JP2009276089A - Stirring method of liquid composition, detection method and detection kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method for a magnetic biosensor for effectively stirr a liquid composition even if a reaction container is small and for outputting an inspection result of high reliability and high sensitivity in a short time. <P>SOLUTION: The detection method for detecting a magnetic marker using a detection element constituted of a detection part and a non-detection part includes the process (1) of bringing the detection element into contact with the liquid composition containing the magnetic marker and gel particles, the process (2) of subjecting the gel particles to volume phase transfer and the process (3) of detecting the magnetic marker present in the vicinity of the detection part. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for stirring a liquid composition, and a detection method and detection kit for shortening detection time and improving detection sensitivity in a magnetic biosensor that magnetically detects a target substance in the liquid composition.

  A biosensor is a measurement device that utilizes the molecular recognition ability of a living body or a biomolecule. In vivo, examples of combinations of substances having affinity for each other include enzyme-substrate, antigen-antibody, DNA-DNA, and the like. In the biosensor, one of these combinations is selectively measured on the other substance by being used while being fixed or supported on a substrate.

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. To expand the range of use, small, lightweight, and highly sensitive biosensors that can be installed or carried anywhere. Is desired.
Currently, as one of the high-sensitivity sensing methods, a magnetic sensor is used to detect the presence and concentration of the target substance in the liquid composition by detecting the presence and number of magnetic markers located near the surface of the detection unit. Research on biosensors has been actively promoted and is also used for solid-phase analysis.

  FIG. 6 shows an example of a solid phase analysis method using a conventional magnetic marker. In the method shown in FIG. 6, first, the first capturing body component 3a (antigen antibody) capable of specifically recognizing and capturing the target substance 5 (referred to as an epitope in the case of an antigen-antibody reaction) in advance in the detection unit. In the case of reaction, this is called primary antibody). Next, the liquid composition containing the target substance is brought into contact. By this operation, the target substance is specifically captured by the first capturing component. Next, the second capture component 4a that can specifically recognize and capture the target substance specifically captured by the first capture component (referred to as a secondary antibody in the case of an antigen-antibody reaction) A magnetic marker 9 having By this operation, the second capturing body component is captured by the target substance specifically captured by the first capturing body component immobilized on the detection unit (sandwich method), and as a result, as shown in FIG. The magnetic marker is immobilized in the vicinity of the detection unit via the target substance.

  As a different method, a magnetic marker having a second capture component is added to a liquid composition containing a target material in advance to form a “target substance-second capture component” complex. As a result, the magnetic marker is immobilized in the vicinity of the detection unit via the target substance as shown in FIG. 6 by bringing the complex into contact with the first capturing component immobilized on the detection unit. Is also possible.

Then, by measuring the number of magnetic markers immobilized on such a detection unit by some method, it is possible to calculate the number and concentration of target target substances.
The following methods have been proposed as biosensors using such a magnetic detection method.

  An immunoassay in which a magnetic substance as a label is bound to a target substance contained in a liquid composition by an antigen-antibody reaction, the label is magnetized, and the label is detected by a SQUID (superconducting quantum interferometer) as a magnetic sensor A method is disclosed (Patent Document 1). The magnetic marker used here discloses that the magnetic particles of 20 to 40 nm are coated with a polymer and that the size is defined as 40 to 100 nm contributes to the improvement of the sensitivity of the SQUID. Although the SQUID has extremely high detection sensitivity, it is necessary to perform detection in a cryogenic environment using liquid helium, and there is a problem that it is difficult to reduce the size and weight, and requires a high running cost.

Therefore, in order to solve the above problems, a biosensor using a magnetic sensor that is small and can be measured at room temperature has been proposed. There are various types of small magnetic sensors capable of detecting magnetic particles, such as Hall effect elements (Patent Document 2), magnetoresistive elements (Patent Document 3), and magnetic impedance elements (Patent Document 4). It is done.
JP 2001-033455 A WO03-067258 US Pat. No. 5,981,297 JP-A-10-234694

  By the way, in the biosensor, in order to realize a purpose of detecting a small amount of a target substance and a reduction in running cost, the amount of liquid composition necessary for one item of inspection is being reduced. In order to obtain an accurate test result even with a reduced amount of the liquid composition, it is necessary to make the reaction container for bringing the detection unit and the liquid composition into contact as small as possible.

  However, in the case of a biosensor using such a small reaction vessel, it is physically difficult to insert and rotate a stirring rod for mixing the liquid composition, and the reaction efficiency in solid-liquid reaction is low. In combination, a fatal problem may occur in terms of detection time and detection sensitivity.

Therefore, the present invention provides a method for stirring a liquid composition that can stir the liquid composition even if the reaction vessel is small.
The present invention also provides a detection method for a magnetic biosensor capable of outputting a highly reliable and sensitive test result in a short time because the liquid composition can be stirred even when the reaction vessel is small. And a detection kit.

  As a result of intensive studies to solve the above problems, the present inventor has obtained a liquid composition containing a magnetic marker and gel particles, and by transferring the volume phase of the gel particles, the liquid can be effectively liquid even if the reaction vessel is small. The present inventors have found that the composition can be agitated and that the detection time in the magnetic biosensor can be shortened and the detection sensitivity can be improved.

A liquid composition stirring method that solves the above problem is a liquid composition stirring method containing a magnetic marker and gel particles, wherein the gel particles undergo volume phase transition.
A detection method for solving the above-described problem is a detection method for detecting a magnetic marker using a detection element including a detection unit and a non-detection unit. (1) The detection element, the magnetic marker, and a gel particle And (2) a step of volume phase transition of the gel particles, and (3) a step of detecting a magnetic marker present in the vicinity of the detection unit.

  A detection kit for solving the above-described problem is a detection kit used in a detection method for detecting a magnetic marker using a detection element including a detection unit and a non-detection unit, the detection element and the magnetic marker And a liquid composition containing gel particles.

The present invention can provide a method for stirring a liquid composition that can stir the liquid composition even if the reaction vessel is small.
The present invention also provides a detection method for a magnetic biosensor capable of outputting a highly reliable and sensitive test result in a short time because the liquid composition can be stirred even when the reaction vessel is small. And a detection kit can be provided.

Hereinafter, the present invention will be described in detail.
The stirring method according to the present invention is a stirring method of a liquid composition containing a magnetic marker and gel particles, wherein the gel particles undergo volume phase transition.

  The detection method according to the present invention is a detection method for detecting a magnetic marker using a detection element composed of a detection unit and a non-detection unit, and includes (1) the detection element, the magnetic marker, and gel particles. A step of contacting the liquid composition, (2) a step of volume phase transition of the gel particles, and (3) a step of detecting a magnetic marker present in the vicinity of the detection unit.

  The detection kit according to the present invention is a detection kit for use in a detection method for detecting a magnetic marker using a detection element composed of a detection unit and a non-detection unit, the detection element, the magnetic marker, and a gel It has the liquid composition containing particle | grains, It is characterized by the above-mentioned.

The relationship between the zeta potential φ _{1 of the} magnetic marker and the zeta potential φ ₂ of the gel particles is preferably φ ₁ φ ₂ ≧ 0.
Wherein the average particle diameter D ₁ of the magnetic marker, it is preferable that the average relationship particle diameter D ₂ of the gel particles is D ₁ ≦ D _2.

The gel particles are preferably hydrogel particles.
It is preferable that the hydrogel particles contain a temperature-responsive polymer as a constituent component.
FIG. 1 or FIG. 2 shows a schematic diagram of an example of a magnetic marker and a detection element used in the detection method of the present invention. 2 is a magnetic structure, 3 is a first target substance supplement, 4 is a second target substance supplement, 5 is a target substance, 7 is a detection unit, and 8 is a non-detection unit.

In FIG. 1, the magnetic marker 9 has a form having the second target substance supplement 4 on the surface, and FIG. 2 shows the form in which the magnetic marker 9 has the target substance 5 on the surface.
Moreover, as a structure of a detection part and a non-detection part, it can also be set as a structure as shown in FIG. The structure shown in FIG. 3 is a structure in which the base material 1 has a region that becomes a part of the detection unit and a region that becomes a part of the non-detection unit. In the case of the structure as shown in FIG. 3, a portion consisting of the first target substance supplement and a region of the base material 1 where the first target substance supplement 3 is formed on the surface is used as the detection unit 7. 1 is a structure in which a non-detection unit 8 is a portion formed of a region other than the detection unit 7 in 1 and a layer formed on the surface of the region. In addition, when the layer formed in the surface of area | regions other than the said detection part 7 among the base materials 1 is multiple layers, the layer of the outermost surface among the said multiple layers is made into the coating layer.

Further, the magnetic marker may be formed by forming a coating layer 16 on the surface of the magnetic structure as shown in FIG. 4 and forming a first target substance supplement on the coating layer.
In the present invention, a detection element including a detection unit and a non-detection unit is brought into contact with a liquid composition containing a magnetic marker and gel particles, and the presence / absence and number of magnetic markers located near the surface of the detection unit are detected. The present invention relates to a detection method for detecting the presence and concentration of a target substance in a liquid composition.

  FIG. 5a is a diagram schematically showing an example of stirring of magnetic labels by volume phase transition of gel particles in the presence of gel particles. Moreover, 5b is a figure which shows typically the state of the magnetic label in the state in which a gel particle does not exist. In the present invention, as shown in FIG. 5 (a), a gel phase is subjected to volume phase transition, whereby liquid is generated by convection caused by mechanical stirring accompanying the volume change of the gel particles and exchange of solvent molecules into and out of the gel particles. By increasing the mobility of the magnetic marker in the composition, the contact frequency between the magnetic marker and the detection unit is increased, and thus the detection time is shortened and the detection sensitivity is improved.

(Gel particles)
The gel particles of the present invention are characterized by exhibiting a phenomenon (volume phase transition) that suddenly changes from a swollen state to a contracted state due to changes in the external environment such as temperature and pH.

  Furthermore, in this invention, it is preferable that a gel particle is a hydrogel. In general, a hydrogel is defined as a state in which a three-dimensionally crosslinked polymer network contains a large amount of water and swells, but in the present invention, in a broader sense, a hydrophilic network containing a large amount of water. It means an association of functional polymers.

Examples of the environmental change imparted for volume phase transition of the gel particles include temperature change, pH change, and light wavelength change.
The gel particles exhibiting a volume phase transition due to a temperature change will be described.

As the polymer constituting such gel particles, it is preferable to contain a temperature-responsive polymer whose affinity for water changes at least in response to a temperature change.
The temperature-responsive polymer in the present invention is a polymer compound having a lower critical solution temperature. The critical eutectic temperature is a temperature at which the solubility of a substance starts to decrease. In particular, a case where the solubility decreases at a specific temperature or higher is called a lower critical eutectic temperature. Such a temperature-responsive polymer preferably has a thermosensitive site in the polymer chain, and in particular, poly (N-substituted acrylamide), which is a polymer of an N-substituted acrylamide derivative, as the thermosensitive site. However, other than poly (N-substituted acrylamide), another substance can be used as a heat-sensitive site as long as the same effect can be expected.

Next, gel particles that exhibit a volume phase transition due to a change in pH will be described.
As the polymer constituting such gel particles, a charged polymer containing at least a charged functional group such as an amino group or a carboxyl group is preferable. For example, in the case of a chargeable polymer having an amino group or a carboxyl group, each functional group is dissociated or undissociated at any pH region. When the charged functional group is dissociated, the gel particles swell based on the increase in osmotic pressure, and conversely, when the ionic pressure is not dissociated, the gel particles contract based on the decrease in osmotic pressure. Arise. On the other hand, for example, in the case of an amphoteric charged polymer having both an amino group and a carboxyl group, the isoelectricity in a certain pH range is due to the dissociation balance between the positively charged functional group and the negatively charged functional group. Has a point. That is, in the case of gel particles having an amphoteric chargeable polymer as a constituent component, the gel particles are in a contracted state at the isoelectric point, and in other conditions, the gel particles are in a swollen state. However, the charged functional group and the charged polymer in the present invention are not limited to the exemplified compounds, and any compound can be applied as long as the object of the present invention can be achieved.

Next, gel particles that exhibit volume phase transition due to changes in the wavelength of light to be irradiated will be described.
As the polymer constituting such gel particles, a photoresponsive polymer whose affinity for water changes at least in response to a change in the light wavelength to be irradiated is preferable. Examples of such a polymer include azobenzene-containing compounds and polymers thereof. Usually, trans azobenzene having no substituent has hydrophobicity. In the trans azobenzene, the π bond of the azo group is excited by ultraviolet light irradiation (λmax = 320 nm), and isomerization into a cis isomer occurs. Thereby, it has hydrophilicity. The azobenzene-containing compound and the polymer thereof can be preferably applied as the photoresponsive polymer in the present invention because the molecular polarity is greatly different between the trans isomer and the cis isomer. However, the photoresponsive site in the present invention is not limited to an azobenzene-containing compound and a polymer thereof, and any compound can be applied as long as the object of the present invention can be achieved.

(Target substance supplement)
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 liquid composition that reacts with the detection element, and is captured by the target substance capturing body on the surface of the detection unit.

  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 it may be detected by directly capturing the detection target with the capture body, or the target substance and the detection target are different, and the target substance capture body detects the target substance. Alternatively, the detection target may be indirectly detected. 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 a chemical substance (biological substance) present in the living body, and the size is not limited. However, 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. Furthermore, the liquid composition may be a sample itself containing the detection target substance, and is prepared through various processes such as extraction, separation, dilution, and purification of the target substance. May be. The liquid composition is prepared using a liquid medium according to the type of the target substance, for example, water or a buffer solution, a mixture of water and a water-soluble organic solvent, or the like.

  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. Note that these are combinations. Therefore, when “A-B” is described as a target substance-target substance capturing body combination, when A is used as the target substance and B is used as the target substance capturing body, B is used as the target substance, and as the target substance capturing body. Both cases where A is used are shown.

(Magnetic marker)
The magnetic marker used in the present invention has at least a magnetic structure and a second target substance capturing body or target substance that captures a target substance present on the surface of the magnetic structure. Such a magnetic marker should just satisfy | fill the physical property and characteristic as a label | marker for target substance detection in the state which makes a capture body which the detection part has on the surface capture a target substance. Therefore, the magnetic structure can be selected from magnetic paraffin and magnetic paraffin (magnetic beads) exhibiting normal paramagnetism and superparamagnetism.

As a magnetic material constituting the magnetic structure, for example, a metal oxide can be used. Among metal oxides, iron oxide particles such as ferrite and magnetite, which are commonly used as magnetic structures for magnetic markers, have sufficient magnetism under physiological activity conditions, and deteriorate due to oxidation or the like in a solvent. Is preferable because it is difficult to occur. 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 substituted 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, a magnetic structure having a laminated structure can be obtained by using a base made of a magnetic material as a core and forming a coating layer (shell) described later on the base. As such a core-shell magnetic structure, for example, a particle made of a metal oxide was used as a substrate, and the surface of the particle of the substrate made of the metal oxide was selected from resins such as styrene, dextran, and acrylamide. The core-shell particle which coat | covered the polymer layer (resin layer) is mentioned. Here, the styrene resin is a resin made of styrene and a styrene derivative, and the same applies to dextran resins and acrylamide resins. 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, a laminated structure including a metal oxide and a resin layer can be used as a magnetic structure for a magnetic marker. For example, a laminated structure in which the core-shell particles are used as a substrate and a coating layer is further formed on the substrate surface can be used as a magnetic structure. In addition to the core-shell type, particles made of magnetic material are dispersed in styrene, dextran, acrylamide and other resins, and fine particles made of magnetic material are supported on the surface of the resin particles. Particles can also be used as the magnetic structure of the present invention. Examples of such magnetic structures that are commercially available include Dynabeads that are commercially available from Dynal, micromer-M and nanomag-D that are commercially available from Micromod, and Esters that are commercially available from Merck. There are poles etc., and these can also be used.

  The size of the magnetic structure can be variously selected depending on the shape, size, or application of the detection element, but generally the one having a diameter of 3 nm to 500 μm is preferable. More preferably, it has a diameter of 3 nm to 10 μm, and still more preferably has a diameter of 5 nm to 1 μm. The diameter or average particle diameter of the magnetic structure can be measured by a dynamic light scattering method.

  The magnetic marker preferably has a capturing body that captures the target substance on the surface of the magnetic structure. In addition, the target substance possessed by the magnetic marker that the target substance capturing body (hereinafter referred to as the first target substance capturing body) of the detection unit specifically recognizes and captures the first region of the target substance in the liquid composition. It is preferable that the capturing body (hereinafter, second target substance capturing body) specifically recognizes and captures the second region of the target substance in the liquid composition. Here, the second region is a region different from the first region, and indicates at least a part of a region other than the first region in the target substance. In such a case, since both the magnetic marker and the detection unit have the target substance capturing body on the surface, the target substance and the detection unit and the target substance and the magnetic marker interact with each other. That is, both the first target substance capturing body existing on the surface of the magnetic marker and the second target substance capturing body existing on the detection unit surface capture the target substance. Therefore, the magnetic marker is fixed in the vicinity of the surface of the detection unit via the target substance, and the detection unit can detect the magnetic marker and easily detect the target substance.

  Even if the magnetic marker does not have the second target substance capturing body on the surface, the magnetic marker has the target substance on the surface of the magnetic structure, and the target substance is captured on the surface of the detection unit. It is also possible to detect the target substance by capturing the body. In such a case, it is preferable that the target substance that the magnetic marker has on the surface has at least two areas of an area that is immobilized on the magnetic marker and an area that is captured by the target substance capturing body present on the surface of the detection unit.

(Coating layer)
As described above, the magnetic marker and the detection element according to the present invention can have a structure composed of a laminate of a plurality of layers. A laminate comprising a plurality of layers can be obtained, for example, by forming at least one layer on the surface of the substrate. In such a case, the outermost layer of the laminate including a plurality of layers including the substrate is called a coating layer. Therefore, when forming one layer with respect to the surface of a base | substrate, the said layer is called a coating layer.

As the material of these coating layers, for example, a material selected according to the purpose from an inorganic material such as glass, an organic material such as resin, a semiconductor material such as silicon, and a metal material can be used.
Furthermore, when the target substance or the target substance capturing body is a biological substance, it is desirable that these coating layers are hydrophilic layers.

In general, biological substances such as proteins are hydrophobic, and therefore, when the coating layer is hydrophobic, non-specific adsorption to the coating layer may easily occur due to “hydrophobic interaction”.
For example, when the target substance is adsorbed non-specifically other than the first target substance-capturing body on the detection unit surface, the second target substance-capturing body in which the non-specifically adsorbed target substance exists on the surface of the magnetic marker Captures the magnetic marker signal as noise. Such noise can be a factor that lowers detection sensitivity and accuracy. In addition, when the second target substance capturing body on the surface of the magnetic marker or the target substance on the surface of the magnetic marker is adsorbed nonspecifically on the non-detection part or detection part directly without passing through the target substance Similarly, it can be a factor that lowers detection sensitivity and accuracy.

  On the other hand, when the coating layer is hydrophilic, it is possible to reduce “hydrophobic interaction”, which is one of the causes of non-specific adsorption of biological substances, thereby reducing non-specific adsorption of biological substances. It becomes possible.

(Relationship between magnetic markers and gel particles)
The zeta potential is a physical property value defined as the potential at the sliding surface of the electric double layer formed by the surface potential. Since it represents the charged state of the solid surface, it can be used as an important index in explaining the aggregation of particles and the driving force when the particles adsorb to the interface.

In the present invention, the relationship between the zeta potential φ ₁ of the magnetic marker and the zeta potential φ ₂ of the gel particles is characterized by φ ₁ φ ₂ ≧ 0. When the zeta potential has the same sign, an electrostatic repulsive force acts on the magnetic marker and the gel particle, and thus it can be co-dispersed in the liquid composition without aggregating each other. On the contrary, when the zeta potential has an opposite sign, the magnetic marker and the gel particles are associated with each other due to electrostatic attraction, which may cause noise in the detection method of the present invention. . When the zeta potential of the magnetic marker and the gel particle is 0, there is no electrostatic interaction between the particles, so the dispersion system becomes unstable when co-dispersed, but the dispersion system is maintained. Applicable only if any measures are taken. As an example of any measures for maintaining the dispersion system, there is a method of adding a polymer dispersant or a low molecular dispersant to the dispersion system, but the present invention is not limited to this.

In the present invention, the relationship between the zeta potential φ ₂ of the gel particles and the zeta potential φ ₃ of the detection element is φ ₂ φ ₃ ≧ 0. Thus, when the zeta potential has the same sign, an electrostatic repulsive force acts on the gel particles and the detection element, so that it is possible to suppress nonspecific adsorption of the gel particles to the detection element. . Furthermore, when the zeta potential of the magnetic marker and the zeta potential of the gel particle have the same sign, nonspecific adsorption of the magnetic marker to the detection element can be suppressed. On the other hand, when the zeta potential has an opposite sign, it is difficult to suppress non-specific adsorption because an electrostatic attractive force acts on the gel particles, the magnetic marker, and the detection element. When the zeta potential of any one of the gel particles, magnetic marker, and detection element is 0, or any zeta potential is 0, it may be difficult to suppress nonspecific adsorption electrostatically. It is applicable only when some measure to suppress specific adsorption is taken. An example of any measure for suppressing non-specific adsorption includes a method of adding a polymer dispersant or a low molecular dispersant to the dispersion system, but the present invention is not limited thereto.

In the present invention, the relationship between the average particle diameter D ₁ of the magnetic marker and the average particle diameter D ₂ of the gel particles is characterized by D ₁ ≦ D ₂ . More preferably, 5D ₁ ≦ D ₂ , and even more preferably 10D ₁ ≦ D ₂ . The purpose of the present invention is to increase the mobility of the magnetic marker in the liquid composition by stirring the liquid composition by volume phase transition of the gel particle. When the diameter is small, it is difficult and difficult to obtain a sufficient stirring effect. However, the average particle size of the gel particles in the present invention means the particle size in the swollen state.

(Detection element)
The detection element used in the present invention comprises a detection unit having a target substance capturing body on its surface and a non-detection unit. When the target substance is captured by the target substance capturing body, the magnetic marker is fixed in the vicinity of the detection unit. When the magnetic marker is positioned in the vicinity of the detection unit, the detection unit recognizes the magnetic marker, and the target substance is detected by using a change in signal due to the presence or absence of the magnetic marker.

  In the present invention, the detection part of the detection element is a part having a function of measuring the presence / absence / amount of the target substance in the liquid composition according to the presence / absence / amount of the magnetic marker, and the target capturing the target substance on the surface It has a substance trap. In addition, the non-detection part indicates a part other than the detection part in the detection element. The non-detection part and detection part which a detection element has should just adjoin, a part may touch, and the non-detection part may be located in the circumference of a detection part.

  The detection part and the non-detection part may be constituted by a single layer, or may be constituted by a laminate of a plurality of layers. In addition, the surface potential of the detection unit or non-detection unit may be such that the detection unit on the surface of the detection unit or non-detection unit immobilizes a molecule having an active group in advance on the surface of the detection unit, and then immobilizes the capturing body with the active group. Is preferred.

(Detection kit)
The detection kit of this embodiment comprises a magnetic marker and a detection element, and comprises the above-described detection element. That is, the presence or absence of the target substance in the liquid composition is detected by taking the target substance into the detection unit surface of the detection element by contact with the liquid composition, and detecting the presence or number of magnetic markers present near the detection unit surface. The kit can detect the concentration.

(Detection method)
In the detection method of the present invention, the presence / absence and the number of magnetic markers located in the vicinity of the detection unit surface are detected, and the presence / absence and concentration of the target substance in the liquid composition are detected by comparison with a calibration curve prepared in advance. Hereinafter, an example of the detection method will be described below.

(First detection method example)
A first target substance capturing body is immobilized on the surface of the detection unit. Then, a liquid composition is made to contact a detection part. At this time, if the desired target substance is present in the liquid composition, the first target substance capturing body captures the target substance. After washing the liquid composition on the surface of the detection unit and removing unnecessary substances, the solution containing the magnetic marker and gel particles in which the second target substance capturing body for capturing the target substance is immobilized on the surface is washed. It contacts with the detection part surface of a subsequent detection element. Next, the gel particles are subjected to a volume phase transition by an arbitrary environmental change such as a temperature change, a pH change, or a change in light wavelength to be irradiated. It should be noted that the number of volume phase transitions is not limited as long as the present invention can be effectively implemented, even if it is one time or a plurality of times.

  Next, the surface of the detection unit is washed to remove the magnetic marker and gel particles that are not bound to the detection unit. Thereafter, the target substance can be indirectly detected by detecting the magnetic marker.

  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 capturing body is a secondary antibody. At this time, capturing of the target substance in the first target substance capturing body means an antigen-antibody reaction.

(Second detection method example)
Similar to the first example, the first target substance capturing body is immobilized on the surface of the detection unit. Next, the target substance is immobilized on the surface of the magnetic marker. After the liquid composition containing the magnetic marker having the target substance immobilized thereon is brought into contact with the surface of the detection unit, the surface of the detection unit is washed to remove the magnetic marker that has not bound to the detection unit. Thereafter, the target substance can be indirectly detected by detecting the magnetic marker.

  Examples of such a system include a case where the target substance is an antigen and the first target substance capturing body is an antibody, and a case where the target substance is an antibody and the first target substance capturing body is an antigen.

  In any of the first and second examples, any means may be used as the detection means as long as it is a method for detecting a magnetic marker existing in the vicinity of the detection unit surface. Among them, a method using a magnetic field effect when a magnetic marker is present on the detection unit surface is preferable, and in particular, a magnetoresistive effect element, a Hall effect element, and a superconducting quantum interferometer element can be suitably used.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.
(Synthesis of magnetic structure)
Magnetite hydrophobized with oleic acid (particle size 8 nm) is dispersed in a mixed solution of styrene and glycidyl methacrylate, and the dispersion is further mixed with an aqueous sodium dodecyl sulfate solution to obtain a mixed solution. This mixture was emulsified using an ultrasonic homogenizer, and then subjected to nitrogen bubbling for 30 minutes, and 2,2′-azobis (2-methylpropionamidine) dihydrochloride (2,2′-Azobis (2-methylpropionamide) dihydrochloride (hereinafter referred to as V-50) is added and polymerized at 70 ° C. Two hours after the start of the polymerization, glycidyl methacrylate is added, and further polymerized at 70 ° C. for 20 hours to obtain the magnetic structure 1. After the magnetic structure 1 is purified by centrifugation, the magnetic structure 1 is added to an aminoethanethiol aqueous solution and reacted at room temperature and pH 9 for 20 hours. The magnetic structure 2 in which amino groups and hydroxyl groups coexist on the surface. Get.

  Further, the magnetic structure 1 is added to an aqueous mercaptoacetic acid solution and reacted at room temperature and pH 9 for 20 hours to obtain a magnetic structure 3 in which carboxyl groups and hydroxyl groups are mixed on the surface. When the average particle diameter of the magnetic structure 2 in water is evaluated by DLS8000 (Otsuka Electronics), it is confirmed that the magnetic structure 2 is 275 nm. Further, when the zeta potential was evaluated by ZEECOM (Microtech Nithion), it was confirmed to be +12 mV. Similarly, when the average particle diameter of the magnetic structure 3 in water is evaluated, it is confirmed to be 283 nm. Moreover, when zeta potential was evaluated, it was confirmed that it was −15 mV.

(Synthesis of hydrogel particles)
N-isopropylacrylamide, methylenebisacrylamide, glycidyl methacrylate and azobisisobutyronitrile are dissolved in toluene, and nitrogen substitution is performed for 30 minutes by nitrogen bubbling. Further, this solution is mixed with a sodium dodecyl sulfate aqueous solution to obtain a mixed solution. The mixture is emulsified with a shearing homogenizer, then subjected to nitrogen bubbling for 30 minutes, and heated to 60 ° C. for polymerization. Two hours after the start of the polymerization, acrylamide, methylenebisacrylamide and glycidyl methacrylate dissolved in ethanol are added, and further polymerized at 60 ° C. for 20 hours to obtain gel particles. After the gel particles are purified by centrifugation, the gel particles are added to an aminoethanethiol aqueous solution and reacted at room temperature and pH 9 for 20 hours to obtain aminated gel particles in which amino groups and hydroxyl groups are mixed on the surface. Further, the gel particles are added to an aqueous mercaptoacetic acid solution and reacted at room temperature and pH 9 for 20 hours to obtain carboxylated gel particles in which carboxyl groups and hydroxyl groups are mixed on the surface. After purification by centrifugal separation, classification is performed by sedimentation and centrifugal separation to obtain the following evaluation results.
Hereinafter, physical properties of gel particles 1 to gel particles 5 having different average particle diameters produced by appropriately changing the shear rate of the shear type homogenizer will be shown.

(1) Gel particle 1
It is confirmed that the average particle size in water at 34 ° C. is 12 μm and the zeta potential is +8 mV, the average particle size in water at 38 ° C. is 5 μm and the zeta potential is +19 mV.
(2) Gel particle 2
It is confirmed that the average particle size in water at 34 ° C. is 3 μm and the zeta potential is +7 mV, the average particle size in water at 38 ° C. is 1.4 μm and the zeta potential is +21 mV.
(3) Gel particle 3
It is confirmed that the average particle size in water at 34 ° C. is 1.5 μm and the zeta potential is +7 mV, the average particle size in water at 38 ° C. is 0.8 μm and the zeta potential is +21 mV.
(4) Gel particle 4
It is confirmed that the average particle size in water at 34 ° C. is 0.3 μm and the zeta potential is +9 mV, the average particle size in water at 38 ° C. is 0.15 μm and the zeta potential is +24 mV.
(5) Gel particle 5
It is confirmed that the average particle size in water at 34 ° C. is 9 μm and the zeta potential is −12 mV, the average particle size in water at 38 ° C. is 5 μm, and the zeta potential is −29 mV.

(Confirmation of stirring effect based on volume phase transition of gel particles)
A dispersion liquid in which the magnetic structure 2 is dispersed in distilled water is dropped on a slide glass, covered with a cover glass, placed on a hot plate, and observed in a dark field mode of an optical microscope. It is confirmed that no change is observed in the mobility of the magnetic structure 2 when the temperature of the hot plate is raised from 34 ° C. to 38 ° C. and lowered from 38 ° C. to 34 ° C.

  A dispersion obtained by dispersing the magnetic structure 2 and the gel particles 1 in distilled water is dropped onto a slide glass, covered with a cover glass, placed on a hot plate, and observed in a dark field mode of an optical microscope. When the temperature of the hot plate is raised from 34 ° C. to 38 ° C. and lowered from 38 ° C. to 34 ° C., a rapid change in the mobility of the magnetic structure 2 is confirmed.

  A dispersion in which the magnetic structure 2 and the gel particles 2 are dispersed in distilled water is dropped onto a slide glass, covered with a cover glass, placed on a hot plate, and observed in a dark field mode of an optical microscope. When the temperature of the hot plate is raised from 34 ° C. to 38 ° C. and lowered from 38 ° C. to 34 ° C., a rapid change in the mobility of the magnetic structure 2 is confirmed.

  A dispersion liquid in which the magnetic structure 2 and the gel particles 3 are dispersed in distilled water is dropped on a slide glass, covered with a cover glass, placed on a hot plate, and observed in a dark field mode of an optical microscope. When the temperature of the hot plate is raised from 34 ° C. to 38 ° C. and lowered from 38 ° C. to 34 ° C., a gradual change is confirmed in the mobility of the magnetic structure 2.

  A dispersion in which the magnetic structure 2 and the gel particles 4 are dispersed in distilled water is dropped onto a slide glass, covered with a cover glass, placed on a hot plate, and observed in a dark field mode of an optical microscope. When the temperature of the hot plate is raised from 34 ° C. to 38 ° C. and lowered from 38 ° C. to 34 ° C., it is confirmed that no significant change is observed in the mobility of the magnetic structure 2.

  When a dispersion liquid in which the magnetic structure 3 and the gel particles 3 are dispersed in distilled water is dropped on a slide glass, placed on a hot plate with a cover glass, and observed in the dark field mode of an optical microscope, an aggregate is formed. To be confirmed.

(Preparation of magnetic marker)
In the presence of water-soluble carbodiimide (WCS), the magnetic structure 2 is reacted with a phosphate buffer solution in which an anti-PSA antibody (secondary antibody) is dissolved to obtain a magnetic marker by immobilizing the antibody on the surface of the magnetic structure 2 Purify by centrifugation. When the zeta potential of the magnetic marker is measured, it is confirmed to be +9 mV.

Example 1
In this example, a biosensor that detects prostate specific antigen (PSA) using the magnetic marker of the present invention will be described. A magnetoresistive effect element is used as a biosensor detection method.

  In order to carry the primary antibody on the surface of the Au film formed on the detection part of the biosensor, the surface of the Au film is first subjected to hydrophilic pretreatment and then treated with an aminosilane coupling agent. Furthermore, a primary antibody that captures a desired antigen by binding between the amino group derived from the aminosilane coupling agent and the peptide chain using a cross-linking agent such as glutaraldehyde for immobilizing the primary antibody is a detection part of the biosensor. It is fixed to. Thus, when the detection part which fix | immobilized the primary antibody was immersed in the dispersion liquid of the gel particle 1 and the dispersion liquid of the gel particle 5 respectively for a fixed time, it was observed that only the gel particle 5 adsorb | sucks to a detection part, It is predicted that the zeta potential value of the detection unit is positive.

Using this biosensor, detection of PSA (isoelectric point 7.5) known as a marker for prostate cancer can be attempted according to the following protocol.
(1) The detection part of the biosensor is immersed in phosphate buffered saline containing PSA which is an antigen (target substance) and incubated for 5 minutes.
(2) Unreacted PSA is washed with phosphate buffered saline.
(3) The detection part of the biosensor after steps (1) and (2) is immersed in a magnetic marker and a phosphate buffer containing gel particles 1 to 5 respectively, and incubated for 3 minutes. Meanwhile, on the hot plate, the operation of raising the temperature of the phosphate buffer from 34 ° C. to 38 ° C. and lowering the temperature from 38 ° C. to 34 ° C. is performed for one cycle.
(4) Unreacted magnetic markers and gel particles are washed with a phosphate buffer.

As a comparative experiment, the same operation is performed for the phosphate buffer containing only the magnetic marker not containing gel particles in the step (3).
According to the above protocol, in the experiment using the gel particles 1 to the gel particles 3, it is confirmed that a large number of magnetic markers are fixed to the detection part with almost no aggregation. In the experiment using the gel particle 4 and the blank experiment, it was confirmed that the magnetic marker was fixed to the detection unit. However, the number of the magnetic marker clearly fixed was different from the experiment using the gel particle 1 to the gel particle 3. It is confirmed that there are few. In the experiment using the gel particles 5, it is confirmed that the magnetic marker and the aggregate of the gel particles 5 are attached to the detection part.

  In experiments using gel particles 1 to 4 and blank experiments, it is possible to detect the appropriate signal intensity from the magnetic marker by the magnetoresistive effect element, and to detect PSA as the target substance. Is done. However, in the experiment using the gel particles 1 to 3, the signal intensity detected from the magnetic marker is larger than the experiment using the gel particles 4 and the blank experiment, and the target substance can be made highly efficient in a short time. The possibility of being detected is suggested.

Example 2
The detection method is changed from the magnetoresistive element of Example 1 to the Hall effect element, and evaluation is performed in the same manner as in Example 1. As a result, it is confirmed that the same experimental result as in Example 1 can be obtained.

Example 3
The detection method is changed from the magnetoresistive element of Example 1 to a superconducting quantum interferometer element, and evaluation is performed in the same manner as in Example 1. As a result, it is confirmed that the same experimental result as in Example 1 can be obtained.

  Since the present invention can stir a liquid composition even if the reaction vessel is small, and can output a highly reliable and highly sensitive test result in a short time, as a detection method for a magnetic biosensor. Can be used.

It is a figure which shows typically an example of the relationship between a magnetic label | marker and a detection element. It is a figure which shows typically an example of the relationship between the detection element which consists of a non-detection part and a detection part, and the magnetic label | marker which has a target substance on the surface. It is a figure which shows typically an example of the relationship between the detection element which consists of a non-detection part which has a coating layer on the surface, and a detection part, and a magnetic label. It is a figure which shows typically an example of the relationship between the detection element which consists of a non-detection part which has a coating layer on the surface, and a detection part, and the magnetic label | marker which has a coating layer on the surface. It is a figure which shows typically an example of the stirring of the magnetic label | marker by the volume phase transition of a gel particle in the state in which a gel particle exists. It is a figure which shows typically the state of the magnetic label in the state where a gel particle does not exist. It is a figure for demonstrating the solid-phase analysis method using the conventional magnetic label | marker.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Base material 2 Magnetic structure 3 1st target substance capture body 3a 1st capture body component 4 2nd target substance capture body 4a 2nd capture body component 5 Target substance 7 Detection part 8 Non-detection part 9 Magnetic marker DESCRIPTION OF SYMBOLS 10 Substrate 14 Second target substance capturing body 16 Coating layer 17 Shrinked gel particles 18 Swelled gel particles

Claims (15)

  A method for stirring a liquid composition comprising a magnetic marker and gel particles, wherein the gel particles undergo volume phase transition. The method for stirring a liquid composition according to claim 1, wherein the relationship between the zeta potential φ _{1 of the} magnetic marker and the zeta potential φ ₂ of the gel particles is φ ₁ φ ₂ ≧ 0. Wherein the average particle diameter D ₁ of the magnetic marker, stirring method according to claim 1 or 2 Liquid composition according average relationship particle diameter D ₂ of the gel particles is characterized by a D ₁ ≦ D _2.   The method for stirring a liquid composition according to any one of claims 1 to 3, wherein the gel particles are hydrogel particles.   The method for stirring a liquid composition according to any one of claims 1 to 4, wherein the hydrogel particles contain a temperature-responsive polymer as a constituent component.   In the detection method of detecting a magnetic marker using a detection element comprising a detection unit and a non-detection unit, (1) a step of bringing the detection element into contact with a liquid composition containing the magnetic marker and gel particles (2) a step of volume phase transition of the gel particles, and (3) a step of detecting a magnetic marker existing in the vicinity of the detection unit. And zeta potential phi ₁ of the magnetic marker, the a is φ ₁ φ ₂ ≧ 0 relationship zeta potential phi ₂ of the gel particles, the relationship of zeta potential phi ₃ zeta potential phi ₂ and the detection element of the gel particles The detection method according to claim 6, wherein φ ₂ φ ₃ ≧ 0. Wherein the average particle diameter D ₁ of the magnetic marker according to claim 6 or 7 detecting method, wherein the average relationship particle diameter D ₂ of the gel particles is D ₁ ≦ D _2.   The detection method according to claim 6, wherein the gel particles are hydrogel particles.   The detection method according to claim 6, wherein the hydrogel particles contain a temperature-responsive polymer as a constituent component.   A detection kit for use in a detection method for detecting a magnetic marker using a detection element comprising a detection part and a non-detection part, the liquid composition comprising the detection element, the magnetic marker and gel particles A detection kit comprising: And zeta potential phi ₁ of the magnetic marker, the a is φ ₁ φ ₂ ≧ 0 relationship zeta potential phi ₂ of the gel particles, the relationship of zeta potential phi ₃ zeta potential phi ₂ and the detection element of the gel particles The detection kit according to claim 11, wherein φ ₂ φ ₃ ≧ 0. Wherein the particle diameter D ₁ of the magnetic marker, claim 11 or 12 detection kit according relationships particle diameter D ₂ of the gel particles is characterized by a D ₁ ≦ D _2.   The detection kit according to claim 11, wherein the gel particles are hydrogel particles.   The detection kit according to claim 11, wherein the hydrogel particles contain a temperature-responsive polymer as a constituent component.

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