JP2009128234A - Material detecting device and material detecting method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive and sensitive device for detecting a material to be measured. <P>SOLUTION: This material detecting device has a vessel 1001 for holding an analyte 1002. The shape of the vessel 1001 is substantially cylinder shape, and a columnar recessed part is disposed in the upper part of the vessel 1001 while it matches with the center axis of the vessel 1001. A binding object material capable of being specifically bound to the material to be measured is solidified on a side surface of the columnar recessed part. In other words, the side surface of the recessed part is a binding region 1003, the bottom part of the vessel 1001 is a detecting region 1009, and a detecting device 1004 is disposed as a detecting means under the vessel 1001. The detecting region 1009 is made narrower than the binding region 1003. The periphery of the vessel 1001 is provided with a releasing means 1007 for releasing, from the binding region 1003, a complex of a label material and the material to be measured specifically bound to the binding region 1003. The detecting region 1009 has a collecting means 1008 for collecting label material. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a substance detection apparatus for detecting a measurement target substance contained in a specimen. Furthermore, the present invention relates to a substance detection method for detecting a measurement target substance in a specimen.

  In recent years, the need for highly sensitive immunological tests has increased due to the focus on preventive medicine. Various methods have been proposed for immunological tests. One of the methods is as follows. First, in an immunological test, a bound substance that specifically binds to a measurement target substance that is a measurement target is immobilized on a support. Then, after the measurement target substance is specifically bound to the bound substance, the measurement target substance is detected (see Patent Document 1). Here, the measurement target substance is a substance such as an antigen. At this time, an antibody that specifically binds to an antigen is used as the substance to be bound.

  In the substance detection method as described above, a method of forming a complex by reacting a measurement target substance with a labeling substance has also been proposed. In this case, the complex is specifically bound to the bound substance by the measurement target substance. Then, the substance to be measured is indirectly detected by detecting the labeling substance in the complex. At this time, by using a substance that can be easily detected as the labeling substance, detection is possible even if the amount of the substance to be measured is small. Therefore, such a method is expected as a highly sensitive immunoassay method.

  However, the labeling substance is not necessarily specifically bound to the bound substance by the substance to be measured. For example, a labeling substance that does not form a complex with the substance to be measured (hereinafter referred to as an unnecessary labeling substance) may be non-specifically bound to the support or the bound substance. When such an unnecessary labeling substance exists, it becomes impossible to correctly detect the measurement target substance. In particular, when the measurement target substance contained in the sample is small, the influence of the unnecessary labeling substance is increased, and the sensitivity of detection of the measurement target substance is greatly reduced.

A substance detection method for solving this problem is described in Patent Document 2 and Patent Document 3. These substance detection methods are as follows. First, a measurement target substance in a specimen is reacted with a labeling substance to form a complex. In the binding step, the complex is specifically bound to a binding region (a region where a substance to be bound is immobilized). Next, unnecessary labeling substances in the specimen are removed by a washing process. Then, the complex is dissociated from the binding region and again bound to another binding region. Thereafter, a further cleaning step is performed. Thus, by repeating the binding step and the washing step a plurality of times, unnecessary labeling substances that are non-specifically bound to the binding region are removed from the specimen. Thereafter, the labeling substance in the complex specifically bound to the binding region is detected. According to this method, by repeating the binding step, the amount of unnecessary labeling substance that is non-specifically bound can be reduced. And the sensitivity of detection of a measurement object substance can be raised.
U.S. Patent No. 5981297 Japanese Patent No. 2606722 Japanese Patent No.2657672

  In the method described in Patent Document 2 or 3, the efficiency of specific binding between the substance to be measured and the substance to be bound must be high. This is because, when the efficiency of specific binding is low, the amount of the complex that specifically binds to the binding region decreases each time the binding step is repeated. Thus, when the amount of the complex decreases, the detection sensitivity decreases. In order to increase the efficiency of specific binding, the binding region must be widened. This is because the number of substances to be specifically bound is dependent on the number of substances to be bound. Furthermore, there is a limit to increasing the density of the substance to be bound to the binding region.

  In order to detect the labeling substance in the complex bound to the binding region, it is necessary to install a detection device as a detection means over the entire binding region. Therefore, when the coupling region is widened as described above, the detection device is installed over a wide region. The substance detection device configured as described above is expensive. In particular, when the detection device is manufactured using a semiconductor process, the size of the coupling region greatly affects the price of the substance detection apparatus.

  An object of the present invention is to provide a substance detection apparatus and a substance detection method that are cheaper than before and have a high detection sensitivity for a substance to be measured in view of the problems of the background art.

  To achieve the above object, the present invention forms a complex of a measurement target substance and a labeling substance contained in a specimen, and indirectly detects the measurement target substance in the specimen by detecting the labeling substance. In the substance detection apparatus, a binding region in which the complex is specifically bound by the measurement target substance, a dissociation means for dissociating the complex or the labeling substance from the binding area, and a labeling substance A detection region for detection, wherein the detection region is narrower than the combined region.

  The present invention also provides a complex forming step of forming a complex of a measurement target substance and a labeling substance contained in a specimen, and a binding region in which the complex is specifically bound by the measurement target substance. A binding step for specifically binding a body; a washing step for removing the non-specifically bound labeling substance from the binding region; and the specifically bound complex or the labeling substance possessed by the complex Dissociating from the binding region, collecting the complex dissociated from the binding region or the labeling substance into the detection region, and detecting the labeling substance in the detection region It is characterized by having.

  According to the present invention, it is possible to detect a measurement target substance at low cost and with high sensitivity.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 is a diagram illustrating a conceptual diagram of an embodiment of a substance detection device of the present invention. The substance detection apparatus of this example includes a container 1001 that holds a specimen 1002 containing a measurement target substance. The shape of the container 1001 is a substantially cylindrical shape, and a cylindrical recess is provided on the top of the container 1001 so as to coincide with the central axis of the container 1001. That is, the shape of the upper surface of the container 1001 is a ring shape. The cylindrical recess extends from the upper surface of the container 1001 downward. Further, the width (cylinder diameter) of the bottom portion of the container 1001 becomes narrower as it gets deeper. The area of the bottom surface of the container 1001 is made smaller than the side area of the cylindrical recess.

  A bound substance that can specifically bind to the measurement target substance is solid-phased on the side surface of the cylindrical recess in the container 1001. That is, the side surface of the recess is a coupling region 1003.

  Further, the bottom of the container 1001 is a detection region 1009, and a detection device 1004 is provided below the container 1001 as detection means. The detection area 1009 is narrower than the binding area 1003. As a result, the detection device 1004 can be made small. Thus, by making the detection device 1004 small, an inexpensive substance detection apparatus can be obtained.

  The detection device 1004 is connected to the detection signal processing device 1005. The detection signal processing device 1005 processes the detection signal sent from the detection device 1004. By doing so, the presence and amount of the substance to be measured are measured.

  Dissociation means 1007 is installed around the container 1001. The dissociation means 1007 is a means for dissociating the complex of the substance to be measured and the labeling substance specifically bound to the binding region 1003 from the binding region 1003. Also, below the container 1001, a collection unit 1008 for collecting a complex of the measurement target substance and the labeling substance or the labeling substance in the detection region 1009 is installed. Further, a removing means 1006 for removing unnecessary labeling substances is installed above the container 1001. The unnecessary labeling substance means a labeling substance that has not formed a complex with the substance to be measured. Further, unnecessary labeling substances include labeling substances bound to substances other than the measurement target substance. When detecting the measurement target substance, the detection target substance is indirectly detected by detecting the labeling substance. Therefore, when there is an unnecessary labeling substance as described above, the measurement target substance cannot be detected correctly. The removing means 1006 is a means for removing such an unnecessary labeling substance.

  As the removing unit 1006, the dissociating unit 1007, and the collecting unit 1008, an external force applying unit that moves the measurement target substance, the labeling substance, and the complex can be used. When the labeling substance is a magnetic substance, a coil that generates a magnetic field can be used as the external force applying means. When the labeling substance is a charged substance, an electrode that generates an electric field can be used as the external force applying means. The external force applying means used as the removing means 1006, the dissociating means 1007, and the collecting means 1008 may be the same or different.

  The container 1001 need not have the shape and configuration described in the above embodiment. For example, the shape of the container 1001 may be a rectangular parallelepiped, the coupling region 1003 may be provided on the side surface, and the detection region 1009 may be provided on the bottom surface. The shape of the container 1001 may be any shape as long as the detection region 1009 can be narrower than the coupling region 1003. Of course, the coupling region 1003 need not be configured on the side of the container.

  The detection device 1004 may be installed anywhere as long as it can detect the labeling substance (including the labeling substance in the complex) collected in the detection region 1009. The removing means 1006 may be installed anywhere as long as unnecessary labeling substances can be removed. The dissociation means 1007 may be installed anywhere as long as the complex specifically bound to the binding region 1003 can be dissociated. Further, the collecting means 1008 may be installed anywhere as long as the labeling substance or the complex can be collected in the detection region 1009. The arrangement of the detection device 1004, the removing unit 1006, the dissociating unit 1007, the collecting unit 1008, and the like is preferably changed appropriately depending on the shape of the container 1001, the physical properties of the measurement target substance and the labeling substance, and the like.

  The substance to be measured may be any substance as long as it specifically binds to the binding region 1003 and can bind to the labeling substance. Examples of such substances include DNA and antigens. When the substance to be measured is DNA, DNA complementary to the substance to be measured is used as the substance to be immobilized on the binding region 1003. When the substance to be measured is an antigen, an antibody that specifically binds to the antigen is used as the substance to be bound. The labeling substance preferably has a bound substance that specifically binds to the substance to be measured. By doing so, the substance to be measured and the labeling substance are specifically bound. Therefore, a complex can be easily formed.

  As the labeling substance, it is desirable to use a substance capable of obtaining a larger detection signal than directly detecting the measurement target substance. As such a substance, a substance that generates fluorescence, a radionuclide, a magnetic substance, a substance that generates electrochemiluminescence such as a ruthenium complex, or a charged substance is used. Among these, a magnetic substance or a charged substance is more preferably used because it can be easily moved by magnetic force or electrostatic force.

  Any device can be used as the detection device 1004 as long as it can detect the labeling substance. When the labeling substance is magnetized, as the detection device 1004, a magnetic sensor such as a magnetoresistive film, a Hall element, a coil, a magnetic impedance element, or a fluxgate element is used. Further, when the labeling substance is a charged substance, for example, a field effect transistor (FET) is used. In the FET, the magnitude of the current flowing between the source and the drain changes as the charge on the surface of the gate electrode changes. Therefore, it is possible to detect the labeling substance by collecting the charged labeling substance on the surface of the gate electrode. In this way, the substance to be measured is indirectly detected by detecting the labeling substance.

  The detection device 1004 may be provided at a location away from the detection region 1009. For example, when the labeling substance is a substance that emits light, radiation, or the like, the detection device 1004 can be provided at a location away from the detection region 1009. When the labeling substance emits light, an optical detection device is used as the detection device 1004. Then, the optical characteristics of the labeling substance present in the detection region 1009 are measured. When the labeling substance is a radionuclide, a detection device that detects radiation is used. The labeling substance can be detected by measuring the radiation emitted from the labeling substance.

  The inner wall of the container 1001 excluding the binding region 1003 is preferably configured so that the measurement target substance and the labeled object do not adsorb. By doing so, an unnecessary labeling substance can be effectively removed. In addition, the measurement target substance, the labeling substance, and the complex can be prevented from being adsorbed to a region other than the binding region 1003. This can increase the sensitivity of detection of the measurement target substance.

  In the case of using an antibody having a thiol group as a substance to be bound, by forming a gold film in the binding region 1003, the antibody is immobilized on the gold film surface via the thiol group. At this time, the substance to be measured is an antigen, and the labeling substance has an antibody that binds to the antigen. Therefore, it is desirable to cover the inner wall of the container 1001 excluding the bonding region 1003 with silicon nitride, silicon oxide, or the like. Silicon nitride and silicon oxide are materials that are difficult to adsorb thiol groups, antigens, and antibodies.

  When the labeling substance is charged, the inner wall of the container 1001 other than the binding region 1003 may be charged with the same polarity as the charge of the labeling substance. By doing so, the labeling substance can be prevented from adsorbing to a place other than the binding region 1003. Alternatively, by covering the container 1001 with a blocking material, it is possible to prevent the labeling substance from adsorbing to a place other than the binding region 1003.

  Hereinafter, the substance detection method in the present invention will be described. The substance detection method includes a complex formation step, a binding step, a washing step, a dissociation step, a collection step, and a detection step.

  In the complex formation step, the labeling substance and the measurement target substance in the specimen 1002 are combined to form a complex. This is performed by injecting a labeling substance into the specimen 1002. The complex formation step may be performed in advance in the container 1001 or in a container different from the container 1001. When the complex formation step is performed in different containers, the sample 1002 containing the complex is injected into the container 1001 after the complex is formed. Moreover, you may implement a composite_body | complex formation process after a joining process or during a joining process.

  In the binding step, the measurement target substance is specifically bound to the binding region 1003. Here, the measurement target substance includes a measurement target substance forming a complex with the labeling substance. In this case, the complex specifically binds to the binding region 1003 by the measurement target substance.

  After the complex formation step and the binding step are performed, a labeling substance that does not form a complex with the measurement target substance in the specimen 1002 or a binding force of specific binding (hereinafter abbreviated as a specific binding force). There is a labeling substance or the like that is non-specifically bound to the binding region 1003 with a force different from (). In the washing process, such an unnecessary labeling substance is removed by the removing means 1006.

  As the removing means 1006, for example, there is means for replacing the specimen 1002 using a buffer solution. By doing so, unnecessary labeling substances in the specimen 1002 can be removed. At this time, together with the flow of the buffer solution, it is possible to discharge the unnecessary labeling substance that is non-specifically bound with a force weaker than the specific binding force.

  When the unnecessary labeling substance is charged or magnetized, an external force applying means such as electrostatic force or magnetic force can be used as the removing means 1006. Then, unnecessary labeled beads are removed by applying an external force to the unnecessary labeling substance. When it is difficult for an external force to act on an unnecessary labeling substance, particles having a sufficient external force may be bound to the labeling substance. Examples of such particles include magnetic substances and charged substances.

  When the labeling substance is a magnetic substance, it is possible to generate a magnetic force by the removing means 1006 and collect unnecessary labeling substances in the upper part of the container 1001. Unnecessary labeling substances collected in this way can be removed from the specimen 1002 by using a dropper or the like. Alternatively, a buffer solution or the like can be poured into the container 1001 to wash away unnecessary floating labeling substances or unnecessary labeling substances that have come off the binding region 1003.

  Unnecessary labeling substances are removed from the detection region 1009 so as not to affect the detection signal during the detection process. At this time, an unnecessary labeling substance is preferably removed from the specimen 1002. By doing so, it is possible to reduce noise signals from unnecessary labeling substances when detecting the labeling substances. That is, the sensitivity of detection of the measurement target substance can be increased. However, if it is difficult to remove from the specimen 1002, it may be held in a region that does not affect the detection process.

  After the removal step, a dissociation step is performed. In the dissociation step, the complex specifically bound to the binding region 1003 by the measurement target substance is dissociated from the binding region 1003. At this time, the dissociation step may be performed by dissociating specific binding between the measurement target substance and the labeling substance. That is, the labeling substance in the complex may be dissociated from the binding region 1003.

  The dissociation means 1007 for dissociating the complex or the labeling substance is performed by an external force applying means for applying an external force to the labeling substance. It is preferable that the magnitude of the external force to be applied is approximately equal to the specific binding force. When the specific binding force varies, the magnitude of the external force is set to be approximately equal to the largest of the specific binding forces. By doing so, the complex specifically bound to the binding region 1003 and the labeling substance can be dissociated.

  By controlling the magnitude of the external force as described above, unnecessary labeling substances that are non-specifically bound with a force larger than the specific binding force are bound to the binding region 1003. Therefore, it is desirable that the binding region 1003 and the detection region 1009 be different regions. By doing so, an unnecessary labeling substance non-specifically bound to the binding region 1003 with a force larger than the specific binding force does not affect the noise signal during the detection process.

  As described above, the labeling substance non-specifically bound to the binding region 1003 with a force different from the specific binding force can be removed by the removal step and the dissociation step. Therefore, it is possible to increase the sensitivity of detection of the labeling substance.

  In the collection step, the complex and the labeling substance dissociated in the dissociation step are collected in the detection region 1009 by the collection means 1008. The collecting means 1008 may be anything as long as the dissociated complex and the labeling substance can be collected in the detection region 1009.

  In the detection step, the labeling substance (including the labeling substance in the complex) collected in the detection region 1009 is detected by the detection device 1004. Then, the presence / absence or amount of the target substance is indirectly detected by detecting the presence / absence or amount of the labeling substance. As the detection device 1004 for detecting the labeling substance, various devices can be used as described above.

(The labeling substance is an example of a magnetic substance)
Hereinafter, an example in the case of using magnetic beads as the labeling substance will be described. If the magnetic beads are magnetized in the complex formation step, the magnetic beads in the specimen 1002 are aggregated due to magnetostatic coupling. Therefore, the formation efficiency of complex formation will fall. Therefore, it is preferable that the magnetic beads be a substance exhibiting superparamagnetism. Superparamagnetism is a property that magnetizes when a magnetic field is applied and behaves like a non-magnetic material when no magnetic field is applied. That is, the magnetic beads can be prevented from aggregating due to magnetostatic coupling during the complex forming process. When moving the magnetic beads, a magnetic field may be applied to the magnetic beads to magnetize the magnetic beads. Further, in the detection step, when detecting the magnetic beads, the magnetic beads may be magnetized. By doing so, magnetic beads can be easily detected.

When using magnetic beads, the external force applying means for moving the magnetic beads is preferably means for applying a magnetic field. When the magnetic field H is applied to the magnetic beads, the force F _H acting on the magnetic beads is expressed by the following equation (1).

ここで、Mは磁気ビーズの磁化である。

Here, M is the magnetization of the magnetic beads.

The magnetization M of a material exhibiting superparamagnetism is expressed as a function of the magnetic field H. Further, in a relatively small magnetic field, the relationship between the magnetization M and the magnetic field H is almost linear. That is, the magnetization M of the magnetic beads exhibiting superparamagnetism is expressed as M = χH in a small magnetic field. Here, χ is the magnetic susceptibility of the magnetic beads. Accordingly, the force applied to the magnetic beads depends on the magnetic susceptibility, the magnitude of the magnetic field, the gradient of the magnetic field, and the like. A force F _H is applied to the magnetic beads in a direction from a small magnetic field to a large magnetic field. Therefore, when magnetic force is used as the removing means 1006, the dissociating means 1007, and the collecting means 1008, a magnetic field having an appropriate distribution is applied according to the magnetic characteristics of the magnetic beads.

  For example, it is assumed that unnecessary magnetic beads need to be collected on the side surface of the container 1001 in the removal step. At this time, a magnetic field that increases with distance from the coupling region 1003 may be applied to the inside of the container 1001. This can be easily achieved by providing a permanent magnet or an electromagnet on the outer side surface of the container 1001. The magnitude of the magnetic force applied at this time is set such that the complex specifically bound to the binding region 1003 does not dissociate. Thereafter, unnecessary magnetic beads collected on the side surface of the container 1001 may be removed to the outside of the container 1001 by another removing means 1006.

  As the dissociation means 1007, a permanent magnet or an electromagnet installed on the outer side surface of the container 1001 can be used. In this case, the magnitude of the applied magnetic force is set to be as large as the specific binding force. By doing so, the complex specifically bound to the binding region 1003 can be dissociated.

(An example of a charged substance in the labeling substance)
Hereinafter, an example in the case of using a charged substance as the labeling substance will be described. The substance detection apparatus at this time will be described with reference to FIG. The substance detection apparatus includes a container 1001 that holds a specimen 1002. The shape of the container 1001 is the same as that described with reference to FIG.

  A bound substance that can specifically bind to the measurement target substance is solid-phased on the outer peripheral side surface of the container 1001. That is, the side surface on the outer peripheral side of the container 1001 is a coupling region 1003. A cylindrical outer electrode 2007 is provided along the side surface of the container 1001 so that electric charges can be applied to the coupling region 1003. A cylindrical central electrode 2008 is provided in a cylindrical concave portion of the container 1001. The outer electrode 2007 and the center electrode 2008 are electrically connected to the power supply 2009 through a switch. When a voltage is applied, the polarity of both electrodes 2007 and 2008 is set so that the polarity is reversed.

  The bottom of the container 1001 is a detection region 1009, and a detection device 1004 is provided below the container 1001 as detection means. The detection area 1009 is configured to be narrower than the binding area 1003. In order to process the detection signal detected by the detection device 1004, the detection device 1004 is connected to the detection signal processing device 1005.

  Any material can be used as the material of the center electrode 2008 and the outer electrode 2007 as long as the charge is uniformly induced on the surface of the electrode. However, if the labeling substance or the complex is a material that binds nonspecifically, it may cause a decrease in detection sensitivity. In that case, it is desirable to cover both electrodes 2007 and 2008 with a protective film. By doing so, a decrease in detection sensitivity can be prevented.

  FIG. 2B is a cross-sectional view when the substance detection device is cut along the broken line AA ′ shown in FIG. The cross-sectional shape of the container 1001 is a ring shape, and a central electrode 2008 is installed at the center thereof. An outer electrode 2007 is installed on the outer periphery of the container 1001. Here, the radius of the center electrode 2008 is a, and the radius of the inner wall of the outer electrode 2007 is b. The heights of both electrodes 2007 and 2008 are sufficiently larger than the radii a and b.

  When the voltage V is applied to both electrodes 2007 and 2008 configured as described above, the magnitude of the electric field generated inside the container 1001 is expressed by the following equation (2).

ただし、rは容器1001の中心軸からの距離を表す。電界の向きは、容器1001の中心軸から容器1001側面に向かって、放射状に広がる方向となる。もちろん両電極2007,2008間の極性を反転させることによって、電界の向きは反転させることが出来る。

Here, r represents the distance from the central axis of the container 1001. The direction of the electric field is a direction that spreads radially from the central axis of the container 1001 toward the side surface of the container 1001. Of course, by reversing the polarity between the electrodes 2007 and 2008, the direction of the electric field can be reversed.

Here, the labeling substance 2011 is that the charged q _2. The charge q ₂ of the labeling substance 2011 can be changed by the pH, salt concentration, etc. of the specimen 1002. Biological substances such as antibodies and antigens can also change the charge amount as described above.

Further, the size of the labeling substance 2011 is assumed to be sufficiently smaller than the distance between the outer electrode 2007 and the center electrode 2008, that is, (b−a). A voltage V is applied to the outer electrode 2007 so that a charge having the same polarity as the charge q ₂ of the labeling substance 2011 is induced. At this time, a force in the direction toward the center electrode 2008 is applied to the labeling substance 2011 bonded to the binding region 1003. The magnitude of the force F is the magnitude indicated by the following expression (3).

上記のように、両電極2007,2008を構成したことにより、結合領域1003に結合されている標識物質2011には、ほぼ等しい力を印加することが出来る。また、印加する力の大きさは電圧によって簡単に制御可能である。したがって解離手段として磁力を用いる場合、両電極2007,2008間に与える電圧Vを適当な大きさに制御すれば良い。このとき、標識物質2011に印加する力の大きさは、特異的結合力とほぼ同等な大きさとする。そうすることで、結合領域1003と特異的に結合している標識物質2011を解離させることができる。

As described above, since both electrodes 2007 and 2008 are configured, substantially equal force can be applied to the labeling substance 2011 bonded to the binding region 1003. Further, the magnitude of the applied force can be easily controlled by the voltage. Therefore, when magnetic force is used as the dissociation means, the voltage V applied between the electrodes 2007 and 2008 may be controlled to an appropriate level. At this time, the magnitude of the force applied to the labeling substance 2011 is approximately equal to the specific binding force. By doing so, the labeling substance 2011 specifically bound to the binding region 1003 can be dissociated.

  Further, when it is necessary to collect unnecessary labeling substances 2011 in the center of the container 1001 in the removal step, the force F represented by the above formula (3) can be used. At this time, the magnitude of the force F is controlled to be smaller than the specific binding force. By doing so, the labeling substance 2011 floating in the specimen 1002 and the labeling substance 2011 bound nonspecifically with a force smaller than the specific binding force can be collected on the surface of the central electrode 2008. Thereafter, if necessary, unnecessary labeling substances 2011 collected on the surface of the center electrode 2008 may be removed from the container 1001.

(Example 1)
Next, examples of the substance detection apparatus and substance detection method in the present invention will be described. In this embodiment, prostate specific antigen (PSA) is used as the measurement target substance, and magnetic beads are used as the labeling substance.

  FIG. 3 is a diagram for explaining the substance detection apparatus in the present embodiment. The substance detection apparatus includes a container 3001 that holds a specimen 3002. The shape of the container 3001 is a cylindrical shape, and a cylindrical recess is provided so as to coincide with the central axis of the container 3001. That is, the shape of the upper surface of the container 3001 is a ring shape. The cylindrical recess extends downward from the upper surface of the container 3001. The diameter of this recess is 0.6 mm. A coil 3006 is installed inside the cylindrical recess. The bottom surface of the container 3001 is a circle having a diameter of 1.6 mm, and the height of the container 3001 is 14 mm.

A gold film is formed on the side surface of the cylindrical concave portion inside the container 3001. A primary antibody that specifically binds to PSA is immobilized on the gold film surface. That is, the side surface of the concave portion inside the container 3001 is a coupling region 3003. The area of the coupling region 3003 is about 20 mm ² .

  A coil 3011 is provided on the outer side surface of the container 3001. The coil 3011 is connected to a power source 3012 so that a magnetic field can be generated. The coil 3011 is wound around the iron core 3013. As for the shape of the iron core 3013, a portion around which the coil 3011 is wound is a cylindrical shape. Then, the shape is such that it becomes thinner as it approaches the side surface of the container 3001.

  Further, the coil 3011 is configured to be able to move up and down along the side surface of the container 3001. Further, the coil 3011 can rotate with respect to the central axis of the container 3001 along the side surface of the container 3001. The coil 3011 is used as a removing unit, a dissociating unit, and a collecting unit.

  As a collecting means, a coil 3014 is installed below the container 3001. The coil 3014 is connected to a power source. Further, a coil 3008 wound around an iron core 3009 is installed above the container 3001 as a removing means. The coil 3008 is connected to a power supply 3010.

  In this embodiment, one coil 3011 is used, but a plurality of similar coils may be provided. By doing so, a magnetic field can be applied to a wide area inside the container 3001.

  The bottom of the container 3001 is a detection region 3026, and a detection device 3004 is provided on the bottom surface of the container 3001 as detection means. The shape of the detection device 3004 is a square with a side length of 0.7 mm. FIG. 4 is a diagram showing a detailed configuration of the detection device 3004. The detection device 3004 is composed of a plurality of magnetoresistance effect elements 3021. The magnetoresistive elements 3021 are arranged on the same plane with 512 columns × 512 rows with a gap. A selection transistor 3020 is formed below the magnetoresistive effect element 3021. One magnetoresistive element 3021 is connected to one select transistor 3020. An upper electrode 3022 is formed on the magnetoresistive element 3021. The upper electrode 3022 is configured as the lower surface of the container 3001. The upper electrode 3022 is electrically connected to the detection signal processing device 3005.

  The magnetoresistive effect element 3021 is formed of a multilayer film of Ta / MnPt / CoFe / Ru / CoFeB / MgO / CoFeB / Au. The magnetoresistive element 3021 has magnetic anisotropy in the direction in the film plane. For this reason, the electric resistance of the magnetoresistive effect element 3021 changes depending on the magnitude of the magnetic field in the direction in the film plane. Therefore, the magnetic beads 3019 can be detected by detecting the magnitude of the stray magnetic field in the direction in the film plane.

The area of the surface of the magnetoresistive effect element 3021 is 0.4 μm × 1.3 μm per element. Therefore, the surface area of all magnetoresistive elements 3021 is 0.13 mm ² . This is significantly smaller than the area of the coupling region 3003 (about 20 mm ² ). If the surface area equivalent to the area of the coupling region 3003 is filled with the magnetoresistive effect element 3021, the magnetoresistive effect element 3021 is required to be 100 times or more that in the present embodiment. This is a factor that significantly increases the cost of the substance detection apparatus.

  A protrusion made of silicon nitride 3023 is formed on the inner side of the container 3001 of the upper electrode 3022. The silicon nitride 3023 is formed corresponding to a region having a gap between the magnetoresistive effect elements 3021. Silicon nitride 3023 is a material that hardly adsorbs antigens or antibodies. Thus, the magnetic beads 3019 can be collected immediately above the magnetoresistive effect element 3021 during the collecting process.

  As the magnetic beads 3019, those showing superparamagnetism are used. The diameter of the magnetic bead 3019 is 100 nm, and a secondary antibody that specifically binds to PSA 3017 is formed on the surface of the magnetic bead 3019.

  Hereinafter, the substance detection method in a present Example is demonstrated. First, as a complex formation step, a specimen 3002 and magnetic beads 3019 are injected into a container 3001 and stirred for about 20 minutes. At this time, PSA 3017 and magnetic beads 3019 in the specimen 3002 form a complex via the secondary antibody.

  Next, as a coupling step, a current is passed through the coil 3006 by the power source 3007. Then, a magnetic field is formed in the container 3001 so as to become weaker as the distance from the coupling region 3003 increases. Therefore, the magnetic beads 3019 are attracted to the surface of the coupling region 3003 having a stronger magnetic field. At this time, as shown in FIG. 5, the complex of PSA 3017 and magnetic beads 3019 is specifically bound to the primary antibody 3016 by PSA 3017. That is, the magnetic beads 3019 are specifically bound to the binding region 3003 via the primary antibody 3016, the PSA 3017, and the secondary antibody 3018.

  In addition, the magnetic beads 3019 that have not formed a complex in the complex formation step do not bind to the binding region 3003 or are non-specifically bound to the binding region 3003. Such magnetic beads 3019 are unnecessary magnetic beads that cause a reduction in detection sensitivity.

  Next, as a removing step, application of the magnetic field by the coil 3006 is stopped. Then, unnecessary magnetic beads not bound to the binding region 3003 are diffused into the specimen 3002 by Brownian motion. Then, on the surface of the binding region 3003, magnetic beads 3019 that are specifically bound by the PSA 3017 and unnecessary magnetic beads that are non-specifically bound remain.

  Next, a current is supplied from the power source 3012 to the coil 3011. Then, a magnetic field that becomes stronger as the distance from the coupling region 3003 increases. When there is no iron core 3013, the strength of the magnetic field H generated by passing the current I through the coil 3011 is expressed by the following equation (4).

ここで、コイル3011の半径をαとした。またコイル3011の中心軸上において、コイル3011からの距離をｚとした（図６参照）。ただし、Nはコイルの巻き数である。

Here, the radius of the coil 3011 is α. In addition, the distance from the coil 3011 on the central axis of the coil 3011 is z (see FIG. 6). Here, N is the number of turns of the coil.

The magnetization M of the magnetic bead 3019 exhibiting superparamagnetism is expressed as M = χH in a small magnetic field. Accordingly, the force F _H applied to the magnetic bead 3019 at a distance z from the coil 3011 by passing the current I through the coil 3011 is expressed by the following equation (5) from the equations (1) and (4). It is estimated as follows.

またコイル3011内部に鉄心3013を設けた場合、鉄心3013の先端部を小さくすることによって、より大きな磁力を得ることが可能である。本実施例においては、コイル3011が巻かれている部分の鉄心3013の断面の直径を1cmとした。また、鉄心3013の先端部の直径を1mmとし、コイルの巻き数Nを100とした。さらに、鉄心3013の先端部から結合領域3003に結合している磁気ビーズ3019までの距離zが1 [mm]となるように構成されている。本実施例で用いた磁気ビーズ3019の磁化率χは2×10^-22である。このとき、コイル3011に流す電流Iを80 [mA]とした。

When the iron core 3013 is provided inside the coil 3011, it is possible to obtain a larger magnetic force by reducing the tip of the iron core 3013. In this embodiment, the diameter of the cross section of the iron core 3013 where the coil 3011 is wound is 1 cm. The diameter of the tip of the iron core 3013 was 1 mm, and the number of turns N of the coil was 100. Furthermore, the distance z from the tip of the iron core 3013 to the magnetic beads 3019 bonded to the binding region 3003 is set to 1 [mm]. The magnetic bead 3019 used in this example has a magnetic susceptibility χ of 2 × 10 ⁻²² . At this time, the current I flowing through the coil 3011 was set to 80 [mA].

  In this way, the current is controlled so that the magnetic beads 3019 specifically bound by the PSA 3017 are not dissociated. And by moving the coil 3011 up and down and rotating, unnecessary magnetic beads that are non-specifically bound to the unnecessary magnetic beads floating in the specimen 3002 with a force weaker than the specific binding force, Pull to coil to 3011. Then, unnecessary magnetic beads are collected on the top of the container 3001 by moving the coil 3011 to the top of the container 3001. Thereafter, a current is passed through the coil 3008 using the power supply 3010 to generate a magnetic field from the coil 3008. Then, unnecessary magnetic beads are attracted to the iron core 3009 provided in the coil 3008. At this time, unnecessary magnetic beads are bound to the iron core 3009 by bringing the iron core 3009 into contact with the specimen 3002. Then, the iron core 3009 is moved away from the container 3001 while the magnetic field generated by the coil 3008 is generated. By doing so, unnecessary magnetic beads can be removed from the container 3001.

  After removing unnecessary magnetic beads from the specimen 3002, a dissociation step is performed. In the dissociation step, a magnetic field is generated again from the coil 3011. The magnitude of the magnetic field generated at this time is set to be approximately equal to the specific binding force between the PSA 3017 and the primary antibody 3016. This is implemented by controlling the current flowing through the coil 3011. In this embodiment, the current flowing through the coil 3011 is 700 [mA]. By doing so, the complex of PSA 3017 and magnetic beads 3019 that are specifically bound is dissociated from the binding region 3003. At this time, unnecessary magnetic beads that are non-specifically bound to the binding region 3003 with a binding force larger than the magnetic force applied to the magnetic beads 3019 are not dissociated. Thus, unnecessary magnetic beads can be efficiently removed by the removal step and the dissociation step.

  Next, as a collection step, the complexes dissociated in the dissociation step are collected in the detection region 3026. For this purpose, the coil 3011 is moved below the container 3001 while a magnetic field is generated from the coil 3011. By doing so, the dissociated complex is collected below the container 3001. Thereafter, the magnetic field is stopped from the coil 3011 and the magnetic field is generated by the coil 3014, whereby the complex is collected in the detection region 3026. As described with reference to FIG. 4, a protrusion made of silicon nitride 3023 is formed at the bottom of the container 3001. Therefore, in the collecting step, the composite is collected on the surface of the magnetoresistive effect element 3021.

  Finally, as a detection step, the magnetic beads 3019 in the complex collected in the detection region 3026 are detected. Since the magnetic bead 3019 exhibits super paramagnetism, no magnetization occurs in the absence of a magnetic field. Therefore, in order to detect the magnetic bead 3019, it is necessary to magnetize the magnetic bead 3019. For this purpose, a bias magnetic field may be applied to the magnetic beads 3019. FIG. 7 is a view showing a state when a bias magnetic field 3024 is applied to the magnetic beads 3019. As described above, a bias magnetic field 3024 in a direction perpendicular to the film surface of the magnetoresistive effect element 3021 is applied to the magnetic beads 3019. The magnitude of the bias magnetic field 3024 is preferably such a magnitude that the magnetization of the magnetic beads 3019 to be used is not saturated. The magnitude of such a magnetic field is about 500 [Oe] to 2000 [Oe]. Such a bias magnetic field 3024 can be generated by the coil 3014.

  Magnetic beads 3019 magnetized by the bias magnetic field 3024 generate a stray magnetic field 3025. The stray magnetic field 3025 has a component in a direction parallel to the film surface of the magnetoresistive element 3021. Therefore, the resistance of the magnetoresistive effect element 3021 changes due to the stray magnetic field 3025. By measuring the amount of change in resistance, the amount of magnetic beads 3019 can be detected. The amount of PSA 3017 can be measured from the amount of magnetic beads 3019.

(Example 2)
Another embodiment of the substance detection apparatus and substance detection method of the present invention will be described below. In this example, charged beads are used as the labeling substance, and PSA is used as the measurement target substance.

  FIG. 8 is a conceptual diagram of the substance detection apparatus in the present embodiment. The substance detection apparatus includes a container 4001 that holds a specimen 4002 containing a measurement target substance. The shape of the container 4001 is a substantially cylindrical shape. The bottom surface of the container 4001 is a circle having a diameter of 1.6 mm, and the height of the container 4001 is 14 mm.

  A discharge port 4010 for discharging the fluid in the container 4001 is formed in the upper part of the container 4001. A valve 4009 for opening and closing the discharge port 4010 is attached to the discharge port 4010. In addition, an injection port 4012 for injecting a specimen 4002, a buffer solution, or the like is formed in the lower part of the container 4001. A valve 4011 for opening and closing the injection port 4012 is attached to the injection port 4012.

A gold film is formed on the side surface of the container 4001. A primary antibody capable of specifically binding to PSA is formed on the gold film. In this way, the coupling region 4003 is configured on the side surface of the container 4001. The area of the coupling region 4003 is 100 mm ² .

  A cylindrical outer electrode 4006 is attached to the side surface of the container 4001 so as to surround the coupling region 4003. A cylindrical center electrode 4008 is provided along the center axis of the container 4001. The center electrode 4008 has a height from the top surface of the container 4001 to the vicinity of the bottom surface of the container 4001. The diameter of the center electrode 4008 is 0.3 mm. The center electrode 4008 is electrically connected to the outer electrode 4006 via the power supply 4007.

  In the center electrode 4008, a ring-shaped moving electrode is installed with its center axis aligned. This ring-shaped moving electrode is cut into two on the plane including the central axis, and each has a semi-ring shape. The two moving electrodes 4021 and 4022 can move up and down along the center electrode 4008, respectively.

  A detection device 4004 is provided as a detection means on a part of the bottom surface of the container 4001. Therefore, a part of the bottom surface of the container 4001 becomes the detection region 4018. As described above, the binding region 4003 and the detection region 4018 are different regions. The detection device 4004 is connected to the detection signal processing device 4005. As the detection device 4004, an FET (Field Effect Transistor) is used. Ten thousand FETs are provided, each connected to a selection transistor. The FET is electrically connected to the detection signal processing device 4005.

FIG. 9 is a diagram illustrating a detailed configuration of the FET. The FET has a source 4013, a drain 4014, and a gate electrode 4015. The source 4013 and the drain 4014 are spaced apart from the surface of the silicon substrate 4016. A gate oxide film 4020 is formed above the source 4013 and the drain 4014. A gate electrode 4015 is formed on the gate oxide film 4020. The area of the gate electrode 4015 is 5000 nm ² . Therefore, the total area of the gate electrode 4015 is 50 μm ² . Thus, the detection region 4018 is significantly narrower than the binding region 4003. Therefore, it is not necessary to increase the number of FETs, and the substance detection device can be manufactured at a low cost.

  When the charge on the gate electrode 4015 changes, the current flowing between the source 4013 and the drain 4014 changes. That is, when there is a charged bead 4019 on the gate electrode 4015, the quantity of the labeled bead 4019 can be detected by measuring the change in the magnitude of the current. In this way, it is possible to indirectly measure the concentration of PSA 4017.

  Hereinafter, the substance detection method in a present Example is demonstrated. A negatively charged substance is used as the labeling bead 4019. First, as a complex formation step, the specimen 4002 and the labeled beads 4019 are injected into the container 4001 from the injection port 4012. At this time, a secondary antibody that specifically binds to PSA 4017 is immobilized on the surface of the labeled bead 4019. Then, stirring is performed for about 20 minutes, and PSA 4017 and labeled beads 4019 are sufficiently reacted to form a complex.

  Next, a negative polarity is given to the center electrode 4008 by the power source 4007 as a coupling step. By doing so, the labeled beads 4019 can be collected in the binding region 4003. At this time, the labeled beads 4019 that have formed a complex with PSA 4017 are specifically bound to the primary antibody by PSA 4017. In this way, the complex is specifically bound to the binding region 4003 by PSA 4017.

  Next, as a removing step, the electric charge of the center electrode 4008 is removed, and the electrostatic force applied to the labeled beads 4019 is removed. Then, unnecessary labeled beads that are not bound to the binding region 4003 are dispersed in the container 4001 by Brownian motion. Then, the valve 4011 attached to the injection port 4012 is opened, and the phosphate buffer solution is injected from the injection port 4012. Accordingly, the valve 4009 attached to the discharge port 4010 is opened, and the mixed solution of the specimen 4002 and the phosphate buffer solution is discharged from the discharge port 4010. In this way, unnecessary labeled beads floating in the specimen 4002 are discharged out of the container 4001. Further, unnecessary labeled beads non-specifically bound to the binding region 4003 with a weak force due to the flow of the mixed solution inside the container 4001 are dissociated from the binding region 4003 and discharged out of the container 4001. As described above, the inside of the container 4001 is sufficiently washed to remove unnecessary magnetic beads from the inside of the container 4001. Thereafter, the valve 4009 and the valve 4011 are closed.

  After removing unnecessary magnetic beads, a dissociation step is performed. In the dissociation process, a positive polarity is given to the center electrode 4008 by the power source 4007. In this way, a force in the direction toward the center electrode 4008 is applied to the labeled bead 4019 in the complex. The magnitude of the force applied at this time is approximately the same as the specific binding force between PSA 4017 and the primary antibody. Then, the complex is dissociated from the binding region 4003. At this time, unnecessary labeled beads that are non-specifically bound with a binding force larger than the magnetic force applied to the labeled beads 4019 are not dissociated. Thus, unnecessary labeled beads can be removed by the removal step and the dissociation step. At this time, the dissociated complex is attracted to the vicinity of the center electrode 4008.

  Next, a collecting process is performed. In advance, the moving electrodes 4021 and 4022 are stationary at the top of the container 4001. In the collection process, first, application of voltage to the center electrode 4008 is stopped. Then, the moving electrode 4021 is positively charged. At the same time, the moving electrode 4021 is moved from the upper part of the container 4001 toward the lower part along the center electrode 4008. At this time, the composite drawn to the center electrode 4008 is collected together with the center electrode 4008 in the lower part of the container 4001. Similarly, the moving electrode 4022 is positively charged, and the moving electrode 4022 is moved from the upper part of the container 4001 toward the lower part. By doing so, the composites collected near the center electrode 4008 are collected at the bottom of the container 4001. Thereafter, the charges of the moving electrodes 4021 and 4022 are removed, and the gate electrode 4015 of the FET is positively charged. In this way, the composite can be collected on the surface of the gate electrode 4015, that is, the detection region 4018.

  After the collection process, a detection process is performed. In the detection step, first, charges induced in the gate electrode 4015 of the FET are removed. Then, current flows from the source 4013 toward the drain 4014. The magnitude of the current at this time is read by the detection signal processing device 4005. The magnitude of the current flowing at this time varies depending on the total amount of charges on the surface of the gate electrode 4015. Therefore, the amount of labeled beads 4019 in the complex can be detected by measuring the magnitude of the current. Thereby, the amount of PSA 4017 can be detected indirectly.

  According to the substance detection method of each Example demonstrated above, the area | region which installs a detection device can be made small, maintaining high detection sensitivity. And it is possible to manufacture a substance detection apparatus cheaply by reducing the area | region which installs a detection device.

The conceptual diagram which shows an example of the substance detection apparatus of this invention. (A) is a conceptual diagram which shows an example of the substance detection apparatus of this invention. (B) is sectional drawing when a substance detection apparatus is cut | disconnected in broken line AA 'illustrated by (a). The schematic diagram of the substance detection apparatus which is one Example of this invention. The schematic diagram of the detection device used in the substance detection apparatus shown in FIG. The figure explaining a mode that a labeling substance couple | bonds with a detection area | region specifically with a measuring object substance. The conceptual diagram explaining the magnetic field produced by a coil. The conceptual diagram explaining the bias magnetic field applied when detecting a magnetic bead, and the stray magnetic field which arises from a magnetic bead. The schematic diagram of the substance detection apparatus which is one Example of this invention. Sectional drawing of the detection device using FET which is a detection means of a labeled substance.

Explanation of symbols

1002,3002,4002 specimens
1003,3003,4003 Bonding region
1004,3004,4004 Detection device
1006 Removal methods
1007 Dissociation means
1008 Collection means
1009,3026,4018 Detection area
2011 Labeling substance
3017,4017 PSA
3019 magnetic beads
3021 magnetoresistive element
4019 labeled beads

Claims (7)

In the substance detection apparatus for detecting the measurement target substance in the sample indirectly by forming a complex of the measurement target substance and the labeling substance contained in the sample, and detecting the labeling substance,
A binding region in which the complex is specifically bound by the substance to be measured;
Dissociation means for dissociating the complex or the labeling substance from the binding region;
A detection region for detecting the labeling substance,
The substance detection apparatus, wherein the detection area is narrower than the binding area.   The substance detection apparatus according to claim 1, wherein the dissociation unit dissociates the complex or the labeling substance using magnetic force.   The substance detection apparatus according to claim 1, wherein the dissociation unit dissociates the complex or the labeling substance using electrostatic force.   The substance detection apparatus according to claim 1, wherein the binding region and the detection region are in different regions.   The substance detection apparatus according to claim 1, further comprising a collection unit configured to collect the complex and the labeling substance in the detection region. A complex forming step for forming a complex of the measurement target substance and the labeling substance contained in the specimen;
A binding step of specifically binding the complex to a binding region where the complex is specifically bound by the substance to be measured;
A washing step of removing the non-specifically bound labeling substance from the binding region;
A dissociation step of dissociating the complex bound specifically or the labeling substance of the complex from the binding region;
Collecting the complex or the labeling substance dissociated from the binding region into a detection region;
A substance detection method comprising: a detection step of detecting the label substance in the detection region.   The substance detection method according to claim 6, wherein the labeling substance is removed from the sample in the washing step.

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