JP2009128169A - Detection method, detection cartridge and detection kit of target material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method, a detection cartridge and a detection kit of a target material, having excellent detection efficiency. <P>SOLUTION: This detection method of the target material in a specimen includes processes for: (1) forming a complex wherein a nonmagnetic particle is held by a magnetic particle through a target material by bringing the nonmagnetic particle equipped with a first target material capturing body into contact with the magnetic particle equipped with a second target material capturing body and the target material; (2) extracting each complex into an accumulation domain by applying an electric field; (3) detecting the amount of the magnetic particles extracted into the accumulation domain by a magnetometric sensor; and (4) calculating the amount of the target material from the amount of the magnetic particles. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a target substance detection method, a detection cartridge, and a detection kit for detecting a target substance in a specimen. The present invention particularly relates to a detection method, a detection cartridge, and a detection kit for a target substance that can be suitably applied to a so-called biosensor using a specific molecular recognition ability of a substance derived from a living body or a similar substance.

  A biosensor is a measuring device that utilizes the excellent molecular recognition ability of living organisms and biomolecules. In vivo, for example, there are enzyme-substrate, antigen-antibody, DNA-DNA, etc. as a combination of substances having affinity for each other. A biosensor carries one of these combinations on a substrate and uses it for the other. It uses the principle that it can selectively measure any substance. In recent years, biosensors are expected to have a wide range of applications not only in the medical field, but also in the environment and foodstuffs. In order to expand the range of use, biosensors that are small, lightweight, highly sensitive, and highly efficient are desired. Yes.

  Currently, as one of the methods for detecting such an interaction between biomolecules, research on a magnetic detection method using magnetic particles as a label is being actively pursued.

  FIG. 1 shows an example of a conventional solid-phase analysis method using a magnetic label. In the method shown in FIG. 1, first, a first target substance capture capable of specifically recognizing and capturing one region of a target substance (referred to as an epitope in the case of an antigen-antibody reaction) on the substrate surface in advance. The body (called the primary antibody in the case of an antigen-antibody reaction) is fixed. Next, the sample liquid containing the target substance is brought into contact. By this operation, the target substance is specifically captured by the first target substance capturing body. Next, a second target substance capturing body that can specifically recognize and capture the other region of the target substance specifically captured by the first target substance capturing body (second in the case of an antigen-antibody reaction). Magnetic particles having a secondary antibody) are introduced into the liquid. By this operation, the second target substance capturing body is captured by the target substance specifically captured by the first target substance capturing body immobilized on the substrate surface. As a result, as shown in FIG. The magnetic particles are immobilized on the substrate surface through the substance.

  Also, as a different method, magnetic particles having a second target substance capturing body are added in advance to a sample liquid containing the target substance to form a “target substance-second target substance capturing body” complex. By bringing the complex into contact with the first target substance capturing body immobilized on the substrate, as a result, as shown in FIG. 1, the magnetic particles are immobilized on the substrate surface via the target substance. Is also possible.

  Then, the concentration of the target substance can be calculated by measuring the number of magnetic particles immobilized on the substrate surface by some method.

  As a method for detecting the magnetic particles immobilized on the surface of the substrate, a method using a SQUID (superconducting quantum interferometer) (Patent Document 1), a method using a semiconductor Hall element (Patent Document 2), and a magnetoresistive effect element method (Patent Document 3) is disclosed.

  In addition, the following method has been proposed as a method of using the characteristics of magnetic particles not for use as a label as described above but for other purposes.

  Patent Document 4 discloses a method of attaching a magnetic substance to a biopolymer, applying a magnetic attraction force thereto, and pulling it toward the probe biopolymer fixed on the substrate. Such a method is aimed at speeding up hybridization.

Patent Document 5 discloses an apparatus in which particles equipped with a probe are maintained in a reaction part by a capturing means. The capturing means is an electrode and an electromagnet, and the particles are maintained in a reaction part that reacts the probe with a target substance by application. Such a configuration is intended to provide an apparatus capable of arranging a plurality of types of probes at a desired position of the reaction unit according to the types and capable of detecting many types of substances at a time.
Japanese Patent Laid-Open No. 2001-033455 International Publication No. 03/067258 Pamphlet U.S. Patent No. 5981297 JP 2003-159057 A JP 2002-281967 A

  The target substance detection method based on the detection of magnetic particles disclosed in Patent Documents 1 to 3 includes a step of binding reaction between a target substance capturing body and a target substance fixed in advance on a solid phase on the solid phase. Therefore, there is a problem that it takes time to sufficiently advance the reaction on the solid phase, and there is a problem that a sufficient number of bonds for detection cannot be obtained when the solid phase area is limited and small.

  Therefore, an object of the present invention is to provide a method for further reducing reaction time and increasing sensitivity, that is, improving detection efficiency, a cartridge used in the method, and a detection kit in a target substance detection method using a magnetic material. It is in.

The first detection method of the present invention is a method for detecting a target substance in a specimen, and (1) a non-magnetic particle having a first target substance capturing body and a magnetic having a second target substance capturing body. Contacting the particles with the target substance in a fluid to form a complex in which the nonmagnetic particles are held on the magnetic particles via the target substance;
(2) attracting the composite to the accumulation region by applying an electric field;
(3) a step of detecting the amount of magnetic particles possessed by the complex attracted to the accumulation region by a magnetic sensor;
(4) calculating the amount of the target substance based on the amount of magnetic particles possessed by the complex;
It is a detection method of the target substance characterized by including.

The second detection method of the present invention is a method for detecting a target substance in a specimen,
(1) A non-magnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body, and the target substance are contacted in a fluid, and the magnetic particles are passed through the target substance. Forming a composite with the non-magnetic particles held therein,
(2) drawing the composite to the first integration region by applying an electric field;
(3) removing the magnetic particles not forming the complex;
(4) drawing the composite to the second integration region by applying a magnetic field;
(5) a step of detecting the amount of magnetic particles of the composite attracted to the second accumulation region by a magnetic sensor;
(6) calculating the amount of the target substance based on the amount of magnetic particles of the complex;
It is a detection method of the target substance characterized by including.

A third detection method of the present invention is a method for detecting a target substance in a specimen,
(1) A non-magnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body, and the target substance are contacted in a fluid, and the magnetic particles are passed through the target substance. Forming a composite with the non-magnetic particles held therein,
(2) drawing the composite to the first integrated region by applying a magnetic field;
(3) drawing the composite to the second integration region by applying an electric field;
(4) detecting the amount of magnetic particles of the composite attracted to the second accumulation region by a magnetic sensor;
(5) calculating the amount of the target substance based on the amount of magnetic particles of the complex;
It is a detection method of the target substance characterized by including.

The detection cartridge of the present invention is a detection cartridge for detecting a target substance in a specimen,
A liquid holding region for holding a liquid containing a complex in which nonmagnetic particles are held on magnetic particles via the target substance;
Comprising at least one or more integration regions for integrating the composite,
At least one of the integrated regions is provided in a space that can be detected by a magnetic sensor detection element.

Furthermore, the detection kit of the present invention is a detection kit for detecting a target substance in a specimen,
A non-magnetic particle having a first target substance capturing body;
A magnetic particle having a second target substance capturing body,
The detection kit is characterized in that the non-magnetic particles are charged in an aqueous solution whose pH is approximately the isoelectric point of the magnetic particles.

  The present invention provides a method for detecting a target substance, a detection cartridge, and a detection kit that are excellent in detection efficiency.

  A preferred embodiment of the present invention will be described in detail.

  First, after describing the detection method in the present invention, each component, each form of the detection cartridge and the detection kit will be described in detail.

<Detection method>
(First embodiment)
A first embodiment of the detection method according to the present invention is a method for detecting a target substance in a specimen,
(1) A nonmagnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body and a target substance are brought into contact with each other in a fluid, and the magnetic particles are nonmagnetic via the target substance. Forming a composite with retained particles;
(2) attracting the composite to the accumulation region by applying an electric field;
(3) a step of detecting the amount of magnetic particles of the composite attracted to the accumulation region by a magnetic sensor;
(4) a step of calculating the amount of the target substance based on the amount of the magnetic particles included in the complex.

  FIG. 2 is a diagram schematically showing a first embodiment of the detection method according to the present invention.

  FIG. 2A shows a case where a nonmagnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body, and a target substance are brought into contact with each other in a fluid, and the magnetic substance is passed through the target substance. It is the figure which showed typically the process (1) which forms the composite_body | complex with which the nonmagnetic particle was hold | maintained to particle | grains. In this step, the nonmagnetic particles provided with the first target substance capturing body, the magnetic particles provided with the second target substance capturing body, and the target substance are mixed in the liquid to prepare a mixed solution. The reaction time is appropriately determined according to the concentration of particles and target substance, the amount of liquid, the stirring method and the like.

  In the present invention, since the complex is formed in the liquid phase (in the fluid), the reaction time can be shortened as compared with the conventional method in which the complex formation reaction is performed in the solid phase. It is desirable that the mixed liquid is introduced into a liquid holding region of the detection cartridge described later. As a result, the complex is held in the liquid holding region. The mixed liquid may be introduced into the liquid holding area after being mixed and adjusted in a container different from the detection cartridge, that is, in an area different from the liquid holding area, or may be mixed and adjusted in the liquid holding area. Good. In addition, it is preferable that pH of the adjusted liquid mixture is a value close | similar to the isoelectric point of a magnetic particle apart from the isoelectric point of the nonmagnetic particle mentioned later. By doing so, in a liquid mixture, it can prepare so that a nonmagnetic particle may have a surface charge and a magnetic particle may have a surface charge as much as possible.

FIG. 2B is a diagram schematically showing a step (2) of drawing the composite to the accumulation region by applying an electric field. In this step, the mixed solution containing the complex is brought into contact with the accumulation region, and an electric field is applied to draw the complex to the accumulation region. Therefore, it is desirable that the accumulation region is disposed in contact with the liquid holding region. As will be described later, the application of an electric field means applying a potential or a current so that the surface charge of the accumulation region has a polarity opposite to the polarity of the surface charge of the nonmagnetic particles in the mixed solution. Therefore, nonmagnetic particles are selectively attracted to the accumulation region. On the other hand, most of the magnetic particles not forming the composite remain in the liquid. As will be described later, this is because the surface charge amount of the magnetic particles according to the present invention is smaller than the surface charge amount of the non-magnetic particles in the liquid for accumulation. The electric field application time, potential, current value, and the like are appropriately determined depending on the selectivity between the nonmagnetic particles and the magnetic particles, the particle concentration, and the like. You may have the process of removing the magnetic particle which is not forming the composite_body | complex remaining in a liquid in the state which applied the electric field after this operation. In particular, in the case where detection is performed in a state in which a complex as described below is dispersed in a liquid, a step of removing magnetic particles remaining in the liquid and not forming a complex after step (2). It is preferable to have.
The removal method is the simplest method for washing with a solvent, but is not limited to this as long as magnetic particles that do not form a complex can be selectively removed, such as removal using a magnetic field.

FIG. 2C is a diagram schematically showing the composite held in the accumulation region by performing the steps (1) and (2). Here, the step (3) of detecting the amount of magnetic particles attracted to the accumulation region by a magnetic sensor is performed. This detection step may be performed with an electric field applied or may be performed with the electric field canceled as long as the amount of magnetic particles can be detected by a magnetic sensor. That is, the composite may be held in the accumulation region or may be dispersed in the liquid. However, when the magnetic sensor has a high sensitivity in the vicinity of the detection element surface, such as a magnetoresistive effect element, it is desirable to perform the detection step while keeping the vicinity of the detection element surface as an integration region and holding the complex in the integration region. Furthermore, the target substance in the specimen can be detected by performing the step (4) of calculating the quantity of the target substance based on the quantity of the magnetic particles. Here, with regard to the relationship between the amount of magnetic particles and the amount (concentration) of the target substance, the relationship between the magnetic particles and the concentration is obtained in advance using a standard sample having a plurality of known concentrations. If a calibration curve is obtained and a function of magnetic particles and concentration is obtained, the amount (concentration) of the target substance can be obtained from the amount of magnetic particles at the time of actual measurement using this function.
(Second embodiment)
A second embodiment according to the present invention is a method for detecting a target substance in a specimen,
(1) A nonmagnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body and a target substance are brought into contact with each other in a fluid, and the magnetic particles are nonmagnetic via the target substance. Forming a composite with retained particles;
(2) drawing the composite to the first integration region by applying an electric field;
(3) removing the magnetic particles not forming the complex;
(4) drawing the composite to the second integration region by applying a magnetic field;
(5) a step of detecting the amount of magnetic particles of the composite attracted to the second accumulation region by a magnetic sensor;
(6) calculating the amount of the target substance based on the amount of the magnetic particles of the composite.

  FIG. 3 is a diagram schematically showing a second embodiment according to the present invention.

  FIG. 3A illustrates a case where a nonmagnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body, and a target substance are brought into contact with each other, and the magnetic particles are non-contacted via the target substance. It is the figure which showed typically the process (1) which forms the composite_body | complex with which the magnetic particle was hold | maintained. This step is a step of adjusting the mixed liquid as in step (1) in the first embodiment.

  FIG. 3B is a diagram schematically showing a step (2) of drawing the composite to the first integration region by applying an electric field. In this step, the mixed solution is brought into contact with the first accumulation region and an electric field is applied to draw the complex to the first accumulation region. The application of the electric field is as described in the step (2) of the first embodiment. Moreover, it has the process of removing the magnetic particle which is not forming the composite_body | complex remaining in a liquid in the state which applied the electric field after this operation.

  FIG. 3C is a diagram schematically showing the composite held in the first accumulation region by performing the steps (1), (2), and (3). Next, the electric field application to the first integrated region is released. FIG. 3D is a diagram schematically showing a state in which the electric field application is canceled and the composite holding force in the first accumulation region is weakened. As a result, the complex becomes redispersible in the liquid holding region. If redispersion is difficult, stirring may be performed. Further, a dispersion liquid may be introduced. As will be described later, when the detection cartridge has a flow channel shape, it is effective to flow the dispersion liquid and promote re-dispersion. The dispersion is different in the same composition as the liquid mixture and the washing liquid as long as the complex formation, particularly the binding between the second capturing body provided in the magnetic particles and the target substance can be suitably maintained. The composition may be used.

  FIG. 3 (e) is a diagram schematically showing a step (4) of drawing the composite to the second accumulation region by applying a magnetic field. In this step, the composite is attracted to the second accumulation region by applying a magnetic field. Therefore, it is desirable that the second accumulation region is also disposed in contact with the liquid holding region. As will be described later, magnetic field application means that a magnetic field having a distribution in which a force that causes magnetic particles to move toward the accumulation region is applied. Therefore, the magnetic particles are selectively attracted to the second accumulation region. On the other hand, most of the non-magnetic particles that do not form a composite remain in the liquid holding portion. The magnetic field application time and the magnetic field strength are appropriately determined depending on the selectivity between the nonmagnetic particles and the magnetic particles, the particle concentration, and the like. You may have the process of removing the nonmagnetic particle which is not forming the composite_body | complex remaining in a liquid in the state which applied the magnetic field after this operation. The removal method is the simplest method for washing with a solvent, but is not limited to this as long as it can selectively remove non-magnetic particles that do not form a complex, such as removal using an electric field. As described above, the composite is selectively attracted to the second accumulation region by this step.

  FIG. 3F is a diagram schematically showing the composite held in the second accumulation region by performing the steps (1), (2), (3), and (4). Here, a step (5) of detecting the amount of magnetic particles attracted to the second accumulation region by a magnetic sensor is performed. This detection step may be performed with the magnetic field applied or may be performed with the magnetic field released as long as the amount of magnetic particles can be detected by the magnetic sensor. That is, the complex may be held in the detection region or may be dispersed in the liquid. However, when the sensitivity of the magnetic sensor is high in the vicinity of the detection element surface, such as a magnetoresistive effect element, the detection step is performed with the vicinity of the detection element surface as the second integration region and the composite held in the second integration region. It is desirable to do. According to the present invention, the complex is selectively attracted to the second accumulation region, resulting in a concentration effect. Therefore, as described later, if the detection element surface is the second integrated region, it is possible to improve sensitivity as compared with a sensor using a conventional detection element having the same detection element area.

Furthermore, as in the first embodiment, the target substance in the sample can be detected by performing the step (6) of calculating the quantity of the target substance based on the quantity of the magnetic particles.
(Third embodiment)
A third embodiment according to the present invention is a method for detecting a target substance in a specimen,
(1) A nonmagnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body and a target substance are brought into contact with each other in a fluid, and the magnetic particles are nonmagnetic via the target substance. Forming a composite with retained particles;
(2) drawing the composite to the first integrated region by applying a magnetic field;
(3) drawing the composite to the second integration region by applying an electric field;
(4) a step of detecting the amount of magnetic particles of the complex attracted to the second accumulation region by a magnetic sensor;
(5) calculating the amount of the target substance based on the amount of magnetic particles of the composite.

  FIG. 4 is a diagram schematically showing a third embodiment according to the present invention.

  FIG. 4 (a) shows a case where a nonmagnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body, and a target substance are brought into contact with each other, and the magnetic particles are non-contacted via the target substance. It is the figure which showed typically the process (1) which forms the composite_body | complex with which the magnetic particle was hold | maintained. This step is a step of adjusting the mixed liquid as in step (1) in the first and second embodiments.

  FIG. 4B is a diagram schematically showing a step (2) of drawing the composite to the first integration region by applying a magnetic field. In this step, the mixed solution is brought into contact with the first accumulation region and a magnetic field is applied to draw the complex to the first accumulation region. The application of the magnetic field is as described in the step (4) of the second embodiment.

  Moreover, you may have the process of removing the nonmagnetic particle which has not formed the composite_body | complex remaining in a liquid in the state which applied the magnetic field after this operation.

  FIG. 4C is a diagram schematically showing the composite held in the first accumulation region by performing the steps (1) and (2). Next, the magnetic field application to the first integrated region is released. FIG. 4D is a diagram schematically showing a state where the application of the magnetic field is canceled and the composite holding force in the first accumulation region is weakened. As a result, the complex becomes redispersible in the liquid holding region. If redispersion is difficult, stirring may be performed. Further, a dispersion liquid may be introduced. As will be described later, when the detection cartridge has a flow channel shape, it is effective to flow the dispersion liquid and promote re-dispersion. In addition, the dispersion may have the same composition as the mixed liquid and the cleaning liquid, as long as the complex formation, in particular, the binding between the first capturing body provided in the nonmagnetic particles and the target substance can be suitably maintained. Different compositions may be used. In addition, when the step (3) is performed while the dispersion is held in the liquid holding region, the pH of the dispersion is close to the isoelectric point of the magnetic particles, and the nonmagnetic particles are likely to have a surface charge. Is desirable.

FIG. 4E is a diagram schematically showing a step (3) of drawing the composite to the second integration region by applying an electric field. In this step, the composite is attracted to the second accumulation region by applying an electric field. The application of the electric field is as described in the step (2) of the first embodiment.
Therefore, it is desirable that the second accumulation region is also disposed in contact with the liquid holding region. Moreover, you may have the process of removing the magnetic particle which is not forming the composite_body | complex remaining in a liquid in the state which applied the electric field after this operation. The removal method is the simplest method for washing with a solvent, but is not limited to this as long as magnetic particles that do not form a complex can be selectively removed, such as removal using a magnetic field. As described above, the composite is selectively attracted to the second accumulation region by this step.

  FIG. 4F is a diagram schematically showing the composite held in the second accumulation region by performing the steps (1), (2), and (3). Here, as in the second embodiment, the step (4) of detecting the amount of magnetic particles attracted to the second accumulation region by the magnetic sensor is performed. This detection step may be performed with an electric field applied or may be performed with the electric field canceled as long as the amount of magnetic particles can be detected by a magnetic sensor. That is, the complex may be held in the second detection region or may be dispersed in the liquid. However, when the sensitivity of the magnetic sensor is high in the vicinity of the detection element surface, such as a magnetoresistive effect element, the detection step is performed with the vicinity of the detection element surface as the second integration region and the composite held in the second integration region. It is desirable to do. According to the present invention, the complex is selectively attracted to the second accumulation region, resulting in a concentration effect. Therefore, as described later, if the detection element surface is the second integrated region, it is possible to improve sensitivity as compared with a sensor using a conventional detection element having the same detection element area.

Further, similarly to the first embodiment, the target substance in the specimen can be detected by performing the step (5) of calculating the quantity of the target substance based on the quantity of the magnetic particles.
(Concentration effect)
FIG. 5 is a diagram schematically showing the relationship between the binding rate and the concentration of the target substance in the conventional solid phase reaction. In the present invention, the binding rate is the ratio of the number of bound substances finally bound to the target substance to the binding site on the solid phase (for example, the number of solid phase antibodies). In the schematic diagram, the region (A) is a region where the number of binding sites starts to be insufficient and the coupling rate converges to a constant value. The region (C) is a region where the binding rate converges to a constant value due to the lower limit of sensitivity due to the measurement method and the like, and the lower limit of the number of target substances present in the reaction system. Therefore, mainly, the target substance is detected and the concentration is calculated using the region (B) in the meantime.

  When a general biological reaction is used, the concentration of the target substance and the binding rate are in a proportional relationship in the region (B). That is, if the detection elements have the same number of binding sites, the binding rate decreases proportionally as the target substance concentration decreases. This means that the number of binding sites to which the target substance is not bound increases, and the absolute number of bound substances also decreases in proportion. Therefore, in order to achieve high sensitivity by a conventional method using a solid phase reaction, it is necessary to increase the detection element area and increase the number of binding sites to ensure the absolute number of bound substances.

  In this detection method, this combined substance, that is, a complex is formed in a liquid phase in advance, and this complex is selectively attracted to the accumulation region. Therefore, if the detection element surface is used as an integration region, an absolute number of combined substances is secured on the detection element surface. As a result, the sensitivity can be improved as compared with a sensor having the same number of binding sites, for example, a conventional detection element having the same detection element area.

<Target substance capturing body>
The target substance capturing body used in the present invention is a substance related to the selection of the target substance in the specimen, and can be selected according to the target substance.

  Here, before describing the target substance capturing body of the present invention, the target substance in the present invention will be described. The target substance is contained in the specimen and has a region captured by the target substance capturing body. The present invention is an invention that can be suitably applied to a so-called biosensor utilizing the specific molecular recognition ability of a substance derived from a living body or a similar substance. Therefore, it is desirable that the target substance is a biological substance such as sugar, protein, amino acid, antibody, antigen or pseudoantigen, vitamin, or gene, and related substances or artificially synthesized pseudobiological substances.

  In the present invention, it is only necessary that the detection target can be detected using the target substance. Therefore, the detection target itself is the target substance, and detection may be performed by capturing the detection target directly by the capturing body. Further, the target substance and the detection target may be different, and the detection target may be indirectly detected by detecting the target substance with the capturing body. An example of the latter is a case where a target substance is generated due to the presence of a detection target. Therefore, the detection target is not limited to the biological material, and the size is not particularly limited.

  Furthermore, the specimen may be a sample itself containing the detection target substance, or may be prepared through various processes such as target substance extraction processing, separation processing, dilution processing, and purification processing. Good. The specimen is prepared using a liquid medium corresponding to the type of the target substance, such as water or a buffer solution, or a mixture of water and a water-soluble organic solvent. Note that the present invention can also be suitably applied to the case where the analysis target substance is indirectly detected by detecting a decomposition product or product obtained directly or indirectly from the analysis target substance as the target substance.

  Examples of the target substance capturing body used in the present invention are those that can capture the above-described examples of the target substance, and include proteins such as enzymes, antibodies and antigens, DNA, RNA, sugar chains, etc. It is not limited to.

  Examples of the target substance-target substance capturing body combination in the present invention include antigen-antibody, enzyme-substrate, DNA-DNA, DNA-RNA, DNA-protein, RNA-protein, sugar chain-protein and the like. There is no particular limitation as long as it has a specific binding relationship.

  In the detection method of the present invention, the target substance capturing body included in the nonmagnetic particles is used as the first target substance capturing body, and the target substance capturing body included in the magnetic particles is used as the second target substance capturing body. Further, that the non-magnetic particle or the magnetic particle has the target substance capturing body means that the target substance capturing body is immobilized to such an extent that the target substance capturing body can be maintained in a state of being bound to these particles in the detection method. Examples of such immobilization methods include immobilization by chemical bonding and physical adsorption.

  The target substance detection method according to the present invention is characterized in that the first target substance capturing body and the second target substance capturing body capture spatially different regions of the target substance. Examples of such a system include a case where the target substance is an antigen, the first target substance capturing body is a primary antibody, and the second target substance capturing body is a secondary antibody. At this time, the primary antibody and the secondary antibody each capture another region in the antigen. As shown in FIG. 6A, if the antigen region captured by the primary antibody and the antigen region captured by the secondary antibody are different antigenic determinants, the present invention is applicable. In addition, the antigen regions that each captures may be the same antigenic determinant. For example, as shown in FIG. 6B, when the antigen forms a multimer, even the same antigenic determinant is applicable to the present invention. In other words, the effect of the present invention can be obtained if the first target substance capturing body and the second target substance capturing body spatially capture different regions of the target substance, not based on antigenic determinants. Become.

<Nonmagnetic and magnetic particles>
(Non-magnetic particles)
The present invention attracts nonmagnetic particles to the accumulation region by applying an electric field, which is one of the methods for attracting the accumulation region. This utilizes the surface charge of non-magnetic particles, and any non-magnetic particles have surface charge in a mixed solution, have a target substance trapping body, and can form a complex. Can also be used. For this reason, the nonmagnetic particles are preferably charged particles.

  Metal oxide has the property of being positively charged at a pH lower than the isoelectric point and negatively charged at a pH higher than the isoelectric point, and is preferably used in the present invention as a material for nonmagnetic particles. There is something that can be done. Therefore, a nonmagnetic metal oxide is preferable as a material for the nonmagnetic particles of the present invention, and silicon oxide, aluminum oxide, and the like can be easily formed into fine particles and are preferably used. Moreover, you may use as a nonmagnetic particle what formed the coating layer on the surface of arbitrary particle | grains so that it may mention later. In addition, the nonmagnetic particle said here contains not only the particle itself but also a structure in which the particle material contained and the coating layer are combined.

The size of the non-magnetic particles can be variously selected depending on the shape, size, or application of the accumulation region, but generally a particle having a diameter of several tens of nanometers to several hundreds of micrometers is suitable. is there.
(Magnetic particles)
In the present invention, the amount of magnetic particles is detected by a magnetic sensor. That is, the magnetic particles satisfy physical properties and characteristics as a label for detection. Further, it can be attracted to the integrated region by applying magnetism, which is one of the methods for attracting the integrated region. Therefore, it can be used by selecting from normally used paramagnetic and superparamagnetic magnetic fine particles.

As the magnetic material constituting the magnetic particles, for example, a metal oxide can be used. Among metal oxides, iron oxides such as ferrite and magnetite, which are generally used as magnetic labels, are preferable because they have sufficient magnetism under physiologically active conditions and are less susceptible to deterioration such as oxidation in a solvent. . The ferrite is selected from magnetite (Fe ₃ O ₄ ), maghemite (γ-Fe ₂ O ₃ ), and a composite in which a part of these Fe is replaced with other atoms. Other atoms include Li, Mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, Ge, Zr, Nb, Mo, Cd, In, Sn, At least one of Ta and W can be mentioned.

  In addition, the above-described magnetic material in which a resin is compounded is generally used as a magnetic label, and can also be used for the magnetic particles in the present invention. As an example of the composite, there is a particle having a laminated structure in which a base made of a magnetic material is used as a core and a shell layer (coating layer) is formed on the base. As such core-shell magnetic particles, for example, a resin selected from a resin made of styrene, dextran, acrylamide, or the like on the surface of particles of a substrate made of a metal oxide, using the particles made of the metal oxide as a substrate. Examples include core-shell particles coated with a layer. In addition, a resin obtained by copolymerizing at least two monomers among the respective monomers forming the styrene resin, the dextran resin, and the acrylamide resin is also included in the styrene resin, the dextran resin, the acrylamide resin, and the like. In addition to the core-shell type, as a composite, particles made of a magnetic material are dispersed in a styrene, dextran, acrylamide, or other resin, or fine particles made of a magnetic material on the surface of a resin particle. And particles on which is supported. These particles can also be used as the magnetic particles of the present invention. Examples of such magnetic particles that are commercially available include Dynabeads commercially available from Dynal, micromer-M and nanomag-D commercially available from Micromod, and Estapol commercially available from Merck. These can also be used.

  The size of such magnetic particles can be variously selected depending on the shape, size, or application of the integrated region, but generally has a diameter of several tens of nanometers to several hundreds of micrometers. Those are preferred.

  Furthermore, as will be described later, the present invention realizes selective integration by the difference in surface charge between non-magnetic particles and magnetic particles when an electric field is applied. Therefore, it is desirable that the surface charge amount of the magnetic particles is smaller than the surface charge amount of the non-magnetic particles in the liquid for accumulation. Furthermore, it is desirable that the pH of the liquid used for accumulation, particularly the aqueous solution, is close to the isoelectric point of the magnetic particles, and that the nonmagnetic particles are charged at this pH.

Such control of the surface charge of the magnetic particles in the aqueous solution can also be controlled by forming a coating layer, which will be described later. The magnetic particles referred to here include not only the particles themselves but also a structure in which the particle material contained and the coating layer are combined.
(Coating layer)
The nonmagnetic particles according to the present invention may have a coating layer on the surface, and the surface charge of the nonmagnetic particles may be derived from this coating layer. Therefore, any material such as an inorganic material such as glass, an organic material such as resin, a semiconductor material such as silicon, or a metal material may be used as the material of the coating layer as long as it has an appropriate surface charge in a liquid that is integrated by applying an electric field. It is also possible. In particular, as described above, the metal oxide has a property of being positively charged at a pH lower than the isoelectric point and negatively charged at a pH higher than the isoelectric point, and is suitable for a coating layer. Also, surface charges can be imparted to the non-magnetic particles by imparting ionic functional groups to the surface. For example, when a large number of carboxyl groups are added, the isoelectric point generally decreases, and the neutral region tends to be negatively charged. On the other hand, when a large number of amino groups are added, the isoelectric point generally increases, and the neutral region is likely to be positively charged. These functional groups can be imparted by coating with an ionic surfactant, coating with a polymer having an ionic functional group, or the like. In addition, if the particles are oxides, they can be coated with a silane agent having an ionic functional group, and if the particles are gold, silver, copper, platinum, etc., they can be coated with a thiol compound having an ionic functional group or an aminated compound. It becomes. It is also effective to introduce a polymerization initiating group on the particle surface and polymerize a polymer film having an ionic functional group.

  If the coating layer is formed in this way, the effect of the present invention can be obtained even when a weak material having no surface charge is used.

  The magnetic particles according to the present invention may also have a coating layer on the surface, and the surface charge of the magnetic particles may be derived from this coating layer. Therefore, the material of the coating layer is an inorganic material such as glass, an organic material such as a resin, a semiconductor material such as silicon, as long as the surface charge is reduced in a liquid that accumulates nonmagnetic particles by applying an electric field. Any material such as a metal material can be used. As described above, the metal oxide has an isoelectric point. Therefore, the surface charge of the magnetic particles can be reduced by using a metal oxide having an isoelectric point close to the pH of the liquid at the time of accumulation by applying an electric field. In addition, by adding an ionic functional group to the surface, it is possible to control the isoelectric point of the magnetic particles and reduce the surface charge. It is also possible to use a method in which the amount of ionic functional groups is directly introduced and a material in which the amount ratio of anionic functional group to cationic functional group is controlled is coated.

These coating layers are preferably hydrophilic layers. In general, biological substances such as proteins are known to non-specifically adsorb to various substances. Also in the present invention, non-specific adsorption on the surface of the detection element and aggregation between the particles via the biological substance may cause problems. For example, when magnetic particles other than the complex formed through the target substance are adsorbed nonspecifically on the detection element surface, noise is generated. Further, when a non-magnetic particle and a magnetic particle form a pseudo complex without using a target substance, the signal from the complex also becomes noise, which can be a factor of lowering detection sensitivity and accuracy. However, if the coating layer is a hydrophilic layer, it is possible to reduce the “hydrophobic interaction” that is one of the causes of non-specific adsorption of biological substances, and to reduce the non-specific adsorption of biological substances. It becomes possible. Furthermore, dispersibility in an aqueous solution is increased, and aggregation can be reduced.
(Complex)
A complex in which non-magnetic particles are held on magnetic particles means that the first and second target substance capturing bodies capture the target substance, and the magnetic particles and non-magnetic particles that bind to these capturing bodies are integrated. Refers to the complex.

<Liquid holding area>
In the present invention, the liquid holding region means a region holding a liquid containing a complex in which nonmagnetic particles are held on magnetic particles via a target substance. The detection method in the present invention includes a step of forming the complex. In this step, nonmagnetic particles, magnetic particles, and a target substance are mixed in a liquid to prepare a mixed liquid. This mixed liquid is introduced into the liquid holding area, and as a result, the complex is held in the liquid holding area. The mixed liquid may be introduced into the liquid holding area after being mixed and adjusted in a container different from the detection cartridge, that is, in an area different from the liquid holding area, or may be mixed and adjusted in the liquid holding area. Good. In the detection method according to the present invention, it is also possible to re-disperse the complex or the like accumulated in the accumulation region in the liquid holding part. Therefore, the liquid holding part may be a so-called well-shaped container or a channel-shaped container as long as it can hold liquid such as a mixed liquid, a cleaning liquid, and a dispersion liquid satisfactorily. . In particular, in the case where cleaning, redispersion, and the like are performed by introducing a dispersion, the shape of the flow path is desirable.

<Integration area>
In the present invention, the accumulation region means a region where the complex is accumulated. The accumulation area is provided in the liquid holding area or in contact with the liquid holding area. The detection method in the present invention includes a step of drawing the complex to the accumulation region by applying an electric field or a magnetic field. Therefore, in the case of integration by applying an electric field, the integration region is a region that is formed by the surface of the electric field generation unit or in which the electric field generation unit is disposed in the vicinity. Further, in the case of accumulation by a magnetic field, the accumulation region is a region where the magnetic field generation means is arranged in the vicinity or composed of the magnetic field generation means. As long as the composite can be suitably integrated by applying an electric field from these electric field generating means and applying a magnetic field from the magnetic field generating means, the accumulation region may be a liquid / liquid interface, a space in an arbitrary liquid phase, as well as a solid surface. Absent. When it is desired to increase the area of the accumulation region, the surface of the porous body or the vicinity of the surface may be the accumulation region, and the shape is not limited.

  When the detection method has each step of attracting the complex to each accumulation region by providing two accumulation regions, the accumulation region that first draws the complex in the order of the two steps is the first accumulation region, Thereafter, the accumulation region where the composite is attracted is defined as a second accumulation region.

<Electric field application>
In the present invention, an electric field is applied as one method for attracting the composite to the accumulation region. That is, applying an electric field means applying a potential or current so that the surface charge of the accumulation region has a polarity opposite to the polarity of the surface charge of the nonmagnetic particles in the mixed liquid. For example, when a non-magnetic particle having a charge q is placed in a uniform electric field E formed between parallel flat plates having a large area, the force FE acting on the non-magnetic particle is the product of charge amount and electric field q E Given in. When a voltage V is applied to parallel plates arranged at a distance d, the electric field E created between the parallel plates is given by V / d. Therefore, the force FE acting on the nonmagnetic particles having the charge q is q V / d. By forming an accumulation region between a non-magnetic particle and a flat plate having a polarity opposite to the polarity of the non-magnetic particles, the non-magnetic particles can be collected in the accumulation region. If other force such as magnetic force or gravity is applied to the non-magnetic particles in the direction away from the accumulation region, the non-magnetic particles must be collected in the accumulation region by applying a force larger than that force. In this case, as described above, it can be easily realized by appropriately adjusting the voltage applied between the parallel plates. Alternatively, non-magnetic particles having a large charge amount may be used, and the interval between the parallel plates may be adjusted. In the above description, the parallel plate has been described as an example. However, the shape of the electrode for forming the electric field is not limited to this, and for example, an electric field can be applied to nonmagnetic particles such as a cylindrical one. Anything can be used.

  The application of the electric field is continued until the nonmagnetic particles are sufficiently collected in the accumulation region. Therefore, the time for applying the electric field is determined by various forces applied to the nonmagnetic particles, the viscosity of the solution, and the like. When collecting non-magnetic particles to the accumulation region within a short time, it is realized by applying a large electric field.

<Magnetic field application>
In the present invention, a magnetic field is applied as one method for attracting the composite to the accumulation region. A force acts on the magnetic particles having magnetization M depending on the gradient of the magnetic field strength, and the magnetic particles try to move toward the larger magnetic field strength. When the magnetic field is H, the force FH applied to the magnetic particles is expressed by the following equation.

In other words, magnetic field application means that a magnetic field having a distribution in which a force that moves the magnetic particles toward the accumulation region works is applied. For example, it is possible to apply a magnetic field to magnetic particles by passing a current through a circular coil and generating a magnetic field from the coil. In this case, the accumulation region is preferably formed between the circular coil and the magnetic particles, and the magnetic particles are preferably located in the vicinity of the central axis of the circular coil. If the radius of the circular coil is a, the distance from the center point of the circular coil to the magnetic particle is z, the magnitude of the current flowing through the coil is I, and the number of turns of the coil is N, the magnetic coil generates a magnetic particle The magnitude of the applied magnetic field is expressed by the following equation.

Here, when the magnetic particles exhibit superparamagnetism, the magnitude of the magnetization M of the magnetic particles depends on the magnitude of the applied magnetic field. In particular, when the applied magnetic field is not large, the magnitude of the magnetization M is proportional to the applied magnetic field H and is represented by M = χH. Here, χ is called magnetic susceptibility and shows a different value depending on the material of the magnetic material. Also, the magnitude of the magnetic field that satisfies this relational expression varies depending on the material of the magnetic material, but is often 80 [kA / m] or less, for example.

  As described above, in the range where the magnitude of the magnetization of the magnetic particles is proportional to the magnitude of the applied magnetic field, when a magnetic field is applied by the circular coil, a magnetic force FH as shown in the following equation acts on the magnetic particles.

Here, it is assumed that an electric field is applied to the composite having magnetic particles and a non-magnetic substance by a parallel plate and a magnetic field is applied by a circular coil, and the forces acting on the composite are in antiparallel directions. In such a state, when collecting the complex in the accumulation region where the force due to the magnetic field is applied, it is sufficient to set FH> FE, and conversely, the complex is accumulated in the accumulation region where the force due to the electric field is applied. When collecting, FE> FH should be satisfied.

  The application of the magnetic field is continued until the nonmagnetic particles are sufficiently collected in the accumulation region. Therefore, the time for applying the magnetic field is determined by various forces applied to the magnetic particles and the viscosity of the solution. When collecting magnetic particles in the accumulation region within a short time, it is realized by applying a large magnetic field.

  The magnetic field is applied by magnetic field generating means. Therefore, the magnetic field generating means is preferably an electromagnet. Further, if a mechanism for changing the distance from the accumulation region is provided, it is also possible to control the magnetic field application with a permanent magnet. Therefore, both of the magnetic field generating means can be used as long as the application and release of the magnetic field can be suitably controlled.

<Magnetic sensor>
As the magnetic sensor, any sensor may be used as long as it detects a magnetic label. Among them, a method using a magnetic field effect when a magnetic label is present on the detection region surface is preferable, and in particular, a magnetoresistive effect element, a Hall effect element, and a superconducting quantum interferometer element can be suitably used.

  As the magnetoresistive effect element, any of an anisotropic magnetoresistive effect element, a giant magnetoresistive effect element, and a spin tunnel magnetoresistive effect element may be used, but a spin tunnel magnetoresistive effect element that can expect a large detection signal is more suitable. preferable. Among them, the one using an MgO thin film as the tunnel film is more preferable because a large detection signal can be obtained. Such a spin tunnel magnetoresistive element is described in Japanese Patent Application Laid-Open No. 2006-80116.

A transition metal element is mainly used for the magnetic material used for the magnetoresistive effect element. In order to improve oxidation resistance or to make the structure amorphous, a material to which an element such as V, W, Cr, B, or Ti is added is also used. In particular, when the tunnel barrier film is an MgO thin film, the magnetic film in contact with the MgO thin film is preferably CoFeB. The basic film configuration of the giant magnetoresistive effect element and the spin tunnel magnetoresistive effect element includes three layers of a magnetization free layer, a nonmagnetic layer, and a magnetization fixed layer. An element whose nonmagnetic layer is made of a conductor such as Cu or Cr is called a giant magnetoresistive element, and an element whose nonmagnetic layer is made of a dielectric film such as Al ₂ O ₃ or MgO is called a spin tunnel magnetoresistive element. . These magnetization fixed layers may be made of a magnetic material having a coercive force larger than the coercivity of the magnetic film constituting the magnetization free layer. However, in order to fix the magnetization direction of the magnetization fixed layer, the magnetic film is antiferromagnetic. A spin valve film formed by contacting a magnetic film and magnetically exchange-coupled may be used. For the antiferromagnetic film, for example, a conductor such as MnPt or MnIr or an insulator such as NiO can be used.

  Materials such as Si, Bi, and GaAs can be used for the Hall effect element. Furthermore, a multilayer film having a HEMT (High Electron Mobility Transistor) structure such as AlGaN / GaN may be used.

<Detection cartridge>
The detection cartridge according to the present invention is a detection cartridge for detecting a target substance in a specimen, and a liquid holding region for holding a liquid containing a complex in which nonmagnetic particles are held on magnetic particles via the target substance; At least one integrated region for integrating the composite is provided, and at least one integrated region is disposed on the surface of the magnetic sensor detection element.

  As described above, since the present invention forms a complex in the liquid phase, the reaction time can be shortened. The detection cartridge according to the present invention includes a liquid holding region that holds the liquid containing the complex. In addition, by providing at least one accumulation region in which the complex is accumulated, the complex held in the liquid holding region can be directly accumulated. In the present invention, the detection step can be performed while holding the complex in the integration region by setting the vicinity of the surface of the detection element as the integration region. In particular, the effect is great when a magnetic sensor element having high sensitivity in the vicinity of the detection element surface, such as a magnetoresistive effect element, is used. Therefore, it is desirable that at least one integrated region of the detection cartridge according to the present invention is disposed on the surface of the magnetic sensor detection element. Although the integrated region is provided in the detection cartridge, the magnetic sensor element may be provided in the cartridge or disposed outside, and at least one integrated region is disposed on the surface of the magnetic sensor detection element at the time of detection. Just do it.

  The detection cartridge according to the present invention is preferably provided with an electric field generating means or a magnetic field generating means in the vicinity of or in contact with the integrated region.

<Detection kit>
A detection kit according to the present invention is a detection kit for detecting a target substance in a specimen, comprising: a non-magnetic particle having a first target substance capturing body; and a magnetic particle having a second target substance capturing body. And the non-magnetic particles are charged in an aqueous solution having a pH substantially equal to the isoelectric point of the magnetic particles.

  If the relationship between the non-magnetic particles and the magnetic particles is the above relationship, the non-magnetic particles are charged in an aqueous solution whose pH is approximately the isoelectric point of the magnetic particles. Therefore, it can be integrated in the integrated region by applying an electric field. On the other hand, the surface charge of the magnetic particles is small and remains in the liquid. That is, it is possible to selectively accumulate nonmagnetic particles in the accumulation region by applying an electric field and remove magnetic particles that do not form a composite.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and detection having similar functions and effects such as materials, composition conditions, reaction conditions, and the like. The method, the detection cartridge, and the detection kit can be freely changed within a range that can be obtained.

  Hereinafter, the present invention will be described in more detail with reference to examples.

(Example 1)
In this example, silica particles having a primary antibody that captures PSA (Prostate specific antigen) are used as nonmagnetic particles having a first target substance capturing body, and second target substance capturing is further performed. PSA is detected by combining magnetite particles with a secondary antibody that captures PSA as magnetic particles with a body.
(1) Production of nonmagnetic particles First, nonmagnetic particles having a primary antibody that captures PSA are produced as a first target substance-capturing body.

The silica particles are heat-treated in a dry N ₂ atmosphere and then dispersed in anhydrous toluene. To this silica particle / toluene dispersion, aminopropyltrimethoxymethoxysilane, which is a silane coupling agent, is added to introduce amino groups on the surface of the silica particles. However, if the introduction amount of amino groups relative to the silica particles is too large, the isoelectric point as silica changes greatly, so the introduction amount of amino groups depends on the pretreatment of silica particles (drying conditions, etc.) The conditions (concentration, mixing ratio, etc.) in the ring process are appropriately controlled. Furthermore, using a cross-linking agent such as glutaraldehyde, the amino group and the peptide chain are chemically bonded to immobilize the primary antibody.

As a result, silica particles provided with a primary antibody for capturing PSA can be obtained as the first target substance capturing body. The silica particles are dispersed in a pH 6.5 phosphate buffer to prepare a nonmagnetic particle dispersion. In addition, since the isoelectric point of silica is around 2.0, the nonmagnetic particles according to this example are negatively charged in an aqueous solution having a pH near neutral.
(2) Production of magnetic particles Next, magnetic particles having a secondary antibody that captures PSA as a second target substance capturing body are prepared.

The magnetite particles are heat-treated in a dry N ₂ atmosphere and then dispersed in anhydrous toluene. To this magnetite particle / toluene dispersion, aminopropyltrimethoxymethoxysilane, which is a silane coupling agent, is added to introduce amino groups on the surface of the magnetite particles. However, if the introduction amount of amino groups is too large relative to the magnetite particles, the isoelectric point of the magnetite will change greatly, so the introduction amount of amino groups depends on the pretreatment of the magnetite particles (drying conditions, etc.) The conditions (concentration, mixing ratio, etc.) in the ring process are appropriately controlled. Further, using a cross-linking agent such as glutaraldehyde, the amino group and the peptide chain are chemically bonded to immobilize the secondary antibody.

As a result, magnetite particles provided with a secondary antibody that captures PSA can be obtained as the second target substance capturing body. The magnetite particles are dispersed in a pH 6.5 phosphate buffer to prepare a magnetic particle dispersion. In addition, since the isoelectric point of magnetite is around 6.5, the charged amount of the magnetic particles according to this example is small in an aqueous solution having a pH near neutral.
(3) Production of Detection Cartridge Next, a detection cartridge having a liquid holding area and an accumulation area is produced. In the present embodiment, the integrated region is provided on the surface of the magnetic sensor detection element, and a detection cartridge capable of accumulating nonmagnetic particles in the integrated region is produced by applying an electric field.

FIG. 7 schematically shows a spin tunnel magnetoresistive element. In this embodiment, this spin tunnel magnetoresistance effect element is used as a detection element. Then, an insulating layer and an electrode 1 are provided on the element surface, and the surface is used as an integrated region (FIG. 8). FIG. 9 is a diagram schematically showing a cross section of the detection cartridge according to the present embodiment. The electrode 1 and the electrode 2 are disposed in contact with the liquid holding region, and further connected to the power supply unit. Electrode 1 and electrode 2 are in contact with the mixed solution. However, although not shown, it is preferable that an insulating layer is formed on the surface of the electrode, and the electrode is in contact with the mixed solution through the insulating layer. This electrode 1 corresponds to an electric field generating means. Although not shown, a measurement unit may be connected to the detection element. Moreover, it is desirable that a stirring mechanism for the mixed solution is provided.
(4) Detection of PSA Using the non-magnetic particles, magnetic particles, and detection cartridge prepared in (1), (2), and (3) above, it is known as a prostate cancer marker by performing the following operations. It is possible to detect the PSA.
(A) The non-magnetic particle dispersion and the magnetic particle dispersion are mixed and reacted in a phosphate buffer solution of pH 6.5 containing PSA as an antigen (target substance) to prepare a mixture. The mixed liquid is introduced into the liquid holding area of the detection cartridge. As a result, the complex in which the nonmagnetic particles are held by the magnetic particles via the target substance is held in the liquid holding region.
(B) A current or voltage is applied from the power supply unit so that the surface potential of the electrode 1 becomes a positive potential, and the composite is drawn to the integrated region.
(C) Unreacted PSA and magnetic particles are washed with a phosphate buffer.
(D) The amount of the composite magnetic particles attracted to the accumulation region is detected by a magnetic sensor.
(E) The amount of the target substance is calculated from the amount of the magnetic particles.

  In this example, since the complex is formed by a liquid phase reaction in the operation (a), the reaction time can be shortened as compared with the conventional method in which the solid phase reaction is performed on the surface of the sensor element. In particular, when the target substance concentration is low, the effect is large. If this method is used, even when the target substance is at a low concentration, the reaction time until sufficient complex formation can be reduced to 1/10 or less.

(Example 2)
In this example, PSA is detected by combining nonmagnetic particles and magnetic particles similar to those in Example 1. Further, the present embodiment is an example in which the detection cartridge has a flow channel shape, and the first integration region and the second integration region, which are two integration regions, are formed in different regions. In the first integration region, the composite is integrated by applying an electric field, and in the second integration region, the composite is integrated by applying a magnetic field.
(1) Production of non-magnetic particles First, silica particles including a primary antibody that captures PSA are produced as a first target substance-capturing body in the same manner as in Example 1, and a silica particle dispersion is further produced. .
(2) Production of magnetic particles Next, magnetite particles comprising a secondary antibody that captures PSA as a second target substance capturing body are prepared in the same manner as in Example 1, and a magnetite particle dispersion is further prepared. .
(3) Production of Detection Cartridge Next, a detection cartridge having a liquid holding area, a first accumulation area, and a second accumulation area is produced. In the present embodiment, a detection cartridge is produced in which the second integrated region is provided in a space that can be detected by the magnetic sensor detection element.

  FIG. 10 is a diagram schematically showing a cross section of the detection cartridge according to the present embodiment. The electrode 1 and the electrode 2 are disposed in contact with the liquid holding region, and further connected to the power supply unit. Electrode 1 and electrode 2 are in contact with the mixed solution. However, although not shown, it is preferable that an insulating layer is formed on the electrode surface, and the electrode is in contact with the mixed solution through the insulating layer. This electrode 1 corresponds to an electric field generating means. A first integrated region is formed on the surface of the electrode 1.

In order to efficiently accumulate a sufficient amount of the composite, the electrode 1 should have a large area. Furthermore, it is desirable that the area has sufficient accumulation of non-magnetic particles. For example, when 200 μl of nonmagnetic particles having a diameter of 100 nm are introduced at a concentration of 1 × 10 10 particles / ml and accumulated in the accumulation region, the occupied area is expected to be 20 mm ² . Therefore, the area of the electrode 1 is desirably 20 mm ² or more. In order to increase the integration efficiency, the electrode 1 may have a porous shape.

Further, a magnet is disposed in the vicinity of the detection element. This magnet corresponds to the magnetic field generating means. A second integrated region is formed in the vicinity of the magnetic field generating means. Moreover, although not shown in figure, the measurement part may be connected to the detection element. Moreover, it is desirable that a stirring mechanism for the mixed solution is provided.
(4) Detection of PSA Using the non-magnetic particles, magnetic particles, and detection cartridge prepared in (1), (2), and (3) above, it is known as a prostate cancer marker by performing the following operations. It is possible to detect a PSA.
(A) The non-magnetic particle dispersion and the magnetic particle dispersion are mixed and reacted with a phosphate buffer having a pH of 6.5 containing PSA as an antigen (target substance) to prepare a mixture. The mixed liquid is introduced into the liquid holding area of the detection cartridge. As a result, the complex in which the nonmagnetic particles are held by the magnetic particles via the target substance is held in the liquid holding region.
(B) A current or voltage is applied from the power supply unit so that the surface potential of the electrode 1 becomes a positive potential, and the composite is drawn to the first integrated region.
(C) The PSA and magnetic particles not forming the complex are washed with a phosphate buffer.
(D) The current or voltage application of the electrode 1 is stopped. Then, re-dispersion of the complex accumulated in the first accumulation region starts. In the case where redispersion is difficult, a phosphate buffer solution is again flowed as a dispersion to promote redispersion.
(E) A magnetic field is applied by a magnet to draw the composite to the detection element surface (second integrated region). The operation (d) and the operation (e) may be performed simultaneously. Moreover, you may perform both operation, flowing a dispersion liquid. In this embodiment, the first accumulation region is disposed on the liquid inlet side, and the second accumulation region is disposed on the liquid discharge port side. Therefore, the complex redispersed from the first accumulation region can be efficiently reintegrated in the second accumulation region by the dispersion introduced from the liquid inlet.
(F) Washing non-magnetic particles not forming a complex with a phosphate buffer.
(G) A magnetic sensor detects the amount of the magnetic particles of the composite attracted to the second accumulation region.
(H) The amount of the target substance is calculated from the amount of the magnetic particles.

  In this example, as in Example 1, since the complex is formed by the liquid phase reaction in the operation (a), the reaction time is shortened compared to the conventional method in which the solid phase reaction is performed on the detection element surface. Can be realized. Further, in the operations (b) and (c), the complex is once accumulated in the first accumulation region, and in the operations (d), (e), and (f), the complex is formed on the second accumulation region, that is, the detection element surface. Are further selectively collected. Therefore, sensitivity can be improved as compared with a sensor using a conventional detection element having the same detection element area. In particular, when the target substance concentration is low, the effect is large. If this method is used, even when the target substance has a low concentration, the detection sensitivity can be increased 10 times or more.

(Example 3)
In this example, PSA is detected by combining nonmagnetic particles and magnetic particles similar to those in Example 1. Further, the present embodiment is an example in which the detection cartridge has a flow channel shape, and the first integration region and the second integration region, which are two integration regions, are formed in different regions. In the first integration region, the composite is integrated by applying a magnetic field, and in the second integration region, the composite is integrated by applying an electric field.
(1) Production of non-magnetic particles First, silica particles including a primary antibody that captures PSA are produced as a first target substance-capturing body in the same manner as in Example 1, and a silica particle dispersion is further produced. .
(2) Production of magnetic particles Next, magnetite particles comprising a secondary antibody that captures PSA as a second target substance capturing body are prepared in the same manner as in Example 1, and a magnetite particle dispersion is further prepared. .
(3) Production of Detection Cartridge Next, a detection cartridge having a liquid holding area, a first accumulation area, and a second accumulation area is produced. In this embodiment, a detection cartridge in which the second integrated region is provided on the surface of the magnetic sensor detection element is manufactured.

FIG. 11 is a diagram schematically showing a cross section of the detection cartridge according to the present embodiment. A magnet is disposed in the vicinity of the first integration region. This magnet corresponds to the magnetic field generating means. As in Example 2, the magnet area should be large in order to efficiently accumulate a sufficient amount of the composite. Further, in order to increase the integration efficiency, the magnet may have a porous shape. The electrode 1 and the electrode 2 are disposed in contact with the liquid holding region, and further connected to the power supply unit. Electrode 1 and electrode 2 are in contact with the mixed solution. However, although not shown, it is preferable that an insulating layer is formed on the surface of the electrode, and the electrode is in contact with the mixed solution through the insulating layer. Further, the electrode 1 is disposed on the detection element surface. This electrode 1 corresponds to an electric field generating means. A second integrated region is formed on the surface of the electrode 1. Moreover, although not shown in figure, the measurement part may be connected to the detection element. Moreover, it is desirable that a stirring mechanism for the mixed solution is provided.
(4) Detection of PSA Using the non-magnetic particles, magnetic particles, and detection cartridge prepared in (1), (2), and (3) above, it is known as a marker for prostate cancer by performing the following operations. It is possible to detect a PSA.
(A) The non-magnetic particle dispersion and the magnetic particle dispersion are mixed and reacted with a phosphate buffer containing PSA as an antigen (target substance) to prepare a mixture. The mixed liquid is introduced into the liquid holding area of the detection cartridge. As a result, the complex in which the nonmagnetic particles are held on the magnetic particles via the target substance is held in the liquid holding region.
(B) A magnetic field is applied by a magnet to draw the composite to the first accumulation region.
(C) Wash the PSA and non-magnetic particles not forming a complex with a phosphate buffer.
(D) Stop application of magnetic field from the magnet. Then, re-dispersion of the complex accumulated in the first accumulation region starts. If redispersion is difficult, a phosphate buffer solution of pH 6.5 is poured again as a dispersion to promote redispersion.
(E) A current or voltage is applied from the power supply unit so that the surface potential of the electrode 1 becomes a positive potential, and the complex is drawn to the detection element surface (second integrated region). The operation (d) and the operation (e) may be performed simultaneously. Moreover, you may perform both operation, flowing a dispersion liquid. In this embodiment, the first accumulation region is disposed on the liquid inlet side, and the second accumulation region is disposed on the liquid discharge port side. Therefore, the complex redispersed from the first accumulation region can be efficiently reintegrated in the second accumulation region by the dispersion introduced from the liquid inlet.
(F) Washing the magnetic particles not forming the complex with a phosphate buffer.
(G) A magnetic sensor detects the amount of the magnetic particles of the composite attracted to the second accumulation region.
(H) The amount of the target substance is calculated from the amount of the magnetic particles.

In this example, as in Example 1, since the complex is formed by the liquid phase reaction in the operation (a), the reaction time is shortened compared to the conventional method in which the solid phase reaction is performed on the detection element surface. Can be realized. Further, in the operations (b) and (c), the complex is once accumulated in the first accumulation region, and in the operations (d), (e), and (f), the complex is formed on the second accumulation region, that is, the detection element surface. Are further selectively collected.
Therefore, sensitivity can be improved as compared with a sensor using a conventional detection element having the same detection element area. In particular, when the target substance concentration is low, the effect is large. If this method is used, even when the target substance has a low concentration, the detection sensitivity can be increased 10 times or more.

Example 4
In this embodiment, by forming a coating layer on the surfaces of nonmagnetic particles and magnetic particles, the chargeability of the particle surfaces is controlled and PSA is detected.

(1) Preparation of non-magnetic particles First, non-magnetic particles having a primary antibody for capturing PSA and having a coating layer on the surface are prepared as a first target substance capturing body.

The silica particles are heat-treated in a dry N ₂ atmosphere and then dispersed in anhydrous toluene. To this silica particle / toluene dispersion, 2- (4-chloromethylphenyl) ethyltrimethoxysilane as a silane coupling agent is added to introduce a chloromethyl group into the silica particles. This reaction can be confirmed by detecting Cl atoms by XPS. Silica particles introduced with chloromethyl groups are dispersed in water, and sodium dithiocarbamate is added to react with the chloromethyl groups, thereby introducing UV graft polymerization starting points on the silica particle surfaces. This reaction can be confirmed by detecting N and S atoms by XPS. Next, silica particles and acetone are weighed in a reaction vessel, and the silica particles are dispersed in acetone by ultrasonic treatment. Next, glycidyl methacrylate is measured in the reaction vessel, and the inside of the reaction vessel is replaced with nitrogen. Next, UV graft polymerization proceeds by irradiating a UV lamp having a wavelength of 312 nm to 577 nm for 2 hours at room temperature, and polyglycidyl methacrylate is grafted onto the surface of the silica particles. This reaction can be confirmed by an increase in particle diameter based on the dynamic light scattering method.

  The coating layer can be formed by the above operation. This coating layer is a hydrophilic layer composed of a graft layer of polyglycidyl methacrylate, is excellent in the ability to prevent nonspecific adsorption to many proteins, and has an amino group, so that it is positively charged in an aqueous solution.

  Next, a primary antibody that captures PSA is introduced onto the surface of the nonmagnetic particles provided with the above-described coating layer. First, disperse nonmagnetic particles in water, add aminoethanethiol and dithiothreitol, adjust the pH to 5 using an aqueous hydrochloric acid solution or an aqueous sodium hydroxide solution, and react. ) Introduce an amino group and a thiol group. Next, N-Succinimidyl 3- (2-pyridyldithio) propionate is added and reacted at room temperature for 5 hours to introduce active ester groups on the surface of the nonmagnetic particles. A primary antibody that captures PSA can be immobilized as a first target substance capturing body by reacting the active ester group with the amino group of the antibody. The unreacted active ester group may be capped by adding ethanolamine. The silica particles are dispersed in a pH 6.5 phosphate buffer to prepare a nonmagnetic particle dispersion.

(2) Production of magnetic particles Next, magnetic particles having a secondary antibody that captures PSA and having a coating layer on the surface are produced as a second target substance capturing body.

  First, magnetite particles are sufficiently dispersed in anhydrous toluene. Thereafter, a precursor of the following atom transfer radical polymerization initiating group (n = 6) is added under a nitrogen atmosphere, and the hydroxyl group of the magnetite particle and the trichlorosilyl group of the precursor are reacted to form an atom transfer radical on the surface of the magnetite particle. A polymerization initiating group is introduced.

  Next, the magnetite particles into which the atom transfer radical polymerization initiating group has been introduced are dispersed in methanol. Thereafter, ethyl 2-bromoisobutyrate is added as a free polymerization initiator, and CuBr, 2,2'-bipyridyl is added. After removing oxygen in the reaction system by freeze vacuum degassing, it is replaced with nitrogen, and HEMA (2-hydroxyethyl methacrylate) monomer is reacted for a predetermined time by atom transfer radical polymerization. By adding a large amount of mercaptoacetic acid: mercaptoethanol = 1: 100 (molar ratio) in the reaction system, carboxyl groups and hydroxyl groups were mixed at the ends of the polymer chains (PHEMA) grafted on the magnetite particles. State.

It can be confirmed that a chain transfer agent has been introduced by detecting S atoms using X-ray photoelectron spectroscopy. By using a time-of-flight secondary ion mass spectrometer to detect ions derived from mercaptoacetic acid and mercaptoethanol bonded to the polymer terminal, it can be confirmed that two types of polymer terminal are formed. Moreover, when the molecular weight and molecular weight distribution of PHEMA produced from ethyl 2-bromoisobutyrate added as a free polymerization starting species were measured, the number average molecular weight was 5000 and the molecular weight distribution was 1.05. When the film thickness and weight of the polymer chain grafted on the surface of the magnetite particle are measured, it is found that the graft density of the polymer chain is 0.4 molecule / nm ² .

  The coating layer can be formed by the above operation. This coating layer is a hydrophilic layer made of a PHEMA layer, has excellent non-specific adsorption preventing ability for many proteins, and has a hydroxyl group, so that the charge amount is small in an aqueous solution near neutrality.

The surface of the magnetite particles is reacted with water-soluble carbodiimide (WSC) and N-hydroxyl succinimide (NHS) to convert the carboxyl group at the end of the PHEMA layer into an active ester group. The magnetite particles are added to a phosphate buffer solution in which a secondary antibody is dissolved, and the amino group of the antibody reacts with the active ester group of the magnetite particles to immobilize the secondary antibody that captures PSA. The unreacted active ester group may be capped by adding ethanolamine.
The magnetite particles are dispersed in a phosphate buffer having a pH of 6.5 to produce a magnetic particle dispersion.

(3) Production of Detection Cartridge Next, a detection cartridge having a liquid holding area, a first accumulation area, and a second accumulation area is produced. In this embodiment, a detection cartridge in which the second integrated region is provided on the surface of the magnetic sensor detection element is manufactured.

  FIG. 12 is a view schematically showing a cross section of the detection cartridge according to the present embodiment. The electrode 1 and the electrode 2 are disposed in contact with the liquid holding region, and further connected to the power supply unit. Electrode 1 and electrode 2 are in contact with the mixed solution. However, although not shown, it is preferable that an insulating layer is formed on the electrode surface, and the electrode is in contact with the mixed solution through the insulating layer. This electrode 1 corresponds to an electric field generating means.

  As in Example 2, the electrode 1 should have a large area in order to efficiently accumulate a sufficient amount of the composite. In order to increase the integration efficiency, the electrode 1 may have a porous shape.

Further, a coil is disposed in the vicinity of the detection element. A magnetic field can be applied by passing a current through this coil. That is, this coil corresponds to the electric field generating means.
Moreover, although not shown in figure, the measurement part may be connected to the detection element. Moreover, it is desirable that a stirring mechanism for the mixed solution is provided.

(4) Detection of PSA Using the non-magnetic particles, magnetic particles, and detection cartridge prepared in (1), (2), and (3) above, it is known as a prostate cancer marker by performing the following operations. It is possible to detect the PSA.
(A) The non-magnetic particle dispersion and the magnetic particle dispersion are mixed and reacted with a phosphate buffer having a pH of 6.5 containing PSA as an antigen (target substance) to prepare a mixture. The mixed liquid is introduced into the liquid holding area of the detection cartridge. As a result, the complex in which the nonmagnetic particles are held by the magnetic particles via the target substance is held in the liquid holding region.
(B) A current or voltage is applied from the power supply unit so that the surface potential of the electrode 1 becomes a negative potential, and the composite is drawn to the first integrated region.
(C) The PSA and magnetic particles not forming the complex are washed with a phosphate buffer.
(D) The current or voltage application of the electrode 1 is stopped. Then, re-dispersion of the complex accumulated in the first accumulation region starts. In the case where redispersion is difficult, a phosphate buffer solution is again flowed as a dispersion to promote redispersion.
(E) A magnetic field is applied by passing an electric current through the coil, and the composite is drawn to the detection element surface (second integrated region). The operation (d) and the operation (e) may be performed simultaneously. Moreover, you may perform both operation, flowing a dispersion liquid.
(F) Washing non-magnetic particles not forming a complex with a phosphate buffer.
(G) A magnetic sensor detects the amount of the magnetic particles of the composite attracted to the second accumulation region.
(H) The amount of the target substance is calculated from the amount of the magnetic particles.

  In this example, as in Example 1, since the complex is formed by the liquid phase reaction in the operation (a), the reaction time is shortened compared to the conventional method in which the solid phase reaction is performed on the detection element surface. Can be realized. Further, in the operations (b) and (c), the complex is once accumulated in the first accumulation region, and in the operations (d), (e), and (f), the complex is formed on the second accumulation region, that is, the detection element surface. Are further selectively collected. Therefore, sensitivity can be improved as compared with a sensor using a conventional detection element having the same detection element area. In particular, when the target substance concentration is low, the effect is large. If this method is used, even when the target substance has a low concentration, the detection sensitivity can be increased 10 times or more.

It is the figure which showed an example of the solid-phase analysis method using the conventional magnetic label | marker. It is the figure which showed typically 1st embodiment of the detection method by this invention. It is the figure which showed typically 2nd embodiment of the detection method by this invention. It is the figure which showed typically 3rd embodiment of the detection method by this invention. It is the figure which showed typically the coupling | bonding rate in the conventional solid-phase reaction. It is the figure which showed typically the state in which a target substance capture body captures a target substance. It is the figure which showed typically the cross section of the magnetoresistive effect element. It is the figure which showed typically the cross section of the magnetoresistive effect element provided with the electrode on the surface. FIG. 3 is a diagram schematically showing a cross section of a detection cartridge in Example 1. FIG. 6 is a diagram schematically showing a cross section of a detection cartridge in Example 2. FIG. 10 is a diagram schematically showing a cross section of a detection cartridge in Example 3. FIG. 10 is a diagram schematically showing a cross section of a detection cartridge in Example 4.

Claims (9)

A method for detecting a target substance in a specimen, comprising: (1) a non-magnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body, and the target substance in a fluid. Forming a complex in which the non-magnetic particles are held on the magnetic particles via the target substance, and
(2) attracting the composite to the accumulation region by applying an electric field;
(3) a step of detecting the amount of magnetic particles possessed by the complex attracted to the accumulation region by a magnetic sensor;
(4) calculating the amount of the target substance based on the amount of magnetic particles possessed by the complex;
A method for detecting a target substance, comprising: A method for detecting a target substance in a specimen,
(1) A non-magnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body, and the target substance are contacted in a fluid, and the magnetic particles are passed through the target substance. Forming a composite with the non-magnetic particles held therein,
(2) drawing the composite to the first integration region by applying an electric field;
(3) removing the magnetic particles not forming the complex;
(4) drawing the composite to the second integration region by applying a magnetic field;
(5) a step of detecting the amount of magnetic particles of the composite attracted to the second accumulation region by a magnetic sensor;
(6) calculating the amount of the target substance based on the amount of magnetic particles of the complex;
A method for detecting a target substance, comprising: A method for detecting a target substance in a specimen,
(1) A non-magnetic particle having a first target substance capturing body, a magnetic particle having a second target substance capturing body, and the target substance are contacted in a fluid, and the magnetic particles are passed through the target substance. Forming a composite with the non-magnetic particles held therein,
(2) drawing the composite to the first integrated region by applying a magnetic field;
(3) drawing the composite to the second integration region by applying an electric field;
(4) detecting the amount of magnetic particles of the composite attracted to the second accumulation region by a magnetic sensor;
(5) calculating the amount of the target substance based on the amount of magnetic particles of the complex;
A method for detecting a target substance, comprising:   The detection method according to claim 1, wherein the first target substance capturing body and the second target substance capturing body capture spatially different regions of the target substance. A detection cartridge for detecting a target substance in a specimen,
A liquid holding region for holding a liquid containing a complex in which nonmagnetic particles are held on magnetic particles via the target substance;
Comprising at least one or more integration regions for integrating the composite,
A detection cartridge, wherein at least one of the integrated regions is provided in a space that can be detected by a magnetic sensor detection element.   The detection cartridge according to claim 5, further comprising an electric field generation unit or a magnetic field generation unit in the vicinity of or in contact with the accumulation region. A detection kit for detecting a target substance in a sample,
A non-magnetic particle having a first target substance capturing body;
A magnetic particle having a second target substance capturing body,
A detection kit, wherein the non-magnetic particles are charged in an aqueous solution whose pH is approximately the isoelectric point of the magnetic particles.   The detection kit according to claim 7, wherein at least one of the magnetic particles and the nonmagnetic particles has a coating layer on a surface thereof.   The detection kit according to claim 8, wherein the coating layer has an ionic functional group.

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