dipole moment.

  When the nanobead is configured to include a dielectric or a metal, the nanobead receives a force under an electric field and a magnetic field. That is, the nanobeads can be controlled to perform a desired motion while maintaining the electric field and the magnetic field inhomogeneous.

  For example, by applying a surface coating treatment with a dielectric or metal component to the nanobead, the nanobead is moved to a desired position by receiving a force inside the nonuniform electric field and magnetic field and controlling the nonuniform electric field and magnetic field. be able to.

  The nanobeads may also include a fluorescence component. When the nanobead is configured to include a fluorescent component, it can be processed relatively easily when finally determining the presence or absence of an antigen.

  That is, the presence or absence of an antigen in a sample can be easily confirmed by irradiating light in a wavelength band in which a fluorescent component reacts. That is, when the fluorescent component reacts with the irradiated light, it can be determined that the specific antigen is present in the sample.

  In step S110 of generating antibody nanobeads by combining the first antibody and nanobeads, a preparatory work for determining the presence or absence of an antigen in the sample is performed. That is, in order to determine the presence or absence of an antigen, an antibody capable of binding to the antigen is prepared.

  Furthermore, in order to easily determine the presence or absence of an antigen at the final stage, the antibody is prepared by binding nanobeads in advance. The nanobeads thus bound to the antibody can be used to force the movement of the nanobeads and have the nanobeads have fluorescent properties when detecting an antigen.

  Next, in step S120 of generating the antigen-antibody nanobead by combining the generated antibody nanobead and the antigen, in order to determine whether the antigen is present in the sample, the antigen present in the sample and the antibody nanobead are used. Combine. Such binding produces antigen-antibody nanobeads.

  That is, if no antigen is present in the sample, antigen-antibody nanobeads will not be produced, so that it should not be possible to detect the antigen at a later stage. On the other hand, when an antigen is present in the sample, antigen-antibody nanobeads are generated by such binding, and the antigen can be detected at a later stage.

  Further, in step S120 of generating antigen-antibody nanobeads, at least one of an electric field and a magnetic field may be applied to the antibody nanobeads. In principle, in order to generate antigen-antibody nanobeads, it is necessary to depend on the Brownian movement of antigen and antibody nanobeads. However, it is inconvenient to produce antigen-antibody nanobeads depending only on Brownian motion because it requires a long time and a large amount of sample.

  Therefore, by applying an electric field and a magnetic field to the antigen and the antibody nanobeads, the antibody nanobeads having a dielectric material and a metal component can be moved more actively, thereby increasing the number of generations of the antigen-antibody nanobeads. That is, antigen-antibody nanobeads are generated more actively and efficiently.

  Next, in step S130, at least one of an electric field and a magnetic field is applied to the generated antigen-antibody nanobeads to bind the generated antigen-antibody nanobeads and the second antibody, whether or not the antigen is present in the sample. This is done to detect the nanobeads that are the basis for the determination.

  According to a general method, antigen-antibody nanobeads bind to the second antibody while performing Brownian motion, and this is detected to determine whether the antigen is present in the sample. However, relying solely on Brownian motion has a number of disadvantages as described above.

  Therefore, by applying at least one of an electric field and a magnetic field to the antigen antibody nanobeads and applying a force to move the antigen antibody nanobeads closer to the second antibody, a larger number of antigen antibody nanobeads can be obtained. Provides an opportunity to react with other antibodies.

  Here, in the step S130 of binding the generated antigen-antibody nanobead and the second antibody, at least one of the applied electric field and magnetic field may be non-uniform.

  When the electric field and the magnetic field are uniformly formed, the antigen-antibody nanobeads maintain an aligned state. Therefore, the electric field and the magnetic field are formed unevenly, and a force is applied to move the antigen-antibody nanobeads.

  A force capable of moving to the antigen-antibody nanobeads may be applied to locate the second antibody. Thereby, the frequency | count of reaction increases and the detection of an antigen can be made easier.

  Here, the second antibody may be attached to a fixed position in the step S130 of binding the generated antigen-antibody nanobead and the second antibody. In order to easily detect the antigen-antibody nanobeads bound to the second antibody, it is necessary to know the position of the antigen-antibody nanobeads, so that the antigen can be more easily attached by attaching the second antibody to the fixed position. Can be detected.

  Furthermore, the force applied by applying at least one of an electric field and a magnetic field to the antigen-antibody nanobeads may be controllable within a range where the antigen-antibody nanobeads already attached to the second antibody cannot be removed.

  Next, in step S140 of detecting the antigen antibody nanobeads bound to the second antibody, the presence or absence of the antigen in the sample can be confirmed by detecting the antigen antibody nanobeads.

  Various methods may be used to detect antigen-antibody nanobeads. Generally, fluorescent nanobeads are used as nanobeads, and in order to detect antigen-antibody nanobeads, light in the wavelength band where fluorescent nanobeads react is irradiated. Then, a fluorescent reaction may be caused.

  Furthermore, there are various methods for detecting using the characteristics of the nanobeads, and any method capable of detecting using the characteristics of the nanobeads can be applied to the present invention without limitation.

  FIG. 2 is a block diagram for explaining an antigen detection apparatus using an antigen detection method according to an embodiment of the present invention.

  Referring to FIG. 2, an antigen detection apparatus 200 using an antigen detection method according to an embodiment of the present invention generates an antigen-antibody nanobead by binding an antibody nanobead formed of a first antibody and a nanobead to an antigen. A mixing region 210, a detection region 220 for binding the antigen-antibody nanobead and the second antibody, and at least one of an electric field and a magnetic field in at least one of the mixing region and the detection region. The electromagnetic field generator 230 may be included.

  The nanobeads used in the antigen detection apparatus 200 may include at least one of a dielectric and a metal. That is, the surface of the nanobead may be coated with at least one of a dielectric and a metal.

  By coating at least one of a dielectric and a metal on the surface of the nanobead, the nanobead can receive a force in an electric field and a magnetic field by using a dipole moment of the dielectric. Depending on the characteristics of the components, forces can be applied in electric and magnetic fields.

  Furthermore, the nanobeads used in the antigen detection apparatus 200 may include a fluorescent component. That is, by including a fluorescent component, it is possible to detect an antigen in the sample by irradiating light with which the fluorescent component reacts.

  The mixed region 210 for generating the antigen-antibody nanobeads by combining the antibody nanobeads formed of the first antibody and the nanobeads with the antigen is a solution containing a sample for determining whether the antigen is present and the antibody nanobeads May be a region where and are mixed together.

  In solution, antigen and antibody nanobeads can be bound by Brownian motion in principle. Furthermore, due to at least one of the electric field and magnetic field applied by the electromagnetic field generator 230, the antigen and antibody nanobeads have more opportunities for reaction than if they depend only on general Brownian motion.

  Next, in the detection region 220 for binding the antigen-antibody nanobead and the second antibody, the solution containing the antigen-antibody nanobead described above is introduced and bound to the second antibody.

  Here, the second antibody may be attached to the detection region 220. That is, the antigen-antibody nanobeads bind to the second antibody attached to the detection region by Brownian motion.

  Furthermore, at least one of the electric field and magnetic field applied by the electromagnetic field generator 230 allows the antigen-antibody nanobeads to move closer to the second antibody, and thus has more opportunities for binding.

  Next, the electromagnetic field generator 230 for forming at least one of an electric field and a magnetic field in at least one of the mixed region 210 and the detection region 220 applies force to the antibody nanobeads in the mixed region 210, thereby It can be actively mixed to induce binding. In addition, active binding to the second antibody can be induced by applying force to the antigen-antibody nanobeads in the detection region 220.

  Here, at least one of the electric field and the magnetic field from the electromagnetic field generator 230 may be non-uniform. That is, antibody nanobeads and antigen antibody nanobeads are subjected to forces by non-uniform electric and magnetic fields, and movement of antibody nanobeads and antigen antibody nanobeads can provide more opportunities for binding.

  FIG. 3 is a conceptual diagram for explaining a microfluidic chip using an antigen detection method according to an embodiment of the present invention.

  Referring to FIG. 3, a microfluidic chip 300 using an antigen detection method according to an embodiment of the present invention generates an antigen-antibody nanobead by binding an antibody nanobead formed of a first antibody and a nanobead to an antigen. In order to form at least one of an electric field and a magnetic field in at least one of the mixed region 310, the detection region 320 for binding the antigen-antibody nanobead and the second antibody, and at least one of the mixed region 310 and the detection region 320. The electromagnetic field generator 330 may be included.

  Since the mixing region 310, the detection region 320, and the electromagnetic field generator 330 have already been described, detailed description thereof will be omitted.

  The electromagnetic field generator 330 may be formed of at least one of an electrode array and a micro coil array. This is not only to generate at least one of the electric field and the magnetic field non-uniformly, but also to increase the efficiency of antigen detection by controlling the electric field and the magnetic field.

  The second antibody attachment region 321 is a region to which the second antibody is attached, and moves to the region to which the second antibody is attached by applying force to the antigen-antibody nanobeads. Thereby, the opportunity of the coupling | bonding of a 2nd antibody and an antigen antibody nano bead increases. That is, the efficiency of antigen detection can be improved.

  Although the present invention has been described with reference to the embodiments, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention described in the following claims. You should understand that there is.

DESCRIPTION OF SYMBOLS 200 Antigen detection apparatus 210 Mixing area 220 Detection area 230 Electromagnetic field production | generation part 300 Microfluidic chip 310 Mixing area 320 Detection area 321 2nd antibody adhesion area 330 Electromagnetic field generation part

Claims (20)

Binding the first antibody and nanobeads to produce antibody nanobeads;
Combining the produced antibody nanobeads with an antigen to produce antigen-antibody nanobeads;
Applying at least one of an electric field and a magnetic field to the generated antigen-antibody nanobeads to bind the generated antigen-antibody nanobeads to a second antibody;
Detecting the antigen-antibody nanobeads bound to the second antibody;
An antigen detection method comprising the steps of:   The method of claim 1, wherein the nanobead includes at least one of a dielectric and a metal.   The method according to claim 2, wherein the dielectric has a dipole moment.   The method of claim 1, wherein the nanobead includes a fluorescence component.   The method of claim 1, wherein the step of generating the antigen-antibody nanobeads includes applying at least one of an electric field and a magnetic field to the antibody nanobeads.   The method of claim 1, wherein at least one of the applied electric field and magnetic field is non-uniform in the step of binding the generated antigen-antibody nanobead and the second antibody.   The method for detecting an antigen according to claim 1, wherein, in the step of binding the generated antigen-antibody nanobead and the second antibody, the second antibody is attached to a fixed position. A mixing region for binding an antibody nanobead formed of a first antibody and a nanobead to an antigen to produce an antigen-antibody nanobead;
A detection region for binding the antigen-antibody nanobead and the second antibody;
An electromagnetic field generator for forming at least one of an electric field and a magnetic field in at least one of the mixing region and the detection region;
An antigen detection apparatus comprising:   The antigen detecting apparatus according to claim 8, wherein the nanobead includes at least one of a dielectric and a metal.   The antigen detection apparatus according to claim 9, wherein the dielectric has a dipole moment.   The antigen detection apparatus according to claim 8, wherein the nanobead includes a fluorescent component.   The antigen detection apparatus according to claim 8, wherein at least one of an electric field and a magnetic field of the electromagnetic field generation unit is non-uniform.   9. The antigen detection apparatus according to claim 8, wherein the second antibody is attached to the detection region. A mixing region for binding an antibody nanobead formed of a first antibody and a nanobead to an antigen to produce an antigen-antibody nanobead;
A detection region for binding the antigen-antibody nanobead and the second antibody;
An electromagnetic field generator for forming at least one of an electric field and a magnetic field in at least one of the mixing region and the detection region;
A microfluidic chip comprising:   The microfluidic chip of claim 14, wherein the nanobead includes at least one of a dielectric and a metal.   The microfluidic chip according to claim 15, wherein the dielectric has a dipole moment.   The microfluidic chip of claim 14, wherein the nanobead includes a fluorescent component.   The microfluidic chip according to claim 14, wherein the second antibody is attached to the detection region.   The microfluidic chip according to claim 14, wherein at least one of an electric field and a magnetic field of the electromagnetic field generator is non-uniform.   The microfluidic chip according to claim 14, wherein the electromagnetic field generator is formed of at least one of an electrode array and a micro coil array.

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